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Fertility in Belgian Blue Bulls (De vruchtbaarheid van Belgisch Witblauwe stieren)

Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Diergeneeskundige Wetenschappen aan de Faculteit Diergeneeskunde,

Universiteit Gent, september 2006

door

Geert Hoflack Merelbeke 2006

Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine

Ghent University

Promotoren: Prof. Dr. Ann Van Soom Copromotor: Prof. Dr. Dr. h.c. Aart de Kruif

Prof. Dr. Geert Opsomer

ISBN-NUMMER: 90 - 5864 - 101 - 5

EAN: 9789058641014

FACULTY OF VETERINARY MEDICINE Department of Reproduction, Obstetrics and Herd Health

Fertility in Belgian Blue Bulls (De vruchtbaarheid van Belgisch Witblauwe stieren)

Geert Hoflack

Thesis to obtain the academic degree of Doctor of Veterinary Science (Phd)

Faculty of Veterinary Medicine, Ghent University

2006

Promotor: Prof. Dr. Ann Van Soom

Prof. Dr. Geert Opsomer

Co-promotor: Prof. Dr. Dr. h.c. Aart de Kruif

De uitgave van dit proefschrift werd mede mogelijk gemaakt door de financiële steun van :

Aan Bram en Tim

Aan Ingeborgh

Aan Jacques

TABLE OF CONTENTS

List of abbreviations

General Introduction .................................................................................................................. 3

Aims of the Study..................................................................................................................... 15

Chapter 1: Assessing Bull Fertility: The Breeding Soundness Evaluation.............................. 19

Chapter 2: Breeding Soundness and Libido Examination of Belgian Blue and Holstein Friesian Artificial Insemination Bulls in Belgium and The Netherlands ..................................................................................................... 49

Chapter 3: Quality comparison of Belgian Blue and Holstein - Friesian sperm...................... 71

Chapter 3A: Comparison of Sperm Quality of Belgian Blue and Holstein Friesian Bulls 73

Chapter 3B: Objective Sperm Motility Comparison of Belgian Blue and Holstein Friesian Bulls .................................................................................. 99

3B1 Validation and Usefulness of the Sperm Quality Analyzer (SQA II-C) for Bull Semen Analysis ................................................. 101

3B2 Comparison of Computer Assisted Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein Friesian Bulls ....................................................................... 125

Chapter 4: Testicular Dysfunction is Responsible for Low Sperm Quality in Belgian Blue Bulls ............................................................................................... 151

General Discussion................................................................................................................. 175

Summary .............................................................................................................................. 205

Samenvatting.......................................................................................................................... 213

Dankwoord ............................................................................................................................. 219

Curriculum Vitae.................................................................................................................... 227

Bibliography........................................................................................................................... 229

LIST OF ABBREVIATIONS

AAA All Abnormal Acrosomes

AHS Abnormal Headshapes

AI Artificial Insemination

ALH Amplitude of the Lateral Head displacement

AM Abnormal Midpieces

AT Abnormal Tails

BB Belgian Blue

BCF Beat Cross Frequency

BSE Breeding Soundness Evaluation

CASA Computer Assisted Sperm Analysis

CV Coefficient of Variation

D Deferred potential breeder

DH Detached Heads

DPD Distal Protoplasmic Droplet

EF East Flemish

HF Holstein Friesian

HF-BB Holstein Friesian – Belgian Blue crossbred

HTR Hamilton Thorne

IVP In Vitro Production

LVS Low VSL cut-off value

LVV Low VAP cut-off value

M Missing values

mh muscular hypertrophy

MED Medium rapid moving spermatozoa

MOT Totally Motile spermatozoa

MVV Medium VAP cut-off value

N Normal sperm cells

ns not significant

NPV Negative Predictive Value

NRR Non-Return Rate

NRRc Non-Return Rate corrected

OAH Other Abnormal Heads

PMOT Progressively Motile spermatozoa

PPD Proximal Protoplasmic Droplet

PPV Positive Predictive Value

PSS Physiological Saline Solution

RAP Rapid moving spermaozoa

S Satisfactory potential breeder

SC Scrotal Circumference

SD Standard Deviation

SMI Sperm Motility Index

SQA Sperm Quality Analyzer

St dev Standard Deviation

STR Straightness

TALP Tyrode solution with Albumin, Lactate and Pyruvate

TSO Total Sperm Output

U Unsatisfactory potential breeder

VAP Velocity Average Path

VSL Velocity Straight Line

GENERAL INTRODUCTION

General Introduction

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The cattle population in Belgium is estimated to consist of approximately 520.000 dairy cows, mainly Holstein Friesian (HF), and 480.000 beef cows, mainly Belgian Blue (BB). The double-muscled BB beef breed, which is famous for its low feed conversion ratio, its high percentage of lean meat and its advantageous carcass classification, stems from the Durham Shorthorn. The latter breed was introduced in Belgium in 1841 in a first organized attempt to improve the Belgian cattle breeds, and it was crossed with local dairy breeds in order to increase muscularity and precocity. Although the Durham fell into disfavour as its crosses were too fat, this breed left its traces, resulting in a breed called the “Blue of Limon” which was further mixed with local breeds. During the “Great depression” which started in 1880, Belgian agriculture shifted from cereal farming to livestock production, as a result of massive cereal imports from North America. In the Condroz, a breed was selected for musculature, and as it was superior to the Durham cross-breeds, it became very influencial. In 1938, selection for a white colour was started, resulting in the “Mid and Upper Belgian White”. However, food shortage after the Second World War directed Belgian cattle selection towards even better muscled animals, and the breed evolved to the “Mid and Upper Belgian Breed”, allowing colour diversity. The post-War era coincided with the development of artificial insemination. This allowed for the massive dispersion of the genes of ‘Gédéon du Vieux-Château de Maurenne’, heterozygous carrier of the mh gene and founder of the present BB breed. Soon after him, homozygous animals were born, and the development of the caesarean section allowed the initial dual-purpose breed to evolve to the specialized BB beef breed as we know it today (Hanset, 1996; Coopman et al., 2001).

In cattle livestock, reproduction is the essential prerequisite for production and thus for potential economic gain. Good fertility of both bulls and cows is imperative for adequate reproductive performance. Male fertility is an important factor influencing the reproductive efficacy since, in cattle, a single bull generally breeds 20 - 100 cows per season (Chenoweth, 1986b). In case of artificial insemination (AI), a single ejaculate is divided in at least 300 insemination doses, which results in bulls producing several 100.000 AI doses per year, and good sperm quality in these diluted straws is important to obtain pregnancies. Despite the bull’s pivotal role in reproduction, cows generally receive more interest, both from a scientific and a practical point of view (Chenoweth, 1997a; Parkinson, 2004). However, as no individual herd member bears as much responsibility for the herd fertility as the sire (Barth, 1997; Hoflack and de Kruif, 2003), knowledge of a bull’s reproductive capacity is of paramount importance to achieve breeding success.

In Belgium, AI is predominantly used in HF, while in the BB, natural service is also frequently used. In contrast to BB cow fertility, which is monitored well to control the negative effects of the routinely applied caesarean section on fertility, a profound investigation of BB bull fertility is only rarely performed. However, disappointing pregnancy


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rates frequently occur in breeding herds using natural service BB bulls. To avoid a poor pregnancy outcome, specific breeding management practices based on empirical farming experience rather than on scientific research are applied, including practices such as limiting the number of females to 15 – 25 per bull (depending on the bull’s age), applying a prolonged breeding season from May to October, and supplementing the bull’s diet with a daily portion of approximately 700 g concentrates / 100 kg body weight (Hoflack and de Kruif, 2003). However, to minimize herd fertility problems and given the bull’s responsibility to herd fertility, a thorough assessment of the BB bulls’ fertility potential might identify possible bull fertility weaknesses and prevent herd fertility problems (Barth, 1997, Hoflack and de Kruif, 2003). To date, this has not been performed in the BB breed. Moreover, detailed data on the general BB sperm quality have not been described yet. Nonetheless, BB bull fertility is under suspicion, since it has been reported that scrotal circumference in young BB bulls is rather low and this might negatively influence semen characteristics (Barth, 1997; Hanset, 2000). Fertility of BB bulls should definitely be thoroughly investigated. The easiest way to directly assess BB bull fertility would be to look at the breed average non-return rates (to service) and compare them to another breed, such as the HF breed which is a popular breed in Belgium of which much AI and literature data are available. Consequently, the HF breed can be used as the ‘gold standard’ to which the BB breed is compared. However, as BB cows generally calve by means of caesarean section and HF cows per vias naturales, post partum complications might bias the non-return rate breed comparison. Furthermore, the currently used insemination doses of both breeds differ considerably, and the insemination dose of BB bulls is moreover adjusted depending on the quality of the fresh ejaculate (personal observations), rendering non-return rate breed comparisons idle. Hence, other fertility estimates should be used.

Breeding soundness evaluations (BSE) of bulls have been used extensively over the past 50 years to assess a bull’s reproductive potential prior to breeding (Ott, 1986; Barth, 1997). The evaluation of a bull’s breeding soundness potential consists of a general physical soundness examination, a genital tract examination of both the external and internal genitalia, and a semen quality evaluation (Bruner and Van Camp, 1992; Chenoweth et al., 1994; Garner, 1997). It provides a reliable, quick and cost-effective method for screening and classifying bulls in terms of fertility, allowing to exclude bulls with impediments to fertility and to select for bulls with traits favourable for high fertility (Chenoweth et al., 1994).

Although the BSE assesses several important characteristics necessary for good fertility, it does not at all deal with the willingness and eagerness of a bull to mount and attempt service (= libido) and with the ability to complete service (= mating ability), since during breeding soundness evaluations worldwide, semen is generally collected by means of electro-ejaculation, although the latter practice has been banned in some countries due to animal welfare issues. Notwithstanding the fact that bulls are classified as satisfactory potential


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breeders, it is very well possible that such bulls are incapable of impregnating cows when the will and ability to service cows is absent (Chenoweth, 1986a, Barth et al., 2004). Furthermore, it was demonstrated that bulls with higher sex-drive obtained better pregnancy rates compared with lower sex-drive bulls (Blockey, 1978; Makarechian and Farid, 1985; Blockey, 1989, Farin et al., 1989), although this effect is most evident over short mating periods and is partially nullified in case of a long breeding season (Silva-Mena et al., 2002; Parkinson, 2004). Hence, bull libido and mating ability must be considered as important contributing factors to good male fertility. Consequently, in addition to the BSE, and to avoid the selection of bulls unwilling or unable to serve cows, tests for bull libido and/or serving capacity should be included (Chenoweth, 1986a; Chenoweth et al., 1994; Barth, 1997; Garner, 1997; Parkinson, 2004). To evaluate libido and serving capacity, observing natural breeding is the most efficient but very time consuming (Bruner and Van Camp, 1992). Hence, various other testing methods have been investigated, such as pasture and corral trials with restrained or unrestrained, estrual and nonestrual females, where bulls are tested either individually or group wise for different periods of time (Chenoweth, 1986a; Barth, 1997). During such serving capacity tests, the number of services within a short period of time is counted. This yields sufficient information, since a successful test requires both good libido and mating ability (Chenoweth, 1997b).

The evaluation of potential breeding soundness of a bull consists of several aspects, among which semen quality evaluation is a substantial element (Bruner and Van Camp, 1992; Chenoweth et al., 1994; Garner, 1997). In all, semen analysis is probably the most relevant procedure to evaluate male fertility potential (Chong et al., 1983; Vantman et al., 1989; Comhaire et al., 1992, Rijsselaere et al., 2002, Phillips et al., 2004). Several methods can be used to evaluate the quality of a fresh ejaculate or of frozen-thawed semen, but subjective evaluation using standard optical microscopy is by far most commonly used (Rijsselaere et al., 2003; Christensen et al., 2005). The semen parameters that are routinely examined using standard optical microscopy are the concentration, the percentage of motile spermatozoa and their morphology (Neuwinger et al., 1990a, Phillips et al., 2004). Concentration can be determined by means of a counting chamber. Motility, both total and progressive, is generally estimated subjectively on a pre-warmed glass slide. Morphology can be assessed using different techniques, but supravital staining procedures such as eosin – nigrosin staining are commonly used and allow both a live-dead assessment and a morphology differentiation (Hancock, 1951; Bangham and Hancock, 1955; Barth and Oko, 1989).

However, the subjective evaluation of sperm count, motility and morphology has been shown to be relatively inaccurate and imprecise as a result of subjectivity and variability (Davis and Katz, 1993, Christensen et al., 1999; 2005). Visual sperm motility assessment is difficult and is influenced by the temperature and the level of training and skills of the investigator, leading to high variability among laboratories and observers examining the same


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specimens (Knuth et al., 1989; Davis and Katz, 1993). Hence, the subjective visual assessment of semen motility of the same sperm samples yielded coefficients of variation of 20% between technicians, and up to 37% between laboratories (Jequier and Ukombe, 1983; Dunphy et al., 1989; Neuwinger et al., 1990b). The need for standardization of semen analysis to reduce this high variability has been demonstrated repeatedly (Chong et al., 1983; Davis and Katz, 1993; Verstegen et al., 2002).

Different systems to overcome this variability, such as turbidimetry, laser-doppler-spectroscopy and photometric methods have been proposed (Johnston et al., 1995; Iguer-ouada and Verstegen, 2001a). However, these techniques are too imprecise or too complicated to be applied routinely, even for human purposes (Comhaire et al., 1992). Since the eighties, the Sperm Quality Analyzer (SQA) was introduced as a practical and inexpensive device to evaluate overall semen quality (Bartoov et al., 1981). The SQA does not require parameter settings, reducing a large source of bias. It uses light passing through a capillary tube containing the motile semen sample and registers fluctuations in optical density, which result from moving particles. These fluctuations are registered by a photometric cell and converted digitally to a numerical output, the sperm motility index (Bartoov et al., 1991; Johnston et al., 1995). Previous versions of this system were validated both in human (Bartoov et al., 1991; Johnston et al., 1995; Mahmoud et al., 1998; Martinez et al., 2000) and in domestic animal species (Bartoov et al., 1981; Mc Daniel et al., 1998; Parker et al., 2000; Iguer–ouada and Verstegen, 2001b; Neuman et al., 2002; Fukui et al., 2004), but an upgrade version of the SQA (the SQA-IIC: Medical Electronic Systems Ltd., Tirat Carmel, Israel) has not yet been validated for bull semen analysis.

Another promising technology, computer assisted semen analysis (CASA), was introduced successfully both in human and in different animal species including the bull (Vantman et al., 1989; Günzel – Apel et al., 1993; Farrell et al., 1995; 1996). CASA systems procure detailed, accurate, and highly repeatable data on different semen motility parameters simultaneously, such as total and progressive motility, slow, medium and rapid moving spermatozoa, linearity of sperm movement, the beat cross frequency, the amplitude of the lateral head displacement and several velocity parameters. This accuracy and repeatability largely reduces the subjectivity and overcomes the variability inherent to the routine microscopical semen examination (Verstegen et al., 2002). However, several parameters related to internal image settings, such as minimum contrast, frame acquisition rate, analysis time, the number of fields and cells analyzed or related to semen handling and processing, such as semen concentration, diluent, analysis temperature, the chamber used, and the sperm equilibration time before assessment all influence the CASA outcome (Verstegen et al., 2002; Rijsselaere et al., 2003; 2005). Consequently, these semen handling procedures and parameter settings should all be standardized after validation and described in detail to enable the comparison of results obtained by different laboratories (Davis and Katz, 1993; Verstegen et


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al., 2002). When using the same CASA standards, subtle differences in sperm motility can be observed.

Compared to semen motility, the accurate assessment of morphology has been demonstrated to be even more problematic. Jequier and Ukombe (1983) reported between 5 and 85% of abnormal spermatozoa when several professional observers assessed the same semen sample and between 25 and 40% spermatozoa with an abnormal morphology when highly experienced technicians performed the analysis, demonstrating the high variability inherent to subjective morphology evaluation. Morphological evaluation not only depends on the fixation and staining technique, the quality of the microscope, and the morphology classification used, but most importantly on the observer’s experience and skills. Another problem related to several staining procedures, among which the eosin–nigrosin staining, is the fact that some spermatozoa show partial staining making interpretation of the live-dead assessment difficult (Hancock, 1957). In case of the eosin–nigrosin staining, differences between membrane intact (i.e. white) and membrane damaged (i.e. red) spermatozoa are sometimes very subtle, since some spermatozoa merely show a slight pink discoloration. Furthermore, the generally low number of morphologically evaluated spermatozoa out of a heterogeneous ejaculate adds to the variation in results. Hence, subjective morphology assessment is to some extent considered inaccurate and imprecise (Davis and Katz, 1993; Coetzee et al., 1999; Hallap et al., 2005), partially because too few spermatozoa are evaluated (Kuster et al., 2004).

The spermiogram of a bull is in essence a retrospective snapshot of the health of the seminiferous epithelium at the time of spermiogenesis of the observed sperm cells, the health of the epididymis during transit and storage of these spermatozoa, but also of the health of the accessory sex glands at the time of ejaculation. Hence, the spermiogram reflects the testicular function at the moment of sperm production, although this can be partially influenced by the ability of the epididymal epithelium to retrieve a proportion of abnormal spermatozoa and to influence spermatozoa during transit, as well as by the semen collection protocol (Barth and Oko, 1989). Sperm abnormalities, and in particular primary sperm abnormalities are the result of dysfunctional testicular tissue. This testicular dysfunction can be caused by hereditary predispositions (Chenoweth, 2005) or by the deleterious influence of adverse environmental conditions (possibly exacerbating hereditary predispositions), such as disturbances in testicular heat regulation, systemic illness, injury and/or pain, toxicity, nutritional deficiencies and environmental changes (Barth and Oko, 1989; Barth, 1997; Johnson, 1997). Hence, assessing sperm morphology is an attempt to evaluate the testicular health and the impact of deleterious influences on the produced sperm. To avoid the subjectivity and variability inherent to sperm morphology assessment, it might be more efficacious to immediately evaluate the testicular health by means of a histopathological examination of a testicular biopsy. For instance, in case of persistently poor semen quality, testicular biopsies could be


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sampled to directly evaluate the condition of the testicular tissue, in order to get a better view of the (severity of the) problem and to be able to formulate a prognosis (Heath et al., 2002; Papa and Leme, 2002). Similarly, at slaughter, testicular tissue can be collected and examined in order to explain certain sperm abnormalities that were generally encountered in these bulls’ ejaculates (Settergren and McEntee, 1992).

Since practice suggests that BB bull fertility should be questioned, and as non-return rates can not be indisputably interpreted, it might be very interesting to evaluate BB bulls using the above-mentioned techniques. Comparing BB results to results of HF bulls, the other breed predominant in Belgium acting as the fertility ‘gold standard’ will allow to fully qualify the BB results.


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AIMS OF THE STUDY

Aims of the Study

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The general aim of the present thesis was to elucidate whether BB bulls contribute to BB herd fertility problems which are frequently encountered in practice. We hypothesized that BB bull fertility is suboptimal and is (partly) responsible for disappointing pregnancy results, magnifying possible detrimental effects on fertility of the generally applied caesarean section deliveries. We furthermore wanted to inventory which were the most important male factors for the BB subfertility and, if possible, to find their etiology. As sperm morphology and motility largely affect bull fertility, special attention was paid to these attributes. Furthermore, objective methods for sperm motility assessment were investigated. We examined a cross section of the sexually active BB bull population and compared these BB bulls to similarly examined Holstein Friesian bulls in order to have a ‘fertility reference’. To increase the efficiency of data gathering, we chose to examine bulls residing at artificial insemination centres.

The more specific aims of the present study were:

1. To compare the breeding soundness and libido of Belgian Blue and Holstein Friesian

bulls used for artificial insemination in Belgium and The Netherlands, to find out if male fertility problems were present, and if so, to define these problems (Chapter 2).

2. To compare the sperm quality of Belgian Blue and Holstein Friesian bulls, with special attention to morphological differences (Chapter 3A)

3. To test the usefulness of the Sperm Quality Analyzer (SQA II-C) for bull semen motility analysis (Chapter 3B1)

4. To investigate differences in sperm motility and sperm motion patterns of Belgian Blue and Holstein Friesian ejaculates by means of computer assisted sperm motility analysis (Chapter 3B2)

5. To investigate whether testicular degeneration is responsible for breed differences in Belgian Blue and Holstein Friesian bull fertility and to examine possible causes for degeneration (Chapter 4).

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CHAPTER 1

ASSESSING BULL FERTILITY: THE BREEDING SOUNDNESS EVALUATION

Modified from: Assessing Bull Fertility: The Breeding Soundness Evaluation G. Hoflack, G. Opsomer, D. Maes, A. de Kruif, A. Van Soom The Flemish Veterinary Journal 2006; 75: 216 - 227

Chapter 1: Assessing Bull Fertility

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ABSTRACT

Fertility is of paramount importance in bovine livestock management. While in Belgium cow fertility is generally monitored quite well, a profound investigation of bull fertility is only rarely performed, which sometimes results in a poor pregnancy outcome. Screening bulls for fertility and finally selecting the bulls with the highest fertility potential can however be easily done by means of a standardized breeding soundness evaluation, in which general health, reproductive health and sperm quality are assessed. Although this is not performed in Belgium, there are several indications that this might overcome part of the fertility problems encountered in Belgian Blue (BB) breeding herds. This paper describes how to perform a breeding soundness evaluation and how to interpret the results, with some specific considerations concerning BB bulls. This allows to exclude bulls with impediments to fertility and to select for bulls with traits favourable for high fertility. However, in spite of recent substantial advances in sperm quality assessment, accurately predicting fertility outcome of an ejaculate or of a bull remains an elusive goal.


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INTRODUCTION

Reproduction in cattle livestock is the essential prerequisite for production and thus for potential economic gain. Good fertility of both bulls and cows is imperative for adequate reproductive performance. Male fertility is an important factor influencing the reproductive efficacy since, in cattle, a single bull is generally bred to 20 - 100 cows (Chenoweth, 1986b). Despite the bull’s pivotal role in beef reproduction, cows generally receive more interest, both from a scientific and a practical point of view (Chenoweth, 1997a; Parkinson, 2004). However, no individual herd member bears as much responsibility for fertility as the herd sire (Barth, 1997; Hoflack and de Kruif, 2003). Hence, knowledge of a bull’s reproductive capacity is of paramount importance to achieve breeding success.

Post breeding assessments

Several methods can be used to assess bull fertility. The most logical and accurate method is to assess pregnancy rates (Wiltbank and Parish, 1986; Koops et al., 1995). This is more efficacious in order to estimate male fertility than the assessment of the number of produced calves, since the latter parameter is largely influenced by the cow and her reproductive performance. However, the assessment of pregnancy rates is very labour intensive since it requires a rectal or ultrasonographic examination of a high number of cows sired by a particular bull (Fissore et al., 1986, Phillips et al., 2004). The latter renders this method rather impractical, particularly in the case of artificial insemination with bulls that sire a huge number of females (Foote, 2003; Rodriguez-Martinez, 2003).

In order to reduce labour, non-return rates to service (oestrus) instead of pregnancy rates can be determined. The non-return rate is the percentage of cows inseminated with semen of a particular bull that were not re-inseminated within a specific time frame afterwards, and this can be considered as a ‘preliminary’ pregnancy rate (den Daas, 1997). However, this method remains very time consuming since one has to wait until a high number of cows have been inseminated and until data of subsequent inseminations of these cows become available. Furthermore, this method is imprecise and overestimates the true conception rate, because inseminated non pregnant cows which are finally culled or naturally served are considered as not returning to oestrus and thus pregnant as a result of the insemination (den Daas, 1997). This falsifies the results to some extent. However, to this very day, non-return rates are the golden mean between reliability and practicality, and are therefore used by all artificial insemination centres all around the world to quantify bull fertility (CRV Holding, personal communication; Rodriguez-Martinez, 2003; Phillips et al., 2004).


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Pre breeding assessments

The main disadvantage of post breeding bull fertility evaluations is that they assess the fertility of any given bull after this bull has been bred to his female counterparts. In case of an infertile bull, damage has been done before fertility results become available. Hence, prediction of fertility prior to breeding rather than post breeding could largely increase reproductive efficacy (Rodriguez-Martinez, 2003). To this purpose, breeding soundness evaluations of bulls have been used over the past 50 years and are widely accepted (Ott, 1986; Barth, 1997). This manuscript deals with the practical application of breeding soundness evaluations in bulls. Additionally, several laboratory assessments have been studied in an attempt to predict fertility (for review see Rodriguez-Martinez, 2003).

THE BREEDING SOUNDNESS EVALUATION

The evaluation of potential breeding soundness of a bull consists of a general physical examination, a reproductive examination which contains an examination of the external and internal genitalia (including a scrotal circumference measurement), and a semen quality evaluation (Bruner and Van Camp, 1992; Chenoweth et al., 1994).

1. General physical examination

The aim of this examination is to ensure that no clinical aberrations which might negatively influence the bull’s fertility are present. This includes an examination of the general health, the musculoskeletal system (gait, feet and legs), the eyes and an oral examination (Ott, 1986).

It is obvious that the general health of a breeding bull is important, since sick animals will be less active and fever should be avoided since this will negatively influence semen quality. Moreover, a bull suffering from an infectious disease might infect the female herd, which in turn might harm the reproductive performances of the cows. Additionally to legally obliged screenings, such as for tuberculosis, brucellosis and leucosis, particular attention should be paid to bovine viral disease virus and infectious bovine rhinotracheitis, since these infections easily spread from bulls to cows with possibly devastating effects on pregnancy results (personal observations).

Besides the general health, a sound conformation of the feet and legs (free of bow leggedness, cow hocks, sickle hocks, and post leggedness) of a bull is imperative to obtain a good breeding outcome (Larson, 1986). Bulls with rear leg impairment may not move around freely to detect cows in oestrus or may be unable to mount successfully, since during copulation most of the bull’s weight is borne on the hind legs and feet (Bruner and Van


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Camp, 1992; Barth, 1997). Furthermore, it has been demonstrated that even in bulls without clinical symptoms of lameness, joint lesions should be taken into consideration as a contributory cause of reproductive failure (Persson et al., 2004). Subclinical feet and leg problems might result in rear leg discomfort, leading to fewer mounting attempts of these bulls as well as spending more time in recumbency, which may interfere with normal testicular temperature and thus with sperm quality (Ott, 1986; Hopkins and Spitzer, 1997). Even more emphasis should be placed on the fact that quite some foot and leg problems (e.g. corkscrew claw defect, interdigital fibromas, weak pasterns, post leggedness as well as sickle hocks) have a hereditary basis and will be passed on to the (female) offspring of an affected bull. The latter will finally reduce longevity of the cow herd and increase labour and veterinary expense (Ott, 1986, Barth, 1997). Hence, a thorough examination of a bull’s hooves and feet prior to the breeding season is an absolute requirement. This examination furthermore allows to detect and treat infectious claw disorders, such as digital dermatitis, interdigital dermatitis, and interdigital phlegmon, which could otherwise be passed on to the cow herd (Kahn and Line, 2005). A detailed review of the pathological conditions resulting in lameness, which consequently can interfere with fertility, is given in the book ‘Lameness in cattle’ by Greenough and Weaver (1997).

Since bulls rely mostly on vision to detect cows in heat, it is logical that a thorough examination of the eyes and vision of the bull is performed. Furthermore, a bull should be free of mouth abnormalities or deformities and have adequate teeth to allow him to sufficiently feed during the breeding season to avoid excessive weight loss. Body condition should therefore be monitored, and both over fitted and under fitted bulls should be avoided (Ott, 1986, Bruner and Van Camp, 1992; Barth, 1997).

2. Reproductive examination

The reproductive examination consists of an exam of the external genitalia (i.e. penis, prepuce, scrotum, testes and epididymides) including a scrotal circumference measurement, and a rectal palpation to assess the internal genitalia (i.e. prostate, vesicular glands, ampullae ductus deferentes, inguinal rings and pelvic lymph nodes).

2.1. External genitalia

2.1.1. The prepuce

The prepuce should initially be visually inspected for evidence of preputial prolapse. Bos indicus bulls and bulls of breeds using Brahman (e.g. Santa Gertrudis, Beefmaster, Brangus) generally have pendulous sheaths, whereas polled breeds are prone to hereditary weakness of the (retractor and protractor) prepuce muscles. All these conditions predispose to preputial eversion, which can result in preputial trauma. This condition may decrease the


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breeding potential of a bull. When examining the external orifice, careful attention should be paid to precipitated crystals on the hairs, since they suggest the presence of urinary calculi predisposing bulls to urethral obstruction and even rupture, making a bull unsuitable for breeding (Figure 1). Furthermore, the prepuce (and penis) should be examined for balanoposthitis, since this condition finally leads to pain and reluctance to mate (Ott, 1986, Bruner and Van Camp, 1992).

Figure 1: Urinary calculi, which can finally lead to urethral rupture, are a common finding in young Belgian Blue bulls. Specific management practices, such as access to free water, NaCl supplementation, water quality assessment, and higher roughage diets can help to prevent this problem.

Next to the visual inspection, a thorough palpation of the entire external preputial sheath (from the external orifice to the scrotal neck) should be performed to examine whether scars, lacerations, adhesions, stenosis or preputial enlargements are present. Moreover, several penile abnormalities such as fibropapillomas, abcesses and hematomas can often already be detected during this palpation (Bruner and Van Camp, 1992).

2.1.2. The penis

The best way to examine the penis is immediately after natural mating prior to penile withdrawal into the prepuce, or before and after semen collection by means of an artificial vagina (Figure 2), since in case of electro-ejaculation and/or manual protrusion, artificial deviations may occur. The extended penis should be examined for the presence of fibropapillomas, hair rings, persistent frenula (Figure 3), and penile deviations. Phimosis (inability to extend the penis) and paraphimosis (inability to withdraw the penis), both secondary conditions, are unacceptable for breeding bulls. Urolithiasis with urethral rupture and penile hematoma (‘broken’ or ‘fractured’ penis) are both presented as a large subcutaneous swelling cranial to the scrotum and warrant exclusion from breeding (Ott, 1986, Bruner and Van Camp, 1992; Hopkins, 1997).


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Figure 2: The bull’s penis can be thoroughly examined for abnormalities, such as fibro-papillomas, hair rings, persistent frenula, and penile deviations during false mounts or immediately prior to (or after) semen collection by means of an artificial vagina.

Figure 3: Persistent frenulum of the penis of a Belgian Blue breeding bull. This condition is encountered in approximately 4 – 7% of young Belgian Blue bulls.

2.1.3. The scrotum and its contents: the testes and epididymides

Both a visual inspection and palpation of the scrotum (and its contents) should be performed. This visual inspection of both scrotal size and shape should be done in a warm environment on a relaxed bull, since under these circumstances the scrotum will be maximally pendulous (Figure 4).


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Figure 4: Both a visual inspection of scrotal size and shape, and a palpation of the scrotum and its contents should be performed. A distinct scrotal neck free of fatty deposits should be noticeable.

In order to provide sufficient thermoregulation, a distinct scrotal neck free of fatty deposits should be present. Straight-sided and wedge-shaped scrotums, as well as normal scrota with fatty deposits in the scrotal neck are associated with impaired testicular thermoregulation, which can result in abnormal sperm production (Barth, 1997; Johnson, 1997; Van Camp, 1997; Warner, 2004). Furthermore, the external scrotal surface should be free of scabs, lacerations, dermatitis and frostbite since these conditions can alter scrotal temperature and subsequently influence the sperm morphology (Bruner and Van Camp, 1992).

Abnormalities, such as cryptorchidism, unilateral testicular hypoplasia/atrophy, orchitis, scrotal hernia, and scrotal hematomas and masses will result in an abnormal scrotal shape, and such bulls should be classified as unsatisfactory potential breeders (Bruner and Van Camp, 1992).

After the visual inspection, scrotal-, testicular- and epididymal palpation should be performed. The thickness of the scrotal wall and the fat content of the scrotal neck should be assessed, and the testicular cords should be checked for the presence of fat, abscesses, varicoceles or viscera in case of a scrotal hernia (Barth, 1997).

Careful palpation of the testes examining the size, shape, symmetry and consistency should be performed to detect possible abscesses, calcification, hematoceles and (rare) neoplasms. During this palpation, both testicular consistency and resilience should be assessed. Healthy breeding bulls should have (very) firm testes, with resilience similar to (soft) rubber. Although testicular palpation remains subjective, tonometers are only rarely used to determine consistency, since tonometer measurements are not strongly correlated to semen quality (Bruner and Van Camp, 1992; Barth, 1997). However, too hard or too soft testes are abnormal and suggest degeneration, which can finally lead to fibrosis. In this case


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the testes will shrink on subsequent evaluations, and this is in contrast to testicular hypoplasia in which case the testes are always small (Bruner and Van Camp, 1992). The mean testicular size in mature dairy and beef bulls is approximately 14 – 16 cm length and 7 – 8 cm diameter. Consequently, testicular length is roughly 2 times its diameter (Larson; 1986). Abnormalities, such as cryptorchidism, unilateral testicular hypoplasia/atrophy, orchitis, scrotal hernia, and scrotal hematomas and neoplasms will be accompanied by a loss of testicular symmetry (Bruner and Van Camp, 1992). The testes must move freely within the scrotum and it should be easily possible to slide a testicle upward without invagination of the scrotal tip, which is indicative of testicular adhesions, so that the corpus epididymis of the opposite testicle, which is situated on the medial side of the testes, can be palpated.

Furthermore, the epididymal head (caput epididymidis), a flat and firm structure on the craniodorsal surface of the testicle, and the epididymal tail (cauda epididymidis), ventral on the testicle and generally protruding well beyond the ventral limits of the testicle should also be carefully palpated for size, shape, symmetry and consistency (Larson, 1986; Ott, 1986, Bruner and Van Camp, 1992; Barth, 1997). The most common abnormalities involve inflammation (epididymitis) or loss of patency. The latter situation can be the result of (inherited) segmental aplasia, tumours, abscesses and spermatoceles, in which case the efferent tubules are defective. The epididymal tail distal to this occlusion will be empty, flaccid, and soft. When occlusion is finally accompanied by break-down of the epididymal lumen, sperm will enter the surrounding tissues and prompt an inflammatory reaction, resulting in the formation of nodular masses or sperm granulomas (Bruner and Van Camp, 1992). Epididymitis is often secondary to orchitis or seminal vesiculitis, and in case of aplasia, the corresponding vesicular gland or ampulla is often also absent (Hopkins, 1997).

2.2. Scrotal circumference measurement

The most important part of the scrotal examination is the scrotal circumference measurement (Hopkins and Spitzer, 1997), as scrotal circumference, a highly heritable trait, is positively correlated to daily sperm output, normal sperm morphology and sperm motility, and consequently to pregnancy rates (Makarechian and Farid, 1985; Ott, 1986; Bruner and Van Camp, 1992; Barth, 1997). Each gram of functional testicular tissue has the same amount of tubular epithelium resulting in a constant sperm production per gram of testis weight without breed differences. Hence, in order to predict the potential sperm production of a bull, it suffices to weigh his testicles. However, high correlations between paired testis weight and scrotal circumference have been demonstrated. Consequently and for practical purposes, measuring the scrotal circumference of a bull is, in essence, weighing his testicles (Spitzer and Hopkins, 1997). This can be done by pulling a scrotal tape around the testicles until snug at the site of maximal circumference, after both testicles were carefully forced ventrally into the bottom of the scrotum until no scrotal wrinkles are any longer evident (Figure 5).


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Figure 5: Scrotal circumference measurement of a Belgian Blue breeding bull.

During this measurement, the testicles should be immobilized by placing the thumb and fingers on opposite sides of the scrotal neck, avoiding putting thumbs or fingers between the testes, which results in separation of the testes, thus falsifying the measurement (Bruner and Van Camp, 1992). It is recommended to repeat this measurement to check for accuracy. The scrotal circumference thresholds for all breeds, regardless of genotype or environment, are listed in Table 1 (Chenoweth et al., 1992; 1994; Hopkins and Spitzer, 1997). These are the minimal acceptable measures suitable for all breeds, notwithstanding the fact that breed differences in scrotal circumference at a given age have been demonstrated (Michaux and Hanset, 1981; Chenoweth et al., 1984; Coulter et al., 1987; Bruner et al., 1995; Chenoweth et al., 1996).

Table 1: Minimum scrotal circumference (in cm) in bulls relative to the bull’s age, as advised by the 1993 society for Theriogenology guidelines, to be classified as a satisfactory potential breeder

Age (months) Minimum scrotal circumference (cm)

> 12 - 15 30 > 15 - 18 31 > 18 - 21 32 > 21 - 24 33

> 24 34


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2.3. Internal genitalia

The last step in the reproductive examination of a bull is a rectal palpation of the internal genital tract, during which the dorsal transverse ridge of the prostate gland, the ampullae of the ductus deferens, the vesicular glands, and the internal inguinal rings are examined. Immediately after entering the rectum, the pelvic urethra can be identified as a firm cylindrical structure on the pelvic floor.

2.3.1. The prostate

Approximately 7 cm cranial to the anus, the corpus of the prostate can be identified as a transverse ridge crossing the pelvic urethra. It is very rare to detect prostatic abnormalities (Larson, 1986; Ott, 1986; Bruner and van Camp 1992).

2.3.2. The seminal vesicles

Craniolateral to the prostate, on both sides, the seminal vesicles can be palpated as grape – like turgid, easily mobile clusters, approximately 2 – 6 cm in width and 6 – 15 cm in length. A common finding here, which can result in high numbers of white blood cells and even in pus in the semen, is seminal vesiculitis, generally unilateral but possibly bilateral. This generally produces no external signs of illness, but results in increased size and firmness of the glands (finally leading to fibrosis), loss of lobulation and pain on palpation. This condition is very difficult to treat and can evolve to abscessation, intrapelvic adhesions and even, although extremely rare, peritonitis. Secondary infections of the ampullae, epididymides and testes can result from vesiculitis.

Exceptionally, congenital defects such as aplasia or hypoplasia can occur, but this is often accompanied by aplasia of other segments of the reproductive tract (e.g. the epididymis) (Larson, 1986; Ott, 1986; Bruner and van Camp 1992; Barth, 1997; Cavalieri and Van Camp, 1997; Hopkins, 1997).

2.3.3. The ampullae of the ductus deferens

These smooth tubular structures of approximately 0.5 – 0.8 cm in diameter and 6 – 15 cm in length can be found directly cranial to the prostate and in between the vesicular glands by rubbing the fingers over the pelvic floor. This procedure should be painless, but in case of a rare ampullitis, generally secondary to seminal vesiculitis, this is no longer true. Hypoplasia or aplasia of the ampullae very seldomly occur (Larson, 1986; Bruner and van Camp 1992; Barth, 1997).

2.3.4. The internal inguinal rings

These slit-like openings can be palpated by examining both sides of the abdominal wall approximately 15 cm downwards, after passing the hand over the pelvic brim. No structures


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other than the spermatic cord, which leaves the abdomen through these openings, should be palpable in these rings. These rings should admit no more than 2 (4 cm) to 3 (6 cm) fingers in yearling and adult bulls, respectively. Large rings predispose the bulls to inguinal hernias, a condition that can be diagnosed through rectal examination. Due to the risk for genetic transfer of this condition, such bulls should be excluded from breeding (Larson, 1986; Bruner and van Camp 1992).

3. Semen collection and evaluation

3.1. Semen collection

The final part of the breeding soundness evaluation is the collection and analysis of a semen sample. Several methods for the collection of semen exist, such as transrectal massage, electro-ejaculation, and the use of external and internal artificial vaginas (Barth, 1997; Barth et al., 2004). The method most commonly used for bull semen collection during breeding soundness evaluations throughout the world is electro-ejaculation (Elmore, 1994). However, in Belgium there is little expertise with this technique. Moreover, this practice has been banned in several countries due to animal welfare concerns (Barth et al., 2004). Collection by means of an artificial vagina yields the best sperm quality (Spitzer and Hopkins, 1997). In addition, this method allows for an evaluation of libido and mating ability, which are very important attributes necessary for adequate breeding efficiency that are not routinely tested during the breeding soundness evaluation (Ott, 1986; Barth, 1997). Hence, collection of semen by means of an artificial vagina is preferable to all other collection techniques (Larson, 1986). However, in some cases of injured bulls, the only humane sperm collection technique is transrectal massage (Barth, 1997). This can easily be accomplished by massaging the ampullae, prostate and urethra until urethral contractions begin, after which one tries to massage in synchrony with these contractions. In general, semen can be collected in a 37°C sperm collection vial by a second person within a few minutes after the start of the massage procedure. However, lack of penile protrusion frequently occurs, resulting in contaminated samples. Furthermore, not all bulls can be collected with this technique, and semen quality is generally poorer compared to other collection techniques (Barth, 1997; Palmer et al., 2005).

3.2. Semen evaluation

Several methods can be used to evaluate the quality of a semen sample, but subjective evaluation using standard optical microscopy is by far most commonly used. The semen parameters that are routinely examined using standard optical microscopy are the volume, the concentration, the percentage of motile spermatozoa and the morphological grading of the sperm cells (Neuwinger et al., 1990; Rodriguez-Martinez, 2003; Phillips et al., 2004). However, volume and density of the sperm are unreliable characteristics when semen is


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collected by electro-ejaculation, since they are largely influenced by many factors other than the bull. Nevertheless, when bulls produce several ml of highly concentrated semen, it assures the fact that the bull is capable of producing good ejaculates (Barth, 1997).

The volume of each ejaculate can be read from the graded collection tube immediately after collection.

Concentration is generally estimated by evaluating the colour, opacity and viscosity of the sample. A creamy, thick and viscid ejaculate is considered very good and corresponds to a concentration of ≥ 750 million sperm per ml. Good ejaculates, containing 400 – 750 million spermatozoa per ml, look like slightly viscid milk. Skim-milk like, non-viscid samples contain 250 – 400 million sperm per ml and are considered fair, whereas poor ejaculates (< 250 million sperm per ml) have a watery translucent appearance (Elmore, 1994; Barth, 1997). However, concentration can also be more accurately determined by means of a counting chamber, e.g. a Bürker counting chamber (Merck, Leuven, Belgium). In this case, the concentration of the ejaculates is determined by diluting 10 µl of semen in 990 µl 1M HCl and by counting the number of sperm cells in 1/100 mm3, which corresponds to 40 small squares.

Semen motility is best evaluated immediately after collection. Gross motility can be determined on a wet mount of neat semen at 100 x magnification. Generally, the following scoring system is used: 1 = cells present without of with very little motion; 2 = prominent individual cell motion without swirls; 3 = slow swirls; 4 = rapid dark swirls (Barth, 1997). Individual cell motion should be discernable when assessing gross motility (≥ 2). Total and progressive individual motility are subjectively assessed to the nearest 5% by placing 10 µl of diluted semen (10 µl aliquot of pure semen in 790µl physiological saline solution) on a pre-warmed glass slide at 37°C under a coverslip, and by examining 5 different microscopic fields all in the centre of the coverslip, under a 200 x phase-contrast microscope (Hopkins and Spitzer, 1997). This procedure is best repeated twice to ensure the correctness of the estimate. Progressive motility should be at least 30% (= fair), ≥ 50% to be good and ≥ 70% to be very good (Chenoweth et al., 1994). Next to these percentages, a velocity score (1 – 4) can also be attributed to the sample: 1 = very slow semen, 2 = slow semen, 3 = rapid semen, 4 = extremely rapid semen.

Sperm morphology is the most reliable criterion to qualify an ejaculate, since it is least influenced by the collection process (Garner, 1997), and since no other sperm criterion is more closely related to fertility than morphology (Elmore, 1994). Morphology can be assessed using different techniques, but supravital staining procedures such as eosin-nigrosin staining are commonly used and allow both a morphology differentiation and a live-dead assessment (Barth and Oko, 1989; Elmore, 1994; Hopkins and Spitzer, 1997). This live-dead assessment is based on the physical intactness (i.e. structural integrity) of the membranes,


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only allowing the stain to penetrate the damaged sperm cells, resulting in both eosin penetrated (dead) and unstained (live) spermatozoa. To this purpose, a drop of semen is mixed with a few drops of stain with a glass slide on another glass slide, after which a smear of this mixture is prepared by dragging the first glass slide at a 30 – 40 ° angle across a third slide. Then, the eosin-nigrosin stained smears are air-dried and assessed under a 1000 x light microscope, using immersion oil. At least 100 spermatozoa should be evaluated for the live-dead assessment and the morphology evaluation (Barth, 1997; Kuster et al., 2004). Individual spermatozoa should be classified based on the 1993 Society for Theriogenology guidelines (Chenoweth et al., 1992; 1994; Hopkins and Spitzer, 1997). This classification systems logs abnormalities and orders sperm cells as either normal cells or abnormal cells. Abnormal sperm cells are further classified as primary abnormalities (i.e.underdeveloped forms, double forms, acrosome defects, narrow heads, nuclear pouches or vacuoles or diadem defects, pear-shaped heads, abnormal contour, small and free abnormal heads, abnormal midpieces (pseudodroplets, rough midpieces and segmental aplasia), proximal droplets, folded or coiled tails and accessory tails) which are of testicular origin, or secondary abnormalities (i.e. small normal heads, giant or short broad heads, free normal heads, detached or folded or loose acrosomal membranes, simple bent tails, abaxial tails, terminally coiled tails and distal cytoplasmic droplets) which are considered to originate after the sperm cells have left the testis (Barth and Oko, 1989; Hopkins and Spitzer, 1997). When multiple abnormalities are observed in the same sperm cell, only one abnormality is logged. Primary abnormalities are given first priority in classification. At least 70% of the spermatozoa should have a normal morphology (Chenoweth et al., 1994; Elmore, 1994). Other cells should also be determined, but are generally not discernable on eosin–nigrosin stains. When neutrophils are present in the ejaculate, white irregular bodies three times the size of a sperm head are noticeable, and their presence can be easily confirmed using a white blood cell stain, such as Diff Quick (Barth, 1997; Hopkins, 1997).

When bulls pass the general physical and reproductive examination, and when they also equal or exceed the minimum thresholds for scrotal circumference, sperm motility (gross motility ≥ 2, progressive motility ≥ 30%) and sperm morphology (≥ 70% normal spermatozoa), they are classified as satisfactory potential breeders. When they fail for one (or more) reason(s), the bulls are classified either as deferred or unsatisfactory potential breeders. Bulls are only attributed to the latter category when genetic faults or one (or more) other severe problem(s) occur, or when a problem is irreversible. Deferred bulls are likely to improve with time or therapy and should be scheduled for a retest (Chenoweth et al., 1994; Hopkins and Spitzer, 1997).


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4. And what about libido?

Although the breeding soundness evaluation assesses several important characteristics necessary for good fertility, it does not at all deal with the willingness and eagerness of a bull to mount and attempt service (= libido) and with the ability to complete service (= mating ability), since in general semen is collected by means of electro-ejaculation. Notwithstanding the fact that bulls are classified as satisfactory potential breeders, it is very well possible that such bulls are incapable of impregnating cows when the will and ability to service cows is absent (Chenoweth, 1986a, Barth et al., 2004). Furthermore, it was demonstrated that bulls with higher sex-drive obtained better pregnancy rates compared with lower sex-drive bulls (Blockey, 1978; Makarechian and Farid, 1985; Blockey, 1989, Farin et al., 1989), although this effect is most evident over short mating periods and is partially nullified in case of a long breeding season (Silva-Mena et al., 2002; Parkinson, 2004). Hence, bull libido and mating ability can be considered as important contributing factors to good male fertility. Consequently, in addition to the breeding soundness evaluation, to avoid the selection of bulls unwilling or unable to serve cows, tests for bull libido and/or serving capacity should be included (Chenoweth, 1986a; Chenoweth et al., 1994; Barth, 1997; Garner, 1997; Parkinson, 2004).

Observing natural mating is the simplest and least expensive method to assess willingness and ability to service cows, but these traits can also be evaluated to some extent when semen is collected by means of an artificial vagina (Ott, 1986; Bruner and Van Camp, 1992; Figure 6).

Figure 6: Apart from penile abnormalities, both libido and mating ability can be assessed during semen collection by means of an artificial vagina, which results in the most reliable semen quality compared to other collection methods.


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In case of semen collection by electro-ejaculation or massage, neither libido nor mating ability can be evaluated and other methods to test these characteristics should be implemented in order to fully evaluate reproductive potential. For this purpose, various testing methods have been investigated, such as pasture and corral trials with restrained or unrestrained, estrual and nonestrual females, where bulls were tested either individually or group wise for different periods of time (Barth, 1997). In general, several bulls (e.g. 5) of comparable age, to avoid invalid results due to social dominance, are tested simultaneously in a small corral with several (e.g. 2 or 3) sedated and restrained, nonestrual cows of which the vagina was lubricated with a sterile lubricating jelly to reduce vaginal trauma as a result of repeated breedings. The expression of sex drive, the ability to serve, the reaction time (the elapsed time between exposure of the bull to suitable stimuli and first service), the number of mounts (without ejaculation) and the number of services (= serving capacity) completed within a stipulated time period can then be observed (Chenoweth, 1986a; Barth, 1997). It is obvious that a serving capacity test, counting the number of services within a short period of time, yields sufficient information, since a successful test requires both good libido and mating ability (Chenoweth, 1997b). The simultaneous testing of several bulls is done to assess social interactions between dominant and more timid bulls, which is important in multi-sire breeding programs as dominant bulls can exert an inhibitory effect on submissive bulls from a distance, which results in more services being performed by the dominant bulls (Chenoweth, 1997b, Fordyce et al., 2002). However, this can negatively influence pregnancy outcome when the dominant bull is subfertile or becomes subfertile through over-use (Parkinson, 2004). Since dominance is related to bull age and weight, the use of mixed age bulls should be avoided (Makarechian and Farid, 1985; Barling et al., 1997). Contrastingly, beneficial social effects on bull sex-drive expression can also occur, but primarily in young bulls, in which dominance is of little or no significance (Blockey, 1979; Barling et al., 1997; Chenoweth, 1997b). However, in Belgium single-sire mating systems are the general rule. Hence, individual testing of bulls then is as appropriate. However, since corral trials with restrained females are impossible in the Benelux countries for practical, sanitary and ethical reasons, other methods to estimate libido and mating ability have to be used. The measurement of the reaction time, defined as the elapsed time between exposure to stimuli and first service, can be used to test libido (Chenoweth, 1986a), while mating ability can be assessed when semen is collected with an artificial vagina (Ott, 1986, Barth et al., 2004). Older bulls tend to mount less, although no difference in the number of services can be demonstrated. This probably is the result of a greater mating experience (Chenoweth et al., 1984).

The breeding soundness evaluation is generally used for naturally serving beef bulls, although it also applies to dairy and beef bulls used for artificial insemination purposes with cryopreserved semen (Ott, 1986; Barth, 1997). It provides a reliable, quick and cost-effective method for screening and classifying bulls in terms of fertility in order to minimize the use of


36

subfertile bulls and bulls of questionable fertility (Chenoweth et al., 1994). These evaluations are reliable for detecting bulls that have the potential for high fertility and those that are clearly unsatisfactory (Barth, 1997). However, it does not predict the fertility of bulls classified as satisfactory potential breeders; it merely identifies bulls with a high or low probability of having reduced fertility, thus classifying bulls as having a high or low risk to develop fertility problems. Hence, other more specialized semen evaluation tests were developed in an attempt to predict the fertility outcome based on qualitative measures of the ejaculate (Rodriguez-Martinez, 2003).

SPECIALIZED SEMEN EVALUATION TESTS

In all, semen analysis is probably the most relevant procedure to evaluate male fertility potential (Chong et al., 1983; Vantman et al., 1989; Comhaire et al., 1992, Rijsselaere et al., 2002, Phillips et al., 2004). Several methods can be used to evaluate the quality of a fresh ejaculate or of frozen-thawed semen, but subjective evaluation using standard optical microscopy is by far most commonly used (Rijsselaere et al., 2003, Christensen et al., 2005). Although literature on the relation between subjectively assessed sperm quality and fertility is contradictory, several authors reported a relation between the percentages of morphologically normal or abnormal sperm (Chenoweth, 1980; Wiltbank and Parish, 1986; Coulter and Kozub, 1989; Pangewar and Sharma, 1989; Larsen et al., 1990; Söderquist et al., 1991; Fitzpatrick et al., 2002; Holroyd et al., 2002; Phillips et al., 2004) or motility (Lindford et al., 1976; Chenoweth, 1980; Pangewar and Sharma, 1989; Christensen et al., 1999; 2005) and fertility. However, subjective visual assessment of sperm morphology and motility has been demonstrated to be inaccurate and imprecise, partially as a result of the generally small number of analyzed spermatozoa and evidenced by high coefficients of variation both within and between technicians, as well as between laboratories (Jequier and Ukombe, 1983; Dunphy et al., 1989; Neuwinger et al., 1990). To overcome this high variability and lack of accuracy, other more standardised and objective semen evaluation methods have been developed. The goal of these tests is to evaluate one or more sperm attributes necessary to reach, bind, penetrate and fertilize an oocyte, such as motility, normal morphology, capacitation, hyperactivation, zona pellucida binding, acrosome reaction, zona pellucida penetration, fusion with the oocyte, and DNA decondensation.

These tests include numerous cell function assessments by means of fluorescent stains, such as plasma membrane integrity, capacitation status, the acrosome reaction, mitochondrial function, apoptosis, and DNA condensation status, which are generally evaluated by flow cytometry enabling the analysis of large numbers of spermatozoa in a short time and the simultaneous assessment of different sperm characteristics when stains are combined (Evenson et al., 1980; De Pauw et al., 2003). Computer assisted sperm analysis provides


37

objective and detailed information on various sperm characteristics, both for motility and morphometry, which cannot be identified by means of conventional light microscopy (Verstegen et al., 2002). Sperm migration tests have also been used as a motility test by different researchers to predict bull fertility, with contradictory results (Murase et al., 1990; Sittal et al., 1996; Verberckmoes et al., 2002). Glycosaminoglycan induced capacitation and acrosome reaction (Ax and Lenz, 1987), and calcium ionophore (A23187) induced acrosome reaction (Whitfield and Parkinson, 1995) were also related to bull semen fertility, as well as several in vitro assays, which evaluate the capacity of sperm to bind to oviductal epithelium explants (De Pauw et al., 2002) or to the zona pellucida (Fazeli et al., 1997; Zhang et al., 1998). Functional tests evaluating the capacity of sperm to penetrate or to in-vitro fertilise the oocyte require the ability of spermatozoa to fulfil all the steps necessary for fertilization and consequently are the ultimate test, showing great promise (Larsson and Rodriguez-Martinez, 2000). Unfortunately, the outcome of these tests is largely influenced by the oocyte batch quality, leading to variability and repeatability problems (den Daas, 1997). Furthermore, these tests should be very well defined and standardized, since only some parameters after in-vitro tests yield moderate to high and significant correlations to in-vivo bull fertility, while most of the assessed parameters don’t (Ward et al., 2001; 2002).

However, all these tests have been thoroughly reviewed and are not within the scope of this article (Verstegen et al., 2002; De Pauw et al., 2003; Rodriguez-Martinez, 2003; Parkinson, 2004). Moreover, although significant correlations between the respective tests and fertility outcome can be demonstrated in many cases, accurate prediction of the fertility of a bull based on these tests still largely remains an elusive goal, and no more than an estimate can be provided (Zhang et al., 1999). Male fertility by itself already is a very complex matter as it depends on a heterogeneous population of spermatozoa (Rodriguez-Martinez, 2003). These spermatozoa are furthermore largely influenced by the female animals, since they interact at various levels of the female genital tract. A recent comprehensive study revealed that differences between bulls and between the ejaculates of any given bull accounted for only 0.38 % of the total variation in non-return rates (Christensen et al., 2005). Combining several laboratory semen assessments, thus testing as much sperm attributes relevant for fertilization and embryo development as possible results in better correlations and hence more accurate predictability of in vivo fertility compared to testing a single attribute, but the prediction of bull fertility based on laboratory assessment of semen still remains utopian (Rodriguez-Martinez, 2003; Parkinson, 2004).


38

BREEDING SOUNDNESS EVALUATIONS OF BELGIAN BULLS

In Belgium, breeding soundness examinations and libido evaluations are only rarely performed, resulting in a lack of data concerning fertility of naturally serving bulls. The two predominant breeds in this region are the BB beef breed, and the Holstein Friesian (HF) dairy breed. The BB breed stems from the Durham Shorthorn, which was introduced in Belgium in 1841 and crossed with local dairy breeds, resulting in a breed called the “Blue of Limon” which was further mixed with local breeds. In 1938, selection for a white colour was started, resulting in the “White breed of Middle and High Belgium”. Almost simultaneously, and from a limited number of ancestors selection for a better muscularity was begun, which eventually led to the present, hyper muscled BB breed, which is famous for its low feed conversion ratio, its high percentage of lean meat and its advantageous carcass classification (Coopman et al., 2001). While in BB, natural service and artificial insemination are both in use, in HF mainly artificial insemination is used for reproduction. Because BB bulls are used for natural service without a preceding breeding soundness evaluation, herd fertility problems frequently occur (Bombeek, 2004). Bull turnover at the farm level is consequentially high, which is due in part to the susceptibility of the bulls to injuries, as well as to the fact that quite a number of bulls are finally culled due to poor pregnancy rates. Usually, when using natural service BB bulls, specific breeding management practices, based on empirical farming experience with BB livestock rather than on scientific research, are applied to avoid the frequently noticed disappointing pregnancy results. This includes practices such as lowering the breeding pressure by limiting the number of females to 15 – 25 per bull (depending on the bull’s age), applying a prolonged breeding season from May to October, and supplementing the bull’s diet with a daily portion of approximately 700 g concentrates / 100 kg body weight (Hoflack and de Kruif, 2003). These recommendations, undertaken to avoid poor pregnancy rates, suggest that subfertility is a problem in the BB bulls, although this has never been studied in detail so far. However, Hanset (2000) already demonstrated that the average scrotal circumference of 13-month-old BB bulls was rather low, namely 31.3. cm, with a median scrotal circumference of 32.0 cm, both of which are low compared to other beef breeds (Coulter et al., 1987; Hanset, 2000). Breed differences in scrotal circumference at a given age are however not uncommon (Michaux and Hanset, 1981; Chenoweth et al., 1984; Coulter et al., 1987; Bruner et al., 1995; Chenoweth et al., 1996). The BB breed is indeed a specific breed, with extreme muscularity but reduced organ size. This has been demonstrated for several organ systems (Ansay and Hanset, 1979). Hence, it might be possible that this is also the case for the reproductive organs, which would result in smaller testicles compared to other breeds and, as a consequence, a lower scrotal circumference relative to the bull’s age (Michaux and Hanset, 1981). However, the scrotal circumference thresholds set by the Society for Theriogenology are minimum values suitable for all bulls, regardless of genotype


39

or environment, and consequently also apply to the BB breed (Chenoweth et al., 1992; 1994; Hopkins and Spitzer, 1997). Moreover, high circumference bulls produce higher fertility offspring, both male and female, that attain puberty at an earlier age, which results in economic profit (Coulter and Foote, 1979; Bruner and Van Camp, 1992; Barth, 1997; Spitzer and Hopkins, 1997). Selection for higher scrotal circumference in the BB breed is therefore advisable. Since scrotal circumference is positively correlated to daily sperm output, normal sperm morphology and motility, and consequently to pregnancy rates, Hanset’s data (2000) are somewhat worrying (Makarechian and Farid, 1985; Ott, 1986; Bruner and Van Camp, 1992; Barth, 1997). In view of these findings, it might very well be possible that a high proportion of the BB bulls have a substandard semen quality and are consequently subfertile. As scrotal circumference is correlated to the fertility of the female offspring, a negative effect of the use of these subfertile bulls on the reproductive performance of female offspring in the BB breed seems inevitable (Barth, 1997). Although no reliable data on this subject are yet available, based on limited data, a general tendency toward a higher age at first calving is noticeable in BB heifers (AWE, personal communication).

Moreover, several other abnormalities which have been described to cause problems for breeding soundness in other beef breeds, are also encountered in the BB breed. It is, for example, common knowledge that the BB breed is susceptible to several heritable feet and leg abnormalities, such as sickle hocks and post leggedness, two problems which can interfere with the bull’s ability to mate (Larson, 1986; Hanset et al., 2003). Furthermore, embryo transplantation was extensively used in this breed to disseminate the best genetics throughout the entire Belgian beef population. This not only led to even higher inbreeding coefficients and subsequent feet and leg problems in BB animals, but indirectly also is the reason that infectious claw disorders, which were otherwise rare in this breed, entered the BB population (personal observations). The predominantly used HF embryo receptors are partially responsible for the introduction of these infectious problems in the BB breed. Additionally, mouth abnormalities, such as brachygnatia inferior and superior, hyper muscled and long tongue, and crooked jaw are frequently encountered heritable abnormalities that can interfere with the ability to sufficiently graze, and intensive selection procedures against these defects have been undertaken (Hanset and de Tillesse, 2000).

In view of these findings, fertility in the BB breed deserves special attention and should therefore be monitored with the utmost care. To minimize herd fertility problems, breeding soundness examinations of BB bulls seem essential, since no individual herd member bears as much responsibility for fertility as the herd sire (Barth, 1997; Hoflack and de Kruif, 2003). Nevertheless, the accurate prediction of bull fertility remains utopian (Rodriguez-Martinez, 2003). Hence, bovine fertility management to date is, in essence, minimizing the risk for infertility (Parkinson, 2004).


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Rodriguez-Martinez H. Laboratory semen assessment and prediction of fertility : still utopia? Reprod. Dom. Anim. 2003; 38: 312 – 318.


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Warner GD. Breeding Soundness Evaluation: Physical Assessment. In: Proceedings of the 37th annual convention of the American Association of Bovine Practitioners, Forth Worth, Texas 2004, 37; p. 63-66.

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CHAPTER 2

BREEDING SOUNDNESS AND LIBIDO EXAMINATION OF BELGIAN BLUE

AND HOLSTEIN FRIESIAN ARTIFICIAL INSEMINATION BULLS IN BELGIUM

AND THE NETHERLANDS

Modified from: Breeding Soundness and Libido Examination of Belgian Blue and Holstein Friesian Artificial Insemination Bulls in Belgium and The Netherlands G. Hoflack, A. Van Soom, D. Maes, A. de Kruif, G. Opsomer, L. Duchateau Theriogenology 2006; 66 (2): 207 - 216

Chapter 2: Breeding Soundness of BB and HF Bulls

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ABSTRACT

Data on breeding soundness and libido evaluations in Belgian Blue (BB) bulls are scarce. The present study compared results of breeding soundness and libido evaluations of young BB bulls to young Holstein Friesian (HF) bulls prior to acceptance into an AI program. Breed differences for breeding soundness exist between BB and HF bulls, as 93.7 % of the young BB bulls failed the breeding soundness examination (BSE) compared to 59.3 % of the HF bulls (P = 0.0005). Within the BB breed, differences were present between bulls of different ages, and bull selection for better fertility with increasing age apparently influenced the results. The number of reasons for which bulls failed the test differed between the age groups in the BB breed, whereas a tendency for more failure reasons in the BB breed was noticed in the breed comparison. The most important reasons for failure were sperm morphology and scrotal circumference (SC), but far more BB bulls failed for these traits compared to the HF breed (82.8 versus 56.0% and 43.8 versus 17.6% in the BB and the HF breed for sperm morphology (P = 0.0005) and SC (P < 0.0001), respectively). The high proportion of BB bulls with a substandard SC and poor sperm morphology might suggest an increased prevalence of testicular hypoplasia or degeneration within this breed. Concerning libido, the reaction time did not differ either between breeds or between age groups within the BB breed, whereas mounting enthusiasm, although not different between the two breeds, did decline with increasing age, probably due to the greater mating experience of the older bulls. All in all, libido did not seem to be different between the breeds.


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INTRODUCTION

Male fertility is an important factor in bovine reproduction since a single bull is generally bred to numerous cows. Hence, evaluation of male fertility prior to breeding is of paramount importance to achieve breeding success. Breeding soundness evaluations (BSE) of bulls have been used extensively for this purpose over the past 50 years (Ott, 1986; Barth, 1997). The evaluation of a bull’s breeding soundness potential consists of a general physical soundness examination, a genital tract examination of both the external and internal genitalia (including a scrotal circumference (SC) measurement), and a semen quality evaluation (Bruner and Van Camp, 1992; Chenoweth et al., 1994; Garner, 1997). This provides a reliable, quick and cost-effective method for screening and classifying bulls in terms of fertility in order to minimize the use of subfertile bulls and bulls of questionable fertility (Chenoweth et al., 1994). The BSE is generally used for naturally serving beef bulls, although it also applies to dairy and beef bulls used for artificial insemination (AI) purposes with cryopreserved semen (Ott, 1986; Barth, 1997).

In the Benelux countries (the region of Europe comprising Belgium, The Netherlands and Luxembourg), BSE are rarely performed. The two predominant breeds in this region are the Belgian Blue (BB) beef breed and the Holstein Friesian (HF) dairy breed. In the BB, natural service and AI are both in use, while in the HF, AI is predominantly used. Because BB bulls are used for natural service without a preceding BSE, herd fertility problems frequently occur. Bull turnover at the farm level is consequentially high, which is due in part to the susceptibility of the bulls to injuries, as well as to the fact that quite a number of bulls are finally culled due to poor pregnancy rates. Usually, specific breeding management practices based on empirical farming experience are applied to avoid poor pregnancy rates. This includes practices such as limiting the number of females to 15 – 25 per bull (depending on the bull’s age), applying a prolonged breeding season from May to October, and supplementing the bull’s diet with a daily portion of approximately 700 g concentrates / 100 kg body weight (Hoflack and de Kruif, 2003). However, to minimize these herd fertility problems, the BSE of these bulls is essential, since no individual herd member bears as much responsibility for fertility as the herd sire (Barth, 1997; Hoflack and de Kruif, 2003).

In addition to a BSE, and to avoid the selection of bulls unwilling or unable to serve cows, tests for bull libido and/or serving capacity may be included (Chenoweth, 1986; Chenoweth et al., 1994; Barth, 1997; Garner, 1997). To evaluate libido, observing natural breeding is the most efficient (Bruner and Van Camp, 1992) but other tests can also be used (Chenoweth, 1986). Since corral trials with restrained females are impossible in the Benelux countries for practical, sanitary and ethical reasons, other methods of measuring libido have to be used. The measurement of the reaction time, defined as the elapsed time between exposure to stimuli and first service can be used to test libido (Chenoweth, 1986), while mating ability


53

can be assessed when semen is collected with an artificial vagina (Ott, 1986; Barth et al., 2004).

The aims of the present study were to evaluate BB beef bulls for breeding soundness and libido in order to assess their fertility potential and to identify possible fertility weaknesses. To achieve these aims, the BB breed was compared with the HF dairy breed, the other predominant breed in the Benelux.

MATERIALS AND METHODS

Breeding soundness evaluation

As HF bulls are mainly used for AI purposes, data for the HF breed was collected at an AI center, on young bulls (average age 13.4 months (range: 11.5 – 17.2)) prior to AI admittance. For BB bulls, this study was similarly conducted at AI centers using bulls originating both from selection centers and from private farms. The average age of the BB bulls was 32.3 months (range: 13.6 – 90.9): 70 bulls were < 24 months, 39 bulls were from 24 to 35 months, 22 bulls were from 36 to 47 months, 10 bulls were from 48 to 59 months, 8 bulls were from 60 to 71 months, 4 bulls were from 72 to 83 months, and 3 bulls were ≥ 84 months.

Between February 2002 and February 2004, 156 BB bulls in Belgium and 91 HF bulls in The Netherlands (since no HF AI bulls are present in Belgium) were examined for breeding soundness in accordance with the 1993 Society for Theriogenology guidelines (Chenoweth et al., 1992; 1994; Hopkins and Spitzer, 1997). To this end, the AI centers were visited once during the spring, once during the summer, once during the autumn and once during the winter. On these occasions, all bulls that had not yet been tested for breeding soundness were examined. No bulls were retested. The BB bulls were purchased on the basis of their linear classification, which is a scoring system describing several physical characteristics of a bull (relating to size, muscular development, meaty type, stand and general appearance), and after a quarantine period of one month they were accepted for AI purposes without further selection. This is in contrast to the procedure for HF bulls, in which the bull calves were purchased on the basis of their expected genetic value. At approximately 11 months of age, these HF bulls were transferred to an AI facility, where, after a quarantine period of one month, semen was collected. Only when these bulls passed a strict semen quality test (2 consecutive ejaculates collected with 3 or 4 days interval ≥ 2 ml containing ≥ 600 x 106 spermatozoa / ml, with ≥ 65% motility, ≥ 80% normal morphology and ≥ 50% intact acrosomes) were they accepted for AI purposes. We tested HF bulls prior to this semen quality test to have an unselected population, comparable to the unscreened BB bulls.


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All semen samples were collected by means of an artificial vagina. Semen motility was evaluated immediately after collection: gross motility on a wet mount of neat semen at 100 x magnification and individual progressive motility on 10 µl of diluted semen (10 µl aliquot of pure semen in 790 µl physiological saline solution) at 37°C, on a warm glass slide under a cover slip at 200 x magnification using a phase contrast microscope. Morphology evaluation was performed by counting 200 spermatozoa at 1000 x magnification under immersion oil on an eosin-nigrosin stained, air-dried slide, determining the percentage of normal spermatozoa. Semen concentration was the average of 2 counts determined by means of a Bürker counting chamber (VWR International, Leuven, Belgium). Bulls were classified as satisfactory potential breeders when they passed all evaluations of the BSE. When they failed for one (or more) reason(s), the bulls were classified either as deferred or unsatisfactory potential breeders. These failing bulls were only attributed to the latter category when severe feet and legs, teeth and mouth, testicular, epididymal or inguinal abnormalities were found or when the SC was more than 2 cm below the threshold relative to the bull’s age (Chenoweth et al., 1994).

Libido testing

For all bulls undergoing the BSE, the libido was assessed during semen collection. For this purpose, the reaction time was recorded, measured as the amount of time between first contact with the manikin or mount animal and the first false mount with the penis erected (expressed in seconds), and the mounting enthusiasm both during these false mounts and during service was scored. Mounting enthusiasm was scored from -2 to +2: -2: bull does not mount, -1: bull mounts by sliding, 0: mounting between sliding and jumping, +1: bull mounts by jumping, +2: bull jumps with great enthusiasm. Since all semen samples were collected with an artificial vagina, mating ability was considered to be satisfactory in all bulls from which semen could be collected.

Determining the subclass and overall BSE scores

The BSE score (satisfactory (S), deferred (D) or unsatisfactory (U); Table 1) is the result of a transformation based on the position of the original score for each constituting characteristic as given in Table 2, relative to threshold values (Table 2). For most of the categorical variables examined during the BSE, an original score with 3 categories was used (0 – 2: 0 = abnormal, 1 = considered normal but with remark(s), 2 = normal). For the traits ‘gait’ and ‘penis’, a more complex scoring system based on 5 (-2 to +2: -2 = lame, -1 = considered normal gait but with remark(s), 0 = normal gait, 1 = smooth gait, 2 = extremely smooth gait) and 4 (-1.5. to +1: -1.5. = irreversible problem, -1 = reversible problem, 0 = normal, 1 = well developed) categories, respectively, was used. For gross motility, 5 categories were used (0 to 4: 0 = aspermia, 1 = semen present but no individual cell motion, 2


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= individual cell motion without swirls, 3 = slow swirls, 4 = rapid swirls), whereas progressive motility and morphology were expressed as percentages. The threshold values for classifying bulls as deferred or unsatisfactory potential breeders, depending on the importance of the trait and severity of the problem, are listed in Table 2. The percentage of missing values (M), when present, is given as well (Table 1). The subclasses determining the overall BSE score, as well as the separately examined characteristics constituting these subclasses − except for those relating to the external (i.e. penis, prepuce, scrotum, testis, epididymis, funiculus spermaticus) and internal (i.e. vesicular glands, ampullae ductus deferentes, prostate, inguinal rings, pelvic lymph nodes) genitalia − are described in Table 1.

Statistical analysis

The main objectives were to evaluate the BSE of BB bulls, to compare the BSE of BB bulls of different age categories and to compare the BSE of young BB and HF bulls. For the second objective, the BB population was subdivided into 3 groups: < 24 months, from 24 to 48 months, and > 48 months. For the third objective, the BSE of the unselected HF group was compared with that of the unselected BB group, namely the first age category.

The original scores, subclass scores, overall BSE score, number of failure reasons, libido and mass motility were compared by means of the Kruskall Wallis test. Finally, SC, % normal cells, and progressive motility were compared by a fixed effects model using the F-test. This was done in SAS 9.1.

RESULTS

Overall BSE score

In total, 247 BSE were performed: 156 on BB bulls and 91 on HF bulls.

Six of the 156 BB bulls were not fully examined: 1 bull (21.11 months) never showed any mounting interest, and 5 other bulls (between 15.39 and 22.65 months) mounted vigorously but were not able to protrude the penis. Since no semen of these 6 bulls could be collected using an artificial vagina, they were all considered unsatisfactory potential breeders and were discarded from further analysis.

The overall BSE results, the BSE subclass scores and the results for the separate constituting traits for the remaining 150 BB and 91 HF bulls are given in Table 1. The separate constituting traits results of the subclass scores for external and internal genitalia are not presented, since no significant differences were noted, either between breeds or between age groups within the BB breed. Markedly more young BB bulls failed the BSE test


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compared to young HF bulls (P = 0.0005), while in the BB group, fewer old bulls (> 48 months) failed the BSE compared to both the younger age groups (P = 0.0243).

BSE subclass scores

A comparison of the subclass scores reveals that SC (P < 0.0001), semen quality (P = 0.0005) and physical soundness (P = 0.0359) were responsible for the breed difference in overall BSE result, whereas external (P = 0.7229) and internal (P = 0.6635) genitalia were not different between the HF and the BB breed (Table 1).

For the three age categories within the BB breed, the fact that the number of BB bulls older than 48 months that fail the BSE for SC is significantly lower compared to the other BB age groups (P < 0.0001) is responsible for the difference for the trait “genitalia” within the breed (P = 0.0007), which in turn, and together with semen quality (P = 0.0461), is responsible for the difference in overall BSE result within the breed (P = 0.0243; Table 1).

Separate constituting BSE traits

Significantly more young BB bulls failed the BSE test due to a below threshold SC compared to HF bulls and a high number of bulls of both breeds (although markedly more BB bulls) failed the BSE due to poor sperm morphology, whereas only a few bulls failed for progressive motility (Table 1). A significantly higher proportion of BB bulls (31.3. and 29.5% for BB bulls < 24 months and from 24 to 48 months of age, respectively) failed both for SC and for sperm morphology compared to HF bulls (13.2%).

In order to further compare the HF and BB bulls, a comparison of the separate constituting breeding soundness characteristics based on the more detailed original scores and not on the position of these scores relative to threshold values is additionally given in Table 2.

Testicular consistency did not differ between the young bulls of the two breeds (P = 0.4548; Table 2). Average percentage of normal sperm (P = 0.0021) and average progressive motility (P < 0.0001) were significantly better in the HF breed (Table 2). Physical soundness consists of several factors, of which the average general health (trait “general examination”; P = 0.0250), the average gait (P = 0.0003) and the average soundness of feet and legs (P = 0.0330) differed between the breeds due to a higher average score for the HF breed (Table 2).

For the three age categories within the BB breed, persistently more young BB bulls failed for gross motility, progressive motility and normal morphology compared to the older age groups (Table 1). A more detailed comparison within the breed based on the original scores (Table 2) reveals that neither average gross motility (P = 0.6344) and progressive motility (P = 0.7842) nor average semen morphology (P = 0.3572) differed between the age

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Table 1: The percentages of bulls classified as satisfactory (S), deferred (D) or unsatisfactory (U) potential breeders out of N assessed bulls for each constituting breeding soundness characteristic (except for external and internal genitalia), for the subclass scores and for the overall breeding soundness score, both for the HF and the three age categories of the BB breed. The percentage of missing values when these occurred is also given (M). Statistical analysis is only presented for the subclass- and overall breeding soundness scores.

Characteristic HF BB < 24 months BB 24 – 48 months BB > 48 months N = 91 N = 64 N = 61 N = 25 S D U M S D U M S D U M S D U M Overall BSE 35.2 49.4 9.9 5.5 1 6.3 82.8 10.9 0.0 2a 16.4 63.9 16.4 3.3 a 32.0 64.0 4.0 0.0 b Physical soundness 91.2 2.2 1.1 5.5 1 87.4 6.3 6.3 0.0 2a 85.3 4.9 8.2 1.6 a 92.0 4.0 4.0 0.0 a General examination 100.0 0.0 0.0 0.0 96.9 3.1 0.0 0.0 100.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 Eyes 100.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 100.0 0.0 0.0 0.0 Mouth 94.5 4.4 1.1 0.0 96.9 3.1 0.0 0.0 93.4 6.6 0.0 0.0 100.0 0.0 0.0 0.0 Gait 94.5 0.0 0.0 5.5 100.0 0.0 0.0 0.0 98.4 0.0 0.0 1.6 96.0 4.0 0.0 0.0 Feet and legs 100.0 0.0 0.0 0.0 93.8 0.0 6.2 0.0 91.8 0.0 8.2 0.0 96.0 0.0 4.0 0.0 Genitalia 72.5 18.7 8.8 0.0 1 48.4 46.9 4.7 0.0 2a 52.5 34.4 13.1 0.0 a 92.0 8.0 0.0 0.0 b External genitalia 89.0 5.5 5.5 0.0 1 90.6 6.3 3.1 0.0 1a 96.8 1.6 1.6 0.0 a 96.0 4.0 0.0 0.0 a Internal genitalia 96.7 3.3 0.0 0.0 1 95.3 4.7 0.0 0.0 1a 96.7 3.3 0.0 0.0 a 100.0 0.0 0.0 0.0 a Scrotal Circumference 82.4 13.2 4.4 0.0 1 56.2 42.2 1.6 0.0 2a 55.7 32.8 11.5 0.0 a 96.0 4.0 0.0 0.0 b Semen evaluation 44.0 56.0 0.0 0.0 1 17.2 82.8 0.0 0.0 2a 36.1 62.3 0.0 1.6 b 32.0 68.0 0.0 0.0 ab Gross motility 100.0 0.0 0.0 0.0 96.9 3.1 0.0 0.0 98.4 1.6 0.0 0.0 100.0 0.0 0.0 0.0 Progressive motility 97.8 2.2 0.0 0.0 95.3 4.7 0.0 0.0 96.8 1.6 0.0 1.6 96.0 4.0 0.0 0.0 Normal morphology 44.0 56.0 0.0 0.0 17.2 82.8 0.0 0.0 36.1 63.9 0.0 0.0 36.0 64.0 0.0 0.0 A different superscript number for the young HF and BB bulls corresponds to a significant breed difference (P < 0.05); a different superscript letter for two age categories of BB bulls corresponds to a pair-wise significant difference (P < 0.05)

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Table 2: Arithmetic means of the original scores for the separate constituting breeding soundness characteristics, both for the HF and for the BB bulls. For the BB bulls, a subdivision for the assessed age categories is made. Both the scoring range and the threshold values for classifying bulls as deferred or unsatisfactory potential breeders for each specific characteristic are given, as well as the number of assessed bulls in each group (N)

Group

HF BB < 24 months BB 24 – 48 months BB > 48 months

Characteristic

Original score

BSE threshold

N = 91

N = 64

N = 61

N = 25

General examination 0 - 2 < 1: deferred 1.71 1 1.54 2a 1.70 ab 1.82 b Eyes 0 - 2 < 1 : deferred 2.00 1 1.99 1a 1.99 a 2.00 a Mouth and teeth 0 - 2 < 1: deferred; 0: failure 1.52 1 1.63 1a 1.60 a 1.78 a Gait -2 - 2 -2: deferred 0.70 1 0.35 2a 0.29 a 0.14 a Feet and legs 0 - 2 < 1: failure 1.37 1 1.25 2a 1.25 a 1.19 a Penis -1.5 - 1 -1: deferred; -1.5: failure -0.03 1 0.00 1a -0.02 a 0.00 a Prepuce 0 - 2 0: deferred 1.95 1 1.98 1a 2.00 a 2.00 a Scrotum 0 - 2 0: deferred 1.81 1 1.79 1a 1.66 b 1.53 b Testes 0 - 2 0: failure 1.87 1 1.97 1a 1.90 a 1.90 a Testicular consistency 0 - 4 no threshold 2.63 1 2.61 1a 2.31 b 2.08 b Epididymis 0 - 2 0: failure 1.88 1 1.84 1a 1.93 ab 2.00 b Funiculus spermaticus 0 - 2 0: deferred 1.96 1 1.98 1a 2.00 a 2.00 a Vesicular glands 0 - 2 0: deferred 1.93 1 1.88 1a 1.97 a 1.96 a Ampullae ductus def. 0 - 2 0: deferred 1.98 1 1.92 1a 1.90 a 2.00 a Prostate gland 0 - 2 0: deferred 1.85 1 1.88 1a 1.89 a 1.92 a Inguinal rings 0 - 2 0: failure 1.99 1 2.00 1a 2.00 a 2.00 a Pelvic lymph nodes 0 - 2 0: deferred 1.98 1 1.92 1a 2.00 a 2.00 a SC – threshold (cm) no limit < 0: deferred. < -2: failure 1.74 1 0.26 2a 0.15 a 2.2 b Gross motility 0 - 4 < 2: deferred 3.69 1 3.49 1a 3.38 a 3.52 a Progressive motility (%) 0 - 100 < 30: deferred 75.54 1 63.11 2a 64.92 a 63.08 a Normal morphology (%) 0 - 100 < 70: deferred 65.03 1 55.70 2a 58.34 a 61.74 a

A different superscript number for the young HF and BB bulls corresponds to a significant breed difference (P < 0.05); a different superscript letter for two age categories of BB bulls corresponds to a pair-wise significant difference (P < 0.05)


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groups within the BB breed. Within the BB breed, average testicular consistency decreased with age, with a significant difference (P = 0.0003) between bulls < 24 months of age and the older BB bulls, and with a tendency (P = 0.0982) for the oldest bulls to produce poorer results. Apart from average testicular consistency and SC (P < 0.0001), only the average scores for general health (trait “general examination”; P = 0.0323), scrotum (P = 0.0143) and epididymis (P = 0.0481) differed within the BB breed (Table 2).

Table 3: The number of (individual constituting breeding soundness) reasons for which bulls fail (both deferred and unsatisfactory classification) the breeding soundness examination, both for the HF and for the BB bulls. For the BB bulls, a subdivision for the 3 assessed age categories is made. The number of assessed bulls per group is given (N). The number (n) and percentage (%) of bulls failing for one or more reasons is listed

Group

Number of reasons HF

BB < 24 months

BB 24 - 48 months

BB > 48 months

N = 91 N = 64 N = 61 N = 25 statistics 1 1a ab b

1 n 34 29 29 14 % 37.36 45.31 47.54 56.00

2 n 16 21 14 2 % 17.58 32.81 22.95 8.00

3 n 2 8 4 1 % 2.20 12.50 6.56 4.00

4 n 2 1 1 0 % 2.20 1.56 1.64 0.00

5 n 0 1 1 0 % 0.00 1.56 1.64 0.00

A different superscript number for the young HF and BB bulls corresponds to a significant breed difference (P < 0.05); a different superscript letter for two age categories of BB bulls corresponds to a pair-wise significant difference (P < 0.05).

The number of reasons for which bulls failed the BSE are given in Table 3, both for the HF bulls and for the three age groups of BB bulls. A tendency (P = 0.0866) toward more failure reasons in the BB breed was noticed in the breed comparison. Within the BB breed, significantly less failure reasons occurred in the oldest bulls compared to the youngest group


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(P = 0.0170), whereas a tendency toward less failure reasons in the oldest bulls was present when they were compared with the BB bulls from 24 to 48 months old (P = 0.0946).

Libido

The reaction time differed neither between HF and BB bulls < 24 months of age (P = 0.9743), nor between the three age groups of BB bulls, although a tendency was noticed within the BB population toward a slower reaction with increasing age (P = 0.0675). Mounting enthusiasm did not differ between young bulls of the two breeds (HF and BB bulls < 24 months of age; P = 0.6208), though on the other hand, significant differences were observed between all three age groups in the BB population (P < 0.0001), thus illustrating an obvious decrease in mounting enthusiasm with increasing age (Table 4).

DISCUSSION

Markedly more BB bulls failed the BSE test compared to HF bulls, which indicates that BB bulls are more subject to fertility associated problems. This was further illustrated by the tendency for more failure reasons in the BB breed. The most obvious significant breed differences occurred for SC and semen quality. Hanset (2000) had already demonstrated that the average SC of 13-month-old BB bulls was rather low, namely 31.3. cm, with a median SC of 32.0 cm, both of which are low compared to other beef breeds (Coulter et al., 1987; Hanset, 2000). In our study, we were not able to measure such young bulls since the youngest BB bull examined was already 13.6 months old. Nevertheless, we concluded that approximately 44% of the BB bulls younger than 48 months had a below threshold SC, as compared to a figure of only 17.6% for the HF bulls. Two reasons for this obvious breed difference are possible: either the SC threshold values as set by the Society for Theriogenology are too high for BB bulls, which would suggest that a breed specific exception as was discussed for Bos Indicus, American Bison and Wagyu bulls might be necessary (Chenoweth et al., 1996; Keen et al., 1999; Sosa et al., 2002), or BB bulls are more prone to testicular hypoplasia or degeneration compared to other breeds. Breed differences in SC at a given age have already been demonstrated (Michaux and Hanset, 1981; Chenoweth et al., 1984; Coulter et al., 1987; Bruner et al., 1995; Chenoweth et al., 1996). The BB breed is indeed a specific breed, with extreme muscularity but reduced organ size. This has been demonstrated for several organ systems (Ansay and Hanset, 1979). Hence, it might be possible that this is also the case for the reproductive organs, which would result in smaller testicles compared to other breeds and, as a consequence, a lower SC relative to the bull’s age (Michaux and Hanset, 1981).

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Table 4: Libido assessment, on the basis of average mounting enthusiasm score and reaction time (in seconds), both for the HF breed and the BB breed. For the BB bulls, a subdivision for the assessed age categories is made. The number of assessed bulls per group (N) is given as well.

Group

Parameter

HF

N = 91 BB < 24 months

N = 64 BB 24 - 48 months

N = 61 BB > 48 months

N = 25

Mounting enthusiasm Average score (mean) 0.98 1 0.91 1a 0.57 b 0.10 c

Reaction time (s) Mean (± SD) 150.32 (± 334.61) 1 175.18 (± 355.57) 1a 186.60 (± 381.75) a 335.67 (± 555.67) a

Median 48.00 1 38.00 1a 46.00 a 109.50 a

A different superscript number for the young HF and BB bulls corresponds to a significant breed difference (P < 0.05); a different superscript letter for two age categories of BB bulls corresponds to a pair-wise significant difference (P < 0.05)


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However, in order to allow a BB specific lower SC threshold, these BB bulls (with smaller testicles compared to other breeds) should still produce good quality semen, which was not the case in the present study. Hence, since low SC were accompanied by poor sperm quality, we suspect the BB breed to be more prone to testicular hypoplasia or degeneration compared to the HF breed, and thus selecting for larger SC in BB bulls instead of lowering the SC thresholds is more appropriate. Since SC is correlated to the fertility of the female offspring, a negative effect of the use of subfertile bulls on the reproductive performance of female offspring in the BB breed seems inevitable (Barth, 1997). However, no reliable data on this subject are yet available. The fact that SC is not used as a parameter for bull selection in the Benelux countries might in part be responsible for the apparent testicular hypoplasia or degeneration within the BB breed. Although SC is also not used for HF bull selection in the Benelux countries, it is an international HF bull selection criterion, and the fact that semen of foreign HF bulls is also used in the Benelux countries might explain the better situation for SC in the HF breed in our study. However, the average SC of the HF bulls in our study is remarkably lower compared to literature data on the HF breed (Hueston et al., 1988), and slightly – albeit significantly – lower compared to New Zealand HF bulls (age corrected average: 31.51 versus 32.25 cm for Benelux and New Zealand bulls, respectively; Ambreed NZ, unpublished data). The fact that almost all BB bulls > 48 months of age pass the BSE for SC might be due to the selection within the BB AI centers, which involves discarding bulls with repeatedly poor semen motility before and after cryopreservation, as well as bulls with poor non-return rates, finally only keeping the “better fertile” bulls with increasing age. This age-dependent fertility selection hypothesis is supported by the finding that fewer reasons for BSE failure were noticed in the oldest BB bulls compared to the younger age groups. Furthermore, persistently more young BB bulls failed for gross motility, progressive motility and normal morphology compared to the older age groups. However, average sperm quality does not differ between the 3 BB age groups. A possible explanation for this might be that sperm quality in older bulls decreases due to testicular degeneration, eventually leading to a quality loss, even in the “better fertile” bulls with an above threshold SC (Kumi-Diaka et al., 1981; Hopkins, 1997; Van Camp, 1997; Brückmann et al., 2000; Brito et al., 2002). This hypothesis is supported by the finding that testicular consistency gradually decreased with age within the BB breed.

Regarding the BSE trait ‘semen evaluation’, significantly more BB bulls failed the BSE as a result of poor sperm quality compared to HF bulls, and the average breed scores for progressive motility and for sperm morphology both indicated a relevant difference in the advantage of the HF breed. The reasons for the generally poor BB semen quality have not yet been established, but several hypothetical causes can be suggested, such as inherited testicular hypoplasia and/or acquired testicular degeneration as a consequence of environmental stresses and the particularly high susceptibility of the BB breed to these stresses. The high proportion


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of BB bulls failing the BSE for SC seems to support this hypothesis, while impaired heat exchange at scrotal level due to the extreme muscularity combined with rather small scrota (average SC 1.74 versus 0.26 cm above threshold, for HF and BB bulls, respectively, Table 2) might increase the heat stress risk. Furthermore, inbreeding has been demonstrated to be a possible cause for poorer seminal traits (Smith et al., 1989), and it is generally accepted that the inbreeding coefficient in the BB population is quite high (Hanset et al., 2003). Although markedly more BB bulls failed the test due to poor sperm morphology compared to HF bulls, this seems to be the BSE bottle neck for both breeds. The substantial influence of sperm abnormalities on BSE classification has already been described for different breeds (Chenoweth et al., 1988; Bruner et al., 1995). In our study, a very strict morphology assessment, based on the guidelines of Barth and Oko (1989), was used. Indeed, any sperm cell that was not completely normal was considered abnormal, leading to a high proportion of bulls refused for this aspect in both breeds. The presence of abnormal spermatozoa in a semen sample indicates impaired spermatogenesis or testicular dysfunction, and a thorough sperm evaluation should therefore be performed. According to Saacke (2004), ejaculates containing high proportions of physically abnormal spermatozoa (and thus compensable defects) probably also contain certain proportions of morphologically normal semen bearing uncompensable defects, such as chromatin abnormalities, which justifies a strict morphology assessment. This resulted in low percentages of spermatozoa with a normal morphology for both breeds, although markedly more BB bulls failed for this item. The HF bulls were all examined for this study prior to the semen quality test that was implemented to decide on their acceptance for AI purposes, which explains the apparently poor morphology result for this breed. Moreover, the rather young age of these bulls might in some cases result in an immature sperm picture with subsequent poor morphology (Almquist and Amann, 1976; Lunstra and Echternkamp, 1982; Johnson, 1997). Of the HF bulls selected for AI use, 81.0% had an above threshold semen morphology (data not shown), which is markedly better than the 44.0 and 17.2% for the unselected HF and BB bulls, respectively. The difference between the latter two groups might, in addition to the significant difference of the SC relative to the bull’s age, partially be explained by the fact that the HF breed has been selected for AI purposes and thus for “good” fertility for many years worldwide (Söderquist et al., 1991). Since a significantly higher proportion of BB bulls < 48 months failed both for SC and for sperm morphology compared to HF bulls, testicular hypoplasia or degeneration seems the most relevant hypothesis to explain the breed difference in sperm quality. However, testicular consistency did not differ between young bulls of the two breeds. Further histological research to clarify this discrepancy is currently being undertaken.

The incidence rate of 7.1% (5 out of 70 discarded from further analysis) young BB bulls with persistent frenulums is remarkably higher than previously reported in other breeds (Carroll et al., 1964; Spitzer et al., 1988; Bruner et al., 1995). This problem was not at all


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encountered in the HF bulls in our study. This fact indicates breed differences, and the high prevalence in this small population of BB bulls of different origin might suggest the omnipresence of this heritable defect within the genetically narrow BB breed (Hanset et al., 2003). The significant HF advantage in the average breed score for gait and soundness of feet and legs, combined with 4 (of 64) young BB bulls failing the BSE due to severe feet and leg abnormalities, suggests that BB bulls might be less mobile than HF bulls. The remaining significant breed difference (i.e. general examination) in the advantage of the HF bulls is in our opinion coincidental.

Within the BB population, a few significant differences between the three age categories were noted, although this should be interpreted with the utmost reserve, since “fertility selection” of the older bulls was performed. Apart from SC and testicular consistency, only general animal health (trait “general examination”), scrotum and epididymis differed within the BB breed, but these differences were merely coincidental.

The results for the BB bulls clearly indicate that fertility associated problems, which might partially explain the observed poor field fertility results of BB natural service bulls, are present within the BB breed. Notwithstanding these problems, the average raw 56 day non-return rate of marketed BB semen over recent years (2001 - 2003) is 70.9 %, versus 71.7 % for HF bulls (CR Delta-VRV, personal communication), which is a negligibly small breed difference in non-return rate given the fresh semen quality differences. Several explanations for this apparent discrepancy can be given. Firstly, many BB bulls were classified as deferred rather than as unsatisfactory potential breeders. Although we did not re-examine bulls, these deferred bulls might have been classified as satisfactory on a subsequent evaluation. Secondly, the semen of several of the assessed BB bulls was never marketed since it was discarded either for inadequate fresh quality or poor freezability. Thirdly, BB straws are filled with an insemination dose of 22 x 106 motile spermatozoa, whereas HF straws generally contain 15 x 106 total spermatozoa/straw prior to freezing (personal observations). Even this lower HF insemination dose seems extremely high, a fact which could possibly compensate for the morphological abnormalities and result in a high non-return rate, since transcervical AI with HF semen doses as low as 2 x 106 spermatozoa/straw did not result in lower pregnancy rates compared to 12 x 106 spermatozoa/straw (Verberckmoes et al., 2005). Although we did not asses the non-return rates of the bulls whose semen was finally marketed, we assume that a distinct negative effect of poor sperm morphology on fertility is mainly present at insemination doses lower than currently used and in natural serving bulls where repeated servings can result in poorly concentrated ejaculates. Furthermore, a recent comprehensive study revealed that differences between bulls and between the ejaculates of any given bull accounted for only 0.38 % of the total variation in non-return rates (Christensen et al., 2005). Altogether, these factors might explain the comparable non-return rates of BB and HF bulls in spite of the fresh semen quality differences.


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When libido was assessed, neither reaction time nor mounting enthusiasm differed between young bulls of the two breeds. This confirms the fact that the libido of BB bulls is not lower than the libido of the HF breed, and that, notwithstanding their extreme muscularity, they are willing and able to mate naturally. The absence of breed differences in sex drive in Bos taurus bulls has already been demonstrated (Chenoweth et al., 1984). However, within the BB population, mounting enthusiasm significantly decreased and a tendency towards a slower reaction time, which was probably due to musculoskeletal or other inhibitory problems adversely affecting libido (Chenoweth, 1986), was noticed with older age. A decline in mounting enthusiasm with increasing age was similarly found in HF bulls (29 bulls, ranging from 54.87 to 128.58 months of age) that were compared with the young HF study population (data not shown), and it has already been described in bulls of different breeds (Chenoweth et al., 1984). This is the result of the greater mating experience of the older bulls (Chenoweth et al., 1984).

To our knowledge, results of breeding soundness and libido evaluations in BB bulls have never been published before. Breed differences between HF and BB bulls for the BSE exist, as markedly more BB bulls failed the BSE test compared to HF bulls and since a tendency towards more failure reasons was noticed in the BB breed. This confirms the fact that BB bulls are more subject to fertility associated problems. The most important reasons for failure were sperm morphology and SC. The latter parameter should definitely be incorporated into bull selection procedures in the Benelux. Within the BB breed, less failure reasons and fewer bulls failing the BSE were noted among the oldest bulls, since with increasing age only the “better” fertile bulls are further kept at the AI centers. Further research to determine whether BB bulls are more prone to testicular hypoplasia or degeneration compared to HF bulls is advisable.

ACKNOWLEDGEMENTS

The authors wish to thank the respective AI centers for their hospitality and for the opportunity to examine their bulls. The authors also wish to thank Dr. M. Coryn for critically reading the manuscript.


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REFERENCES

Almquist JO, Amann RP. Reproductive capacity of dairy bulls: Part XI. Puberal characteristics and postpuberal changes in production of semen and sexual activity of Holstein bulls ejaculated frequently. J. Dairy Sci. 1976; 59: 986 -991.




Barth AD, Arteaga AA, Brito LFC, Palmer CW. Use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation of sex drive and mating ability. Anim. Reprod. Sci. 2004; 84: 315 – 325.

Brito LFC, Silva AEDF, Rodrigues LH, Vieira FV, Deragon LAG, Kastelic JP. Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil. Anim. Reprod. Sci. 2002; 70: 181 – 190.

Brückmann A, Yang X, Schallenberger E. The effects of age on fertility, ejaculate parameters and testosterone, estradiol-17β and luteinizing hormone concentration in breeding bulls. Proceedings of the 14th International Congress on Animal Reproduction 2000; 3: 20.

Bruner KA, Van Camp SD. Assessment of the reproductive system of the male ruminant. Vet. Clin. North Am. Food Anim. Pract. 1992; 8(2): 331 – 345.

Bruner KA, McCraw RL, Whitacre MD, Van Camp SD. Breeding soundness examination of 1952 yearling beef bulls in North Carolina. Theriogenology 1995; 44: 129 – 145.

Carroll EJ, Aanes WA, Ball L. Persistent penile frenulum in bulls. J.A.V.M.A. 1964; 144: 747.

Chenoweth PJ. Libido testing. In Morrow DA (ed): Current Therapy in Theriogenology 2. Philadelphia: WB Saunders, 1986; p 136 - 142.

Chenoweth PJ, Farin PW, Mateos ER, Rupp GP, Pexton JE. Breeding soundness and sex drive by breed and age in beef bulls used for natural mating. Theriogenology 1984; 22: 341 – 349.


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Chenoweth PJ, Farin PW, Mateos ER, Rupp GP, Pexton JE. Relationship between breeding soundness and sex drive classifications in beef bulls. Theriogenology 1988; 30: 227 – 233.

Chenoweth PJ, Spitzer JC, Hopkins FM. A new bull breeding soundness evaluation form. In: Proceedings of the Society for Theriogenology AGM, 1992; p 63 – 70.


Chenoweth PJ, Chase Jr CC, Thatcher MJD, Wilcox CJ, Larsen RE. Breed and other effects on reproductive traits and breeding soundness categorization in young beef bulls in Florida. Theriogenology 1996; 46 (7): 1159 – 1170.



Garner DL. Ancillary tests of bull semen quality. Vet. Clin. North Am. Food Anim. Pract. 1997; 13 (2): 313 – 330.


Hanset R, de Tillesse S, Michaux P, André E. La consanguinité en Blanc-Bleu Belge. Sa genèse et son contrôle. Publication Herd-Book du BBB n° 2003 03-36 (in French).

Hoflack G, de Kruif A. Enkele aspecten van de diergeneeskundige begeleiding van bedrijven met belgisch witblauwe zoogkoeien. Vlaams Diergeneeskundig Tijdschrift 2003; 72: 243 – 255 (in Dutch).

Hopkins FM. Chapter 29: Diseases of the reproductive system of the bull. In Youngquist RS (ed): Current Therapy in Large Animal Theriogenology. Philadelphia: WB Saunders, 1997; p. 237 – 239.


Hueston WD, Monke DR, Milburn RJ. SC measurements on young Holstein bulls. J.A.V.M.A. 1988; 192: 766 – 768.



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Keen JE, Rupp GP, Wittenberg PA, Walker RE. Breeding soundness examination of North American bison bulls. J. Am. Vet. Med. Assoc. 1999; 214 (8): 1212 – 1217

Kumi-Diaka J, Nagaratnam V, Rwuaan JS. Seasonal and age-related changes in semen quality and testicular morphology of bulls in a tropical environment. Vet. Rec. 1981; 108: 13 – 15.

Lunstra DD, Echternkamp SE. Puberty in Beef Bulls: Acrosome Morphology and Semen Quality in Bulls of Different Breeds. J. Anim. Sci. 1982; 55 (3): 638 – 648.



Saacke RG. Sperm Morphology. In: Proceedings of the 37th annual convention of the American Association of Bovine Practitioners, Forth Worth, Texas 2004, 37; p. 67-71.

Smith BA, Brinks JS, Richardson GV. Estimation of genetic parameters among breeding soundness examination components and growth traits in yearling bulls. J. Anim. Sci. 1989; 67 (11): 2892 – 2896.


Sosa JM, Senger PL, Reeves JJ. Evaluation of American Wagyu sires for SC by age and body weight. J. Anim. Sci. 2002; 80: 19 – 22.

Spitzer JC, Hopkins FM, Webster HW, Kirkpatrick FD, Hill HS. Breeding soundness examination of yearling beef bulls. J.A.V.M.A. 1988; 193: 1075 – 1079.


Verberckmoes S, Van Soom A, Dewulf J, Thijs M, de Kruif A. Low dose insemination in cattle with the Ghent device. Theriogenology 2005; 64: 1716 - 1728.


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CHAPTER 3

QUALITY COMPARISON OF BELGIAN BLUE AND HOLSTEIN - FRIESIAN SPERM

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CHAPTER 3A

COMPARISON OF SPERM QUALITY OF BELGIAN BLUE AND HOLSTEIN FRIESIAN

BULLS

Modified from: Comparison of Sperm Quality of Belgian Blue and Holstein Friesian Bulls G. Hoflack, G. Opsomer, A. Van Soom, D. Maes, A. de Kruif, L. Duchateau Theriogenology, In Press

Chapter 3A: Sperm Quality of BB and HF Bulls

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ABSTRACT

Few data are currently available on sperm quality of Belgian Blue (BB) bulls. The present study compared sperm quality of BB to Holstein Friesian (HF) bulls of several age categories, by means of a classical semen evaluation. Volume and concentration, and consequently total sperm output depended largely on age. Gross, total, and progressive motility, % live and % normal spermatozoa were significantly lower in the BB breed. Primary sperm abnormalities, such as nuclear vacuoles, midpiece defects and cytoplasmic droplets which were noticed most frequently, occurred far more in the BB breed. Hence, disturbances in spermiogenesis are deemed to be the cause of the poorer BB sperm quality. Since these sperm abnormalities occur significantly more in the BB breed than in the HF breed, it seems as if the BB breed is genetically predisposed to a higher susceptibility to environmental stresses which are known to interfere with normal spermiogenesis. The small scrota typical of the genetically narrow BB breed might in part be responsible for this, and therefore selection for larger scrota in the BB breed is advisable.


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INTRODUCTION

Male fertility is an important factor in bovine reproduction since a single pasture bull is generally bred to numerous cows. Hence, evaluation of male fertility prior to breeding is of paramount importance to achieve breeding success. To this purpose, breeding soundness evaluations of bulls have been used over the past 50 years and are widely accepted (Ott, 1986; Barth, 1997). The evaluation of potential breeding soundness of a bull consists of several aspects, among which semen quality evaluation is a substantial element (Bruner and Van Camp, 1992; Chenoweth et al., 1994; Garner, 1997). Furthermore, artificial insemination (AI) has become common practice in bovine reproduction, enabling the use of semen of any given bull on a large number of cows. Cryopreserved semen of good quality is imperative in order to obtain acceptable non-return and/or conception rates (Phillips et al., 2004).

Overall, semen analysis is probably the most relevant procedure to evaluate male fertility potential (Chong et al., 1983; Vantman et al.,1989; Comhaire et al., 1992; Rijsselaere et al., 2002; Phillips et al., 2004). Several methods can be used to evaluate the quality of a fresh ejaculate or of frozen-thawed semen, but subjective evaluation using standard optical microscopy is by far most commonly used. The semen parameters that are routinely examined using standard optical microscopy are the concentration, the percentage of motile spermatozoa and the morphological grading of the sperm cells (Neuwinger et al., 1990; Phillips et al., 2004). Concentration can be determined in different ways, such as by means of a hemocytometer, a spectrophotometer, flow cytometrically, and by means of computer assisted sperm analysis (Prathalingam et al., 2006). Motility, both total and progressive, is generally estimated subjectively on a pre-warmed glass slide. Sperm morphology is the most reliable criterion to qualify an ejaculate, since it is least influenced by the collection process (Garner, 1997). Morphology can be assessed using different techniques, but supravital staining procedures such as eosin – nigrosin staining are commonly used and allow both a morphology differentiation and a live-dead assessment (Barth and Oko, 1989). This live-dead assessment is based on the physical intactness (i.e. structural integrity) of the membranes, only allowing the stain to penetrate the damaged sperm cells, resulting in both eosin penetrated (dead) and unstained (live) spermatozoa.

In Belgium, the two predominant cattle breeds are the Belgian Blue (BB) for beef, and the Holstein Friesian (HF) for dairy. The BB breed stems from the Durham Shorthorn, which was introduced in Belgium in 1841 and crossed with local dairy breeds, resulting in a breed called the “Blue of Limon” which was further mixed with local breeds. In 1938, selection for a white colour was started, resulting in the “White breed of Middle and High Belgium”. Based on a limited number of well muscled animals, selection for a better muscularity was started, which finally led to the present hyper muscled BB breed, which is famous for its low feed conversion ratio and its extremely high percentage of lean meat (Coopman et al., 2001).


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In contrast to the HF breed, data on semen quality of BB bulls are scarce. However, recently, a suboptimal semen quality in BB bulls was demonstrated, although this study only reported on a few semen parameters (Hoflack et al., 2006). The aims of the present study were to evaluate and compare the semen quality of the 2 predominant cattle breeds in Belgium, namely the BB beef breed and the HF dairy breed, with special emphasis on sperm morphology.


1. Study population

From February 2002 to February 2004, semen quality of 158 Belgian Blue (BB) bulls in Belgium and 270 Holstein Friesian (HF) bulls in The Netherlands (since no HF AI bulls are present in Belgium) was assessed. All semen samples were collected by means of an artificial vagina. In order to increase the efficiency of data gathering, the semen samples were collected at AI centres. To this end, the AI centres were visited 4 times a year and all the collected semen samples were examined.

The BB AI bulls were purchased from selection centres as well as from private farms based on their linear classification, which is a scoring system describing several physical characteristics of the bull (concerning size, muscular development, meaty type, stand and general appearance), and after a quarantine period of one month, they were accepted for AI purposes without further selection. However, bulls with poor libido or repeated poor semen motility before (< 60% progressive motility) and after (< 30% of total or < 15% of progressive motility) cryopreservation were discarded keeping only “more fertile” bulls with increasing age. Furthermore, aggressive or injured bulls were also eliminated. This selection procedure resulted in 3 groups of BB bulls which could arbitrarily be divided as follows: (1) the unselected youngest bulls (< 2 years), (2) the active breeding bulls between 2 and 4 years old which were intensively selected, and (3) the bulls of proven fertility of over 4 years of age considered to be veterans which survived culling for different reasons.

This was in contrast to the HF bulls, where bull calves were purchased based on their expected genetic value. At approximately 11 months of age, these bulls were transferred to an AI facility, where, after a quarantine period of one month semen was collected. Only when these bulls passed a strict semen quality test (2 consecutive ejaculates collected with 3 or 4 days interval ≥ 2 ml containing ≥ 600 x 106 spermatozoa / ml, with ≥ 65% motility, ≥ 80% normal morphology and ≥ 50% intact acrosomes) were they accepted for AI purposes.

When an accepted bull had produced 3000 straws, which was in average at approximately 14.5 months of age, semen collection was stopped for at least 3 years until the milk production data of his daughters became available. Only those progeny-tested bulls with


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good production indices were kept and semen collection was then restarted. This system resulted in 3 groups comparable to the 3 groups of BB bulls: (1) an unselected young group, (2) a group of selected young bulls that passed an initial semen quality test, and (3) a group of old bulls with good production indices in progeny tests that passed the semen quality test when they were young. Hence 4 comparisons were made:

1: the unselected young HF and BB bulls < 2 years of age, hereafter referred to as “the unselected bulls”;

2: the fertility selected young HF bulls, and the BB bulls between 2 and 4 years of age, hereafter referred to as “the selected bulls”;

3: old HF and BB (> 4 years of age) bulls selected for fertility as well as for other traits, hereafter referred to as “the old bulls”;

4: the overall HF and BB bull study population.

Since HF bulls only produced 3000 straws, after which production was stopped until indices of progeny tests were known, and since we only visited the AI centres 4 times a year, it was rare to have several semen evaluations of a particular HF bull, whereas this was quite common in the BB bulls of which production was only stopped when they were injured or became too aggressive to handle.

2. Classical semen evaluation

Different from the routine procedures of both AI centres, which slightly differed, we examined all ejaculates as described below.

The volume of each ejaculate was read from the graded collection tube immediately after collection.

The concentration of the ejaculates was determined by diluting 10 µl of semen in 990 µl 1M HCl and by counting the number of sperm cells in 1/100 mm3 of a Bürker counting chamber (Merck, Leuven, Belgium). The final semen concentration was the average of 2 - 4 counts. Total sperm output (TSO) was calculated by multiplying the volume and the concentration of each ejaculate.

Semen motility was evaluated immediately after collection. Gross motility was scored from 1 to 5 on a wet mount of neat semen at 100 x magnification (1 = cells present without motion; 2 = individual cell motion without swirls; 3 = slow swirls; 4 = rapid dark swirls; 5 = macroscopically visible rapid dark swirls). Total and progressive individual motility were subjectively assessed to the nearest 5% by placing 10 µl of diluted semen (10 µl aliquot of pure semen in 790µl physiological saline solution) on a pre-warmed glass slide at 37°C under a coverslip, and by examining 5 different microscopic fields all in the centre of the coverslip, under a 200 x phase-contrast microscope (Hopkins and Spitzer, 1997). This procedure was


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repeated twice and was always done by the same experienced observer. Next to these percentages, a velocity score (1 – 3.5) was also attributed to the sample: 1 = very slow semen (e.g. after freeze-thawing), 2 = slow semen, 3 = rapid semen, 3.5 = extremely rapid semen.

The assessment of the percentage of live (i.e. structurally membrane intact) spermatozoa and sperm morphology was done on air-dried, eosin-nigrosin stained smears under a 1000 x light microscope, using immersion oil. Between 200 and 400 spermatozoa were evaluated for the live-dead assessment, whereas the morphology evaluation was performed by counting 200 spermatozoa. Individual spermatozoa were classified according to Barth and Oko (1989) into one of five categories: (1) normal morphology; (2) abnormal head; (3) abnormal midpiece or tail; (4) proximal droplet; or (5) having a distal droplet. When multiple abnormalities were observed in the same sperm cell, only one abnormality was logged. Abnormal heads were given first priority in classification, abnormal midpieces or tails were classified with second priority, proximal droplets with third and distal droplets with least and last priority. These subjective morphology evaluations were all performed by the same experienced observer.

3. Detailed semen evaluation

A more detailed morphology classification based on the 1993 Society for Theriogenology guidelines (Chenoweth et al., 1992; 1994; Hopkins and Spitzer, 1997), albeit slightly adapted (Barth and Oko, 1989), was used on a subset of sperm evaluations. The abnormal sperm cells were classified into one of the following categories: (1) abnormal headshape (including rolled head – nuclear crest – giant head syndrome, microcephalic or macrocephalic heads, small or giant abnormal heads, narrow or broad heads, pear-shaped heads, abnormal contour, underdeveloped forms); (2) ‘all abnormal acrosomes’ (including knobbed acrosome, abnormal acrosome, detached or folded acrosomal membranes); (3) detached heads (both those with normal and abnormal head morphology); (4) ‘other head abnormalities’ (including nuclear pouches or vacuoles or diadem defects, double heads); (5) abnormal midpieces (pseudodroplets, rough midpieces and segmental aplasia); (6) abnormal tails (including tail stump defect, folded or coiled tails, simple bent tails, double tails, corkscrew defect, accessory tails, abaxial tails, terminally coiled tails, and broken tails); (7) proximal and (8) distal cytoplasmic droplets. From this, the proportion of spermatozoa with primary (i.e. underdeveloped sperm, double forms, acrosome defects, narrow heads, nuclear pouches or vacuoles or diadem defects, pear-shaped heads, abnormal contour, small abnormal heads, free abnormal heads, abnormal midpieces, strongly folded or coiled tails, accessory tails, proximal droplets) and secondary (i.e. small normal heads, giant and short broad heads, free normal heads, detached or folded or loose acrosomal membranes, abaxial tails, simple bent tails, terminally coiled tails, distal droplets) abnormalities was deducted (Hopkins and Spitzer, 1997). Primary abnormalities are considered to arise in the testis, while secondary abnormalities originate after the sperm cells have left the testis (Barth and Oko, 1989).


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4. Statistical analysis

When multiple semen evaluations of a bull were available, the data of the respective semen evaluations were averaged to obtain an as accurate as possible picture of the bull’s semen. The response variables that passed a test for normality were analysed by a fixed effects model, first including breed, and in a second model the 6 different categories (3 categories for HF and BB). These variables include concentration, TSO, volume, total motility, progressive motility, % live, % normal, % abnormal heads, % abnormal midpieces or tails, % proximal and % distal droplets for the classical semen evaluation and % abnormal headshape, % ‘all abnormal acrosomes’, % detached heads, % ‘other abnormal heads’, % abnormal midpieces, % abnormal tails, % proximal and % distal droplets, and finally the % primary and % secondary abnormalities for the detailed evaluation.

For all the other variables which could not be considered to be normally distributed, the nonparametric Kruskal-Wallis test was performed, also first globally between the two breeds and next between the 6 categories.

This was done in SAS 9.1. The global significance level for all statistical analyses was 0.05. The comparisonwise significance level for the 6 relevant pairwise comparisons was adjusted by Bonferroni’s multiple comparisons technique and therefore set to 0.008 (0.05/6).

RESULTS

1. Classical semen evaluation

In total, 101 semen evaluations of 68 unselected BB bulls, 216 semen evaluations of 101 selected BB bulls, 84 semen evaluations of 35 old BB bulls, and 119 semen evaluations of 113 unselected HF bulls, 153 semen evaluations of 148 selected HF bulls and 36 semen evaluations of 35 old HF bulls were available. The results of the examined parameters for the respective groups within both breeds, as well as the overall results per breed are listed in Table 1.

1.1. Effect of breed on classical semen evaluation results

Significant (P ≤ 0.0005) differences existed for all the assessed semen parameters when the overall breed results were compared: although volume, concentration and (as a consequence) TSO were higher in the BB breed, gross motility, total and progressive individual motility, velocity, % live and % normal spermatozoa were always higher in the HF breed. The percentages of abnormalities (abnormal heads, midpieces or tails, proximal and distal droplets) were consistently higher in the BB bulls.

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Table 1: Arithmetic mean (± SD) and median (and range) of all assessed classical sperm quality parameters for the HF and BB breed, both for the overall breed results and for the respective groups of both breeds. The number of assessed bulls in each group is also given (N).

Parameter N

Overall BB 204

Overall HF 296

Unselected BB 68

Selected BB 101

Old BB 35

Unselected HF113

Selected HF 148

Old HF 35

Concentration (mL-1) 1150 ± 410 1 982 ± 440 2 1190 ± 445 aα 1152 ± 394 aα 1069 ± 381 aα 766 ± 389 b 1161 ± 415 a 917 ± 365 a

1142 (0 - 2273) 973 (125 - 2325) 1246 (0 - 2273) 1144 (285 - 2250) 1090 (346 - 1948) 706 (125 - 1970) 1110 (310 - 2325) 848 (270 - 1913)

TSO 6328 ± 29421 4026 ± 2891 2 5426 ± 2621 aα 6522 ± 2661 aβ 7522 ± 3754 aβ 2644 ± 1602 b 3975 ± 2017 b 8663 ± 4224 a

6118 (0 - 19360) 3510 (128 - 22840) 5174 (0 - 12664) 6284 (656 - 13840) 6392 (891 - 19360) 2320 (128 - 7092) 3667 (744 - 11296) 7873 (3510 - 22840)

Volume (mL) 5.52 ± 1.95 1 4.15 ±2.40 2 4.48 ± 1.57 aα 5.74 ± 1.76 aβ 6.91 ± 2.09 aγ 3.40 ± 1.19 b 3.44 ± 1.15 b 9.54 ± 2.37 b

5.50 (0.80 - 13.50) 3.50 (0.90 - 16.00) 4.55 (0.80 - 9.00) 5.60 (1.50 - 10.38) 6.80 (1.30 - 13.50) 3.30 (0.90 - 6.40) 3.30 (1.20 - 7.50) 8.90 (6.50 - 16.00)

Total motility (%) 66.51 ± 11.73 1 81.65 ± 9.31 2 66.11 ± 13.30 aα 67.37 ± 10.58 aα 64.85 ± 11.76 aα 79.89 ± 11.02 b 82.55 ± 8.18 b 83.53 ± 6.69 b

68.88 (5.00 - 87.50) 85.00 (35.00 - 95.00) 70.00 (5.00 - 87.50) 69.50 (30.00 - 85.00) 67.00 (30.00 - 80.50) 80.00 (35.00-95.00) 85.00 (40.00 - 95.00) 85.00 (72.00 - 95.00)

Progressive motility (%) 62.73 ± 13.56 1 79.07 ± 10.95 2 62.80 ± 14.85 aα 63.73 ± 12.74 aα 59.77 ± 13.18 aα 76.19 ± 13.28 b 80.36 ± 9.07 b 82.89 ± 7.27 b

65.00 (1.00 - 85.00) 80.00 (30.00 - 95.00) 65.00 (1.00 - 85.00) 66.13 (20.00 - 85.00) 62.50 (25.00 - 78.00) 80.00 (30.00-95.00) 82.00 (35.00 - 95.00) 85.00 (65.00 - 95.00)

Live spermatozoa (%) 75.14 ± 10.79 1 88.79 ± 6.34 2 74.99 ± 11.30 aα 76.16 ± 9.83 aα 72.46 ± 12.22 aα 87.29 ± 7.68 b 90.96 ± 3.83 b 84.49 ± 6.68 b

77.31 (22.00 - 92.00) 90.50 (47.75 - 99.75) 76.88 (22.00 - 88.75) 77.50 (43.88 - 92.00) 76.75 (29.50 - 87.25) 89.75 (47.75 - 99.75) 91.50 (77.25 - 97.67) 83.25 (71.25 - 95.75)

Normal spermatozoa (%) 57.21 ± 17.28 1 71.89 ± 14.26 2 57.56 ± 16.61 aα 58.08 ± 17.18 aα 54.03 ± 18.91 aα 65.51 ± 17.72 b 76.33 ± 10.06 b 73.74 ± 8.28 b

59.00 (3.00 - 90.00) 74.50 (12.50 - 96.00) 60.50 (3.00 - 87.00) 59.50 (4.50 - 86.75) 51.00 (10.50 - 90.00) 68 (12.50 - 96.00) 77.00 (38.00 - 93.00) 76.50 (49.00 - 88.50)

Abnormal heads (%) 13.52 ± 11.75 1 9.52 ± 8.08 2 14.40 ± 10.45 aα 13.77 ± 13.00 aα 11.10 ± 10.18 aα 11.19 ± 9.82 a 8.17 ± 6.61 b 9.79 ± 6.52 a

10.17 (1.00 - 75.00) 7.50 (0.50 - 63.50) 12.00 (1.00 - 46.00) 10.13 (1.50 - 75.00) 7.67 (2.00 - 49.25) 9.50 (1.00 - 63.50) 6.00 (0.50 - 48.50) 9.5 (2.00 - 38.00)

Abnormal midpieces or 20.02 ± 10.48 1 13.31 ± 7.77 2 20.23 ± 11.16 aα 19.08 ± 9.45 aα 22.32 ± 11.82 aα 14.13 ± 8.93 b 12.63 ± 7.30 b 13.54 ± 5.17 b

tails (%) 17.50 (4.00 - 77.00) 11.50 (1.00 - 53.00) 17.50 (5.75 - 77.00) 17.50 (4.00 - 65.50) 17.20 (5.83 - 50.50) 11.50 (1.00 - 41.00) 11.00 (1.50 - 53.00) 12.00 (5.50 - 26.50)

Proximal droplets (%) 6.40 ± 8.09 1 3.81 ± 8.07 2 5.55 ± 7.96 aα 6.24 ± 7.66 aα 8.45 ± 9.35 aα 7.04 ± 12.15 a 1.88 ± 2.22 b 1.54 ± 1.31 b

3.50 (0.00 - 56.00) 1.50 (0.00 - 68.50) 3.00 (0.00 - 56.00) 3.25 (0.00 - 37.00) 5.00 (0.50 - 45.50) 3.00 (0.00 - 68.50) 1.50 (0.00 - 19.00) 1.00 (0.00 - 6.00)

Distal droplets (%) 2.86 ± 2.85 1 1.47 ± 2.79 2 2.26 ± 2.34 aα 2.82 ± 2.50 aαβ 4.11 ± 4.12 aβ 2.13 ± 4.06 a 0.99 ± 1.41 b 1.39 ± 1.50 b

2.00 (0.00 - 17.17) 1.00 (0.00 - 39.50) 1.75 (0.00 - 12.00) 2.00 (0.00 - 11.75) 2.63 (0.00 - 17.17) 1.00 (0.00 - 39.50) 0.50 (0.00 - 11.00) 1.00 (0.00 - 6.00)

Velocity 2.68 ± 0.46 1 2.94 ± 0.45 2 2.76 ± 0.48 aα 2.65 ± 0.45 aα 2.63 ± 0.43 aα 2.81 ± 0.56 a 2.98 ± 0.36 b 3.15 ± 0.28 b

2.88 (1.00 - 3.50) 3.00 (1.50 - 3.50) 3.00 (1.00 - 3.50) 2.75 (1.50 - 3.50) 2.79 (1.50 - 3.25) 3.00 (1.50 - 3.50) 3.00 (2.00 - 3.50) 3.00 (2.50 - 3.50)

Gross motility 3.47 ± 0.74 1 3.88 ± 0.75 2 3.46 ± 0.83 aα 3.51 ± 0.72 aα 3.37 ± 0.61 aα 3.61 ± 0.82 a 4.09 ± 0.58 b 4.18 ± 1.01 b

3.50 (0.50 - 5.00) 4.00 (2.00 - 5.00) 3.50 (0.50 - 5.00) 3.50 (1.25 - 4.75) 3.44 (2.25 - 4.50) 3.88 (2.00 - 4.75) 4.00 (2.50 - 5.00) 4.50 (2.00 - 5.00)

A different superscript number (1, 2) for the overall results of the HF and BB bulls and a different superscript letter (a, b) for the matched comparisons correspond to a significant breed difference (P < 0.05); a different Greek superscript letter (α, β, γ) for the 3 BB age categories corresponds to a pair-wise within breed significant difference (P < 0.05). (TSO: total sperm output = volume x concentration)

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Table 2: Arithmetic mean (± SD) and median (and range) of the (cumulative subclasses of the) assessed detailed sperm morphology classification of the HF and BB breed, both for the overall breed results and for the respective groups of both breeds. The number of assessed bulls in each group is also given (N).

ParameterN

Overall BB 161

Overall HF189

Unselected BB49

Selected BB85

Old BB 27

Unselected HF60

Selected HF95

Old HF 34

%AHS 1.89 ± 2.51 1 1.96 ± 1.94 1 2.47 ± 3.07 aα 1.75 ± 2.43 aα 1.27 ± 1.14 aα 2.33 ± 2.37 a 2.01 ± 1.63 a 1.19 ± 1.73 a

1.33 (0.00 - 19.50) 1.50 (0.00 - 13.00) 1.50 (0.00 - 19.50) 1.25 (0.00 - 17.33) 1.00 (0.00 - 4.50) 2.00 (0.00 - 13.00) 1.50 (0.00 - 8.50) 0.75 (0.00 - 9.00)

%AAA 7.09 ± 5.21 1 4.86 ± 5.02 2 7.58 ± 5.38 aα 6.90 ± 5.33 aα 6.84 ± 4.63 aα 6.50 ± 7.18 a 3.87 ± 2.84 b 4.76 ± 4.54 a

6.00 (0.00 - 35.00) 3.50 (0.00 - 38.50) 6.50 (0.00 - 26.50) 5.50 (0.50 - 35.00) 6.50 (1.00 - 23.00) 5.00 (0.50 - 38.50) 3.50 (0.00 - 13.50) 3.50 (0.50 - 23.50)

% DH 0.53 ± 0.90 1 0.81 ± 1.52 2 0.71 ± 1.29 aα 0.51 ± 0.71 aα 0.30 ± 0.36 aα 1.33 ± 2.24 a 0.58 ± 1.02 a 0.51 ± 0.67 a

0.25 (0.00 - 7.00) 0.50 (0.00 - 11.00) 0.25 (0.00 - 7.00) 0.33 (0.00 - 3.50) 0.25 (0.00 - 1.50) 0.50 (0.00 - 11.00) 0.00 (0.00 - 6.50) 0.50 (0.00 - 3.00)

%OAH 6.38 ± 9.14 1 3.06 ± 4.72 2 7.12 ± 7.28 aα 6.40 ± 10.44 aα 4.98 ± 7.83 aα 2.84 ± 3.25 b 3.06 ± 5.97 b 3.49 ± 2.46 a

3.88 (0.00 - 66.00) 1.50 (0.00 - 43.50) 5.00 (0.00 - 31.50) 3.75 (0.00 - 66.00) 3.38 (0.00 - 40.25) 1.75 (0.00 - 14.00) 1.50 (0.00 - 43.50) 3.00 (0.00 - 9.50)

%AM 13.83 ± 8.04 1 9.07 ± 5.94 2 12.92 ± 6.43 aα 13.53 ± 8.14 aα 16.44 ± 9.92 aα 8.41 ± 5.40 b 9.77 ± 6.75 b 8.28 ± 4.06 b

11.50 (2.00 - 51.50) 7.50 (0.00 - 49.00) 11.50 (3.50 - 29.50) 11.00 (2.00 - 51.50) 12.00 (6.67 - 36.50) 7.00 (0.00 - 27.00) 8.00 (2.50 -49.00) 7.00 (2.50 - 20.00)

%AT 6.76 ± 4.94 1 5.48 ± 4.27 2 7.25 ± 4.17 aα 6.31 ± 4.83 aα 7.30 ± 6.41 aα 6.68 ± 5.81 a 4.72 ± 3.07 a 5.50 ± 3.51 a

5.75 (0.00 - 38.50) 4.50 (0.50 - 36.00) 6.00 (0.50 - 25.00) 5.50 (0.00 - 38.50) 6.50 (1.50 - 33.00) 5.00 (1.00 - 36.00) 4.00 (0.50 - 16.00) 4.50 (2.00 - 15.50)

% PPD 6.63 ± 8.36 1 3.43 ± 8.02 2 6.31 ± 9.12 aα 5.96 ± 7.02 aα 9.34 ± 10.39 aα 7.13 ± 13.21a 1.77 ± 2.37 b 1.54 ± 1.33 b

4.00 (0.00 - 56.00) 1.50 (0.00 - 68.50) 3.00 (0.00 - 56.00) 3.25 (0.00 - 36.00) 5.00 (1.50 - 45.50) 2.50 (0.00 - 68.50) 1.00 (0.00 - 19.00) 1.00 (0.00 - 6.00)

% DPD 2.81 ± 2.74 1 1.19 ± 1.42 2 2.12 ± 2.13 aα 2.89 ± 2.59 aαβ 3.83 ± 3.74 aβ 1.79 ± 1.82 a 0.75 ± 0.85 b 1.40 ± 1.51 b

2.00 (0.00 - 17.17) 0.50 (0.00 - 10.00) 1.50 (0.00 - 8.50) 2.00 (0.00 - 11.00) 2.75 (0.00 - 17.17) 1.25 (0.00 - 10.00) 0.50 (0.00 - 3.50) 1.00 (0.00 - 6.00)

%N 54.08 ± 16.54 1 70.15 ± 13.30 2 53.54 ± 14.71 aα 55.78 ± 17.50 aα 49.69 ± 16.26 aα 63.00 ± 17.07 b 73.52 ± 10.02 b 73.32 ± 8.00 b

55.00 (3.00 - 85.50) 72.50 (12.50 - 90.00) 57.00 (3.00 - 76.00) 57.00 (4.50 - 85.50) 50.63 (10.50 - 79.00) 66.25 (12.50 - 90.00) 74.00 (38.00 - 89.50) 76.25 (49.00 - 85.50)

A different superscript number (1, 2) for the overall results of the HF and BB bulls and a different superscript letter (a, b) for the matched comparisons correspond to a significant breed difference (P < 0.05); a different Greek superscript letter (α, β, γ) for the 3 BB age categories corresponds to a pair-wise within breed significant difference (P < 0.05). (AHS: abnormal headshapes, AAA: all abnormal acrosomes, DH: detached heads, OAH: other abnormal heads, AM: abnormal midpieces, AT: abnormal tails, PPD: proximal cytoplasmic droplets, DPD: distal cytoplasmic droplets, N: normal sperm cells)


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1.2. Effect of sire group and breed on classical semen evaluation results

When the 3 respective groups were compared between breeds, volume, % of total and progressive motility, % of live and % of normal spermatozoa, and % of abnormal midpieces or tails always differed significantly (P ≤ 0.0007) between breeds within all respective groups. These results were always higher for the HF breed, except for % abnormal midpieces or tails which were consistently higher in the BB groups, and for volume, where the BB results, with the exception of the old bulls, were higher. Of the other assessed parameters, concentration only differed between the unselected bulls (P < 0.0001; BB > HF), while TSO differed both between the unselected bulls (P < 0.0001; BB > HF), as well as between the selected bulls (P < 0.0001; BB > HF). The percentage of abnormal heads only differed between the selected bulls (P < 0.0001; BB > HF). The percentage of proximal (P ≤ 0.0003; BB > HF) and distal (P < 0.0001; BB > HF) droplets, as well as the velocity (P < 0.0001; HF > BB) and gross motility (P ≤ 0.0072; HF > BB) differed between breeds both for the selected bulls and for the old bulls.

1.3. Effect of sire age within the BB breed on classical semen evaluation results

When the 3 age groups within the BB breed were compared, only volume, TSO and % distal droplets significantly differed, whereas all the other parameters did not differ amongst ages within the BB breed. Volume significantly increased with age (P ≤ 0.0001), whereas only the youngest BB bulls had a lower TSO compared to both the older BB age groups (P ≤ 0.0059). The oldest BB bulls had significantly more distal droplets compared to the BB bulls < 2 years of age (P = 0.0015).

2. Detailed semen evaluation

Regarding the detailed morphology classification on a subset of 68 semen evaluations of 49 unselected BB bulls, 141 semen evaluations of 85 selected BB bulls, 57 semen evaluations of 27 old BB bulls, and 61 semen evaluations of 60 unselected HF bulls, 95 semen evaluations of 95 selected HF bulls and 34 semen evaluations of 34 old HF bulls, the results are listed in Tables 2 and 3. The results for the parameters of the classical semen evaluation were similar to those of all the semen evaluations (Table 1).

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Table 3: Arithmetic mean ± SD (and median) of the morphological characteristics (except abnormal midpieces, proximal and distal droplets) that significantly differed between breeds, either for the overall breed results or for the respective groups of the HF and BB breed, or between age groups within the BB breed. The number of assessed bulls in each group is also given (N).

Parameter N

Overall BB161

Overall HF189

Unselected BB49

Selected BB85

Old BB 27

Unselected HF60

Selected HF95

Old HF 34

% Knobbed acrosome 0.48 ± 0.79 (0.13) 1 0.19 ± 0.42 (0.00) 2 0.48 ± 0.87 (0.00) aα 0.48 ± 0.75 (0.13) aα 0.47 ± 0.78 (0.38) aα 0.15 ± 0.37 (0.00) b 0.19 ± 0.45 (0.00) b 0.22 ± 0.43 (0.00) a

% Acrosome defect 4.92 ± 3.91 (4.00) 1 3.24 ± 4.01 (2.00) 2 5.41 ± 4.60 (3.75) aα 4.64 ± 3.66 (3.50) aα 4.89 ± 3.34 (4.50) aα 4.16 ± 5.55 (2.50) a 2.52 ± 2.30 (2.00) b 3.65 ± 4.29 (2.50) a

% Nuclear vacuoles 6.37 ± 9.15 (3.88) 1 3.06 ± 4.71 (1.50) 2 7.11 ± 7.28 (5.00) aα 6.39 ± 10.45 (3.75) aα 4.98 ± 7.83 (3.38) aα 2.84 ± 3.25 (1.75) b 3.05 ± 5.96 (1.50) b 3.47 ± 2.43 (3.00) a

% Rolled-/giant head/nuclear crest 0.24 ± 0.43 (0.00) 1 0.39 ± 0.62 (0.00) 1 0.24 ± 0.55 (0.00) aα 0.23 ± 0.35 (0.00) aα 0.23 ± 0.39 (0.00) aα 0.25 ± 0.41 (0.00) a 0.58 ± 0.74 (0.50) b 0.09 ± 0.31 (0.00) b

% Tail stump defects 0.03 ± 0.10 (0.00) 1 0.06 ± 0.37 (0.00) 1 0.03 ± 0.10 (0.00) aα 0.04 ± 0.12 (0.00) aα 0.01 ± 0.03 (0.00) aα 0.15 ± 0.62 (0.00) a 0.02 ± 0.11 (0.00) b 0.03 ± 0.12 (0.00) a

% Folded or coiled tails 1.55 ± 2.38 (1.00) 1 2.07 ± 2.81 (1.00) 1 1.20 ± 1.10 (1.00) aα 1.43 ± 1.58 (1.00) aα 2.55 ± 4.82 (1.38) aα 3.38 ± 4.11 (2.00) b 1.72 ± 1.76 (1.00) a 0.74 ± 0.87 (0.50) b

% Simple bent tails 1.77 ± 2.01 (1.00) 1 1.02 ± 1.28 (0.50) 2 1.76 ± 2.18 (1.00) aα 1.69 ± 1.97 (1.00) aα 2.05 ± 1.81 (1.50) aα 1.28 ± 1.61 (1.00) a 1.07 ± 1.18 (1.00) a 0.41 ± 0.42 (0.50) b

% Accessory tails 2.44 ± 2.37 (2.00) 1 1.97 ± 2.41 (1.00) 2 3.11 ± 3.38 (2.50) aα 2.15 ± 1.66 (1.50) aα 2.14 ± 1.80 (1.88) aα 1.52 ± 1.69 (1.00) b 1.55 ± 1.99 (1.00) b 3.91 ± 3.46 (3.00) a

% Abaxial tails 0.57 ± 1.12 (0.17) 1 0.17 ± 0.43 (0.00) 2 0.87 ± 1.37 (0.33) aα 0.50 ± 1.08 (0.00) aα 0.23 ± 0.41 (0.00) aα 0.17 ± 0.32 (0.00) b 0.14 ± 0.43 (0.00) b 0.26 ± 0.57 (0.00) a

A different superscript number (1, 2) for the overall results of the HF and BB bulls and a different superscript letter (a, b) for the matched comparisons correspond to a significant breed difference (P < 0.05); a different Greek superscript letter (α, β, γ) for the 3 BB age categories corresponds to a pair-wise within breed significant difference (P < 0.05).

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Table 4: Arithmetic mean (± SD) and median (and range) of the percentage primary and secondary sperm abnormalities for the HF and BB breed, both for the overall breed results and for the respective groups of both breeds. The number of assessed bulls in each group is also given (N).

Parameter N

Overall BB 161

Overall HF189

Unselected BB49

Selected BB 85

Old BB 27

Unselected HF60

Selected HF95

Old HF 34

% Primary 38.23 ± 15.94 1 25.02 ± 11.80 2 39.28 ± 14.84 aα 36.40 ± 16.57 aα 42.07 ± 15.57 aα 30.39 ± 14.87 b 22.24 ± 9.48 b 23.32 ± 8.05 b

abnormalities 35.50 (10.00 - 93.00) 23.00 (8.00 - 81.00) 35.00 (15.00 - 93.00) 33.50 (10.00 - 91.50) 42.33 (18.00 - 76.50) 27.50 (10.00 - 81.00) 20.50 (8.00 - 58.50) 21.25 (12.00 - 50.50)

% Secondary 7.43 ± 4.96 1 4.38 ± 3.54 2 6.92 ± 4.14 aα 7.56 ± 5.73 aα 7.98 ± 3.56 aα 6.20 ± 4.93 a 3.64 ± 2.27 b 3.24 ± 2.13 b

abnormalities 6.75 (0.00 - 38.75) 3.50 (0.00 - 28.00) 6.75 (1.00- 17.50) 6.00 (0.00 - 38.75) 7.50 (2.75 - 19.00) 5.50 (0.00 - 28.00) 3.00 (0.00 - 9.50) 2.75 (0.50 - 10.00)


Chapter 3: Sperm Quality of BB and HF Bulls

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2.1. Effect of breed on detailed semen evaluation results

Except for abnormal head shapes, all traits evaluated differed significantly between breeds. This resulted in significantly more ‘all abnormal acrosomes’ (P < 0.0001, due to knobbed acrosomes and abnormal acrosomes), ‘other abnormal heads’ (P < 0.0001, due to nuclear pouches or diadem defects), abnormal midpieces (P < 0.0001), abnormal tails (P = 0.0095, due to simple bent tails, accessory tails and abaxial tails), proximal (P = 0.0003) and distal (P < 0.0001) droplets in the BB breed. Only detached heads were encountered more frequently in the HF breed (P = 0.0458).

The cumulative percentages of both primary (38.2 ± 15.9 versus 25.0 ± 11.8. for BB and HF bulls, respectively) and secondary (7.4 ± 5.0 versus 4.4 ± 3.5 for BB and HF bulls, respectively) abnormalities were significantly higher in the BB breed compared to the HF bulls (P < 0.0001), but the proportion of secondary abnormalities was rather low (Table 4).

2.2. Effect of sire group and breed on detailed semen evaluation results

Significantly more abnormal midpieces were present in the BB breed in all matched breed comparisons (P ≤ 0.0008). Moreover, more ‘other abnormal heads’ were present in the BB breed in all matched breed comparisons (P ≤ 0.0020), except for the matched comparison of the old bulls, while higher percentages of ‘all abnormal acrosomes’ occurred in the BB breed, but only in the breed comparison of the selected bulls (P < 0.0001).

The situation for proximal and distal cytoplasmic droplets in this detailed subset was identical to the results of all semen evaluations, as described above (Table 1). The percentages of abnormal headshapes, detached heads and abnormal tails never differed in any matched group comparison between breeds.

Significantly more abnormalities for several constituting morphology characteristics (Table 3, apart from midpiece defects, proximal and distal droplets: Table 2) were present in the BB breed, both for the unselected and selected bulls (knobbed acrosomes, abnormal acrosomes (tendency: P = 0.0092 for the breed comparison on unselected bulls), nuclear pouches or diadem defects, accessory tails and abaxial tails) compared to the unselected and selected HF bulls, but not for simple bent tails, where only the oldest age comparison resulted in more abnormalities for the BB breed. Other significant differences occurred for rolled head – nuclear crest – giant head syndrome and folded or coiled tails, but the breed advantage differed depending on the age comparison between breeds, rendering these latter differences rather illogical and thus difficult to interpret, and for tail stump defect for which coincidentally only the selected bulls differed, without a difference in the overall breed comparison. The remaining constituting sperm abnormalities were equally present in both breeds. In general, all these specific abnormal spermatozoa were extremely rare (< 1%),


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except for abnormal acrosomes, diadem defects, folded acrosomal membranes, accessory tails, coiled tails, single bents, proximal and distal droplets (all < 10%) and midpiece defects (> 10%). However, for most sperm defects, the percentages of abnormal cells were numerically higher in the BB breed and a larger proportion of BB bulls beared these higher percentages of abnormalities compared to the HF breed (data not shown).

The percentage of primary abnormalities was significantly higher in the BB bulls for all the matched breed comparisons (P ≤ 0.0008). This was also true for the percentage of secondary abnormalities (always P < 0.0001), except for the breed comparison of the youngest bulls, where no significant difference was noticed (P = 0.3715). However, the proportion of secondary abnormalities was always much lower than the primary abnormalities in all groups of both breeds (Table 4).

2.3. Effect of sire age within the BB breed on detailed semen evaluation results

No differences amongst ages within the BB breed were noticed, either for all 8 categories assessed (with the sole exception of the oldest BB bulls having significantly more distal droplets compared to the youngest BB bulls (P = 0.0006)) and for their constituting morphological characteristics, or between the cumulative proportions of primary and secondary sperm abnormalities.

DISCUSSION

The overall breed comparison demonstrates that the semen quality is significantly poorer for BB bulls. For all the important semen quality parameters (total and progressive motility, % live and % normal spermatozoa and % abnormal midpieces or tails), this breed difference is present between all matched groups. Apparently, a high proportion of ejaculated BB spermatozoa are structurally damaged or dead. This higher proportion of structurally damaged (or dead) spermatozoa in the BB breed results in significantly lower percentages of motile spermatozoa compared to HF bulls. Furthermore, the presence of sperm abnormalities is far more common in the BB breed, and these abnormal spermatozoa and in particular abnormal midpieces and tails, and droplets might influence semen velocity (Blom, 1977; Barth and Oko, 1989; Amann et al., 2000). The fact that (both proximal and distal) cytoplasmic droplets occurred equally in the youngest age groups of both breeds (while they were generally more present in the BB breed), can be explained by the rather young age of several of the youngest HF bulls resulting in an immature sperm picture with subsequent poor morphology, evidenced by among other things proximal cytoplasmic droplets (Almquist and Amann, 1976; Lunstra and Echternkamp, 1982; Johnson, 1997; Amann et al., 2000). These droplets probably slow down the affected individual spermatozoa, resulting in a slower velocity of the overall ejaculate. Indeed, velocity and gross motility differ between breeds for


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all groups except for the youngest bulls. However, the latter two parameters are based on a rather rough estimation, and a more detailed computer assisted sperm motility analysis should be performed to see whether kinematic measures of the semen really differ between breeds (Verstegen et al., 2002). Furthermore, gross motility is based on concentration, percentage of progressively motile spermatozoa and velocity of these sperm cells (Barth, 1997). The fact that the concentration was significantly higher in the youngest BB bulls compared to the HF breed, probably as a consequence of the older average age of this youngest BB group since concentration did not differ between breeds in the other groups, might also have resulted in the lack of difference for gross motility between the youngest bulls in the breed comparison. Although the concentration rarely differed between breeds, volume did and rather logically increased with age in both breeds (Garner et al., 1996). Since the BB breed is a specific breed with extreme muscularity but reduced organ size resulting in smaller testicles in comparison with other breeds, one would expect TSO to be significantly lower in the BB breed (Ansay and Hanset, 1979; Michaux and Hanset, 1981). However, since the ejaculation frequency was not always similar in the examined bulls this comparison is hard to interpret and hence, this hypothesis cannot be confirmed. Finally, although the overall breed comparison demonstrates that the percentage of abnormal heads is significantly higher in the BB breed, this difference only recurs in the comparison of selected HF and BB bulls. The latter demonstrates that abnormal heads are quite common in both breeds, although the proportion of abnormal heads is numerically (albeit not significantly) higher in the BB bulls in all matched breed comparisons.

Within the BB breed, none of the assessed semen parameters differed between age groups, except volume, which is logical as volume increases with older age (Garner et al., 1996), and for distal droplets due to a slightly elevated presence of these droplets in the oldest BB bulls. However, a slightly elevated amount of distal droplets is not considered to be pathological (Barth and Oko, 1989; Barth, 1997; Johnson, 1997).

Regarding the detailed morphology classification on the subset of semen evaluations, the same morphological abnormalities were present in both breeds but a higher proportion of BB bulls demonstrated generally higher percentages of abnormal cells compared to the HF breed, and more different abnormalities occurred simultaneously, resulting in higher cumulative proportions of mainly primary abnormalities in the BB breed. Breed differences for percentage of normal (and thus abnormal) spermatozoa were already demonstrated in the bull (Chenoweth et al., 1984; 1996; Söderquist et al., 1996). In the present study, the occurrence of morphologically abnormal sperm only differed significantly between breeds for a few parameters: knobbed and abnormal acrosomes, nuclear pouches or diadem defects, midpiece defects (segmental aplasia as well as “pseudodroplet like” defects), accessory and abaxial tails, and proximal and distal droplets, most of which are related to (genetic, environmentally induced, or a combination of both) disturbances in the spermatogenesis


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(Barth and Oko, 1989; Johnson, 1997). All the abovementioned abnormalities occurred significantly more in the BB breed, except in the breed comparison of the oldest bulls. The age dependent fertility selection, inherent to the AI centres, might be responsible for this finding. The fact that all these anomalies also occur in the HF breed, although at (significantly) lower percentages is not abnormal since it is quite normal to have small numbers of abnormalities in smears of normal semen (Barth and Oko, 1989; Garner, 1997). Hence, the BB breed only differs from the HF breed in the high cumulative proportions and omnipresence of several primary abnormalities and the higher proportions of dead spermatozoa. The omnipresence of the above-mentioned specific abnormalities in the BB breed suggests testicular degeneration (Parkinson, 2004). Indeed, the predominant presence of primary abnormalities compared to the rather low percentages of secondary abnormalities suggests testicular involvement (Barth and Oko, 1989). This can be attributed to a disturbance in spermatogenesis in the affected bulls, which can be due to hereditary predispositions for genetic sperm defects (Chenoweth, 2005), or to the deleterious influence of adverse environmental conditions (possibly exacerbating hereditary predispositions), finally leading to testicular degeneration. These adverse environmental influences include disturbances in testicular heat regulation, systemic illness, injury and/or pain, toxicity, nutritional deficiencies and environmental changes (Barth and Oko, 1989; Barth, 1997; Johnson, 1997). Due to the increased prevalence of sperm abnormalities in general, including nuclear vacuoles in particular in BB semen pictures, we think that this is most likely environmentally induced (Foote, 1999; Chenoweth, 2005). However, based on the limited amount of semen evaluations per bull which are moreover spread in time, we were not able to judge whether these defects were transitory or permanent. Taken into account the history of the bulls in our study and looking at the predominant BB sperm abnormalities similar to those seen after a mild thermal insult, we “per exclusionem” think that disturbances in testicular heat regulation are the most likely (Vogler et al., 1993). The small scrota typical of BB bulls (Hoflack et al., 2006) probably increase this heat stress risk (Cook et al., 1994; Brito et al., 2004). Whether the subsequent testicular degeneration, next to poorer sperm morphology and motility, also resulted in a lowered sperm production (volume and concentration) is hard to judge, since the BB and HF ejaculation frequency differed making it hard to interpret differences. Next to these adverse environmental stresses and a particularly high susceptibility of the BB breed to these spermatogenic stresses, hereditary predispositions for particular sperm abnormalities might also be present in the BB breed. Moreover, inbreeding has been suggested as a possible cause for poorer sperm quality and it is generally accepted that the inbreeding coefficient in the BB population is quite high (Smith et al., 1989; Hanset et al., 2003; Chenoweth, 2005). The better sperm quality in the HF breed is probably partially due to the fact that the HF breed has been selected for AI purposes and thus for “good” fertility for many years worldwide (Söderquist et al., 1991). Although the percentages of secondary abnormalities were generally


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higher in the BB semen, these percentages were negligibly low and probably without clinical and biological relevance for the two breeds.

Notwithstanding these sperm quality differences, the average raw 56 day non-return rate of marketed BB semen over recent years (2001 - 2003) is 70.9 %, versus 71.7. % for HF bulls (CR Delta-VRV, personal communication), which is a negligibly small breed difference in non-return rate given the fresh semen quality differences. Several explanations for this apparent discrepancy can be given. Firstly, not all the ejaculates we examined were marketed, as they were subjected to the quality criteria of the respective AI centres and only good ejaculates were processed. Secondly, most of the morphological sperm abnormalities visualized by standard light microscopy of eosin-nigrosin stained slides are considered to be compensable, with the exception of crater/diadem defects (Saacke et al., 2000; Saacke, 2004). This means that although BB bulls have a higher percentage of crater/diadem defects in their spermiogramme compared to HF bulls, most of the encountered abnormalities are compensable. To avoid fertility problems resulting from this high proportion of (mainly compensable) abnormalities in the BB semen, the semen concentration of BB straws is generally increased to 22 x 106 motile spermatozoa compared to the internationally accepted and in the HF centres implemented 15 x 106 total spermatozoa/straw prior to freezing, in order to compensate for these abnormalities (personal observations). However, a high proportion of morphologically abnormal sperm might be associated by normal appearing sperm bearing uncompensable defects, and this together with the significantly higher proportion of nuclear vacuoles in the BB breed, which is considered an uncompensable trait, probably negatively influences fertility outcome in the BB breed (Saacke, 2004).

No differences whatsoever were present between the age groups within the BB breed. This stresses the fact that morphological grading does not differ between these BB age groups and that poor sperm morphology is an overall breed problem. The AI centre selection effect for better fertile older BB bulls is probably partially nullified by the occurrence of testicular degeneration in these older bulls (Kumi-Diaka et al., 1981; Hopkins, 1997; Van Camp, 1997; Brückmann et al., 2000; Brito et al., 2002; Hoflack et al., 2006).

Although hard to judge because of differences in morphology classifications, the results of the BB breed seem poorer compared to other beef breeds (Bruner et al., 1995; Johnson et al., 1998; Kennedy et al., 2002). The smaller scrota typical of BB bulls compared to other breeds might be responsible for this, and hence, selection for larger scrota in the BB breed seems advisable (Hoflack et al., 2006).

The morphology results for the semen of the HF bulls investigated in the present study are lower compared to previous studies (Parkinson, 1987; Söderquist et al., 1991). Several explanations for these differences can be given: both selected and utterly unselected (not yet accepted for AI purposes) bulls were used in the present study, age differences between bulls


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in the different studies occur, the staining procedures and the morphology assessment differ between studies and subjective morphology assessment itself is to some extent considered inaccurate and imprecise (Davis and Katz, 1993; Coetzee et al., 1999; Hallap et al., 2005). Furthermore, it is generally accepted that fertility in the HF breed is declining and this might also be partially responsible for this, and selection for highly productive albeit better fertile animals, both male and female, should be considered worldwide (Royal et al., 2000, Lucy, 2001).

In conclusion, BB sperm differs from HF sperm due to a lower % of live and normal spermatozoa. This lower % of live spermatozoa influences the % of totally and progressively motile spermatozoa. Semen velocity is lower in the BB breed, as can be expected as a result of the higher proportion of abnormal midpieces and tails, and droplets. The morphological abnormalities encountered in both breeds are similar, but the proportion of morphologically abnormal spermatozoa is higher in the BB breed. The main problems occur for nuclear pouches or diadem defects, abnormal midpieces and both proximal and distal droplets.

ACKNOWLEDGEMENTS

The authors wish to thank the respective AI centers for their hospitality and for the opportunity to examine their bulls. Special thanks to CRV Holding for their kind cooperation.


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Amann RP, Seidel GE, Mortimer RG. Fertilizing potential in vitro of semen from young beef bulls containing a high or low percentage of sperm with a proximal droplet. Theriogenology 2000; 54: 1499 - 1515.

Ansay M, Hanset R. Anatomical, physiological and biochemical differences between conventional and double-muscled cattle in the Belgian Blue and White breed. Livest. Prod. Sci. 1979; 6: 5 - 13.

Barth AD. Evaluation of potential breeding soundness of the bull. In: Youngquist RS, editor. Current Therapy in Large Animal Theriogenology. Philadelphia: WB Saunders, 1997; p. 222 – 236 (Chapter 28).


Blom E. Sperm morphology with reference to bull infertility. First All-India Symp. Anim. Reprod. Ludhianap. (61 -81), 1977.

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Brito LFC, Silva AEDF, Barbosa RT, Kastelic JP. Testicular thermoregulation in Bos indicus, crossbred and Bos taurus bulls: relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production. Theriogenology 2004; 61: 511 - 528.

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Chenoweth PJ, Farin PW, Mateos ER, Rupp GP, Pexton JE. Breeding soundness and sex drive by breed and age in beef bulls used for natural mating. Theriogenology 1984; 22: 341 - 349.

Chenoweth PJ, Spitzer JC, Hopkins FM. A new bull breeding soundness evaluation form. In: Proceedings of the Society for Theriogenology AGM, 1992; p. 63 - 70.

Chenoweth PJ, Hopkins FM, Spitzer JC, Larsen RE. New Guidelines for the Evaluation of Bulls for Breeding Soundness. In: The bovine proceedings, 26 January 1994. p. 105 - 107.

Chenoweth PJ, Chase Jr CC, Thatcher MJD, Wilcox CJ, Larsen RE. Breed and other effects on reproductive traits and breeding soundness categorization in young beef bulls in Florida. Theriogenology 1996; 46 (7): 1159 - 1170.


Coetzee K, Kruger TF, Lombard CJ, Shaughnessy D, Oehninger S, Özgür K, Pomeroy KO, Muller C. Assessment of interlaboratory and intralaboratory sperm morphology readings with the use of a Hamilton Thorne Research integrated visual optical system semen analyzer. Fert. Steril. 1999; 71: 80 - 84.


Cook RB, Coulter GH, Kastelic JP. The testicular vascular cone, scrotal thermoregulation, and their relationship to sperm production and seminal quality in beef bulls. Theriogenology 1994; 41: 653 - 671.


Davis RO, Katz DF. Operational standards for CASA instruments. J. Androl. 1993; 14: 385 - 394.

Foote RH. Bull sperm surface “craters” and other aspects of semen quality. Theriogenology 1999; 51: 767 - 775.

Garner DL. Ancillary tests of bull semen quality. Vet. Clin. North Am. Food Anim. Pract. 1997; 13 (2): 313 - 330.

Garner DL, Johnson LA, Allen CH, Palencia DD, Chambers CS. Comparison of seminal quality in Holstein bulls as yearlings and as mature sires. Theriogenology 1996; 45: 923 - 934.


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Hallap T, Szabolcs N, Håård M, Jaakma Ü, Johannisson A, Rodiguez-Martinez H. Sperm chromatin stability in frozen-thawed semen is maintained over age in AI bulls. Theriogenology 2005; 63: 1752 - 1763.

Hanset R, de Tillesse S, Michaux P, André E. La consanguinité en Blanc-Bleu Belge. Sa genèse et son contrôle. Publication Herd-Book du BBB n° 2003 03 - 36 (in French).

Hoflack G, Van Soom A, Maes D, de Kruif A, Opsomer G, Duchateau L. Breeding soundness and libido examination of Belgian Blue and Holstein Friesian artificial insemination bulls in Belgium and the Netherlands. Theriogenology 2006; in press.

Hopkins FM. Diseases of the reproductive system of the bull. In: Youngquist RS, editor. Current Therapy in Large Animal Theriogenology. Philadelphia: WB Saunders, 1997. p. 237 – 239 (Chapter 29).

Hopkins FM, Spitzer JC. The New Society for Theriogenology Breeding Soundness Evaluation System. Vet. Clin. North Am. Food Anim. Pract. 1997; 13 (2): 283 - 293.

Johnson WH. The significance to bull fertility of morphologically abnormal sperm. Vet. Clin. North Am. Food Anim. Pract. 1997; 13 (2): 255 - 270.

Johnson KR, Dewey CE, Bobo JK, Kelling CL, Lunstra DD. Prevalence of morphologic defects in spermatozoa from beef bulls. J. Am. Vet. Med. Assoc. 1998; 213 (10): 1468 - 1471.

Kennedy SP, Spitzer JC, Hopkins FM, Higdon III HL, Bridges Jr WC. Breeding soundness evaluations of 3648 yearling beef bulls using the 1993 Society for Theriogenology guidelines. Theriogenology 2002; 58: 947 - 961.

Kumi-Diaka J, Nagaratnam V, Rwuaan JS. Seasonal and age-related changes in semen quality and testicular morphology of bulls in a tropical environment. Vet. Rec. 1981; 108: 13 - 15.

Lucy MC. Reproductive loss in high–producing dairy cattle: where will it end? J. Dairy Sci. 2001; 84: 1277 – 1293.

Lunstra DD, Echternkamp SE. Puberty in Beef Bulls: Acrosome Morphology and Semen Quality in Bulls of Different Breeds. J. Anim. Sci. 1982; 55 (3): 638 - 648.

Michaux C, Hanset R. Sexual Development of Double-Muscled and Conventional Bulls. I Testicular Growth. Z. Tierzüchtg. Züchtgsbiol. 1981; 98: 29 - 37.

Neuwinger J, Knuth UA, Nieschlag E. Evaluation of the Hamilton-Thorn 2030 motility analyser for routine semen analysis in an infertility clinic. Int. J. Androl. 1990; 13: 100 - 109.


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Ott RS. Breeding Soundness examination of bulls. In: Morrow DA, editor. Current Therapy in Theriogenology, vol 2. Philadelphia: WB Saunders, 1986. p. 125 - 136.

Parkinson TJ. Seasonal variations in semen quality of bulls: correlations with environmental temperature. Vet. Rec. 1987; 120 (20): 479 - 482.


Phillips NJ, McGowan MR, Johnston SD, Mayer DG. Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires. Anim. Reprod. Sci. 2004; 81: 47 - 61.

Prathalingam NS, Holt WW, Revell SG, Jones S, Watson PF. The precision and accuracy of six different methods to determine sperm concentration. J. Androl. 2006; 27: 257 – 262.


Royal M, Mann GE, Flint APF. Strategies for reversing the trend towards subfertility in dairy cattle. Vet. J. 2000; 160: 53 – 60.


Saacke RG, Dalton JC, Nadir S, Nebel RL, Bame JH. Relationship of seminal traits and insemination time to fertilization rate and embryo quality. Anim. Reprod. Sci. 2000; 60-61: 663-677.

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Söderquist L, Janson L, Haard M, Einarsson S. Influence of season, age, breed and some other factors on the variation in sperm morphological abnormalities in Swedish dairy AI bulls. Anim. Reprod. Sci. 1996; 44: 91 - 98.

Van Camp SD. Common causes of infertility in the bull. Vet. Clin. North Am. Food Anim. Pract. 1997; 13: 203 - 231.



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Vogler CJ, Bame JH, DeJarnette JM, McGilliard ML, Saacke RG. Effects of elevated testicular temperature on morphology characteristics of ejaculated spermatozoa in the bovine. Theriogenology 1993; 40: 1207 - 1219.

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CHAPTER 3B

OBJECTIVE SPERM MOTILITY COMPARISON OF BELGIAN BLUE AND

HOLSTEIN - FRIESIAN BULLS

Modified from: Validation and Usefulness of the Sperm Quality Analyzer (SQA II-C) for Bull Semen Analysis G. Hoflack, T. Rijsselaere, D. Maes, J. Dewulf, G. Opsomer, A. de Kruif, A. Van Soom Reproduction in Domestic Animals 2005; 40: 237 - 244

Modified from: Comparison of Computer Assisted Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein Friesian Bulls G. Hoflack, G. Opsomer, T. Rijsselaere, A. Van Soom, D. Maes, A. de Kruif, L. Duchateau Reproduction in Domestic Animals, In Press

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Chapter 3B1

Validation and Usefulness of the Sperm Quality Analyzer (SQA-II C) for Bull

Semen Analysis

Chapter 3B1: Validation of the SQA-II C

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ABSTRACT

In this study, an upgrade version of the Sperm Quality Analyzer (SQA), the SQA-IIC was tested for the assessment of bull semen quality. In experiment 1, the device showed good repeatability of measurements within and between capillaries, as evidenced by the low coefficients of variation (CVs; <13%) at concentrations between 35 and 705 x 106

spermatozoa/ml. In experiment 2, 10 semen concentrations (1 – 1000 x 106/ml) were stored in HEPES TALP for 48h at room temperature. A time dependent decrease in sperm motility index (SMI) values was noticed. SMI values linearly increased with increasing sperm concentrations, but stagnated around 500, corresponding with a concentration of approximately 50 x 106/ml. For sperm concentrations below 50 x 106/ml, SMI values were highly correlated with concentration (P<0.05) and with semen parameters expressing the overall semen quality (P<0.05; experiment 3). In experiment 4, a correlation of only 0.44 (P<0.05) between SMI values of frozen-thawed semen samples of 35 bulls and the corrected 56 days non return rate (56dNRRc) was demonstrated. Prediction of the 56dNRRc based on the SMI value of a semen sample was inaccurate.

The present study indicates that the SQA-IIC is valuable for a rapid screening of bull semen diluted to a concentration of approximately 50 x 106/ml. Furthermore, the device seems inappropriate for fertility prediction.


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INTRODUCTION

Semen analysis is the procedure most commonly used to evaluate male fertility potential (Chong et al., 1983; Vantman et al., 1989; Comhaire et al., 1992). The conventional semen parameters that are examined are the concentration, the percentage of motile spermatozoa and their morphological grading (Neuwinger et al., 1990b). However, subjective visual measures of sperm count, motility and morphology are inaccurate and imprecise (Davis and Katz, 1993). Furthermore, the subjective motility judgement depends to a great extent on the level of training and skills of the investigator (Knuth et al., 1989), leading to a lack in agreement between different laboratories examining the same specimens (Davis and Katz, 1993). Coefficients of variation (CVs) as high as 33% within technicians and 44% between technicians, and up to 73% between laboratories were reported when assessing sperm concentration (Jequier and Ukombe, 1983; Dunphy et al., 1989; Neuwinger et al., 1990a). Similarly, the subjective visual assessment of semen motility yielded CVs of 20% between technicians, and up to 37% between laboratories (Dunphy et al., 1989; Neuwinger et al., 1990a). The need for standardization of laboratory procedures for semen analysis, to reduce the high variability, has been demonstrated repeatedly (Chong et al., 1983; Davis and Katz, 1993; Verstegen et al., 2002).

Different systems to overcome this variability, such as turbidimetry, laser-doppler-spectroscopy and photometric methods have been proposed (Johnston et al., 1995; Iguer-ouada and Verstegen, 2001a). However, these techniques are too imprecise or too complicated to be applied routinely, even for human purposes (Comhaire et al., 1992). Computer assisted semen analysis (CASA), a promising technology, was introduced both in human and in animal species (Vantman et al., 1989; Günzel – Apel et al., 1993; Farrell et al., 1995). CASA systems procure accurate and highly repeatable data on different semen motility parameters in different species (Farrell et al., 1995; Verstegen et al., 2002). However, these devices are expensive and handling procedures and parameter settings still differ between laboratories, making it difficult to compare results (Davis and Katz, 1993; Verstegen et al., 2002).

Since the eighties, the Sperm Quality Analyzer (SQA) was introduced as a practical and inexpensive device to evaluate overall semen quality (Bartoov et al., 1981). This SQA does not require parameter settings, reducing a large source of bias. It uses light passing through a capillary tube containing the motile semen sample and registers fluctuations in optical density, which result from moving particles. These fluctuations are registered by a photometric cell and converted digitally to a numerical output, the sperm motility index (SMI)(Bartoov et al., 1991; Johnston et al., 1995). The manufacturer states that the SMI reflects the overall semen quality, taking into account the concentration, progressive motility and normal morphology of the semen and this SMI is usable both for human and animal semen samples (User guide).


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Previous versions of this system were validated both in human (Bartoov et al., 1991; Johnston et al., 1995; Mahmoud et al., 1998; Martinez et al., 2000) and in domestic animal species, such as bulls (Bartoov et al., 1981), broiler males (Mc Daniel et al., 1998; Parker et al., 2000), dogs (Iguer – ouada and Verstegen, 2001b), turkey males (Neuman et al., 2002) and rams (Fukui et al., 2004), but to our knowledge the SQA-IIC (Medical Electronic Systems Ltd., Tirat Carmel, Israel), an upgrade version of the SQA, has not yet been validated for bull semen analysis. The aims of this study were to validate this version of the SQA for bull semen analysis and to investigate the effect of concentration and motility on the SMI read-outs. Furthermore, a comparison between SMI values and routinely assessed semen parameters was made. Finally, the usefulness of the SQA-IIC to predict male in vivo fertility, by means of the 56 days non return rate (56dNRR), was determined.


1. Animals

In experiments 1 to 3, one Holstein Friesian (HF), one East Flemish (EF), and one Holstein Friesian-Belgian Blue crossbred (HF-BB) bull from the department of Reproduction, Obstetrics and Herd Health of the Faculty of Veterinary Medicine (Ghent University, Belgium) were used. Whenever an individual ejaculate was used (next to pooled semen) the HF-BB bull was collected due to his superior libido. Additionally, in experiment 3, five bulls residing at an artificial insemination (AI) centre were used, namely three HF and one Belgian Blue (BB) bull of poor semen quality (50 –57 % normal spermatozoa in the original ejaculate) and one BB bull of good semen quality (87% normal spermatozoa in the original ejaculate). For experiment 4, frozen semen of 35 HF bulls with known non return rates (NRR), was obtained from Holland Genetics.

2. Diluent

The diluent used throughout the experiments was HEPES TALP, containing 114 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 2.1 mM CaCl2.2H2O, 0.4 mM MgCl2.6H2O, 2 ml/l phenol-red, 1.99 mM bicarbonate, 1mM pyruvate, 36mM lactate, 10 mM HEPES, 3 mg/ml BSA and 50 mg/l gentamycin.

3. Semen collection, processing and evaluation

Semen of all bulls was collected with an artificial vagina after 3 false mounts. The original concentration of individual and pooled ejaculates was determined by diluting 10 µl of semen in 990 µl 1M HCl and by counting the number of sperm cells in 1/100 mm3 of a Bürker counting chamber. Total and progressive motility were subjectively assessed to the nearest


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5% by placing 10 µl of diluted semen (10 µl of semen in 790 µl of physiological saline solution (PSS) for raw semen and semen concentrations higher then 1 x 109 spermatozoa/ml, 10 µl of semen in 490 µl of PSS for semen concentrations from 500 x 106 to 1 x 109 spermatozoa/ml, a 1/1 dilution in PSS for semen concentrations above 150 x 106 and under 500 x 106 spermatozoa/ml and simply 10 µl of diluted semen at concentrations lower then or equal to 150 x 106 spermatozoa/ml) on a pre-warmed glass slide at 37°C under a coverslip, and by examining 5 different microscopic fields in the centre of the coverslip, under a 200 x phase-contrast microscope. This procedure was repeated twice and was always done by the same experienced observer. The percentage of normal live sperm cells (i.e. membrane intact spermatozoa with a normal morphology) was assessed on air-dried, eosin-nigrosin stained smears. At least 100 spermatozoa were evaluated under a 1000 x light microscope using immersion oil.

4. SQA experiments

SQA measurements were carried out by aspirating mixed (undiluted or diluted) semen into a specially designed capillary tube, which was dried externally and inserted into the SQA II-C, according to the manufacturers instructions. The result was displayed after 45 seconds by means of SMI values. All experiments were carried out at room temperature (20°C).

Experiment 1: Repeatability of measurements

A single ejaculate of the HF-BB bull and a pooled ejaculate of 3 bulls were used in this experiment. Based on the assessed concentration, a series of twofold dilutions in HEPES TALP of these ejaculates was prepared, yielding a range of concentrations from 1410 to 0.5 x 106 spermatozoa/ml. For each concentration, 5 capillaries were measured 5 times, and the CVs for SMI read-outs were calculated within each capillary (on 5 measurements) and between capillaries (on 25 measurements).

Experiment 2: Effect of concentration and (time dependent) motility decline on SMI read-outs

Semen of 3 bulls was collected, pooled, and diluted in HEPES TALP to 1000, 500, 250, 200, 150, 100, 50, 10, 5, and 1 x 106 spermatozoa/ml. SMI read-outs for all 10 concentrations were assessed at 0h, 3h, 6h, 12h, 24h and 48h by measuring one capillary 3 times and by calculating the average SMI, to assess the effect of concentration and (time dependent) motility decline on SMI values. The experiment was repeated 3 times, using a pooled semen sample of the same bulls.


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Experiment 3: Correlation between SMI read-outs and standard semen quality parameters

All data obtained in experiments 1 (pooled as well as individual ejaculate) and 2 were gathered and the average SMI values of 3 measurements of 1 capillary in HEPES TALP, at 0h after collection, for the samples of which all subjective semen parameters were assessed, were kept for further analysis. Additionally, a single ejaculate of 5 individual AI bulls, tested as in experiment 2 but without repeating the experiment, was used (data not shown). In this experiment, for each semen sample of which an SMI value was assessed with the SQA, an individual assessment of both motility and morphology, for a given concentration was performed. Correlations between these average SMI values and the routinely assessed semen quality parameters (concentration, % of total and progressive motility and % of normal live spermatozoa) were calculated.

Experiment 4: Correlation between SMI read-outs and 56dNRR and fertility prediction

A selection of 35 bulls with a wide range in raw 56dNRR was made and 1 straw of frozen-thawed semen of 2 to 10 different ejaculates of these bulls was pooled. Subsequently, 1 capillary of pooled semen was measured 3 times and the average of the resulting 3 SMI read-outs was calculated. All straws were filled at 15 x 106 spermatozoa in a TRIS based diluent as described by Van Wagtendonk-de Leeuw et al. (2000), resulting in a concentration of approximately 60 x 106 spermatozoa/ml. The raw 56dNRR of these bulls was calculated based on 599 to 1749 first and second AIs and corrected for inseminator, day (of the week) of insemination, month of insemination, farm, parity of the inseminated cows, insemination number, interval between calving and insemination and production of the inseminated cows, resulting in the corrected 56dNRR (56dNRRc). Subjective microscopic motility estimations of the pooled semen samples were performed as well.


To assess the repeatability of measurements, CVs of the SMI values were calculated for the 5 consecutive measurements within each capillary and for the 25 measurements (5 capillaries measured 5 consecutive times) between capillaries (Exp 1). Repeated measures analysis of variance was used to assess the effect of concentration and time dependent motility decline on SMI read-outs (Exp 2). In experiment 3, spearman’s correlations between SMI values and traditional, subjectively assessed semen quality parameters were established, since these data were not normally distributed. Limits of agreement were not calculated because the units in which the compared data were expressed, differed. In experiment 4, Spearman’s correlations between the SMI values, % of total motility and % of progressive motility on the one hand and the raw 56dNRR and the 56dNRRc on the other hand, were also


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calculated. Linear regression was not performed since the data were not normally distributed. In addition, 2 x 2 tables were generated based on different threshold values for 56dNRRc and SMI in order to establish predictive values for the 56dNRRc based on the SQA outcome: the positive predictive values (PPV) were defined as “the chance for a frozen-thawed semen sample with a SMI value higher than a given SMI threshold value to produce a 56dNRRc higher than the 56dNRRc threshold value”, whereas the negative predictive values (NPV) were defined as “the chance for a frozen-thawed semen sample with a SMI value lower than a given SMI threshold value to produce a 56dNRRc lower than the 56dNRRc threshold value”. The statistical analyses were performed using procedures in SPSS 11.0 (SPSS Inc. Headquarters, Chicago, IL, USA), except for experiment 4, where the predictive values were calculated using multinomial bootstrapping in @risk 4.5 (Palisade Corporation, Newfield, NY, USA). Values were considered to be statistically significant at the 0.05 level.

RESULTS

Experiment 1: Repeatability of measurements

Twenty-four different sperm concentrations were assessed ranging from 0.5 to 1410 x106 spermatozoa/ml. The percentage of totally and progressively motile spermatozoa ranged from 60 to 85 and from 10 to 85, respectively, and the percentage of normal spermatozoa ranged from 63 to 80. Minimum and maximum mean SMI read-outs for measurements within capillaries were 9.0 and 527.0 respectively, and 14.4 and 513.3 for measurements between capillaries, respectively.

The CV within capillaries varied from 1.29% to 75.90%. For concentrations between 18 and 1135 x 106 spermatozoa/ml, the CV was consistently lower than 15%. The CV between capillaries varied from 2.13% to 58.79%. Between 35 and 705 x 106 spermatozoa/ml, the CV was lower than 10%. The CV within capillaries was lower than 12.89% within this concentration interval. The higher variability within and between capillaries was mainly noticed at the lower concentrations (< 22 x 106 spermatozoa/ml), although extremely high concentrations also resulted in an increasing variability, namely 14.94% and 20.54% at 1410 x 106 spermatozoa/ml within capillaries and 13.79% at 1135 x 106 spermatozoa/ml, and 27.21% at 1410 x 106 spermatozoa/ml between capillaries. Both semen sources (individual HF-BB bull and semen pool of 3 bulls) were equally represented at very high and very low concentrations and the pattern of variability at these extreme concentrations was comparable for both semen sources.


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Experiment 2: Effect of concentration and (time dependent) motility decline on SMI read-outs

Average SMI values within replicates ranged from 0.0 to 505.7. For each concentration, a generally linear decline in SMI read-outs was noticed as time progressed (Fig 1). When SMI values were averaged over all concentrations, SMI values significantly differed at all time points (P<0.05), except between 3 and 6h, where no difference was noticed (P = 0.118). The interaction between time and concentration was also significant (P<0.05). The difference in SMI values between the different concentrations was more obvious at early time points and less pronounced as the experiment progressed in time (Fig 1).

0

50

100

150

200

250

300

350

400

450

500

0 3 6 12 24 48

Time (h)

SM

I

1

5

10

50

100

150

200

250

500

1000

Concentration (x 106/ml)

Fig 1: Effect of time (h), for 10 assessed sperm concentrations, upon SMI values. Each symbol is the average of 3 SMI measurements of pooled bull semen repeated into 3 replicates.

When the SMI values for each concentration were averaged over time, SMI values almost linearly increased from 1 to 50 x 106 spermatozoa/ml, although no significant differences in SMI values were noticed at concentrations below 10 x 106 spermatozoa/ml. SMI values measured at 1 and 5 x 106 spermatozoa/ml, differed significantly (P<0.05) from those measured at 50 to 250 x 106 spermatozoa/ml, whereas no significant difference (P =0.143) between 10 and 50 x 106 spermatozoa/ml could be demonstrated. From 50 to 250 x 106 spermatozoa/ml, no significant differences were noticed in SMI values indicating a


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saturation of the system. At even higher concentrations, the SMI values linearly decreased (Fig 2).

0

50

100

150

200

250

300

1 5 10 50 100 150 200 250 500 1000

Concentration (x 106/ml)

SMI

Fig 2: Effect of sperm concentration upon SMI values, averaged over the different time points (0, 3, 6, 12, 24 and 48 h). Each symbol is the average of 3 SMI measurements of pooled bull semen repeated into 3 replicates, averaged over time.

Experiment 3: Correlation between SMI read-outs and standard semen quality parameters

The fact that morphology was not assessed for every semen sample when repeatability was tested and when the concentration of the samples was too low, resulted in a reduction of the available data from experiment 1 and 2. After selection, only 80 average SMI values were retained of which all subjective semen parameters were assessed, with 14 different concentrations between 10 and 1410 x 106 spermatozoa/ml, within which the percentage of total and progressive motility varied from 60 to 95 and from 50 to 95, respectively, while the percentage of normal live spermatozoa ranged from 37 to 85.

The correlations between the average SMI values and the traditional sperm characteristics evaluated by routine microscopic semen evaluation are shown in Table 1. A relatively high correlation (P<0.05) between SMI values and concentration, and between SMI values and the concentration of progressively motile, normal spermatozoa was noted but only at concentrations below 50 x 106 spermatozoa/ml. Neither motility parameters, nor percentage of normal live spermatozoa were correlated with SMI values.


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Table 1: Spearman’s correlations between the average SMI values of 3 SQA measurements and sperm quality parameters evaluated by standard optical microscopy (for 14 concentrations (10 to 1410 x 106 spermatozoa/ml) of which the percentage of total and progressive motility varied from 60 to 95 and from 50 to 95, respectively and of which the percentage of normal spermatozoa ranged from 37 to 85), as well as with combined semen quality parameters, for different concentration intervals.

Parameters Conc.< 50 50 < conc. < 250 Conc. > 250

N = 80 N = 11 N = 45 N = 24

SMI - sperm concentration 0.63 * 0.10 -0.30

SMI - % total motility 0.50 -0.02 -0.04

SMI - % progressive motility 0.31 -0.04 -0.04

SMI - % normal spermatozoa 0.52 0.21 -0.07

SMI - conc. prog. mot. spermatozoa 0.54 0.07 -0.30

SMI - conc. prog. mot. normal spermatozoa 0.65 * 0.20 -0.28

* P<0.05

(conc. prog. mot. spermatozoa: concentration x % progressively motile spermatozoa; conc. prog. mot. normal spermatozoa: concentration x % progressively motile spermatozoa x % normal spermatozoa)

Experiment 4: Correlation between SMI read-outs and 56dNRR and fertility prediction

The selected raw 56dNRR ranged from 40.1 to 71.7. and the 56dNRRc between 47.6 and 74.2. Percentage of total and progressive motility of the evaluated frozen-thawed semen samples ranged from 20 to 75 in both cases. The average SMI values ranged from 44.7 to 309.0. Correlations between the SMI values, % of total motility and % of progressive motility on the one hand and the raw 56dNRR and the 56dNRRc on the other hand, are shown in Table 2. SMI values were significantly correlated to all NRR parameters. The highest correlation (0.44; r2 < 0.2) was obtained between SMI and 56dNRRc, but even this correlation was only low to moderate. In all cases, correlations between SMI values and NRR were higher than correlations between subjectively estimated motility parameters and NRR, when significant.


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Table 2: Spearman’s correlations between average SMI values of 3 SQA measurements on pooled semen in a TRIS based diluent at a concentration of approximately 60 x 106

spermatozoa/ml, percentage of total motility and progressive motility of 35 bulls on the one hand and the raw 56dNRR and the corrected 56dNRR on the other hand. The range of each parameter is given as well.

Parameter (range) 56dNRR (40.1 - 71.7) 56dNRRc (47.6 - 74.2)

SMI (44.67 - 309.00) 0.38* 0.44 **

% total motility (20 - 75) 0.29 0.35 *

% progressive motility (20 - 75) 0.27 0.35 *

* P<0.05; **P<0.01

(56dNRR: 56 days NRR; 56dNRRc: corrected 56 days NRR, corrected for inseminator, day of the week of insemination, month of insemination, farm, parity of the inseminated cows, insemination number, interval between calving and insemination and production of the inseminated cows).

The positive and negative predictive values for different SMI and 56dNRRc threshold values are given in Table 3. For each SMI threshold value, both PPV and NPV generally increase as the 56dNRRc threshold increases, resulting in the highest average predictive values, both positive and negative, for a 56dNRRc threshold of 68%. While for each 56dNRRc threshold value, the PPV generally increases as the SMI threshold value decreases, this is only the case for the NPV at the lower 56dNRRc thresholds. When using 65 and 68% as 56dNRRc threshold value, NPV is highest for an SMI threshold value of approximately 100. For each 56dNRRc threshold value, the 95% confidence interval for NPV seems to get smaller with higher SMI threshold values, whereas for PPV this is the opposite. Summarising, when 68% is used as threshold value for 56dNRRc, an SMI threshold value of 50 results in the highest PPV with the narrowest 95% confidence interval, whereas an SMI threshold value of 100 results in the highest NPV with the almost narrowest 95% confidence interval. However, these 95% confidence intervals remain too wide, and the average predictive values are too low.


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Table 3: Average positive and negative predictive values (as % and the 95% confidence interval) for frozen-thawed semen samples, to result in a corrected 56dNRR higher or lower than the corrected 56dNRR threshold value, respectively, for different given SMI threshold values.

corrected 56dNRR threshold

60 62 65 68

SMI threshold

50 PPV 43 (28 - 60) 49 (33 - 64) 54 (38 - 70) 60 (44 - 75)

NPV 75 (30 - 99) 75 (31 - 99) 75 (32 - 99) 75 (30 - 99)

80 PPV 36 (19 - 56) 44 (25 - 63) 48 (29 - 67) 56 (36 - 75)

NPV 64 (38 - 87) 64 (39 - 86) 71 (47 - 91) 71 (46 - 91)

100 PPV 30 (12 - 52) 30 (13 - 51) 35 (17 - 57) 45 (26 - 65)

NPV 63 (40 - 83) 74 (52 - 90) 79 (59 - 95) 79 (60 - 94)

150 PPV 25 (8 - 49) 25 (8 - 48) 31 (12 - 55) 44 (24 - 68)

NPV 61 (38 - 79) 70 (49 - 86) 74 (53 - 89) 74 (55 - 89)

200 PPV 22 (4 - 51) 22 (3 - 56) 22 (4 - 54) 44 (15 - 76)

NPV 53 (36 - 70) 60 (42 - 77) 67 (49 - 82) 67 (49 - 82)

250 PPV 25 (10 - 73) 25 (1 - 69) 25 (1 - 70) 25 (5 - 71)

NPV 49 (33 - 65) 54 (37 - 71) 60 (43 - 75) 66 (51 - 80)

(PPV: positive predictive value; NPV: negative predictive value)


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DISCUSSION

The results in experiment 1 show a high repeatability (CV < 15%) of the measurements, both within and between capillaries for a wide range of concentrations. This consistency in SMI read-outs was already demonstrated in human (Bartoov et al., 1981; Martinez et al., 2000), in the dog (Iguer-ouada and Verstegen, 2001b; Rijsselaere et al., 2002) and in broiler males (Mc Daniël et al., 1998). In the present study, very high or very low sperm concentrations resulted in a decreasing repeatability. A higher variability in SMI read-outs at lower sperm concentrations has already been reported (Johnston et al., 1995; Mahmoud et al, 1998), but in the present study a similar trend was observed at very high concentrations. This may partially be due to the relatively low SMI values combined with relatively high standard deviations obtained at very low or very high concentrations. Based on our findings, we suggest that measurements are best performed at concentrations between 35 and 705 x 106

spermatozoa/ml when using the SQA-IIC for bovine semen. To increase the efficiency of the SQA, for further experiments one capillary was measured 3 times and the average SMI was then calculated.

The influence of sperm concentration and motility on SMI reads-outs was demonstrated in experiments 2 and 3. In experiment 2, semen samples at several concentrations were stored and measured over time. An almost linear decrease in SMI values was noticed over concentrations as time progressed, reflecting the descent in motility of the semen samples over time and stressing the effect of motility on SMI values (Iguer–ouada and Verstegen, 2001b; Rijsselaere et al., 2002). The interaction between time and concentration was significant, indicating that this time- and thus motility effect was influenced by the concentration. Indeed, this decline was noticed for all concentrations but the effect was less pronounced at very low or very high concentrations, probably due to the relatively lower SMI values at these concentrations. This indicates the influence of concentration on SMI values. Averaged over time, SMI values almost linearly increased from 1 to 50 x 106 spermatozoa/ml, although no significant differences in SMI values were noticed at concentrations below 10 x 106 spermatozoa/ml. This lack of significance can partially be explained by the low SMI values (< 300) combined with a high variability at these low concentrations. SMI values obtained at 50 x 106 spermatozoa/ml significantly differed from those obtained at low concentrations but not from those obtained at higher concentrations: from 50 to 250 x 106

spermatozoa/ml, no differences in SMI values were noticed, suggesting a saturation of the system. This is probably due to the condensed state of the sperm cells at these high concentrations, resulting in multiple individual collisions, which interfere with and slow down the progressive motility (Vantman et al., 1988; Bartoov et al., 1991; Rijsselaere et al., 2002). This inaccuracy of individual motility assessment at high semen concentrations was also


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noticed in computer assisted semen motility studies (Verstegen et al., 2002), where a similar concentration of approximately 50 x 106 spermatozoa/ml has been advised, to obtain reliable and unbiased kinematic measurements (Vantman et al., 1989; Neuwinger et al., 1990b; Davis and Katz, 1993; Rijsselaere et al., 2003). Although comparable to our findings, this is in contrast with Iguer-ouada and Verstegen (2001b) and Rijsselaere et al. (2002), who also observed a saturation of the SQA but at concentrations above 150 x 106 spermatozoa/ml in dog semen. The saturation in these studies occurred at SMI values of approximately 500 - 600, similar to our findings. In human, Bartoov et al. (1991) noticed a saturation at a total concentration of 140 x 106 human spermatozoa/ml (when more than 40 x 106 of the 140 x 106 spermatozoa/ml were motile), but in their study SMI values remained below 100, probably due to the lower kinematic values of human semen compared to bull and dog semen (Farrell et al., 1996; Rijsselaere et al., 2003). The higher concentrations at saturation in these studies, compared to our results, might partially be explained by the fact that human and dog spermatozoa are smaller than bull spermatozoa (Gravance et al., 1996; Dominguez et al., 1999; Boersma et al., 2001; Rijsselaere et al., 2004), probably permitting a higher concentration of the smaller cells before saturation occurs. At extremely high sperm concentrations (>250 x 106 spermatozoa/ml), SMI values even decreased, presumably due to an overcondensation of the semen. At these concentrations, spermatozoa seem no longer able to move freely in the capillary tube (Mc Daniel et al., 1998), resulting in microscopically noticeable dark swirls as present in a concentrated fresh bull ejaculate, in which individual motility of semen cells can hardly be observed (Barth, 1997). These dark swirls may not allow light to adequately pass through, resulting in low SMI values. Low SMI values at very high sperm concentrations were already reported in broiler and turkey breeder males (Mc Daniel et al., 1998; Neuman et al., 2002), but not in other species, as semen in other species is less concentrated than bull semen (Bartoov et al., 1991; Johnston et al., 1995; Farrell et al., 1996) or diluted to lower concentrations before assessment (Iguer–ouada and Verstegen, 2001b; Rijsselaere et al., 2002; Fukui et al., 2004). A previous version, the Sperm Motility Analyzer, was able to cope with extremely high concentrations, but recent SQA versions with increased sensitivity, enabling the detection of individual motility seem less appropriate (Bartoov et al., 1981; 1991). Consequently, the SQA-IIC appears unreliable to predict bull semen quality at concentrations higher than approximately 50 x 106 spermatozoa/ml. Moreover, since repeatability is only good above 35 x 106 spermatozoa/ml, we recommend a sperm concentration of approximately 50 x 106 spermatozoa/ml for further experiments. This effect of concentration on SMI read-outs is obvious at the beginning of the experiment, when motility is high and comparable for all concentrations, but becomes less pronounced since progressive motility decreases with time, eventually resulting in low SMI values at all concentrations (Iguer–ouada and Verstegen, 2001b; Rijsselaere et al., 2002).


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These findings are confirmed in experiment 3, where correlations between SMI values measured at 0h and concentration are only significant and relatively high (0.63) at concentrations below 50 x 106 spermatozoa/ml. Iguer–ouada and Verstegen (2001b) and Rijsselaere et al. (2002) also reported a high correlation (0.84 and 0.82, respectively) between SMI values and sperm concentration under the saturation level in dog semen. Martinez et al. (2000) reported the loss of correlation at SMI values around 500, which is similar to our findings. In our study, percentages of total and progressive motility were not correlated with SMI values. Rijsselaere et al. (2002) also reported the absence of correlations between SMI values and single motility parameters in dog semen. These authors also found that the percentage of morphologically normal spermatozoa did not correlate with SMI values, which is in agreement with our findings. In human however, an influence of morphology on SMI values was demonstrated (Johnston et al., 1995; Mahmoud et al., 1998). It might be possible that in dog and bull semen, morphology differences between samples are too small to generate an effect on SMI values, since morphology in dog and bull semen is generally much better than in human semen (World Health Organization, 1992; Verberckmoes et al., 2004). However, it should be noted that only a moderate correlation (0.65) between SMI values and the concentration of progressively motile normal spermatozoa, which reflects the overall semen quality, at concentrations below 50 x 106 spermatozoa/ml was obtained. According to the claim of the manufacturer, this overall semen quality parameter is what the SQA measures. Rijsselaere et al. (2002) found a somewhat higher correlation of 0.80 between SMI values and the concentration of progressively motile normal spermatozoa below the saturation plateau of the SQA-IIC in dog semen. It might be possible that bull effects exist, which could influence our results. However, the database available for this correlation experiment is too small to draw any conclusions on this subject. The moderate correlation in our experiment suggests that discrimination by the SQA-IIC, of semen samples with little difference in quality, even when measured at the optimal concentration of 50 x 106 spermatozoa/ml, might be a problem. Indeed, the SMI is determined by a combination of several parameters. A deficient quality of one parameter can be compensated by a good quality of another parameter, biasing accurate quality discrimination of different semen samples (Bartoov et al., 1991; Johnston et al., 1995).

According to the manufacturer, the SMI values obtained with the SQA-IIC represent a semen quality assessment including concentration, progressive motility and percentage of normal spermatozoa. In frozen-thawed bull semen, the concentration is generally kept at 15 x 106 spermatozoa/straw (approximately 60 x 106 spermatozoa/ml). Consequently, only the progressive motility and the percentage of normal spermatozoa vary between straws. The SQA-IIC might therefore be useful to measure the quality of frozen-thawed bull semen and predict pregnancy outcome of these ejaculates. It has already been shown that bull fertility might partially be predicted based on semen motion quality (Farrell et al., 1998). In


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experiment 4, we tested the correlation between SMI values and the 56dNRR, both raw and corrected. This resulted in correlations of 0.38 (r2 = 0.14) and 0.44 (r2 = 0.19), respectively, which are comparable to correlations between SMI values and human in vitro fertilization outcome (Shibahara et al., 1997; Mahmoud et al., 1998). Although a little higher, these findings are in agreement with those of Correa et al (1997), who found a correlation of 0.29 between SMI values and fertilizing competence of bull ejaculates. This small difference and our better correlation might partially be due to the extremely wide range in 56dNRR in our study. Farrell et al. (1998) reported extremely high correlations (0.99, r2 = 0.98) between combined motility parameters measured by CASA on fresh semen and 59dNRR to first service. When only one parameter was taken into account, total motility seemed most appropriate and resulted in a correlation with 59dNRR to first service of 0.58 (r2 = 0.34), which is slightly higher than our results (Table 2). Other research on computerized semen quality measurements and the relation to bull fertility also highlighted the promise of combining several semen quality parameters, whereas single motility parameters do not always correlate with fertility (Budworth et al., 1987; 1988). Although SMI is based on 3 different semen quality parameters (concentration, progressive motility and morphology), only motility and morphology are responsible for differences in fertility since concentration is kept constant in all straws. Nevertheless, the correlation between the SMI and the NRR is rather low. Since a better correlation between SMI and the corrected 56dNRR compared to the raw 56dNRR was demonstrated in this experiment, we only assessed the predictive values for the corrected 56dNRR, based on the SQA outcome. The average positive and negative predictive values for 56dNRRc, based on the comparison of the SMI value of a semen sample with a given SMI threshold value, are rather low, while the 95% confidence intervals are wide, partially due to the fact that the sample size in this experiment was quite small (35 samples). Sixty eight percent seems the most practical 56dNRRc threshold, since this is the average 56dNRRc for The Netherlands over the last years (Anonymous, 2004), and this 56dNRRc threshold value yielded the highest average predictive values, regardless of the SMI threshold value. When 68% is used as threshold value for 56dNRRc, the best SMI threshold values are 50 and 100 for the PPV and NPV, respectively, since this results in the highest average predictive values and the close to narrowest 95% confidence intervals. However, these 95% confidence intervals remain too wide, and the average predictive values are too low. In all, this reduces the practical applicability of the SQA-IIC to predict the NRR, based on the comparison of the SMI value of a semen sample with a given SMI threshold value.

In conclusion, the SQA-IIC appeared valuable for a rapid screening of bull semen samples diluted to a concentration of approximately 50 x 106 spermatozoa/ml. The prediction of the NRR of a frozen-thawed semen sample, based on its SMI value, seems impractical.


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ACKNOWLEDGEMENTS

The authors wish to thank Holland Genetics for providing frozen-thawed semen samples with a wide variety in NRRs. The semen samples with suboptimal NRRs are not available for commercial but only for research purposes. The authors also wish to thank Dr. M. Coryn for critically reading the manuscript.


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REFERENCES

Anonymous. NRS Jaarstatistieken 2003, CR Delta, Arnhem, 2004; p. 57.

Barth AD. Chapter 28: Evaluation of potential breeding soundness of the bull. In Youngquist RS (ed), Current Therapy in Large Animal Theriogenology. Philadelphia: WB Saunders, 1997; p. 222-236.



Boersma A, Raβhofer R, Stolla R. Influence of sample preparation, staining procedure and analysis conditions on bull sperm head morphometry using the morphology analyser integrated visual optical system. Reprod. Dom. Anim. 2001; 36: 222-229.

Budworth PR, Amann RP, Hammerstedt RH. A microcomputer-photographic method for evaluation of motility and velocity of bull sperm. J. Dairy Sci. 1987; 70: 1927-1936.

Budworth PR, Amann RP, Chapman PL. Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility. J. Androl. 1988; 9: 41-54.

Chong AP, Walters CA, Weinrieb SA. The neglected laboratory test. The semen analysis. J. Androl. 1983; 4: 280-282.

Comhaire FH, Huysse S, Hinting A, Vermeulen L, Schoonjans F. Objective semen analysis: has the target been reached? Hum. Reprod. 1992; 7 (Suppl. 2): 237-241.

Correa JR, Pace MM, Zavos PM. Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program. Theriogenology 1997; 48: 721-731.


Dominguez LA, Burgos MH, Fornés MW. Morphometrical comparison of human spermatozoa obtained from semen and swim-up methodology. Androl. 1999; 31: 23-26.



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Farrell P, Trouern-Trend V, Foote RH, Douglas-Hamilton D. Repeatability of measurements on human, rabbit, and bull sperm by computer-assisted sperm analysis when comparing individual fields and means of 12 fields. Fertil. Steril. 1995; 64 (1): 208-210.


Farrell PB, Presicce GA, Brockett CC, Foote RH. Quantification of bull sperm characteristics by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 1998; 49: 871-879.

Fukui Y, Togawa M, Abe N, Takano Y, Asada M, Okada A, Iida K, Ishikawa H, Ohumi S. Validation of the sperm quality analyzer and the hypo-osmotic swelling test for frozen-thawed ram and minke whale (Balaenoptera bonarensis) spermatozoa. J. Reprod. Dev. 2004; 50: 147-154.

Gravance CG, Vishwanath R, Pitt C, Casey PJ. Computer automated morphometric analysis of bull sperm heads. Theriogenology 1996; 46: 1205-1215.

Günzel-Apel AR, Gunther C, Terhaer P, Bader H. Computer-assisted analysis of motility, velocity and linearity of dog spermatozoa. J. Reprod. Fertil. (Suppl.) 1993; 47: 271-278.

Iguer-ouada M, Verstegen JP. Evaluation of the Hamilton Thorn computer-based automated system for dog semen analysis. Theriogenology 2001a; 55: 733-749.

Iguer-ouada M, Verstegen JP. Validation of the sperm quality analyzer (SQA) for dog sperm analysis. Theriogenology 2001b; 55: 1143-1158.

Jequier AM, Ukombe EB. Errors inherent in the performance of a routine semen analysis. Br. J. Urol. 1983; 55: 434.




Martinez C, Mar C, Azcarate M, Pascual P, Aritzeta JM, Lopez-Urrutia A. Sperm motility index : a quick screening parameter from sperm quality analyser-IIB to rule out oligo-


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and asthanozoospermie in male fertility study. Hum. Reprod. 2000; 15 (Suppl. 8): 1727-1733.

McDaniel CD, Hannah JL, Parker HM, Smith TW, Schultz CD, Zumwalt CD. Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index. Poult. Sci. 1998; 77: 888-893.

Neuman SL, Mc Daniel CD, Frank L, Radu J, Einstein ME, Hester PY. Utilisation of a sperm quality analyser to evaluate sperm quantity and quality of turkey breeders. Br. Poult. Sci. 2002; 43: 457-464.



Parker HM, Yeatman JB, Schultz CD, Zumwalt CD, McDaniel CD. Use of a sperm analyzer for evaluating broiler breeder males. Selection of young broiler breeder roosters for the sperm quality index increases fertile egg production. Poult. Sci. 2000; 79: 771-777.

Rijsselaere T, Van Soom A, Maes D, de Kruif A. Use of the Sperm Quality Analyzer (SQA II-C) for the Assessment of Dog Sperm Quality. Reprod. Dom. Anim. 2002; 37: 158-163.


Rijsselaere T, Van Soom A, Hoflack G, Maes D, de Kruif A. Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyzer. Theriogenology 2004; 62: 1292-1306.

Shibahara H, Naito S, Hasegawa A, Mitsuo M, Shigeta M, Koyama K. Evaluation of sperm fertilizing ability using the sperm quality analyzer. Int. J. Androl. 1997; 20: 112-117.

Vantman D, Koukoulis G, Dennison L, Zinaman M, Sherins RJ. Computer-assisted semen analysis: evaluation of method and assessment of the influence of sperm concentration on linear velocity determination. Fertil. Steril. 1988; 49: 510-515.

Vantman D, Banks SM, Koukoulis G, Dennison L, Sherins RJ. Assessment of sperm motion characteristics from fertile and infertile men using a fully automated computer-assisted semen analyzer. Fertil. Steril. 1989; 51: 156-161.


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Van Wagtendonk-de Leeuw AM, Haring RM, Kaal-Lansbergen LMTE, den Daas JHG. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soybean extract. Theriogenology 2000; 54: 57-67.

Verberckmoes S, Van Soom A, de Kruif A. Intra-uterine insemination in farm animals and humans. Reprod. Dom. Anim. 2004; 39: 195-204.

Verstegen J, Iguer-ouada M, Onclin K. Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002; 57: 149-179.

World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Third edition. Cambridge. The Press Syndicate of the University of Cambridge, 1992.

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Chapter 3B2

Comparison of Computer Assisted

Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein -

Friesian Bulls

Chapter 3B2: CASA of BB and HF Sperm

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ABSTRACT

Subjective microscopic sperm motility results have recently been demonstrated to differ between Holstein Friesian (HF) and Belgian Blue (BB) bulls. However, such assessments are rather imprecise. In the present study, sperm motility was assessed objectively by means of the Hamilton Thorne CEROS version 12.2c computer assisted sperm motility analyser (CASA), and differences between the BB and HF breed could also be demonstrated. Higher percentages of both totally (P < 0.0001) and progressively (P < 0.0001) motile spermatozoa were encountered in the HF breed compared to the BB breed. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower beat cross frequency (P = 0.0007) combined with a higher lateral head displacement (P = 0.0015), was the basis for the lower velocity of BB sperm cells. Additionally, BB spermatozoa move less straight forward, resulting in a lower straightness (P < 0.0001). No sperm motility differences were observed between age groups within the BB breed. The breed differences were observed in the examined bull populations residing at artificial insemination centres, in Belgium for the BB bulls and in the Netherlands for the HF bulls, respectively. However, these bull populations are selected for fertility. A similar pattern was observed in an unselected bull population of both breeds, although these differences were mostly non significant for the different CASA parameters. Nevertheless, these data suggest that a genetic component might be responsible for the observed sperm motility breed differences.


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INTRODUCTION

Male fertility is an important factor in bovine reproduction since a single bull is generally bred to numerous cows. To evaluate male fertility potential, semen analysis is the most commonly used procedure (Chong et al., 1983; Vantman et al., 1988; Comhaire et al., 1992; Rijsselaere et al., 2002). Hence, semen evaluation of breeding bulls is of paramount importance (Ott, 1986; Bruner and Van Camp, 1992; Chenoweth et al., 1994; Barth, 1997; Garner, 1997). Although several methods can be used to evaluate the quality of a fresh or frozen-thawed semen sample, subjective evaluation using standard optical microscopy is by far most commonly used (Rijsselaere et al., 2003; Christensen et al., 2005). The conventional semen parameters routinely examined are the concentration, the percentage of motile spermatozoa and their morphology (Vantman et al., 1988; Neuwinger et al., 1990b). However, subjective evaluation of sperm count, motility and morphology has been shown to be relatively inaccurate and imprecise (Davis and Katz, 1993, Christensen et al., 1999; 2005). Furthermore, the subjective motility judgement largely depends on the level of training and skills of the investigator (Knuth et al., 1989), frequently leading to a lack in agreement between different laboratories examining the same specimens (Davis and Katz, 1993). Hence, the subjective visual assessment of semen motility of the same sperm samples yielded coefficients of variation of 20% between technicians, and up to 37% between laboratories (Jequier and Ukombe, 1983; Dunphy et al., 1989; Neuwinger et al., 1990a). The need for standardization of semen analysis to reduce this high variability has been demonstrated repeatedly (Chong et al., 1983; Davis and Katz, 1993; Verstegen et al., 2002).

Computer assisted semen motility analysis (CASA) procures detailed, accurate and highly repeatable data on different semen motility parameters both in human and animal species (Vantman et al., 1989; Günzel-Apel et al., 1993; Farrell et al., 1996), largely reducing the subjectivity and overcoming the variability inherent to the routine microscopical semen examination (Verstegen et al., 2002). However, several parameters, such as semen concentration influence CASA outcome (Verstegen et al., 2002; Rijsselaere et al., 2003; 2005). The latter is due to multiple individual collisions of the condensed sperm cells at high concentrations, which interfere with the progressive motility (Vantman et al., 1988; Bartoov et al., 1991; Rijsselaere et al., 2002; 2003; Hoflack et al., 2005). To avoid this inaccuracy and to obtain more reliable and unbiased kinetic measurements, a concentration between 20 and 50 x 106 spermatozoa/ml has been advised in CASA studies (Budworth et al., 1988; Vantman et al., 1989; Neuwinger et al., 1990b; Davis and Katz, 1993; Farrell et al., 1996; Verstegen et al., 2002; Rijsselaere et al., 2003).

Although literature data on the relation between sperm motility and fertility are contradictory, with some reports acknowledging this relationship (Wood et al., 1986; Kjaestad


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et al., 1993; Correa et al., 1997; Zhang et al., 1998; Januskauskas et al., 2000), while others were not able to demonstrate any relationship (Söderquist et al., 1991b; Andersson et al., 1992; Januskauskas et al., 1999), sperm motility is generally considered to be an important semen quality parameter. Most studies on this relationship used frozen-thawed semen. In studies using subjectively assessed fresh sperm motility to predict fertility outcome, similarly, some found no relation (Fitzpatrick et al., 2002; Holroyd et al., 2002), while others demonstrated that fresh sperm motility is correlated with the fertilizing potential of the inseminate (Christensen et al, 1999; 2005). Furthermore, Farrell et al. (1998) reported very high correlations (0.99, r2 = 0.98) between combined motility parameters measured by CASA on fresh semen and bull fertility, evidenced by the 59 days non-return rate (NRR) to first service. Other reports on the relation between computerized semen-quality measurements and bull fertility also demonstrated that the combination of several semen-quality parameters correlate better with fertility than single motility parameters (Budworth et al., 1987; 1988). Hence, CASA has the potential for a more accurate prediction of fertility than the parameters assessed by the routine microscopical semen evaluation (Farrell et al., 1998; Christensen et al., 1999).

In Belgium, the two predominant cattle breeds are the double muscled Belgian Blue (BB) beef breed, which is famous for its low feed conversion ratio, its high percentage of lean meat and its advantageous carcass classification, and the internationally known Holstein Friesian (HF) dairy breed. In contrast to the HF breed, data on semen quality of BB bulls are scarce. However, recent studies demonstrated a poorer quality of BB sperm in comparison with HF sperm. Concerning motility, both the percentages of totally and progressively motile sperm, as well as sperm velocity were lower in the BB ejaculates (Hoflack et al., 2003; 2006a; 2006b). However, these parameters were subjectively assessed. The aim of the present study was therefore to evaluate and compare several semen motility characteristics of the BB beef breed and the HF dairy breed by means of CASA in order to objectively assess whether the same breed differences occur.


1. Study population

CASA assessment was performed on fresh semen of 41 BB and 49 HF ejaculates of 36 BB and 42 HF bulls, respectively, collected at artificial insemination (AI) centres (in Belgium for the BB bulls and in The Netherlands for the HF bulls, since no HF AI bulls are present in Belgium). All semen samples were collected by means of an artificial vagina and only first ejaculates were used. The examined bulls were all regularly collected (twice a week) in the weeks preceding this study, which was conducted in November 2003 and May 2004 for the BB and HF bulls, respectively.


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The BB AI bulls were purchased from selection centres as well as from private farms based on their linear classification, which is a scoring system describing several physical characteristics of the bull (concerning height, muscular development, meat type (skeletal conformation), stance and general appearance), and after a quarantine period of one month, they were accepted for AI purposes without further selection. However, bulls with poor libido or repeated poor semen motility before (< 60% progressive motility) and after (< 30% of total or < 15% of progressive motility) cryopreservation were discarded keeping only “more fertile” bulls with increasing age. Furthermore, aggressive or injured bulls were also eliminated. This selection procedure resulted in 3 groups of BB bulls which could arbitrarily be divided as follows: (1) the unselected youngest bulls (< 2 years), (2) the active breeding bulls between 2 and 4 years old which were intensively selected, and (3) the bulls of proven fertility of over 4 years of age considered to be veterans which had survived culling for different reasons.

This was in contrast to the HF bulls, where bull calves were purchased based on their expected genetic value. At approximately 11 months of age, these bulls were transferred to an AI facility, where, after a quarantine period of one month semen was collected. Only when these bulls passed a strict semen quality test (2 consecutive ejaculates collected with 3 or 4 days interval ≥ 2 ml containing ≥ 600 x 106 spermatozoa / ml, with ≥ 65% motility, ≥ 80% normal morphology and ≥ 50% intact acrosomes) were they accepted for AI purposes. When an accepted HF bull had produced around 3000 straws, semen collection was stopped for at least 3 years during which the bull’s progeny was tested. This resulted in 2 HF groups comparable to the 2 youngest groups of BB bulls: (1) an unselected young group, and (2) a group of selected young bulls that passed an initial semen quality test. Hence, we made 3 breed comparisons:

1: an ‘overall’ breed comparison using both unselected and selected bulls of both breeds, stratifying for these respective groups.

2: the unselected young HF and BB bulls < 2 years of age, hereafter referred to as “the unselected bulls”;

3: the fertility selected young HF bulls, and the BB bulls between 2 and 4 years of age, hereafter referred to as “the selected bulls”;

Next to these 3 breed comparisons, within breed comparisons of the 3 BB groups (unselected – selected – old BB bulls) were also performed.

2. Computer assisted sperm motility analysis

Immediately after semen collection, a 50 µl semen aliquot was diluted in physiological saline solution at 37 °C to a concentration of approximately 30 x 106 spermatozoa/ml. This concentration is well within the limits to obtain accurate kinetic measurements (Vantman et


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al., 1989; Neuwinger et al., 1990b; Davis and Katz, 1993; Verstegen et al., 2002; Rijsselaere et al., 2003), which are not biased by multiple individual collisions of condensed sperm cells at higher concentrations falsifying the motility results (Vantman et al., 1988; Bartoov et al., 1991; Rijsselaere et al., 2002). Motility analysis was directly performed by means of the Hamilton Thorne CEROS version 12.2c computer-aided semen analyser (Hamilton-Thorne Research, Beverly, USA) using the green light filter. The software settings of the HTR 12.2c used in the present study were largely based on the recommendations of Davis and Katz (1993) and are summarized in Table 1. Preliminary trials using the playback facility to see whether the sperm cells were correctly identified in physiological saline solution and whether their analyzed trajectories were correctly reconstructed, led to these parameter settings, which slightly differ from those advised by the manufacturer. Each ejaculate was assessed twice by loading 7 µl of the diluted semen in a pre-warmed disposable Leja counting chamber (Orange Medical, Brussels, Belgium) on a minitherm stage warmer (37 °C), and by counting at least 1000 cells per analysis to avoid poor repeatability, starting in the left upper corner and ending in the right lower corner of the chamber (Verstegen et al., 2002). The average result of these 2 analyses per ejaculate was used for further data processing. In general, only one ejaculate per bull was assessed by CASA, but in some exceptional cases (5 BB and 7 HF bulls) two ejaculates per bull, collected on different days, were available. In the latter situation, the CASA results of these two ejaculates were averaged to obtain an as accurate as possible picture of these bull’s semen. The assessed parameters were: average pathway velocity (VAP) = the average velocity of the smoothed cell path (µm/s); straight line velocity (VSL) = the average velocity measured in a straight line from the beginning to the end of the track (µm/s); the amplitude of the lateral head displacement (ALH) = the mean width of the head oscillation as the sperm cells swim (µm); the beat cross-frequency (BCF) = frequency of the sperm head crossing the average path in either direction (Hz); the straightness (STR) = average value of the ratio VSL/VAP (%); the percentage of totally motile spermatozoa (MOT; %) and the percentage of progressive spermatozoa (PMOT; %). According to the low (LVV) and medium (MVV) VAP cut-off values, and the low VSL cut-off value (LVS; Table 1), the sperm population was additionally divided into 4 velocity categories: rapid (RAP; % with VAP > MVV), medium (MED; % with MVV > VAP > LVV), slow (SLOW; % with VAP < LVV and VSL < LVS) and static (STATIC; the percentage of spermatozoa which were not moving during the analysis) spermatozoa. To avoid inaccuracy due to wave motion of the sample, an equilibration time of approximately one minute after loading the Leja chamber was adopted, and slow and static cells were not considered to determine the percentage of motile spermatozoa. The percentage of progressive spermatozoa (PMOT; %) includes all sperm cells with both VAP > MVV (= 50 µm/s) and STR > 70%. Of several motility parameters (VAP, VSL, ALH, BCF, STR), the Hamilton Thorne equipment also calculates the within ejaculate standard deviations. These within ejaculate standard deviations were averaged over all bulls


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within each bull group (unselected BB, selected BB, old BB, unselected HF and selected HF bulls) and compared amongst bull groups to assess the homogeneity of (the assessed sperm parameter in) the ejaculates of bulls belonging to a particular bull group.

Table 1: Software settings of the Hamilton Thorne Ceros 12.2c used in the present study for the bull sperm motility assessment.

Parameter Parameter function Value

Frame rate (Hz)

Image capture

60

Number of frames acquired Image capture 30

Minimum contrast Cell detection 20

Minimum cell size (pixels) Cell detection 10

Non motile head size (pixels) Cell detection 5

Non motile head intensity Cell detection 20

Medium VAP cut-off (µm/s, MVV) Progressive cell detection 50

Straightness cut-off (%, STR) Progressive cell detection 70

Low VAP cut-off (µm/s, LVV) Static cell detection 30

Low VSL cut-off (µm/s, LVS) Static cell detection 15

Minimum static intensity limit Cell differentiation 0.5

Maximum static intensity limit Cell differentiation 1.5

Minimum static size limit Cell differentiation 0.5

Maximum static size limit Cell differentiation 3.0

Minimum static elongation limit Cell differentiation 0

Maximum static elongation limit

Cell differentiation 85

3. Subjective semen analysis

3.1. Subjective motility analysis

Simultaneously, the percentages of total and progressive motility were subjectively assessed to the nearest 5% by placing 10 µl of diluted semen (10 µl aliquot of pure semen in 790 µl physiological saline solution) on a pre-warmed glass slide at 37°C under a coverslip, and by examining 5 different microscopic fields all in the centre of the coverslip, under a 200 x phase-contrast microscope (Hopkins and Spitzer, 1997). This procedure was repeated twice by the same experienced observer. Additionally, a velocity score (1 – 4) was attributed to the


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sample: 1 = very slow semen (e.g. after freeze-thawing), 2 = slow semen, 3 = rapid semen, 4 = extremely rapid semen.

3.2. Subjective morphology analysis

Sperm morphology of the examined bulls was also assessed by 2 experienced observers. This was done by counting 200 spermatozoa on air-dried, eosin-nigrosin or eosin-aniline blue stained smears under a 1000 x light microscope, using immersion oil. Individual spermatozoa were classified according to Barth and Oko (1989) into one of five categories: (1) normal morphology; (2) abnormal head; (3) abnormal midpiece or tail; (4) proximal droplet; or (5) having a distal droplet. When multiple abnormalities were observed in the same sperm cell, only one abnormality was logged. Abnormal heads were given first priority in classification, abnormal midpieces or tails were classified with second priority, proximal droplets with third and distal droplets with least and last priority.


The overall breed comparison for all the motility parameters was based on the fixed effects model with normally distributed random error term, including bull group (unselected-selected) as a stratifying factor. The comparisons between the breeds within the respective groups and the comparisons between the 3 age categories of the BB breed were also based on the fixed effects model with normally distributed random error term. The global level of significance for all statistical analyses was 0.05, but for the 5 pairwise comparisons, the comparisonwise significance level was Bonferroni adjusted to 0.01 (0.05/5 comparisons).

The sperm morphology parameters were compared between the two breeds by means of the Mann-Whitney test, since these data were not normally distributed. Furthermore, the Spearman’s rank correlation coefficient was derived to quantify the relationship between the sperm morphology results on the one hand and the CASA parameters on the other hand.

RESULTS

1. Computer assisted sperm motility analysis

The CASA results of the overall breed comparison and of the respective groups within both breeds (3 BB and 2 HF groups) for the assessed motility parameters are listed in Table 2. The results for the within ejaculate standard deviations of all the assessed kinetic measurements are given in Table 3.

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Table 2: Arithmetic mean (± SD) and median (and range) of the motility parameters assessed by CASA for the Holstein Friesian (HF) and Belgian Blue (BB) breed, both for the overall breed results and for the respective groups of both breeds. The number of assessed bulls within each group is also given (N).

Parameter Overall BB Overall HF Unselected BB Selected BB Old BB Unselected HF Selected HF N 29 42 6 23 7 27 15

MOT (%) 70.4 ± 15.2 1 82.9 ± 12.0 2 71.3 ± 6.5 aα 70.5 ± 13.7 aα 58.1 ± 25.6 α 82.1 ± 6.2 a 83.8 ± 5.0 b 74.5 (37.5 - 85.0) 84.0 (67.0 - 91.5) 73.3 (62.3 - 77.3) 74.5 (37.5 - 85.0) 65.5 (5.0 - 81.5) 83.5 (67.0 - 91.5) 85.5 (75.0 - 91.5) PMOT (%) 53.8 ± 14.8 1 68.9 ± 11.7 2 55.4 ± 5.6 aα 53.7 ± 12.6 aα 44.2 ± 20.3 α 67.9 ± 7.2 b 70.1 ± 5.1 b 57.5 (27.0 - 69.5) 69.8 (52.3 - 79.0) 55.3 (49.0 - 63.5) 58.0 (27.0 - 69.5) 51.0 (3.5 - 64.5) 69.0 (52.3 - 79.0) 70.0 (58.5 - 79.0) VAP (µm/s) 114.0 ± 18.3 1 120.9 ± 14.4 2 114.9 ± 5.9 aα 114.5 ± 11.2 aα 115.9 ± 15.8 α 119.6 ± 12.2 a 122.6 ± 9.9 a 115.9 (88.7 - 133.2) 119.7 (99.1 - 150.9) 114.6 (109.2 - 121.7) 115.9 (88.7 - 133.2) 116.0 (84.8 - 131.2) 119.3 (99.1 - 150.9) 120.2 (105.5 - 140.4) VSL (µm/s) 98.8 ± 18.0 1 109.4 ± 14.2 2 100.5 ± 5.9 aα 99.0 ± 12.2 aα 99.1 ± 14.2 α 107.9 ± 11.0 a 111.4 ± 9.8 b 100.6 (75.8 - 118.8) 108.8 (91.1 - 132.1) 98.6 (94.9 - 107.6) 100.6 (75.8 - 118.8) 102.6 (72.8 - 115.9) 107.2 (91.1 - 132.1) 110.4 (95.8 - 129.8) ALH (µm) 5.0 ± 1.0 1 4.5 ± 0.8 2 4.7 ± 0.4 aα 5.1 ± 0.7 aα 5.6 ± 1.4 α 4.4 ± 0.7 a 4.5 ± 0.4 b 5.1 (4.0 - 7.2) 4.4 (3.2 - 6.1) 4.7 (4.2 - 5.2) 5.1 (4.0 - 7.2) 5.7 (3.2 - 7.2) 4.3 (3.2 - 6.1) 4.5 (3.7 - 5.2) BCF (Hz) 36.9 ± 3.7 1 39.0 ± 3.0 2 37.5 ± 1.9 aα 36.6 ± 2.2 aα 35.5 ± 2.1 α 39.1 ± 2.4 a 39.1 ± 2.2 b 37.1 (32.7 - 40.8) 39.3 (32.3 - 42.8) 37.3 (35.0 - 40.8) 37.1 (32.7 - 40.1) 35.3 (33.3 - 39.0) 39.5 (32.3 - 42.8) 39.1 (35.4 - 42.6) STR (%) 84.9 ± 4.0 1 89.2 ± 3.1 2 85.8 ± 1.6 aα 84.7 ± 3.1 aα 84.2 ± 2.8 α 88.9 ± 2.1 b 89.7 ± 1.7 b 85.5 (76.0 - 88.5) 89.5 (83.5 - 92.5) 85.8 (83.8 - 88.0) 85.0 (76.0 - 88.5) 85.5 (80.5 - 87.0) 89.5 (83.5 - 92.5) 89.5 (86.5 - 92.5)


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Table 3: Arithmetic mean (± SD) and median (and range) of the standard deviations of the motility parameters assessed by CASA for the Holstein Friesian (HF) and Belgian Blue (BB) breed, both for the overall breed results and for the respective groups of both breeds. The number of assessed bulls in each group is also given (N).


Parameter Overall BB Overall HF Unselected BB Selected BB Old BB Unselected HF Selected HF

N 29 42 6 23 7 27 15

VAP SD 46.3 ± 5.5 1 43.4 ± 4.3 2 47.3 ± 2.8 aα 45.6 ± 3.1 aα 46.0 ± 3.7 α 43.9 ± 3.3 a 43.0 ± 3.9 a 46.6 (39.8 - 52.2) 43.7 (35.8 - 50.4) 47.2 (44.4 - 52.2) 46.1 (39.8 - 51.2) 46.5 (38.5 - 50.7) 44.3 (35.8 - 50.4) 42.5 (37.2 - 48.9)

VSL SD 47.7 ± 5.8 1 45.9 ± 4.6 1 48.8 ± 2.8 aα 47.1 ± 3.8 aα 46.5 ± 5.4 α 46.3 ± 3.3 a 45.6 ± 3.8 a 47.6 (37.3 - 53.9) 46.0 (37.6 - 51.8) 48.2 (45.6 - 53.9) 47.2 (37.3 - 52.4) 48.4 (35.4 - 52.3) 46.8 (37.6 - 51.8) 45.2 (38.5 - 51.3)

ALH SD 2.8 ± 0.4 1 2.5 ± 0.3 2 2.7 ± 0.2 aα 2.8 ± 0.3 aα 2.9 ± 0.4 α 2.5 ± 0.2 a 2.6 ± 0.3 b 2.8 (2.4 - 3.7) 2.5 (2.1 - 3.1) 2.7 (2.5 - 3.0) 2.8 (2.4 - 3.7) 3.1 (2.1 - 3.2) 2.5 (2.1 - 2.9) 2.6 (2.2 - 3.1)

BCF SD 17.5 ± 1.4. 1 16.9 ± 1.1. 2 17.4 ± 0.4 aα 17.4 ± 0.9 aα 17.4 ± 0.4 α 16.9 ± 0.8 a 16.8 ± 0.8 a 17.6 (14.9 - 18.9) 16.9 (15.3 -18.9) 17.5 (16.8 - 18.0) 17.6 (14.9 - 18.9) 17.5 (17.0 - 18.0) 16.9 (15.4 - 18.9) 17.0 (15.3 - 18.0)

STR SD 17.3 ± 2.1 1 15.3 ± 1.7. 2 17.0 ± 0.7 aα 17.4 ± 1.4. aα 17.2 ± 1.6. α 15.2 ± 1.3. b 15.4 ± 1.3. b 17.5 (14.0 - 20.0) 15.5 (12.5 - 17.3) 17.3 (16.0 - 17.8) 17.5 (14.0 - 20.0) 18.0 (14.0 - 18.5) 15.5 (12.5 - 17.3) 15.5 (12.5 - 17.0)

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Table 4: Arithmetic mean (± SD) and median (and range) of the subjectively assessed motility parameters for the Holstein Friesian (HF) and Belgian Blue (BB) breed, both for the overall breed results and for the respective groups of both breeds. The number of assessed bulls within each group is also given (N).

Parameter N

Overall BB 29

Overall HF 40

Unselected BB6

Selected BB 23

Old BB 7

Unselected HF27

Selected HF 13

Motile % 60.5 ± 18.0 1 79.6 ± 14.7 2 63.3 ± 8.3 aα 59.6 ± 15.1 aα 48.6 ± 21.9 α 79.3 ± 8.1 b 80.6 ± 8.4 b

60.0 (30.0 - 80.0) 80.0 (60.0 - 92.0) 62.5 (52.5 - 75.0) 60.0 (30.0 - 80.0) 55.0 (5.0 - 70.0) 80.0 (60.0 - 90.0) 80.0 (65.0 - 92.0)

Progressive % 56.5 ± 18.8 1 78.7 ± 15.4 2 60.4 ± 8.3 aα 55.5 ± 15.5 aα 46.3 ± 21.1. α 77.7 ± 8.8 b 80.6 ± 8.4 b

60.0 (25.0 - 75.0) 77.5 (55.0 - 92.0) 58.8 (50.0 - 72.5) 60.0 (25.0 - 75.0) 55.0 (4.0 - 65.0) 75.0 (55.0 - 90.0) 80.0 (65.0 - 92.0)

Velocity 2.9 ± 0.4 1 3.5 ± 0.3 2 2.9 ± 0.1 aα 2.9 ± 0.3 aα 2.6 ± 0.4 α 3.5 ± 0.2 b 3.5 ± 0.0 b

3.0 (2.0 - 3.0) 3.5 (3.0 - 3.8) 3.0 (2.8 - 3.0) 3.0 (2.0 - 3.0) 2.8 (2.0 - 3.0) 3.5 (3.0 - 3.8) 3.5 (3.5 - 3.5)



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1.1. Overall breed differences for the CASA results

When the overall results of both breeds were compared, all the assessed parameters (except MED%) differed significantly (Figure 1, Table 2). The percentages of totally and progressively motile spermatozoa (MOT, PMOT; P < 0.0001) and the semen velocity parameters were significantly lower (VAP: P = 0.0224; VSL: P = 0.0005; STR: P < 0.0001) in the BB breed. The ALH was higher (P = 0.0015) combined with a lower BCF (P = 0.0007) for the BB spermatozoa (Table 2). Fewer rapid (P < 0.0001) but more slow (P < 0.0001) and static (P < 0.0001) spermatozoa were present in the BB breed, while a similar proportion of medium velocity sperm (P = 0.2061) occurred in both breeds (Figure 1). The standard deviations of the parameters of which this was assessed (VAP, VSL, ALH, BCF, STR) were consistently significantly higher in the BB breed (P ≤ 0.0102; except for VSL which was borderline significantly higher in the BB breed: P = 0.0574: Table 3).

1.2. Breed differences within the respective bull groups for the CASA results

When comparing the unselected bulls, only the percentage of progressively motile spermatozoa (PMOT; P = 0.0091) was significantly lower in the BB breed. The velocity measures were not significantly different, except for the deducted parameter straightness (STR; P = 0.0062) which was lower in the BB group (Table 2). However, a higher variability


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was noticed in the straightness measures of the BB sperm, evidenced by a significantly higher standard deviation (P = 0.0028: Table 3) compared to the HF bulls.

When the selected bulls were compared, the percentages of totally and progressively motile spermatozoa (MOT, PMOT; P ≤ 0.0008) and some semen velocity measures (VSL: P = 0.0014; BCF: P = 0.0015; STR: P < 0.0001) were significantly lower in the BB breed, while ALH was significantly higher (P = 0.0046) for BB sperm (Table 2). Fewer rapid (P = 0.0003) but more slow (P = 0.0003) spermatozoa were present in ejaculates of the BB breed, while a similar proportion of medium (P = 0.2355) and static (P = 0.0645) sperm were noted in ejaculates of this group of both breeds (Figure 2). The standard deviations of several sperm parameters (ALH SD: P = 0.0028; STR SD: P < 0.0001: Table 3) were always significantly higher in the BB bulls compared to their matches in the HF breed.

1.3. Age differences within the BB breed for the CASA results

No differences whatsoever were present between the different age groups within the BB breed, with the sole exception of the oldest bulls having significantly more static sperm compared to both younger BB groups (Tables 2 and 3, Figure 2).

2. Subjective motility analysis

The results for the parameters of the subjective motility analysis, both for the overall breed comparison and of the respective groups within both breeds (3 BB and 2 HF groups) are summarized in Table 4.

The percentage of totally and progressively motile spermatozoa, as well as the velocity score of these spermatozoa were always significantly (P ≤ 0.0054) lower in the BB breed, both for the overall breed results and for the matched comparisons. No differences were noted between age groups within the BB breed.

3. Subjective morphology analysis

The results on the sperm morphology data of the two breeds are given in Table 5. All the assessed sperm morphology parameters were significantly (P < 0.0001) poorer in the BB bulls compared to HF bulls, evidenced by a lower percentage of normal spermatozoa and a higher percentage of abnormal heads and tails, and proximal and distal droplets in the BB breed.


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Table 5: Arithmetic mean (± SD) and median (and range) of the subjectively assessed sperm morphology parameters, for all the Holstein Friesian (HF) and Belgian Blue (BB) bulls. The number of assessed bulls is also given (N).

% Normal sperm % Abnormal heads % Abnormal tails % Proximal droplets % Distal droplets

All BB bulls (N = 36) 52.9 ± 13.3 16.5 ± 9.9 22.0 ± 9.2 6.7 ± 7.2 1.9 ± 1.6

51.9 (30.0 - 75.0) 12.8 (7.5 - 49.5) 17.8 (11.0 - 46.0) 3.8 (0.0 - 36.0) 1.4. (0.0 - 7.4)

All HF bulls (N = 42) 79.7 ± 7.0 8.3 ± 5.4 9.4 ± 4.3 2.3 ± 2.0 0.3 ± 0.5

81.0 (53.0 - 90.0) 7.0 (2.0 - 34.0) 8.0 (4.0 - 25.0) 2.0 (0.0 - 10.0) 0.0 (0.0 - 1.0)

All these parameters differed significantly between the two breeds (P < 0.05).

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Table 6: The Spearman’s rank correlation coefficients (r) between the subjectively assessed sperm morphology parameters on the one hand and the CASA parameters on the other hand, for all the bulls of both breeds.

Parameters % Normal sperm % Abnormal heads % Abnormal tails % Proximal droplets % Distal droplets

VAP 0.288 -0.218 ns -0.189 ns -0,230 -0.229

VSL 0.426 -0.313 -0.313 -0.343 -0.333

ALH -0.349 0.248 0.294 0.384 0.259

BCF 0.532 -0.504 -0.332 -0.357 -0.416

STR 0.614 -0.503 -0.486 -0.496 -0.440

% MOT 0.631 -0.485 -0.548 -0.292 -0.533

% PROG 0.669 -0.537 -0.556 -0.371 -0.546

% RAPID 0.635 -0,490 -0.532 -0.309 -0.521

% MEDIUM -0.166 ns 0.153 ns 0.055 ns 0.152 ns 0.080 ns

% SLOW -0.571 0.457 0.439 0.303 0.435

% STATIC -0.556 0.423 0.546 0.143 ns 0.535

All the correlation coefficients were significant (P < 0.05), with the exception of those marked by a superscript (ns: not significant).


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4. Correlations between subjective morphology and CASA outcome

The Spearman’s rank correlation coefficients between the sperm morphology outcome on the one hand and the CASA parameters on the other hand are presented in Table 6. Significant correlations (P < 0.05) were always present between all the sperm morphology and sperm velocity measures, except for the correlations between all the sperm morphology parameters and the % MEDIUM sperm and between the % of proximal droplets and the % STATIC sperm (P > 0.05). The correlation between the % of abnormal heads and VAP was borderline significant (P = 0.055), while a tendency (P = 0.097) for correlation was present between the % of abnormal tails and VAP. The highest correlation coefficients were obtained between the percentage of normal spermatozoa and several CASA parameters. The percentage of abnormal tails and distal droplets correlated best although negatively with the percentage of motile, progressively motile and rapid spermatozoa and positively with the percentage of static spermatozoa. The percentage of abnormal heads correlated best albeit negatively with the percentage of progressively motile spermatozoa and with the straightness and beat cross frequency of the sperm cells. Percentage of proximal droplets resulted in the poorest correlation coefficients, of which the correlation with the straightness was the best, but negative.

DISCUSSION

A significant HF breed advantage for subjectively assessed semen motility parameters has recently been demonstrated when HF bulls were compared to BB bulls (Hoflack et al., 2006a; 2006b), and these results were confirmed in the present study. Similarly, in this study objective sperm motility analysis by means of CASA demonstrated an obvious HF breed advantage when the overall CASA results were assessed: the % of totally and progressively motile spermatozoa were higher for HF semen, reflecting the higher proportion of live spermatozoa present in a HF ejaculate (Hoflack et al., 2006a). Additionally, BB spermatozoa apparently move less fluent as evidenced by a lower BCF combined with a higher ALH, compared to HF sperm. This less fluent motility pattern probably results in more resistance due to friction and consequently in a lower velocity. Furthermore, the lower the BCF, the slower the progressive movement will be as a result of less propelling force. Indeed, the BB spermatozoa demonstrated a lower kinetic efficiency as they moved slower and less straight forward compared to HF sperm, since the velocity (VAP, VSL) and direction measures (STR) were significantly lower in the BB ejaculates. This velocity will moreover be influenced by the size of the sperm heads, since small geometrical differences in sperm morphology can result in large differences in sperm hydrodynamics, and subsequently in impaired semen velocity (Dresdner and Katz, 1981). Size and elongation of the sperm heads are two measures


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that were also assessed by the Hamilton Thorne analyzer (data not shown), and these data suggest that BB sperm heads are shorter albeit larger compared to HF spermatozoa. This morphometrical difference might in part be responsible for the slower and less fluent velocity of BB spermatozoa (Dresdner and Katz, 1981). Furthermore, the presence of sperm abnormalities (such as knobbed acrosomes, abnormal acrosomes, nuclear pouches or diadem defects, midpiece defects (segmental aplasia as well as “pseudodroplet like” defects), proximal and distal droplets, and accessory and abaxial tails) is generally more common in the BB breed (Hoflack et al., 2006a), and these abnormalities and in particular abnormal midpieces and tails, and (both proximal and distal) droplets might negatively influence semen velocity (Blom, 1977; Barth and Oko, 1989, Amann et al., 2000). In human sperm, a similar influence of morphology on motility has been demonstrated (Johnston et al., 1995; Mahmoud et al., 1998). Indeed, sperm morphology of the bulls examined in the present study differed significantly between breeds. Moreover, the sperm morphology was significantly correlated to the CASA outcome, suggesting an influence of sperm morphology on motility. Apparently, all the assessed sperm abnormalities negatively influenced sperm motility to some extent. These factors altogether resulted in less rapid and more slow and static (when dead), and subsequently less progressively motile spermatozoa in the BB semen picture. Thus, different subpopulations within an ejaculate exist between breeds. Furthermore, these BB sperm subpopulations were more heterogeneous compared to the HF semen, which was evidenced by the higher standard deviations for most of the assessed CASA parameters in the BB breed.

It is noteworthy to remark that even with a rather small number of ejaculates, the overall breed differences were already this obvious and significant. However, these breed differences were less obvious when the matched breed groups were compared. Some differences were still present when the selected bulls were compared, but only PMOT and STR significantly differed when the unselected bulls of both breeds were compared. However, the observed differences between the small group of unselected BB and HF bulls numerically showed the same pattern for any of the CASA parameters as the overall breed comparison. Since the quantitative relation is exactly the same for the unselected bulls as for the whole population, it might be possible that there is a ‘genetic component’ to the overall breed difference. Moreover, the unselected HF bulls were all rather young which might in some cases result in an immature sperm picture with subsequent poor morphology and consequently have a negative influence on semen velocity, minimizing a possibly inherent breed velocity difference (Almquist and Amann, 1976; Blom, 1977; Lunstra and Echternkamp, 1982; Barth and Oko, 1989; Johnson, 1997; Amann et al., 2000). However, this suggested ‘genetic component’ could very well be the mere result of the worldwide long-term selection for highly fertile HF AI bulls (Söderquist et al., 1991a), a selection that was not performed in the BB breed. In this case, sperm morphological differences would be responsible for the motility differences, as was encountered in the present study. The presented data were however based


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on a limited number of bulls, and the observed differences can consequently be the result of individual bull factors, as was the case in the oldest BB bulls for the percentage of static spermatozoa. Whether a ‘genetic motility difference’ is truly present should be confirmed on a larger scale by examining more ejaculates of a high number of bulls of the two breeds by CASA. In this study, the same CASA device with the exact same parameter settings were used to compare the fresh semen motility results of the two breeds. Generally, AI centres have different CASA equipment and moreover use different parameter settings to evaluate the sperm motility, making large scale comparisons of such motility data irrelevant. Moreover, to elucidate whether the presence of a pure ‘genetic motility difference’ independent of sperm morphology differences exists, bulls of comparable age with a similar (preferably high) percentage of normal spermatozoa of the two breeds should be compared. This was not possible in the present study.

No differences were present between the 3 different age groups within the BB breed, with the sole exception of the oldest bulls having significantly more static sperm. This exception was due to one bull in the oldest group with an extremely high proportion (84.5%; data not shown) of static spermatozoa, without which this difference no longer occurred.

The fact that the sperm motility results did not increase with age, as the BB bulls were selected only keeping the “more fertile” older bulls, might be explained by the fact that sperm quality in older BB bulls decreases due to testicular degeneration, eventually leading to a quality loss, even in the “more fertile” bulls, nullifying the selection effect (Kumi-Diaka et al., 1981; Hopkins, 1997; Van Camp, 1997; Brückmann et al., 2000; Brito et al; 2002).

In conclusion, we can state that sperm motility differs between the BB and HF breed. The generally higher proportion of live spermatozoa in HF ejaculates compared to BB semen (Hoflack et al., 2006a) apparently results in a higher percentage of both totally and progressively motile spermatozoa. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower BCF combined with a higher ALH, is the basis for the lower velocity of BB sperm cells compared to HF semen. Additionally, BB spermatozoa move less straight forward, resulting in a lower STR. These differences appear to be related to the sperm morphology and were obvious in the examined bull populations residing at AI centres. In an unselected rather young bull population, the breed differences for the assessed CASA parameters are numerically albeit not significantly in agreement with the above-mentioned results. Hence, we assume that a genetic component could be responsible for the motility differences between the two breeds.

ACKNOWLEDGEMENTS



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Rijsselaere T, Van Soom A, Tanghe S, Coryn M, Maes D, de Kruif A. New techniques for the assessment of canine semen quality: a review. Theriogenology 2005; 64: 706-719.

Söderquist L, Janson L, Larsson K, Einarsson S. Sperm morphology and fertility in AI bulls. J. Vet. Med. A 1991a; 38: 534 - 543.

Söderquist L, Rodriguez-Martinez H, Janson L. Post-thaw motility, ATP content and cytochrome C oxidase activity of AI bull spermatozoa in relation to fertility. Zentralbl. Veterinarmed. A 1991b; 38: 165 – 174.


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Vantman D, Koukoulis G, Dennison L, Zinaman M, Sherins RJ. Computer-assisted semen analysis: evaluation of method and assessment of the influence of sperm concentration on linear velocity determination. Fertil. Steril. 1988; 49: 510-515.

Vantman D, Banks SM, Koukoulis G, Dennison L, Sherins RJ. Assessment of sperm motion characteristics from fertile and infertile men using a fully automated computer-assisted semen analyzer. Fertil. Steril. 1989; 51: 156-161.

Verstegen J, Iguer-ouada M, Onclin K. Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002; 57: 149-179.

Wood PD, Foulkes JA, Shaw RC, Melrose DR. Semen assessment, fertility and the selection of Hereford bulls for use in AI. J. Reprod. Fertil. 1986; 76: 783 – 795.

Zhang BR, Larsson B, Lundeheim N, Rodriguez-Martinez H. Sperm characteristics and zona pellucida binding in relation to field fertility of frozen-thawed semen from dairy AI bulls. Int. J. Androl. 1998; 21: 207 – 216.

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CHAPTER 4

TESTICULAR DYSFUNCTION IS RESPONSIBLE FOR LOW SPERM QUALITY IN

BELGIAN BLUE BULLS

Modified from: Testicular Dysfunction is Responsible for Low Sperm Quality in Belgian Blue Bulls G. Hoflack, W. Van den Broeck, D. Maes, K. Van Damme, G. Opsomer, L. Duchateau, A. de Kruif, H. Rodriguez-Martinez, A. Van Soom Submitted

Chapter 4: Testicular Histology of BB and HF Bulls

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ABSTRACT

In a previous study, we demonstrated that Belgian Blue (BB) bulls have a higher prevalence of small scrota and poorer semen morphology compared to the Holstein Friesian (HF) breed in Belgium. The present study tested the hypothesis that the underlying reason for these BB traits negative to fertility was testicular degeneration, associated with an eventual hypoplastic background. At culling, sperm quality and testicular histology of BB bulls were assessed and compared to that of HF bulls. Besides semen quality being generally poorer in the BB breed, significantly more degenerative changes were encountered in BB compared to HF testicles (% affected tubules x degeneration severity: 37.7 ± 11.9 versus 29.3 ± 9.9 for BB and HF bulls, respectively; P = 0.053). These results correlated to the percentage of normal spermatozoa (r = -0.44; P = 0.024) and primary abnormalities (r = 0.38; P = 0.053). Moreover, the relative amount of collagen fibres present in the testicular interstitial connective tissue was correlated with % normal sperm (r = -0.47; P = 0.017), primary defects (0.48; P = 0.014), and the degeneration results (r = 0.63; P < 0.001). The % testicular interstitial collagen fibres differed significantly between breeds (10.6 ± 4.0 % for the BB versus 7.6 ± 1.9 % for the HF bulls; P = 0.016). The results suggest that the increased amount of connective tissue in BB testes is responsible for the poorer sperm quality. This condition can be defined as a mild form of testicular hypoplasia, and might, in turn, be responsible for a higher sensitivity to testicular degeneration which is encountered in the BB breed.


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INTRODUCTION

In the Belgian Blue (BB) beef breed, disappointing pregnancy results are often encountered when using natural service, and it has been suggested that BB bulls largely contribute to this problem (Hoflack et al., 2006b). Scrota of BB bulls are rather small (Hanset, 2000; Hoflack et al., 2006b), and it has recently been demonstrated that BB semen quality and in particular sperm morphology is often substandard (Hoflack et al., 2006a). These traits negative to fertility most certainly hamper BB bull fecundity and the combination of small testicles and poor sperm morphology encountered in a high proportion of BB bulls suggests the general presence of testicular hypoplasia or degeneration in the BB breed (Madrid et al., 1988; Hoflack et al., 2006b). However, to date, this suspicion has not yet been scientifically explored.

Early degenerative histological lesions in cases of degeneration of the seminiferous epithelium consist of the loss of scattered germ cells and subsequent vacuolization. Further degeneration is characterized by increased loss of germinal cells with possible accumulation of these cells in the tubular lumen and thinning of the seminiferous epithelium, and finally, as the degenerative process proceeds, in loss of all cells. This will result in softer than normal testes of reduced size (McEntee, 1990b; Blanchard et al., 1991a). These histological alterations are accompanied by obvious decreases in semen quality, notably a lowered sperm concentration, an increase in the proportion of morphological abnormalities and a decrease in motility, finally resulting in azoospermia in case of extreme degeneration (Veeramachaneni et al., 1986; Blanchard et al., 1991a; b; Vogler et al., 1993; Barth and Bowman, 1994).

Although the etiology is seldom established in naturally occurring cases, testicular degeneration can have a multitude of causes. Depending on the etiology causing testicular degeneration, the severity, the frequency and the duration of the causative insult, the above-mentioned process might be reversible (McEntee, 1990b; Blanchard et al., 1991a).

Testicular hypoplasia is a poorly defined gross diagnosis covering a number of underlying histological changes, such as germ deficiencies of various types, germ cell weakness or spermatogenic arrest (McEntee, 1990a).

In several beef cattle breeds, including the BB breed, the double-muscled condition has been associated with both advantageous and disadvantageous traits. One of the drawbacks of this phenotype is its increased stress susceptibility (Arthur et al., 1988; Bellinge et al., 2005). Indeed, BB bulls seem extremely sensitive to trivial environmental stresses, such as environmental temperatures exceeding 30°C, a sudden increase or decrease in the daily concentrate supplementation, lameness, and relocating a bull both within as well as between farms. These stresses can induce a sudden and severe decrease in BB semen motility (AWE,


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personal communication). We hypothesized that these alterations of seminal quality could be the result of acute testicular degeneration, elicited by stress.

The aim of the present study was therefore to examine the testicular histology of culled BB bulls in order to determine whether changes in histology relate to the ante-mortem spermiogram. Furthermore, we wanted to assess the possible etiology of the high susceptibility of BB bulls to trivial environmental stress inducing decreases in sperm quality. To this purpose, the BB breed was compared to the other predominant cattle breed in Belgium, namely the Holstein Friesian (HF) breed.


1. Study population

From March 2005 to November 2005, 19 BB bulls and 7 HF artificial insemination (AI) bulls that were to be culled were followed to the slaughterhouse. A few days prior to slaughter, a semen sample of the bulls was collected by means of an artificial vagina and the semen was analyzed as described below. The HF bulls (average age: 1822 ± 64 days) were all progeny-tested bulls that were culled as a result of poor production indices. Only when these bulls’ ejaculates met the sperm quality criteria set by the HF AI organization, they were enrolled in progeny testing, which implies that the semen quality of these bulls prior to progeny testing was good. Of the 19 BB bulls, 5 bulls were culled due to poor semen quality (and are referred to as BB poor semen bulls: average age: 1546 ± 579 days), 7 bulls were culled as a result of abundant sperm stock and these bulls were considered to be good semen producers (and are referred to as BB good semen bulls: average age: 1393 ± 307 days), and 7 bulls were culled for reasons other than (good or poor) semen quality, such as injuries (n = 2), leg abnormalities (n = 2), herd book refusal (n = 2), and seminal vesiculitis (n = 1), (and are referred to as BB miscellaneous bulls: average age: 1415 ± 842 days).

2. Semen evaluation

Different from the routine procedures of both AI centres, which slightly differed, all ejaculates were examined as described below.

The concentration of the ejaculates was determined by diluting 10 µl of semen in 990 µl 1M HCl and by counting the number of sperm cells in 1/100 mm3 of a Bürker counting chamber (Merck, Leuven, Belgium). The final semen concentration was the average of 2 - 4 counts.

Semen motility was evaluated immediately after collection. Gross motility was scored from 1 to 5 on a wet mount of neat semen at 100 x magnification (1 = cells present without motion; 2 = individual cell motion without swirls; 3 = slow swirls; 4 = rapid dark swirls; 5 =


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macroscopically visible rapid dark swirls). The percentages of total and progressive individual motility were subjectively assessed to the nearest 5% by placing 10 µl of diluted semen (10 µl aliquot of pure semen in 790 µl physiological saline solution) on a pre-warmed glass slide at 37°C under a coverslip, and by examining 5 different microscopic fields all in the centre of the coverslip, under a 200 x phase-contrast microscope (Hopkins and Spitzer, 1997). This procedure was repeated twice and was always done by the same experienced observer. Next to these percentages, a velocity score (1 – 3.5) was also attributed to the sample: 1 = very slow semen (e.g. after freeze-thawing), 2 = slow semen, 3 = rapid semen, 3.5 = extremely rapid semen.

The assessment of the percentage of live (i.e. structurally membrane intact) spermatozoa and sperm morphology was done on air-dried, eosin-nigrosin stained smears under a 1000 x light microscope, using immersion oil. Four hundred spermatozoa were evaluated for the live-dead assessment, whereas the morphology evaluation was performed by counting 200 spermatozoa. Individual spermatozoa were classified as either normal or abnormal, according to the 1993 Society for Theriogenology guidelines (Chenoweth et al., 1992; Chenoweth et al., 1994; Hopkins and Spitzer, 1997). In case of abnormal spermatozoa, all the abnormalities present in these sperm cells were inventoried, which could result in multiple abnormalities in an individual sperm cell. From this, the proportion of primary and secondary abnormalities was deducted (Hopkins and Spitzer, 1997). These morphology evaluations were all performed by the same experienced observer.

3. Testicular histology assessment

3.1. Sample preparation

Immediately after slaughter, both testes of the bulls were collected. A tissue sample was taken from a proximal, medial and distal section of each testicle, and these tissue samples were immediately fixed by immersion in Bouin’s fixative for 24h at room temperature (Hopwood, 1977). Subsequently, the samples were dehydrated in increasing concentrations of ethanol and embedded in paraffin after which two 7 µm thin sections were cut from each sample. The sections were mounted on gelatine-coated slides and dried overnight at 37°C. One section was stained using haematoxylin and eosin (Stevens, 1977) for routine microscopical histology assessment, while the remaining section was stained according a modified Masson technique using a 1% (v/v) Biebrich scarlet solution and a 2,5% (g/v) aniline blue solution without nuclear stain (Bradbury and Gordon, 1977). The latter was done to stain the collagen fibres within the testicular connective tissue.


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3.2. Histological evaluation technique

A simple method was used to quantify and qualify degenerative changes of the tubuli seminiferi. Of each testicle, 5 times one randomly selected mm2 away from the edges of the slide was assessed from a proximal, medial and distal section of the testicle. This resulted in the assessment of 30 mm2 testicular tissue per bull (at 100 x magnification). To quantify the degenerative changes, the total number of tubuli with a lumen showing the slightest degenerative changes (either < 4 cell layers, vacuolisation, or cells or debris in lumen) was counted, divided by the total number of tubuli with a lumen and expressed as the ‘percentage of affected tubules’. In order to qualify the degeneration, only the perpendicularly cut tubuli with a lumen were further assessed since these tubuli allow for the best interpretation of degenerative changes. The latter was done by counting the total number of perpendicularly cut tubuli with a lumen, and the number of these tubuli (1) without spermatids, (2) with regions showing less than 4 cell layers, (3) with regions showing less or equal to 2 cell layers, (4) with regions with 1 cell layer, (5) without cell layers (Sertoli cell only tubuli), (6) with obvious vacuolisation, and (7) with cells or debris in the lumen. The number of perpendicularly cut tubuli demonstrating the above-mentioned degenerative changes were all added and divided by the total number of perpendicularly cut tubuli x 7 (assessed traits) in order to produce a ‘degeneration score’ ranging from 0 to 1. The ‘percentage of affected tubules’ was multiplied with this ‘degeneration score’, resulting in a ‘degeneration index’, ranging from 0 to 100%. These observations were performed in a double blinded way.

3.3. Testicular connective tissue assessment

Additionally, the relative amount of testicular connective tissue (collagen fibres) between the tubuli seminiferi, expressed as area percentage, was objectively determined on the sections stained according the modified Masson technique. Therefore, ten digital micrographs were taken randomly per section with an Olympus DP 50 digital camera mounted on a motorized Olympus BX61 light microscope (Olympus Belgium N.V., Aartselaar, Belgium). Using the software AnalySIS® (Soft Imaging System, Olympus Belgium N.V.), one specific threshold was determined so that the relative amount (percentage) of blue stained connective tissue could be determined on each micrograph. For each bull, 3 sections (proximal, medial and distal section) per testicle were assessed, and per section 10 randomly selected fields away from the edges of the slide were analyzed, resulting in 60 observations per bull.


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The statistical analyses were performed at bull level, the experimental unit. To this aim, the 30 (5 assessments / 3 sections / 2 testicles / bull) and 60 (10 assessments / 3 sections / 2 testicles / bull) observations per bull for the histology results and the connective tissue assessments, respectively, were averaged. As only few individual bulls were available per group, parametric assumptions could not be tested; therefore non-parametric tests were used. First, an overall breed comparison was performed (HF versus the 3 cumulated BB groups) at the 5% significance level. Additionally, pairwise comparisons between only 3 bull groups (the HF bulls, the poor semen and good semen BB bulls) were performed at the Bonferroni-adjusted comparisonwise significance level of 0.016 (0.05/3). One-sided alternative hypotheses were tested since, based on previous observations, we assumed that the BB bulls were performing worse than the HF bulls, and the good semen BB bulls better than the poor semen BB bulls. Finally, the Spearman rank correlation coefficient between several parameters was derived. These analyses were all performed in SAS 9.1.

RESULTS

1. Semen evaluation

The results of the semen evaluations of the 4 groups of bulls are listed in Table 1. The results for concentration, gross motility, total motility, progressive motility, % live, % normal, and % of primary abnormalities were numerically ranked as could be expected: within the BB breed, the poor semen quality bulls had the poorest results, whereas the bulls culled for abundant sperm stock had the best results. The BB bulls culled for reasons other than (bad or good) semen quality had an intermediate position. However, the sperm quality results of the HF bulls were better than the best BB results. These differences were always significant (P ≤ 0.035) when the results of all BB bulls were compared to the HF results, except for velocity where only a tendency (P = 0.098) for better HF results could be demonstrated. For velocity, group results were numerically similar to the other semen trait results, but good semen BB bulls scored (non significantly) higher than HF bulls, although the HF sperm velocity is numerically higher compared to the overall BB results. The percentage of secondary sperm abnormalities was lowest in HF sperm, a little higher in good sperm BB bulls, and highest in miscellaneous BB bulls, but these group differences were not significant (P ≥ 0.021), in contrast to the overall breed difference (P = 0.004). Generally, the group differences were significant (P < 0.016) or marginally significant (P ≤ 0.017) when the poor semen BB bulls were compared to the good semen BB bulls (except for % live spermatozoa, primary and secondary abnormalities) and to the HF bulls (except for % live spermatozoa and secondary


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abnormalities), whereas no significance could be demonstrated when the good semen BB and HF bull results were compared.

2. Testicular histology

The testicular histology results are summarized in Table 2. The total number of tubuli seminiferi per area was significantly higher (P = 0.047) in testicular sections of the BB breed, although no bull group differences were encountered. The proportion of affected tubules did not differ between breeds (P = 0.149), although a (marginally) significantly higher proportion of tubules demonstrated degenerative changes in the poor semen quality BB bulls compared to the good semen BB (P = 0.011) and HF bulls (P = 0.017).

Of the constituting parameters used to determine the degree of degeneration (proportion of perpendicularly cut tubuli without spermatids, with regions showing less than 4 cell layers, with regions showing less or equal to 2 cell layers, with regions with 1 cell layer, without cell layers, with obvious vacuolisation, and with cells or debris in the lumen) several significant breed differences were noticed. When the bull groups were compared, the poor and good BB bulls never differed for these histology traits, and when the HF results differed, this was generally when they were compared with the poor semen BB bulls. The percentage of perpendicularly cut tubuli without spermatids, and those with cells in the lumen did not differ at all between groups or breeds. These results are added for the sake of completeness (Table 2; Figures 1 and 2).

The parameters summarizing degeneration, namely the average degree of degeneration (score: 0 – 1) and the degeneration index (% affected tubules x degeneration score: 0 – 100) demonstrated that degeneration occurred more in the BB testicles as the HF bulls had better results compared to the BB breed (P = 0.021 and P = 0.053 for degeneration score and degeneration index, respectively). This was due to the bad results of the poor semen BB bulls which demonstrated significantly more degeneration compared to the HF bull group (P = 0.009 and P = 0.012 for degeneration score and degeneration index, respectively) and marginally significantly more degeneration compared to the good semen BB bulls (P = 0.017 both for degeneration score and index). Good semen BB bulls and HF bulls did not differ for these parameters (P = 0.124 and P = 0.186 for degeneration score and degeneration index, respectively).

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Table 1: Mean ± standard deviation and median (range) of the assessed sperm parameters for the 3 Belgian Blue (BB) bull groups (bulls with poor sperm quality, bulls culled for miscellaneous reasons, and bulls with good sperm quality) and for the Holstein Friesian (HF) bull group.

BB poor semen BB miscellaneous BB good semen All BB bulls HF

Concentration 401.0 ± 228.5 a 718.9 ± 323.3 822.5 ± 336.0 ab 673.4 ± 337.9 1 1042.1 ± 233.1 b2 312.5 (150.0 - 687.5) 790.0 (252.5 - 1270.0) 797.5 (420.0 - 1482.5) 687.5 (150.0 – 1482.5) 1092.5 (717.5 - 1315.0) Gross motility 2.0 ± 0.4 a 2.8 ± 1.2 3.9 ± 0.9 b 3.0 ± 1.2 1 4.0 ± 0.6 b2 2.0 (1.5 - 2.5) 2.5 (1.0 - 4.5) 4.0 (2.5 - 5.0) 2.8 (1.0 – 5.0) 4.0 (3.0 - 4.8) Total motility 48.0 ± 22.6 a 57.1 ± 27.1 72.1 ± 11.1 ab 60.3 ± 22.4 1 78.6 ± 9.6 b2 50.0 (10.0 - 67.5) 62.5 (5.0 - 85.0) 70.0 (60.0 - 90.0) 65.0 (5.0 – 90.0) 82.5 (65.0 - 87.5) Progressive motility 37.0 ± 24.5 a 52.5 ± 27.0 67.9 ± 11.1 ab 54.1 ± 23.9 1 78.2 ± 10.2 b2 40.0 (5.0 - 62.5) 60.0 (5.0- 80.0) 65.0 (55.0 - 85.0) 60.0 (5.0 – 85.0) 82.5 (62.5 - 87.5) Velocity 2.3 ± 0.5 a 2.8 ± 0.4 3.2 ± 0.3 b 2.8 ± 0.5 1 3.1 ± 0.2 b1 2.0 (1.8 - 3.0) 3.0 (2.0 - 3.3) 3.0 (3.0 - 3.5) 3.0 (1.8 – 3.5) 3.0 (3.0 - 3.5) % Live spermatozoa 62.7± 27.3 a 68.4 ± 27.8 82.4 ± 6.3 a 72.1 ± 22.5 1 86.3 ± 2.9 a2 69.3 (20.3 - 85.8) 81.5 (9.3 - 88.0) 83.0 (70.5 - 91.3) 81.5 (9.3 – 91.3) 87.5 (83.0 - 89.8) % Normal spermatozoa 19.4 ± 10.8 a 30.4 ± 15.4 52.4 ± 13.0 b 35.6 ± 18.8 1 65.9 ± 9.2 b2 15.5 (6.0 - 31.0) 33.0 (9.0 - 52.5) 48.0 (41.5 - 77.0) 37.0 (6.0 – 77.0) 64.0 (54.0 - 80.0) Primary abnormalities 115.2 ± 42.0 a 85.0 ± 20.6 62.3 ± 26.0 ab 84.6 ± 34.8 1 38.4 ± 12.5 b2 90.5 (80.5 - 161.0) 81.0 (64.0 - 116.5) 67.5 (19.0 - 93.5) 81.0 (19.0 – 161.0) 38.5 (21.0 - 58.5) Secundary abnormalities 19.4 ± 13.5 a 32.4 ± 27.1 7.0 ± 4.1 a 19.6 ± 20.4 1 4.1 ± 2.2 a2 21.5 (5.0 - 34.0) 29.0 (4.5 - 88.0) 6.0 (3.0 - 15.0) 15.0 (3.0 – 88.0) 3.5 (1.0 - 7.0)

A different superscript letter (a,b) corresponds to a pair-wise significant (P < 0.0166 = 0.05/3) group difference between BB poor semen, BB good semen and HF bulls. A different superscript number (1, 2) corresponds to a significant (P < 0.05) breed difference.

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Table 2: Mean ± standard deviation and median (range) of the assessed histology parameters for the 3 Belgian Blue (BB) bull groups (bulls with poor sperm quality, bulls culled for miscellaneous reasons, and bulls with good sperm quality) and for the Holstein Friesian (HF) bull group.

BB poor semen BB miscellaneous BB good semen All BB bulls HF

Total number of tubuli /area 10.8 ± 1.9 a 11.7 ± 2.2 10.7 ± 0.8 a 11.1 ± 1.7 1 10.0 ± 0.7 a2 10.8 (8.5 – 13.6) 11.2 (8.3 – 14.1) 10.4 (9.6 – 12.1) 10.8 (8.3 – 14.1) 10.0 (9.2 – 11.1) % Affected tubuli 98.1 ± 2.2 a 90.9 ± 5.3 92.1 ± 4.3 b 93.2 ± 5.1 1 88.8 ± 9.3 ab1 98.8 (95.0 - 100.0) 92.6 (83.8 – 96.0) 93.5 (86.4 – 97.6) 95.0 (83.8 – 100.0) 90.8 (73.0 – 99.2)

% Tubuli without spermatids 43.8 ± 24.4 a 37.9 ± 6.8 34.9 ± 6.3 a 38.3 ± 13.2 1 35.5 ± 5.1 a1 30.2 (24.2 – 81.6) 37.8 (28.4 – 47.5) 34.6 (26.6 – 46.5) 35.6 (24.2 – 81.6) 38.1 (28.7 – 42.3) % Tubuli < 4 cell layers 91.9 ± 8.1 a 73.4 ± 5.8 72.3 ± 13.4 ab 77.9 ± 12.6 1 66.0 ± 14.7 b2 90.1 (81.5 - 100.0) 75.5 (65.7 - 80.8) 71.1 (53.7 – 91.2) 78.1 (53.7 – 100.0) 68.2 (47.6 – 90.2) % Tubuli ≤ 2 cell layers 63.4 ± 24.9 a 28.0 ± 6.6 33.1 ± 19.5 ab 39.2 ± 22.4 1 23.4 ± 16.2 b2 55.8 (40.7 – 97.9) 28.7 (17.0 – 37.7) 24.2 (19.0 – 72.7) 29.4 (17.0 – 97.9) 24.2 (6.7 – 54.7) % Tubuli with 1 cell layer 1.5 ± 2.1 a 0.1 ± 0.3 1.5 ± 2.2 a 1.0 ± 1.8 1 0.0 ± 0.0 b2 0.6 (0.0 - 4.9) 0.0 (0.0 – 0.7) 0.7 (0.0 – 5.7) 0.0 (0.0 – 5.7) 0.0 (0.0 - 0.0) % Tubuli with 0 cell layers 15.3 ± 30.5 a 1.2 ± 1.9 0.8 ± 1.5 a 4.8 ± 15.8 1 0.4 ± 1.1 a2 1.4 (0.0 – 69.7) 0.0 (0.0 - 5.0) 0.3 (0.0 – 4.2) 0.5 (0.0 – 69.7) 0.0 (0.0 – 2.8) % Vacuolated tubuli 83.0 ± 19.9 a 60.2 ± 20.3 64.4 ± 10.4 a 67.8 ± 18.8 1 51.8 ± 18.5 a2 92.7 (50.0 – 96.9) 53.2 (29.9 – 86.1) 65.0 (48.5 – 80.1) 66.2 (29.9 – 96.9) 52.2 (24.8 – 79.2) % Tubuli with luminar cells / debris 60.0 ± 15.3 a 43.9 ± 11.3 44.4 ± 13.0 a 48.3 ± 14.2 1 46.8 ± 14.9 a1 66.0 (33.7 – 72.4) 37.9 (30.1 – 58.5) 44.9 (30.1 – 62.3) 50.8 (30.1 – 72.4) 41.3 (35.6 – 76.9)

Degeneration score 0.51 ± 0.13 a 0.35 ± 0.05 0.36 ± 0.08 ab 0.40 ± 0.11 1 0.32 ± 0.08 b2 0.48 (0.38 - 0.69) 0.33 (0.29 - 0.44) 0.34 (0.29 - 0.51) 0.35 (0.29 – 0.69) 0.31 (0.23 - 0.48) Degeneration index 50.6 ± 13.3 a 32.3 ± 5.8 33.8 ± 9.0 ab 37.7 ± 11.9 1 29.3 ± 9.9 b1 47.9 (36.1 – 69.2) 31.7 (25.6 – 42.2) 32.7 (25.9 – 50.7) 33.3 (25.6 – 69.2) 28.8 (17.6 - 48.1)



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3. Testicular connective tissue assessment

These results are given in Table 3. Testicles of BB bulls (average of all bulls in the 3 groups) contained significantly more connective tissue between the tubuli seminiferi compared to testicles of HF bulls (P = 0.016; Figures 1 and 2). No difference in the amount of connective tissue was present between the poor and good semen BB bulls (P = 0.072), while both BB bull groups (marginally) significantly differed from the HF bulls (P = 0.017 and 0.011, for the poor semen BB - HF and good semen BB - HF comparisons, respectively). Surprisingly the lowest amount of connective tissue in the BB breed was noticed in the miscellaneous BB bulls culled for other reasons than sperm quality.

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Table 3: Mean ± standard deviation and median (range) of the percentage of testicular connective tissue for the 3 Belgian Blue (BB) bull groups (bulls with poor sperm quality, bulls culled for miscellaneous reasons, and bulls with good sperm quality), for all the examined BB bulls, and for the Holstein Friesian (HF) bull group.

Percentage BB poor semen BB miscellaneous BB good semen All BB bulls HF

mean ± st dev 14.1 ± 5.6 ab 8.2 ± 2.1 10.4 ± 2.4 a 10.6 ± 4.0 1 7.6 ± 1.9 b2

median (range) 14.1 (7.3 – 22.2) 7.9 (5.8 – 11.3) 10.1 (6.5 - 13.3) 10.1 (5.8 – 22.2) 8.5 (4.9 – 10.0)



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Figure 1: Testicular section of a Holstein Friesian bull stained according a modified Masson technique. Few degenerative changes are noticeable and little interstitial connective tissue (blue) is present.

Figure 2: Testicular section of a Belgian Blue bull stained according a modified Masson technique. Rather severe degeneration in a high proportion of tubules, and high amounts of interstitial connective tissue (blue) is noticeable. The tubules are smaller than in Figure 1.


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4. Correlations

The assessed correlation coefficients are shown in Table 4. The percentage of testicular connective tissue was significantly correlated with the percentage of affected tubules, the degeneration score and the degeneration index. Of the semen quality parameters that were explored (concentration, progressive motility, % live and % normal spermatozoa, and % primary abnormalities), the percentage of normal spermatozoa was significantly negatively correlated with the degeneration index and with the percentage of testicular connective tissue. The cumulative proportion of primary abnormalities was significantly correlated with the percentage of testicular connective tissue, and borderline with the degeneration index. Concentration was significantly negatively correlated with the degeneration index, while a tendency was present for negative correlation with the percentage of testicular connective tissue.

Table 4: Spearman’s rank correlation coefficients and P-value of the assessed traits.

Parameters Correlation coefficient P value

% connective tissue - % affected tubuli 0.598 0.0012

% connective tissue - degeneration score 0.641 0.0004

% connective tissue - degeneration index 0.632 0.0005

degeneration index - concentration -0.409 0.0383

degeneration index - % progressive motility -0.270 0.1824

degeneration index - % live spermatozoa -0.123 0.5511

degeneration index - % normal spermatozoa -0.442 0.0238

degeneration index - % primary abnormalities 0.384 0.0526

% connective tissue - concentration -0.360 0.0708

% connective tissue - % progressive motility -0.271 0.1807

% connective tissue - % live spermatozoa -0.250 0.2185

% connective tissue - % normal spermatozoa -0.465 0.0167

% connective tissue - % primary abnormalities 0.475 0.0141


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DISCUSSION

The spermiogram of a bull is in essence a historical picture, reflecting the health of the seminiferous epithelium at the time of spermiogenesis of the observed sperm cells, the health of the epididymis during transit and storage of these spermatozoa, and to some extent the health of the accessory sex glands at the time of ejaculation. Hence, the spermiogram represents the testicular function at the moment of sperm production, and sperm abnormalities and in particular primary sperm abnormalities are the result of dysfunctional testicular tissue (Barth and Oko, 1989). The data of the present study generate evidence which suggests that the inferior sperm quality encountered in BB bulls is caused by an increased tendency / susceptibility to testicular degeneration in this breed.

At first, a significantly inferior semen quality of the BB bulls compared to the HF bulls was demonstrated in the present study. Furthermore, within the BB breed obvious quality differences corresponding to the group in which the bulls were classified were numerically noticeable. The assessed bull group comparisons demonstrated a significantly poorer sperm quality in the poor sperm BB bull group compared to the other groups, but no significant differences could be demonstrated between the HF bulls and the good sperm BB bulls. Given the large numerical sperm quality differences between the latter groups, we are inclined to think that this lack of significance is due to the small number of bulls (maximum 7) per group, rather than to the fact that no sperm quality differences exist. Moreover, the sperm quality results of this limited database are numerically in agreement with the results of a large scale field study on semen quality of these 2 breeds (Hoflack et al., 2006a).

Decreased sperm concentration and motility, in combination with an increase in morphological abnormalities, as demonstrated in several BB bulls in the present study, has been associated with testicular degenerative lesions (Veeramachaneni et al., 1986). We found indeed that the HF bulls scored obviously better for data on testicular histology compared to the BB breed, as evidenced by the (marginally) significant advantage of the HF breed compared to the average of all bulls in the 3 BB groups for degeneration score and index, suggesting less severe and fewer degenerative changes in HF testicles. As for semen quality, this breed difference was largely due to the poor semen BB bulls, which demonstrated (marginally) significantly poorer results compared to both the good semen BB bulls and the HF bulls, while the latter 2 groups were not significantly different. Although not statistically assessed, the testicular health of the miscellaneous BB bulls apparently coincided better with the good semen quality BB bulls compared to the poor semen quality BB bulls. This is probably due to the fact that of the 7 bulls present in the BB miscellaneous group, only 2 bulls were injured. The other 5 bulls were culled for other conditions which, in contrast to injuries, do not inflict stress and subsequent testicular degeneration. However, semen quality


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obviously differed between the good semen quality BB and miscellaneous BB group. These 2 injured bulls negatively influenced the sperm quality outcome (data not shown).

Although the degenerative changes were significantly more severe in testes of BB compared to HF bulls (Figures 1 and 2), the proportion of affected tubules was not significantly different between the two breeds. This is due to the fact that high proportions of tubules demonstrated degenerative changes in all bulls since even slightly degenerated tubules were considered affected, although in many cases these degenerative changes were rather mild. The presence of degeneration in the HF bulls, producing semen of acceptable quality is not abnormal, since it is generally acknowledged that focal areas of degeneration occur in all testes (Mc Entee, 1990b). Similarly, in the BB breed, the degeneration was generally focal albeit more severe. However, the poor semen quality BB bulls demonstrated a higher proportion of degenerated tubules compared to both the good semen BB and HF bulls, in addition to more severe degenerative changes, confirming the poor testicular health in this BB bull group.

The total number of tubuli seminiferi per area was significantly higher for BB compared to HF testicles. This suggests that the average diameter of BB tubuli seminiferi is smaller compared to HF tubules, and this was also observed subjectively. A small seminiferous tubule diameter has been associated with lowered sperm production and hypoplastic testes (Krishnalingam et al., 1982). Although more tubules are present in a given volume of BB testicular tissue compared to HF bulls, BB testicles are generally smaller compared to other breeds and certainly compared to the HF breed, resulting in less seminiferous epithelium (Michaux and Hanset, 1981; Coulter et al., 1987; Hanset, 2000; Hoflack et al., 2006b). The latter, in addition to the small tubule diameter results in lower sperm concentrations in BB ejaculates as evidenced in the present study.

Testicular sections of BB bulls contained more connective tissue between the tubuli seminiferi compared to HF bulls (Figures 1 and 2), and this breed difference was present when both the poor and good semen BB bulls, which did not differ from each other, were compared to the HF bulls (when marginal significances of these small groups were also considered significant). Moreover, this amount of connective tissue was significantly correlated to the parameters expressing degenerative changes (% of affected tubuli, degeneration score and index), and furthermore to the percentage of normal spermatozoa and primary abnormalities. The latter correlation coefficients are slightly higher than the correlation coefficients between the degeneration index on the one hand and the percentage of normal spermatozoa and primary abnormalities on the other hand. We consequently think that it might be possible that the high amount of connective tissue in BB bull testicles is responsible for the susceptibility to degenerative changes and the subsequently disturbed sperm morphology. Testicular fibrosis has been described as a histological feature of aging in


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bulls (McEntee, 1990b), but the BB bulls in our study that demonstrated the increased proportion of collagen were generally young (11 / 19 bulls < 4 years; data not shown), suggesting the presence of high amounts of testicular collagen in all BB bulls. However, as the proportion of testicular connective collagen was lower in the miscellaneous BB bulls, this ‘omnipresence’ of high amounts of testicular collagen should be confirmed on higher numbers of BB bulls.

This supposed general presence of more testicular connective tissue in the BB breed compared to the HF breed is of particular interest. A well defined interstitium with large quantities of collagen, similar to the situation of the BB bulls in the present study, has been associated with testicular hypoplasia (Bongso et al., 1981), although a precise definition of the latter condition is lacking (McEntee, 1990a). It seems logical that tubuli seminiferi, surrounded by high amounts of interstitial tissue, as in the examined BB bulls, are less efficiently irrigated with blood and cell sustaining nutrients. This situation could easily lead to cell hypoxia and subsequent degenerative changes in the tubuli seminiferi, evidenced by a higher amount of morphological abnormalities in the semen picture. It has already been demonstrated that BB ejaculates generally contain high percentages of abnormal spermatozoa (Hoflack et al., 2006a; 2006b), and the ejaculates of the bulls in this study confirmed these findings. The currently described high amount of connective collagen content of BB testes might indicate the presence of testicular hypoplasia in BB bulls.

Settergren and Mc Entee (1992) defined hypoplasia as a hereditary condition evidenced by small testicles producing little semen of poor quality or rather normal testicles initially producing normal semen but very sensitive to degenerative changes at a young age. This definition perfectly fits the situation of the BB breed, since the testicles of BB bulls are generally small, and quite some young bulls produce fresh semen of poor quality or semen that cannot be cryopreserved (AWE, personal communication; Hoflack et al., 2006b). Moreover, the omnipresence of focal degenerative changes of the seminiferous epithelium of BB bulls, suggesting high sensitivity to testicular degenerative changes is confirmed by the present results. Hence, the high sensitivity to testicular degeneration, as present in the BB breed, could be caused by testicular hypoplasia (Bongso et al., 1981; Settergren and McEntee, 1992). However, in general it is histologically impossible to diagnose testicular hypoplasia or to distinguish hypoplasia from degeneration (Blanchard et al., 1991b). Since BB bulls generally have small scrota and hence small testicles, the hypoplasia diagnosis seems nevertheless justified. In a previous study on the BB breed, the average scrotal circumference was 32.1 cm for 64 BB bulls ≤ 2 years of age, 34.1 cm for 61 BB bulls between 2 and 4 years of age, and 36.2 cm for the 25 BB bulls > 4 years of age. The highest scrotal circumference was 39.2 cm in a 5.1 year old bull and only 3 bulls (out of 150) had a scrotal circumference surpassing 38 cm (Hoflack et al., 2006b; data not shown). These data clearly demonstrate the generally small nature of the scrota and hence testicles of BB bulls, suggesting that this is a


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breed specific trait, corresponding to testicular hypoplasia. The high inbreeding coefficient of the BB breed, on average 4.65% for BB bulls born in 2004 (Hanset, 2003; 2006), probably partially explains the general nature of these hypoplastic testicles. It has been demonstrated that inbreeding was positively associated with abnormal seminiferous tubules (Carroll and Ball, 1970).

The poor semen BB bulls all demonstrated a high extent of testicular degeneration, which was evidenced by high numbers of primary sperm abnormalities. The most frequently encountered sperm abnormalities in these bulls’ spermiograms were tapered or pear shaped heads, nuclear vacuoles, proximal droplets and midpiece defects (data not shown). These abnormalities were simultaneously present in a high proportion of abnormal spermatozoa of the affected bulls, and were far less encountered (either solely or simultaneously) in bulls of the other (better fertile) groups which all demonstrated less testicular degeneration. The same morphological abnormalities were described in bulls after scrotal insulation (Vogler et al., 1993; Barth and Bowman, 1994). This suggests that the presence of clinically relevant testicular degeneration can be assumed when high numbers of the above-mentioned sperm abnormalities are present in a bull’s spermiogram. It has already been demonstrated that nuclear vacuoles, midpiece defects, and cytoplasmic droplets are omnipresent in spermiograms of BB bulls (Hoflack et al., 2006a), suggesting the general presence of focal testicular degeneration in bulls of the BB breed, which was confirmed in the present study. However, the poor semen BB bulls were largely responsible for the breed differences for degeneration score, degeneration index, and sperm quality in our study. Moreover, the BB breed was compared to ‘fertility selected’ HF bulls as these were progeny tested bulls, which implies sufficient production of good quality semen at a younger age. Furthermore, the good semen quality BB bulls rarely differed from these HF bulls. However, of the unselected HF bull start population, only 11% fail selection due to poor sperm quality, whereas this is 27% in the BB breed. In 89% of cases, HF sperm quality is considered good, while in the BB breed, approximately 18% of the bulls become injured during service, resulting in stress and possibly reduced sperm quality (data not shown). In all, the proportion of good sperm bulls in the BB breed is markedly lower compared to the HF breed. This suggests significantly more BB bulls with marked testicular degeneration compared to the HF breed and adds to the breed difference. The extensive worldwide long-term selection for highly fertile HF AI bulls, a selection that was not performed in the BB breed, is probably partially responsible for this (Söderquist et al., 1991).

In conclusion, it seems that the genetically narrow BB breed is ridden with small testicles probably caused by testicular hypoplasia, which might explain the BB bulls’ high sensitivity to testicular degeneration induced by even trivial environmental stresses. This high sensitivity to testicular degeneration can be explained by the large amount of testicular connective tissue which might hamper blood irrigation of the tubuli seminiferi resulting in


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tissue hypoxia and consequent degenerative changes leading to the production of morphologically abnormal spermatozoa. These conclusions are however based on a small sample of bulls of both breeds and should be confirmed by comparing testicles of large numbers of BB and HF bulls. Nevertheless, selection for larger scrota in the BB breed is absolutely necessary in an attempt to by-pass this situation. However, since BB scrota are generally small and to our knowledge never above 40 cm, the selection for larger scrota might progress less efficiently as in other breeds and might perhaps not be able to overcome the BB problem of high sensitivity to degenerative testicular changes and consequent poor sperm morphology.

ACKNOWLEDGEMENTS



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REFERENCES

Arthur PF, Makarechian M, Price MA. Incidence of dystocia and perinatal calf mortality resulting from reciprocal crossing of double-muscled and normal cattle. Can. Vet. J. 1988; 29: 163 – 167.

Barth AD, Bowman PA. The sequential appearance of sperm abnormalities after scrotal insulation or dexamethasone treatment in bulls. Can. Vet. J. 1994; 35: 93 – 102.


Bellinge RHS, Liberles DA, Iaschi SPA, O’Brien PA, Tay GK. Myostatin and its implications on animal breeding: a review. Anim. Gen. 2005; 36: 1 – 6.

Blanchard TL, Varner DD, Bretzlaff KN, Elmore RG. The causes and pathologic changes of testicular degeneration in large animals. Vet. Med. 1991a; 86: 531 – 536.

Blanchard TL, Varner DD, Bretzlaff KN, Elmore RG. Testicular degeneration in large animals: identification and treatment. Vet. Med. 1991b; 86: 537 – 542.

Bongso TA, Jainudeen MR, Lee JY. Testicular hypoplasia in a bull with XX/XY chimerism. Cornell Vet. 1981; 71: 376 – 382.

Bradbury P, Gordon K. Connective tissues and stains. In Bancroft JD and Stevens A (Editors): Theory and practice of histological techniques. Churchill Livingstone, New York, 1977; p. 104.

Carroll EJ, Ball L. Testicular changes as affected by mating systems in beef cattle. Am. J. Vet. Res. 1970; 31: 241 – 254.

Chenoweth PJ, Spitzer JC, Hopkins FM. A new bull breeding soundness evaluation form. In: Proceedings of the Society for Theriogenology AGM, 1992; p. 63 - 70.

Chenoweth PJ, Hopkins FM, Spitzer JC, Larsen RE. New Guidelines for the Evaluation of Bulls for Breeding Soundness. In: The bovine proceedings, 26 January 1994. p. 105 - 107.



Hanset R. Un défi permanent. Concilier sélection et faible consanguinité. Le Sillon Belge 2006 ; 3208: 10 – 14.


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Hanset R, de Tillesse S, Michaux P, André E. La consanguinité en Blanc-Bleu Belge. Sa genèse et son contrôle. Publication Herd-Book du BBB n° 2003 03 - 36 (in French).

Hoflack G, Opsomer G, Van Soom A, Maes D, de Kruif A, Duchateau L. Comparison of sperm quality of Belgian Blue and Holstein Friesian bulls. Theriogenology, in press 2006a.

Hoflack G, Van Soom A, Maes D, de Kruif A, Opsomer G, Duchateau L. Breeding soundness and libido examination of Belgian Blue and Holstein Friesian artificial insemination bulls in Belgium and the Netherlands. Theriogenology, in press 2006b.


Hopwood D. Fixation and fixatives. In Bancroft JD and Stevens A (Editors): Theory and practice of histological techniques. Churchill Livingstone, New York, 1977; p. 26.

Krishnalingam V, Ladds PW, Entwistle KW, Holroyd RG. Quantitative macroscopic and histological study of testicular hypoplasia in Bos indicus strain bulls. Res. Vet. Sci. 1982; 32: 131 – 139.

Madrid N, Ott RS, Veeramachaneni DN, Parrett DF, Vanderwert W, Willms CL. Scrotal circumference, seminal characteristics, and testicular lesions of yearling Angus bulls. Am. J. Vet. Res. 1988; 49: 579 – 585.

McEntee K. Chapter 13: Scrotum and testis: anatomy and congenital anomalies. In McEntee K (Editor): Reproductive pathology of domestic mammals. San Diego, California: Academic Press Inc., 1990a; p. 224 - 251.

McEntee K. Chapter 14: Scrotum, spermatic cord, and testis: degenerative and inflammatory lesions. In McEntee K (Editor): Reproductive pathology of domestic mammals. San Diego, California: Academic Press Inc., 1990b; p. 252 - 278.

Michaux C, Hanset R. Sexual Development of Double-Muscled and Conventional Bulls. I Testicular Growth. Z. Tierzüchtg. Züchtgsbiol. 1981; 98: 29 - 37.



Stevens A. The haematoxylins. In Bancroft JD and Stevens A (Editors): Theory and practice of histological techniques. Churchill Livingstone, New York, 1977; p. 89.

Veeramachaneni DN, Ott RS, Heath EH, McEntee K, Bolt DJ, Hixon JE. Pathophysiology of small testes in beef bulls: relationship between scrotal circumference, histopathologic


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features of testes and epididymides, seminal characteristics, and endocrine profiles. Am. J. Vet. Res. 1986; 47: 1988 – 1999.


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GENERAL DISCUSSION

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In contrast to the well documented Holstein Friesian (HF) fertility (Royal et al., 2000; Lucy, 2001; Bousquet et al., 2004), Belgian Blue (BB) fertility has only seldomly been described (Hanzen et al., 1994). Furthermore, despite the bull’s pivotal role in reproduction, cow fertility generally receives more interest (Chenoweth, 1997; Parkinson, 2004). The initial goal of this study was to elucidate whether there were indications of BB bull subfertility which might explain the frequently encountered disappointing pregnancy results in BB breeding herds (Bombeek, 2004), and if so, to identify these fertility weaknesses. To this purpose, the standardized bull breeding soundness evaluation (BSE) was performed on BB bulls, and their results were compared to these of HF bulls, which were considered to be ‘the reference’ (Ott, 1986; Barth, 1997). Additionally, bull libido and serving capacity were evaluated to fully qualify male reproductive potential. To increase the efficiency of data gathering, this study was conducted at artificial insemination (AI) facilities (Chapter 2).

BREEDING SOUNDNESS AND LIBIDO EVALUATION

The BB results clearly indicated that fertility associated problems, which might explain the observed poor field fertility results of BB natural service bulls, were present within the BB breed as less than 10 % of unselected BB bulls passed the BSE.

Physical soundness

Concerning physical soundness, 4 of 64 young BB bulls were classified as unsatisfactory potential breeders due to severe feet and leg abnormalities, while no HF bulls failed for this trait, and the average score for gait and soundness of feet and legs was significantly poorer in the BB breed. It is common knowledge that the genetically narrow BB breed is susceptible to several heritable feet and leg abnormalities (Hanset et al., 2003), such as sickle hocks and post leggedness, two problems which can interfere with the bull’s ability to mate (Larson, 1986), and intensive selection procedures against these defects have been undertaken (Hanset and de Tillesse, 2000). In all, this means that HF bulls are better mobile than BB bulls, which can be expected as a result of the muscular hypertrophy and skeletal hypotrophy typical of BB bulls (Ansay and Hanset, 1979), rendering them less agile (Van Camp, 1997).

Libido

Nevertheless, the libido assessment performed during the artificial vagina semen collection procedure clearly demonstrated that double-muscled BB bulls are still willing and able to mate naturally, as no breed differences were noticed, neither for reaction time, nor for mounting enthusiasm. The limited number of females per bull (15 – 25, depending on the bull’s age) and the prolonged breeding season (from May to October) that are generally

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applied for BB breeding herds might rectify possible adverse effects on libido resulting from musculoskeletal or other inhibitory problems in older BB bulls (Hoflack et al., 2006b). Indeed, it has been demonstrated that the better pregnancy rates obtained by higher sex-drive bulls compared with lower sex-drive bulls (Blockey, 1978; Makarechian and Farid, 1985; Blockey, 1989, Farin et al., 1989) are partially nullified in case of a long breeding season (Silva-Mena et al., 2002; Parkinson, 2004).

Scrotal circumference

The most important reasons why BB bulls failed the BSE were sperm quality and small scrota, as approximately 30% of BB bulls < 4 years of age were cursed with both substandard sperm morphology and scrotal circumference (SC). Hanset (2000) already demonstrated that the average SC of yearling BB bulls was rather low compared to that of other beef breeds (Coulter et al., 1987; Hanset, 2000), probably due to the reduced organ size that accompanies the extreme muscularity typical of the BB breed (Ansay and Hanset, 1979; Michaux and Hanset, 1981). Furthermore, Hanset (2000) also described a negative genetic trend evidenced by a declining SC in the BB breed over a 15 year period. Since SC is positively correlated to daily sperm output, normal sperm morphology and motility, and consequently also to pregnancy rates, Hanset’s and our data are worrisome (Makarechian and Farid, 1985; Ott, 1986; Bruner and Van Camp, 1992; Barth, 1997). This might explain the high proportion of BB bulls with a substandard semen quality in our study, and it suggests that a high proportion of BB bulls are subfertile. Furthermore, it has been shown in several studies that high circumference bulls produce higher fertility offspring, both male and female, that attain puberty at an earlier age, resulting in economic profit (Coulter and Foote, 1979; Bruner and Van Camp, 1992; Barth, 1997; Spitzer and Hopkins, 1997). As SC is correlated to the fertility of the female offspring (Barth, 1997), a negative effect of the use of these subfertile bulls on the reproductive performance of female offspring in the BB breed seems inevitable. Although no reliable data on this subject are yet available, based on limited data, a general tendency towards a higher age at first calving is noticeable in BB heifers (AWE, personal communication). Consequently, the continuous use of substandard SC bulls might lead the breed into a downward vicious circle, ultimately ending with infertile animals.

Sperm Quality: Morphology

Belgian Blue –Holstein Friesian breed comparison

Sperm quality, and in particular sperm morphology, was the most important BSE failure reason for BB bulls. When comparing BB to HF sperm by means of a detailed classical semen evaluation (Chapter 3A), we noticed that, apart from fewer live spermatozoa, significantly more abnormal spermatozoa were encountered in BB ejaculates as, although it concerned the

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same morphological abnormalities in both breeds, more different abnormalities were present and for each sperm defect, higher percentages of sperm cells were affected in a higher proportion of BB bulls. Although the specific sperm abnormalities were generally rare (< 10%), except for midpiece defects (> 10%), and only some abnormalities (knobbed and abnormal acrosomes, nuclear pouches or diadem defects, midpiece defects (segmental aplasia as well as “pseudodroplet like” defects), accessory and abaxial tails, and proximal and distal droplets) occurred significantly more in the BB breed, it is the high cumulative proportion of abnormalities in the BB breed that is problematic (Hoflack et al., 2006a).

In our study, a strict morphological sperm assessment was used, leading to a high proportion of bulls failing for this aspect in both breeds. However, slight morphological abnormalities within an ejaculate, when consistent, do not necessarily have consequences on bull fertility (Saacke, 2004). Moreover, in many cases only low percentages of a particular abnormality were present, minimizing its clinical relevance. Unfortunately, there is a deficiency of experimental evidence on the relationship between specific morphological sperm abnormalities and fertility, which is partially due to the coexistence of normal and abnormal sperm cells in an ejaculate (Barth and Oko, 1989). However, in vitro fertility of defective spermatozoa was documented for several specific sperm abnormalities, such as multiple nuclear vacuoles (Thundathil et al., 1998), pyriform-shaped heads (Thundathil et al., 1999), knobbed or abnormal acrosomes (Thundathil et al., 2000; 2001b), and proximal cytoplasmic droplets (Amann et al., 2000; Thundathil et al., 2001a). Similar in vitro production (IVP) trials, using ejaculates containing high proportions of a single or multiple of the frequently encountered discrete BB sperm abnormalities, such as single nuclear pouches (Figure 1) or discrete midpiece defects (Figure 2), might be an important tool to judge the biological relevance of these particular sperm aberrations, i.e. to determine whether both the affected spermatozoa as well as the normal appearing coexisting sperm within the affected ejaculate negatively influence fertility and to assess at which step in the fertilization process (zona pellucida binding, acrosome reaction, ovum penetration, DNA decondensation, cleavage, blastocyst formation) they appear to be defective. Moreover, heterospermic IVP trials, using (pooled or individual) HF and BB sperm labelled with fluorescent viable nuclear stains, such as Hoechst 33342 and SYBR 14, in order to evaluate which breed’s semen was more efficacious in the above-mentioned fertilization processes, might allow to evaluate breed fertility differences (Dziuk, 1996). Sufficient refining of such in vitro models, as demonstrated by Ward et al. (2001) who correlated early cleavage kinetics 33 h post insemination in vitro to in vivo bull fertility, and combining several in vitro semen evaluation methods (Zhang et al., 1999) might enable future researchers to predict in vivo bull fertility.

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Figure 1: Belgian Blue sperm cell with a tapered head containing a single nuclear vacuole, and a proximal droplet.

Figure 2: Discrete defects of the midpiece in Belgian Blue spermatozoa.

The encountered sperm abnormalities can be classified in different ways (Barth and Oko, 1989). To date, the system most commonly used is the primary – secondary classification system (Blom, 1977; Barth and Oko, 1989; Hopkins and Spitzer, 1997). We specifically used this classification system, since we were mainly interested in the etiology of the abnormal sperm morphology in BB bulls. Primary abnormalities are considered to arise in the testis, while secondary abnormalities originate after the sperm cells have left the testis (Barth and Oko, 1989). Our results indicated testicular malfunction in BB bulls, although secondary abnormalities also occurred more frequently in the BB breed, but at rather low and probably

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clinically unimportant proportions. Hence, testicular function of BB bulls (in comparison to HF bulls) was further assessed in our study (see Chapter 4).

However, other classification systems have also been proposed, such as the major – minor and compensable – uncompensable classification system, both of which are used to predict the impact of abnormal sperm on the fertility results (Barth and Oko, 1989, Blom, 1977; 1983; Saacke et al., 2000). The initial retrospective emphasis of the spermiogram on the health of the seminiferous epithelium is now shifted towards the prospective potential fertility of the semen sample itself. Major sperm defects are those that have been correlated to impaired fertility, whereas minor abnormalities are those that seem of minor importance to fertility. When implementing this classification system to our data, the generally higher proportion of major sperm defects in BB ejaculates suggests that BB semen is significantly less fertile compared to HF semen. However, this classification system, although clear and simple to use, has not been widely adopted, and flaws to this scoring system have been discussed (Barth and Oko, 1989).

Consequently, another classification system was developed, and actually continuously gains increasing interest and credibility. In this system, morphological abnormalities excluding sperm from the fertilization process are considered ‘compensable’ defects, since an increase of sperm numbers in the inseminate can overcome this problem, as the oocyte can still become fertilized by a normal spermatozoon present in the inseminate. On the other hand, abnormalities rendering semen able to fertilize but unable to sustain fertilization and/or embryogenesis result in subfertility regardless of the insemination dose, since this ‘abnormal’ spermatozoon will initiate the zona reaction, barring other normal sperm cells from fertilizing the oocyte. Hence, the latter abnormalities are considered ‘uncompensable’ defects (Saacke et al., 2000; Saacke, 2004). The morphological sperm abnormalities that can be visualized by standard light microscopy of eosin-nigrosin stained slides are generally considered to be compensable, with the exception of subtly misshapen heads and crater/diadem defects (Saacke et al., 1998; 2000; Saacke, 2004), although contradictory results have been published on the latter defect (Thundathil et al., 1998). This means that although BB bulls have a higher percentage of crater/diadem defects in their spermiogram in comparison with HF bulls, most of the encountered abnormalities are compensable. To avoid fertility problems resulting from this high proportion of (compensable) abnormalities in BB semen, the sperm concentration of BB straws is generally increased to 22 x 106 motile spermatozoa compared to the internationally accepted and in the HF centres implemented 15 x 106 total spermatozoa/straw prior to freezing (personal observations). Since every ejaculate of any given BB bull is processed to a specifically adjusted different straw concentration, as the percentage of progressively motile spermatozoa determines the actual insemination dose, it is no longer possible to interpret the effect of these sperm abnormalities on fertility, evidenced by the non-return rate (NRR) to service (i.e. the percentage of cows inseminated with semen of a

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particular bull that were not re-inseminated within a specific time frame afterwards, which is considered a ‘preliminary’ pregnancy rate (den Daas, 1997)), let alone to use these NRRs for an ‘in vivo’ breed fertility comparison. The higher BB insemination dose, together with the fact that not all the examined ejaculates were marketed as they were subjected to the quality criteria of the respective AI centres and only ejaculates considered adequate were further processed, resulted in a negligibly small breed difference in the average raw 56 day NRR of marketed BB and HF semen over recent years (2001 – 2003: 70.9 % versus 71.7. % for BB and HF bulls, respectively; CR Delta-VRV, personal communication), notwithstanding the fresh semen quality differences. The true impact of these compensable sperm abnormalities on fertility outcome (evidenced by NRRs) will only become evident when all BB straws contain the exact same, rather low insemination dose, similar to HF straws in order to allow a comparison of the corrected NRRs of the two breeds. Recent research of Verberckmoes et al. (2005) suggests that these insemination doses should be situated beyond 2 x 106 spermatozoa/straw, since the latter dose did not yield differences in pregnancy rates compared to 12 x 106 spermatozoa/straw in HF cattle. However, a high proportion of morphologically abnormal sperm might coexist in the ejaculate with normal appearing sperm bearing uncompensable defects (Thundathil et al., 2000; 2001a), and this together with the significantly higher proportion of nuclear vacuoles in the BB breed, which is considered an uncompensable trait, probably negatively influences fertility outcome in the BB breed to some extent, even at the current high insemination doses (Saacke, 2004).

Within Belgian Blue breed situation

No sperm morphology differences, with the exception of a slightly elevated, non pathological presence of distal cytoplasmic droplets in ejaculates of the oldest compared to the youngest BB bulls, were noticed between the different age groups within the BB breed (Barth and Oko, 1989; Barth, 1997; Johnson, 1997). This emphasizes the omnipresence of abnormal spermatozoa in BB ejaculates, regardless of the bulls’ age. The AI selection for ‘more fertile’ bulls with increasing age, which was evidenced by significantly fewer old bulls failing for SC, is probably nullified by testicular degeneration, occurring more frequently in older bulls and evidenced in our study by a declining testicular consistency with age (Kumi-Diaka et al., 1981; Hopkins, 1997; Van Camp, 1997; Brückmann et al., 2000; Brito et al; 2002). This explains why the semen quality in the BB breed does not improve in the ‘more fertile’ older bulls.

Moreover, no hereditary ‘BB breed specific’ abnormalities were encountered, as has for instance been described in Finnish Yorkshire boars for the ‘short tail’ sperm defect (Andersson et al., 2000, Sukura et al., 2002). It might be interesting to compare BB semen to semen of other beef breeds, and in particular to other beef breeds in which double-muscled animals occasionally occur, such as Piedmontese, Parthenais, Blonde d’Acquitaine, Limousin,

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Charolais and Maine Anjou (Karim et al., 2000). In contrast to the BB breed, where selection for hypermuscularity was performed resulting in the fixation of this condition in the genetic make up of all individuals, other breeds only occasionally produce animals with the double-muscled phenotype since the responsible genetic mutation(s) can be present in either homozygous or heterozygous form or even be absent in other individuals of the breed (Kambadur et al., 1997; Dunner et al., 2003; Marchitelli et al., 2003). This double-muscled condition has been held responsible for several both advantageous and disadvantageous traits (Bellinge et al., 2005). The latter include reduced fertility and a higher incidence of underdeveloped external genitalia, which is confirmed by our results. However, the double-muscled condition can be the result of 6 different mutations of the myostatin gene on chromosome 2 (Grobet et al., 1998; Karim et al., 2000). The comparison of double-muscled bulls of other breeds with BB bulls might clarify whether semen quality in double-muscled individuals of other breeds is equally poor, whether scrota are similarly small, and whether the same morphological sperm defects are predominant. Furthermore, the possibly differing influence of the 6 different genetic mutations of the myostatin gene and of other apparently unknown mutations responsible for the similar double-muscled condition on these fertility traits could also be elaborated (Grobet et al., 1998; Karim et al., 2000). Such investigations might help elucidating whether the BB bull fertility problems encountered in the present study are exclusively determined by the nt821(del11) mutation of the myostatin gene or whether these are also influenced by other genes, as has been demonstrated for the muscular hypertrophy phenotype itself (Dunner et al., 2003). If so, selection for better fertile albeit double-muscled animals might be possible.

Sperm Quality: Motility

Subjective light microscopic motility assessment

Another sperm quality parameter, in addition to sperm morphology, that was significantly lower in the BB breed compared to the HF breed was sperm motility, as evidenced by obvious breed differences for the subjectively estimated percentage of total and progressive motility, as well as for velocity. The higher percentage of eosin positive spermatozoa in BB ejaculates which are considered to be structurally damaged or dead, explains the lower percentages of (totally and progressively) motile spermatozoa (Bangham and Hancock, 1955). Moreover, hypo-osmotic swelling tests (Jeyendran et al., 1984), which assess the biochemical activity (i.e. functional integrity) of the sperm cell membranes (Correa et al., 1997), similarly demonstrated lower percentages of live or active spermatozoa in BB ejaculates (unpublished data).

However, to assure the correctness of our subjectively assessed data, which are considered relatively imprecise (Davis and Katz, 1993, Christensen et al., 1999; 2005), we

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also performed semi-computerized (Chapter 3B1) and computerized (Chapter 3B2) sperm motility measurements.

Semi-computerized motility assessment

In Chapter 3B1, an upgrade version of the Sperm Quality Analyzer (the SQA-IIC: Medical Electronic Systems Ltd., Tirat Carmel, Israel), a practical device that evaluates overall semen quality (Bartoov et al., 1981) and that does not require parameter settings, thus reducing a large source of bias, was validated for bull semen analysis. In agreement with computer assisted semen motility studies, we concluded that semen motility evaluations are best performed at a concentration of approximately 50 x 106 spermatozoa/ml (Vantman et al., 1989; Neuwinger et al., 1990; Davis and Katz, 1993; Rijsselaere et al., 2003). However, even at this optimal concentration, the merely moderate correlation (0.65) between the SQA outcome and the concentration of progressively motile normal spermatozoa indicated that discrimination of semen samples with little difference in quality might be a problem. Indeed, the SQA result is determined by the concentration, the motility and the morphology of the semen sample. A deficient quality of one contributing sperm parameter can be compensated by a good quality of another parameter, biasing accurate quality discrimination of different semen samples. The reverse is likewise true: semen samples with identical SQA values may have different individual sperm characteristics, making it impossible to define the exact semen defect (Bartoov et al., 1991; Johnston et al., 1995). We consequently concluded that the SQA-IIC could not be used to adequately determine subtle motility differences between ejaculates of bulls.

Computerized motility assessment

Alternatively, semen motility of both breeds was compared by means of computer assisted sperm motility analysis (CASA) in Chapter 3B2. CASA procures detailed and accurate, highly repeatable data on several semen motility parameters simultaneously, and was validated both in human and animal species, including the bull (Vantman et al., 1989; Günzel-Apel et al., 1993; Farrell et al., 1996). Especially sperm velocities which are very difficult to score subjectively, can be objectively assessed using this technology. Our CASA results confirmed the results of the subjective light microscopic motility estimation and demonstrated an obvious HF breed advantage for the % of totally and progressively motile spermatozoa. Moreover, CASA also revealed that BB spermatozoa moved less elegant (evidenced by a higher lateral head displacement) compared to HF sperm, resulting in more resistance due to friction and consequently in a lower velocity. Additionally, a lower beat cross frequency of the BB spermatozoa was demonstrated, resulting in less propelling force. These findings probably explain the slower and less straight forward progressive movement of BB spermatozoa. Our results suggested that breed differences in sperm head size (BB

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sperm heads appeared shorter albeit larger compared to HF spermatozoa) and the presence of more BB sperm abnormalities were (partly) responsible for the encountered sperm motility differences. It has been demonstrated that small geometrical differences in sperm morphology can result in large differences in sperm hydrodynamics, and subsequently in impaired semen velocity (Dresdner and Katz, 1981). However, automated sperm head morphometry assessments of BB and HF spermatozoa should be performed to confirm the above-mentioned breed differences in sperm head size (Gravance et al., 1996; Boersma et al., 2001). Additionally, sperm tail morphometry of spermatozoa of the two breeds should also be evaluated, since tail length differences might influence semen motility (Sukura et al., 2002). Anyhow, the presence of particular sperm abnormalities that might interfere with sperm head dimensions (such as knobbed acrosomes, abnormal acrosomes, nuclear pouches or diadem defects), or that might affect the propelling efficiency of the sperm tail (such as midpiece defects, proximal and distal droplets, and accessory and abaxial tails) are far more common in the BB breed (Hoflack et al., 2006a), and might negatively influence semen velocity (Blom, 1977; Dresdner and Katz, 1981; Barth and Oko, 1989, Amann et al., 2000). However, since adenosine triphosphate sources other than midpiece mitochondrial oxidative phosphorylation, such as glycolysis along the length of the principal piece of the sperm tail, seem mainly responsible for sperm flagellar motility, high proportions of even severe midpiece defects will only mildly reduce sperm motility (Turner, 2006). The coexistence within the same ejaculate of a generally low proportion of one particular abnormal form with sperm bearing other morphological abnormalities and with normal forms, some of which might still be functionally deficient, makes it nearly impossible to determine the consequences of one specific morphological abnormality on velocity (Thundathil et al., 2000; 2001a).

Nevertheless, as the motility differences corresponding to the above-mentioned BB motion pattern were numerically encountered in all breed comparisons, these differences seem breed specific. Since CASA motility differences and similarly, a difference in the prevalence of morphological abnormalities were less obvious in the breed comparison of unselected young bulls, we assume that the sperm morphological abnormalities were mainly responsible for the breed motility differences. To determine whether a ‘genetic motility difference’ irrespective of sperm morphology is present between the two breeds, CASA assessment of ejaculates of unselected bulls of comparable age of both breeds, with similar percentages of both live and normal spermatozoa should be performed. If such a genetic motility effect would exist, transmission electron microscopy could be performed to assess the tail ultrastructure, since the tail axoneme consisting of 2 centrally-located single microtubuli and 9 outer doublets connected by dynein arms and radial spokes might be disorganised or (partially) absent, negatively influencing motility (Andersson et al., 2000; Turner, 2006). The high susceptibility of the BB breed to respiratory disease (Bureau et al., 1999) might also be partially determined by similar dysfunctional cilia in the respiratory tract,

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as the axonemal structure is highly conserved in all ciliated and flagellated eukaryotic cells and axonemal disorders can result in clinical signs in all ciliated cell types (Turner, 2006). Moreover, the mitochondrial sheath, the outer dense fibres and the fibrous sheath, which are accessory structures exclusive to the mammalian sperm flagellum might also be defective in BB sperm and thus responsible for a genetic sperm motility difference (Turner, 2006).

Field situation versus artificial insemination centres

All the examined bulls were residing at AI centres, and this may have biased our results in comparison to the actual ‘on farm’ situation, since AI bulls are indirectly selected for fertility as bulls with poor fresh semen quality and/or poor semen freezability, as well as bulls with good quality semen but poor pregnancy results are finally culled. However, the field situation in which bulls of unknown fertility are acquired for natural service was mimicked in all our studies, since an unselected and consequently unbiased group of both breeds was always included, namely the youngest bulls. These BB bulls were purchased uniquely based on their linear classification, while the young HF bulls had not yet passed the initial sperm quality test prior to AI admittance and hence, they were not selected for fertility. Consequently, the most relevant breed comparison in the sperm quality studies is the comparison of the youngest breed groups. However, the unselected HF bulls were all obviously younger compared to the unselected BB bulls, which might in some cases result in an immature HF sperm picture with subsequent poor morphology and consequently have a negative influence on semen velocity (Almquist and Amann, 1976; Blom, 1977; Lunstra and Echternkamp, 1982; Barth and Oko, 1989; Johnson, 1997; Amann et al., 2000). This possible bias due to immaturity in the HF breed might minimize inherent breed motility differences. It has been demonstrated that early collections from young bulls are often substandard and that improvements occur from puberty until 2 years of age (Hallap et al., 2004). Hence, the logical question is how many unselected HF bulls were immature. In chapter 2, the proportion of unselected HF bulls that passed the semen evaluation test was 37% lower in comparison with the selected young HF bulls (81% – 44%; Hoflack et al., 2006b). However, the proportion of unselected HF bulls that never met the criteria of the initial semen test for AI admittance in that period (2002 – 2004) and which apparently had irreversible sperm problems was merely 11% (data not shown). Hence, we can state that the proportion of immature HF bulls throughout these studies was approximately (37 - 11 =) 26 %, and this will most certainly influence the results, rendering biologically inherent breed differences less significant at this young age.

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POSSIBLE CAUSES OF THE SUBSTANDARD BELGIAN BLUE SCROTAL CIRCUM-FERENCE AND SPERM QUALITY

We hypothesized on possible causes responsible for the poorer BB sperm quality and are inclined to think that this is the result of focal testicular degeneration (associated with a hypoplastic background), which is induced by environmental stresses, particularly heat stress of the small ‘hypoplastic-like’ BB testes which allow less heat loss through the small scrotal surface area, in combination with a high stress susceptibility inherent to the BB breed (Figure 1).

Testicular hypoplasia and subsequent degeneration

The high proportion of BB bulls failing the BSE for both small scrota and poor semen quality indeed suggests inherited testicular hypoplasia and/or acquired testicular degeneration. Chapter 4 deals with testicular histology and demonstrated that severe focal testicular degeneration occurs more frequently in the BB breed compared to the HF breed. The degeneration was moreover significantly correlated with sperm morphology, suggesting a causative link. Interestingly, a significantly higher amount of collagen was encountered in the BB testicles resulting in a higher proportion of BB testicular connective tissue compared to HF bulls. Within our small bull sample, this testicular connective tissue amount was always high in the BB bulls, suggesting this to be a breed specific trait. High amounts of interstitial collagen have been associated with testicular hypoplasia (Bongso et al., 1981), and we hence concluded that testicular hypoplasia might be present in all the examined BB bulls. Since this testicular connective tissue amount was significantly correlated to both an overall degeneration estimate (0.63; P < 0.001) and to the sperm morphology (-0.47; P = 0.017), we hypothesize that this higher BB amount of connective tissue, defined as testicular hypoplasia, might hamper blood irrigation of the tubuli seminiferi, resulting in tissue hypoxia, degenerative changes and consequently poor sperm morphology. Testicular hypoplasia has been associated with a high sensitivity to testicular degeneration (Settergren and McEntee, 1992), and this probably explains the high sensitivity of BB bulls to testicular degeneration, evidenced by severe decreases in BB semen motility induced by trivial environmental stresses (AWE, personal communication). It should however be stereologically confirmed whether this increased amount of connective tissue is not associated with more vascular tissue and subsequent blood flow.

188

Figure 1: Possible causes of poor BB sperm quality

Genetic Environmental

Hereditary predisposition Stress: = Testicular Hypoplasia Small scrotum - Disturbances in testicular heat regulation

• Heat • Cold

- Systemic illness - Orchitis - periorchitis - Injury / pain - Trauma (scrotal / testicular) - Nutritional disorders - Toxicity - Environmental changes

• Climate • Location

- Externally administered hormones - Age - Miscellaneous

• Radiation • Ultrasound • Neoplasms • Vascular lesions • Blockage of testicular

spermatozoal outflow • …

Testicular degeneration

Increased connective tissue

Decreased blood irrigation?

Tissue hypoxia?

+

Sperm motility

Defective motility ATP production regulating signalling pathways (Turner, 2006) Principal piece glycolysis Midpiece mitochondrial oxidative phophorylation

Sperm morphological abnormalities - Gross (Chapter 3) - Ultrastructural

* head * axoneme * fibrous sheath * mitochondrial sheath

+

Decreased heat transduction through smaller surface area

Defective genes Proteins missing

Inbreeding

Double muscled breeds’ high stress susceptibility +

?

?

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Contrastingly, one might hypothesize that the frequently encountered degeneration results in testicular fibrosis and smaller testes and that the collagen is not the cause but the result of the degeneration. However, we are inclined to support the former hypothesis because testicular fibrosis is considered to be a histological feature of aging evidenced by firmer testes (McEntee, 1990), whereas the testicular consistency decreased with older age in the BB BSE, indicating degeneration however without fibrosis. Moreover, the high collagen content was also encountered in young BB bulls.

Although the presence of more BB testicular connective tissue should be confirmed on higher numbers of bulls, the generally small BB scrota and testicles support the hypoplasia hypothesis (Hanset, 2000; Hoflack et al., 2006b). Given the scrotal measurements of all 150 BB bulls we examined which never surpassed 39.5 centimetres, small scrota are certainly breed specific.

Triggers inducing testicular degeneration

Several reasons can cause testicular degeneration, such as hereditary predispositions, environmental stresses (possibly exacerbating hereditary predispositions) and the particularly high susceptibility of the BB breed to these stresses (Settergren and Mc Entee, 1992; Chenoweth, 2005; Figure 1).

Environmental stresses

This increased stress susceptibility is a drawback of the double-muscled phenotype, and not particularly limited to the BB breed (Arthur et al., 1988; Bellinge et al., 2005). Adverse environmental influences causing testicular degeneration include disturbances in testicular heat regulation, systemic illness, injury and/or pain, toxicity, nutritional deficiencies and environmental changes (Barth and Oko, 1989; Blanchard et al., 1991; Barth, 1997; Johnson, 1997). Given the history of the bulls and taking the predominant BB sperm abnormalities similar to those seen after a mild thermal insult into account, disturbances in testicular heat regulation seem the most likely (Vogler et al., 1993). Impaired heat exchange at scrotal level due to their extreme muscularity combined with rather small scrota (and hence a small scrotal surface skin area allowing less heat transduction) might increase the heat stress risk in BB bulls (Morcombe et al., 1990; Cook et al., 1994; Setchell, 1998; Thonneau et al., 1998; Hanset, 2000; Brito et al., 2004; Hoflack et al., 2006b). Moreover, the large muscle mass of BB bulls results in higher heat production, which is compounded by a reduced respiratory capacity (Bureau et al., 1999), and thus a lower capacity for heat dissipation, resulting in a higher sensitivity to heat stress (Bellinge et al., 2005). Testicular degeneration caused by thermal injury has been demonstrated to frequently occur with negative effects on testicular function and hence on semen quality (Blanchard et al., 1991; Setchell, 1998). This is the

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result of a higher tissue metabolism at higher temperatures and consequently an increased oxygen demand. However, as testicular blood flow is limited, this increased oxygen demand cannot always be supplied, resulting in testicular tissue hypoxia (Setchell, 1998; Brito et al., 2004). Hence, both the testicular temperature and the blood supply may contribute to the development of potential testicular heat degeneration (Cook et al., 1994; Kastelic et al., 1995; 1996; 1997) and consequently influence semen quality and pregnancy results (Lunstra and Coulter, 1997; Gábor et al., 1998). Testicular heat regulation and possible disturbances herein can be assessed non-invasively by means of scrotal thermography (Coulter et al., 1988), whereas testicular blood flow can be assessed non-invasively using color doppler ultrasound (Pozor and McDonnell, 2004). As we suspect BB bulls to be prone to a deficient testicular and scrotal thermoregulation, these above-mentioned assessments are currently being undertaken in BB bulls.

Genetic influence

Next to these adverse environmental stresses, hereditary predispositions for particular sperm abnormalities might also be present in the BB breed (Chenoweth, 2005). Moreover, inbreeding has been suggested as a possible cause for poorer seminal traits (Smith et al., 1989), and it is generally accepted that the inbreeding coefficient in the BB population is quite high (Hanset et al., 2003). In 2004, the average BB bull inbreeding coefficient was 4.65%, compared to 1.23% in 1990. A similar future evolution will lead to a general bull inbreeding coefficient of 6.95% in 2014 (Hanset, 2006)! The relation between the individual inbreeding coefficient and the sperm quality of BB bulls was not assessed in our study, but it might be very interesting to look into this.

However, as we did not encounter BB breed specific abnormalities, we assume that the poor semen quality typical of the BB breed does not result from genetically defined abnormal spermatogonia, but is the result of negative environmental influences, such as the small scrota and subsequent poorer thermoregulation. These conditions are linked to the breed and specifically to the smaller organs including the testicles in case of hypermuscularity, and are thus ‘indirectly’ genetically defined (Ansay and Hanset, 1979; Michaux and Hanset, 1981), but we assume that BB spermatogonia would produce better quality semen under better ‘environmental’ conditions. This hypothesis could be confirmed, once this technique is fully developed, by heterologous transplantation of spermatogonial stem cells harvested from BB bulls with small scrota and poor semen quality to HF testis producing good quality semen in large scrota as demonstrated in Figure 2 (Izadyar et al., 2003; Hill and Dobrinski, 2006). Quality differences between sperm produced by BB spermatogonia in HF testes (in large scrota), compared to the endogenous BB and HF sperm quality would elucidate whether the

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191

poor BB semen quality originates at spermatogonial stem cell level, is due to the negative environmental influence of small scrota, or to a combination of both.

Figure 2: Heterologous male germ cell transfer, resulting in the production of donor (BB) sperm in the recipient (HF) testes. Donor bull: Belgian Blue Recipient bull: Holstein Friesian Poor semen / small scrotum Good semen / large scrotum Testis stem cell (spermatogonia) harvest Testes stem cell knockout Castration Chemotherapy Testis biopsy Radiation Slaughter

depleted endogenous (HF) spermatogenesis Isolation and culture BB testes stem cell injection: of BB testes stem cells ultrasonographic guidance empty HF tubuli seminiferi Possible sperm quality results: 1. BB sperm-HF testes = BB sperm-BB testes BB sperm quality determined at stem cell level Srotal size no effect 2. HF sperm-HF testes > BB sperm-HF testes > BB sperm-BB testes BB sperm quality determined at stem cell level Scrotal size determines BB sperm quality

HF (tubuli seminiferi) testes containing BB spermatozoa

3. BB sperm-HF testes = HF sperm-HF testes (BB sperm – HF testis) BB sperm quality not determined at stem cell level Only scrotal size determines BB sperm quality

Preliminary successes with this technique have been achieved in cattle (Izadyar et al., 2003; Joerg et al., 2003; Hill et al., 2005). In pigs, this technique already resulted in donor-derived spermatogenesis in the recipient testis (Mikkola et al., 2006), while in goats production of donor-derived live offspring was already achieved (Honaramooz et al., 2003).

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ADVISED FUTURE BREEDING STRATEGY

Selection for larger scrota (resulting from larger testes) in the BB breed seems absolutely necessary to by-pass the generally poor sperm quality. Until now, selection of BB bulls was entirely based on their linear classification, which is a scoring system describing several physical characteristics of a bull, but fertility was not taken into account at all.

This is in contrast to the procedure for HF bulls, which are purchased on the basis of their expected genetic value and progeny tested. These HF bulls need to pass a strict semen quality test before AI admittance. Furthermore, the overall index calculated during progeny testing used to select the HF breeding bulls, consists of 10% fertility traits (based on the 56 days non-return rate, the interval between parturition and first insemination, and the calving interval of the female offspring; Figure 3). Consequently, fertility is (indirectly) taken twice into account in HF bull selection. This AI selection for bulls producing easily cryopreservable semen during many years might be partially responsible for the better results of the HF breed in the BSE (Söderquist et al., 1991).

Figure 3: Calculation of the “overall index” used to decide which progeny tested HF bulls

become breeding bulls.

Overall Index

51 % production traits 49 % health and durability traits

41 % health traits 8 % durability traits

not determined by health traits

7 % birth traits 5 % vitality traits

14 % udder health 5 % exterior traits (legs and frame)

10% fertility

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However, SC itself is not taken into account in HF bull selection, and when comparing to HF literature data (Hueston et al., 1988) and to New Zealand HF bulls (Ambreed NZ, unpublished data), both for which SC is taken into account during bull selection, our HF data are significantly lower, stressing the importance of SC as key bull selection criterion. Moreover, as it is generally accepted that fertility in the HF breed is declining, selection for highly productive albeit better fertile animals, both male and female, should be considered worldwide (Royal et al., 2000; Lucy, 2001). Since bull SC is a highly heritable trait, this seems the ultimate tool to reach this goal (Barth, 1997).

For the BB breed, selecting for larger scrota will probably be less efficient as in other breeds since extremely large scrota are apparently non existent in the BB breed. The only option then is to crossbreed the BB to other breeds with larger scrota. In order to preserve as much breed characteristics as possible, the dual purpose BB breed seems the most obvious for this purpose. Both heterozygote (mh/+) and homozygote (+/+) bulls could be used for this, although heterozygote animals are genetically closer to the double-muscled (mh/mh) animals. Alternatively, other double-muscled breeds with large testes and scrota could be used, or finally as a last option, other conventional beef breeds to alleviate the BB bull fertility problems. Hybrid formation by means of cross breeding different pure bred strains has proven extremely relevant to increase porcine profitability, and a recent study similarly demonstrated its advantageous perspective in bovine (Heins et al., 2004a; 2004b; 2004c; Hansen L, oral communication).

FINAL CONCLUSION

The present thesis described several fertility associated problems encountered in BB bulls, such as a substandard SC and poor sperm quality, which might partially explain the observed poor field fertility results of BB natural service bulls. These results indicate the presence of testicular degeneration, a condition that was indeed quite common in the examined BB bulls. Furthermore, this study demonstrated a high content of connective tissue in BB testicles. The latter might explain the high BB sensitivity to testicular degeneration. Selection for larger scrota in the BB breed should urgently be undertaken.

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Lunstra DD, Coulter GH. Relationship between scrotal infrared temperature patterns and natural-mating fertility in beef bulls. J. Anim. Sci. 1997; 75: 767 – 774.


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McEntee K. Chapter 14: Scrotum, spermatic cord, and testis: degenerative and inflammatory lesions. In McEntee K (Editor): Reproductive pathology of domestic mammals. San Diego, California: Academic Press Inc., 1990; p. 252 - 278.


Mikkola M, Sironen A, Kopp C, Taponen J, Sukura A, Vilkki J, Katila T, Andersson M. Transplantation of normal boar testicular cells resulted in complete focal spermatogenesis in a boar affected by the immotile short-tail sperm defect. Reprod. Dom. Anim. 2006; 41: 124 – 128.

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Reproduction in livestock is an essential prerequisite for production. Since bulls are generally bred to numerous cows, they have a pivotal responsibility in bovine reproduction. In Belgium, the two predominant cattle breeds are the Belgian Blue (BB) beef breed, and the Holstein Friesian (HF) dairy breed. In BB-breeding herds using natural service bulls, disappointing pregnancy results frequently occur. The initial goal of this study was to elucidate the BB bull contribution to this issue. This was done by means of the internationally acknowledged ‘breeding soundness evaluation’ (BSE) of BB bulls, in comparison to HF bulls. Such a bull evaluation screens bulls for potential fertility, in order to select bulls with traits favourable for high fertility and to exclude those bulls with clear impediments to breed properly or with potential sub-fertility, but it is only rarely performed in Belgium.

Therefore, we described how this BSE should be performed and how the results should be interpreted in Chapter 1. The bull evaluation consists of a general physical soundness examination, a genital tract examination of both the external and internal genitalia (including a scrotal circumference (SC) measurement), and a semen quality evaluation. Additionally, the willingness (libido) and ability (serving capacity) of bulls to serve cows should also be determined in order to fully evaluate a bull’s reproductive potential.

In a first original study (Chapter 2), this BSE was applied to young ‘fertility unselected’ BB and HF bulls prior to acceptance into an artificial insemination (AI) program, as data on breeding soundness and libido evaluations in BB bulls are scarce, although several conditions which can negatively influence bull fertility, such as small scrota and several heritable feet and leg abnormalities have been described in the BB breed. Breed differences for breeding soundness between BB and HF bulls were shown, as 93.7 % of the young BB bulls failed the BSE compared to 59.3 % of the HF bulls. Within the BB breed, differences were present between bulls of different ages, and bull selection for better fertility with increasing age improved the results. The most important reasons for failure were a substandard sperm morphology and SC, but far more BB bulls failed for these traits compared to the HF breed (82.8% versus 56.0% and 43.8% versus 17.6% in the BB and the HF breed for sperm morphology and SC, respectively). Concerning libido, the reaction time did not differ either between breeds or between age groups within the BB breed, whereas mounting enthusiasm, although not different between the two breeds, declined with increasing age, probably due to the greater mating experience of the older bulls. All in all, libido did not seem to be different between the breeds. We hence concluded that certain fertility associated problems, which might partially explain the observed poor field fertility results of BB natural service bulls, are present within the BB breed.

As sperm quality was the main reason why BB bulls failed the BSE and since few data are currently available on BB sperm quality, we assessed this in detail in Chapter 3A, by comparing the sperm quality of BB to HF bulls of several age categories by means of a

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classical semen evaluation. Volume and concentration, and consequently total sperm output depended largely on age. Gross, total, and progressive motility, % live and % normal spermatozoa were significantly lower in the BB breed. Primary sperm abnormalities, such as nuclear vacuoles, midpiece defects and cytoplasmic proximal droplets which were noticed most frequently, occurred far more in the BB breed. The breed difference in primary sperm abnormalities was due to the high cumulative proportion of several sperm defects rather than the general high presence of one (or more) particular ‘BB breed specific’ defect(s). Hence, disturbances in spermiogenesis were deemed to be the cause of the poorer BB sperm quality. Since these sperm abnormalities occur significantly more in the BB breed than in the HF breed, it seemed as if the BB breed was genetically predisposed to a higher susceptibility to environmental stresses which are known to interfere with normal spermiogenesis. The small scrota typical of the genetically narrow BB breed, which was the second important reason why BB bulls failed the breeding soundness evaluation, might in part be responsible for this.

This classical semen evaluation, as performed in chapter 3A, is however rather subjective. More objective sperm motility assessments are available, and we consequently evaluated BB and HF sperm motility by semi-computerized (Chapter 3B1) and computerized (Chapter 3B2) methods.

In Chapter 3B1, the Sperm Quality Analyzer IIC (SQA-IIC) was validated for bull semen analysis. This device registers fluctuations in optical density resulting from moving particles in a semen sample by a photometric cell and converts this to a numerical output, the sperm motility index, which takes the concentration, the motility and the sperm morphology into account. Our study demonstrated good repeatability of measurements but pointed out that evaluations should be performed at a concentration of approximately 50 x 106 spermatozoa/ml in order to be reliable, since a saturation of the system occurred at higher concentrations. However, even at this optimal concentration, our results indicated that discrimination of semen samples with little difference in quality by the SQA-IIC might be a problem. This is probably due to the combination of sperm quality parameters that contribute to the sperm motility index, and that the deficient quality of one contributing sperm parameter (e.g. motility) can be compensated by a good quality of another parameter (e.g. concentration), biasing accurate quality discrimination of different semen samples. Moreover, fertility prediction (56 days non-return rate) based on the sperm motility index of a semen sample appeared to be inaccurate. Consequently, we concluded that this device could not be used to adequately determine small motility differences between ejaculates of bulls of the two breeds under investigation.

Alternatively, objectively assessed sperm motility of both breeds by means of the Hamilton Thorne CEROS version 12.2c computer assisted sperm motility analyser (CASA) was compared in Chapter 3B2, and differences between the BB and HF breed, confirming

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the results of the subjective light microscopic motility estimation, were demonstrated. Higher percentages of both totally and progressively motile spermatozoa were encountered in the HF breed compared to the BB breed, reflecting the higher proportion of live spermatozoa present in a HF ejaculate. Additionally, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower beat cross frequency (suggesting less propelling force) combined with a higher lateral head displacement (suggesting more resistance due to friction), was the basis for the lower velocity of BB sperm cells and probably also explains the less straight forward progressive movement of BB spermatozoa. Breed differences in sperm head size and the demonstrated presence of more BB sperm head abnormalities (which might interfere with sperm head dimensions) were (partially) responsible for the encountered sperm motility differences, as small geometrical differences in sperm morphology can result in large differences in sperm hydrodynamics, and subsequently in impaired semen velocity. Furthermore, sperm tail abnormalities and cytoplasmic droplets might affect the propelling efficiency of the sperm tail, negatively influencing semen velocity. No sperm motility differences were observed between age groups within the BB breed. The breed differences were observed in the examined bull populations residing at AI centres, in Belgium for the BB bulls and in the Netherlands for the HF bulls, respectively. However, these bull populations are selected for fertility. A similar numerical pattern was observed in an unselected bull population of both breeds, although these differences were mostly not significant for the different CASA parameters. Nevertheless, these data suggest that a ‘genetic component’ might be responsible for the observed sperm motility breed differences. Whether this ‘genetic component’ is the mere result of sperm morphology differences or also stems from other factors irrespective of sperm morphology is not clear.

The previous studies clearly demonstrate the general presence of small scrota and poor sperm quality in BB bulls. This high proportion of BB bulls with a substandard SC and poor sperm morphology suggests an increased prevalence of testicular hypoplasia or degeneration within this breed. In Chapter 4, we aimed to assess whether testicular degeneration was the underlying reason for these BB traits negative to fertility. At culling, sperm quality and testicular histology of BB artificial insemination bulls were assessed and compared to HF data. In agreement with a generally poorer BB semen quality, more degenerative changes were encountered in BB compared to HF testicles and these results were correlated to the percentage of normal spermatozoa and primary abnormalities. Moreover, both these sperm quality parameters and the degeneration results on the one hand were correlated with the relative amount of collagen fibres present in the testicular connective tissue. The latter was significantly higher in BB compared to HF testicles. This increased amount of connective tissue in BB testes might hamper blood irrigation, resulting in testicular tissue hypoxia, degenerative changes and hence be responsible for the poorer sperm quality. This condition was defined as a mild form of testicular hypoplasia, which might be responsible for the higher

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sensitivity to testicular degeneration in the BB breed compared to HF bulls. Given the history of the bulls, the predominant BB sperm morphological abnormalities, and the decreased heat transduction via the small scrotal surface area, heat injury seems the most important stress factor inducing testicular degeneration in the hypoplastic BB testes.

In view of these findings, selection for larger scrota in the BB breed is advisable. Until now, selection of BB bulls was entirely based on their linear classification. This is in contrast to the selection procedure for HF bulls, where fertility is indirectly taken twice into account, once during a sperm evaluation to decide on AI admittance, and once during progeny testing, as the overall bull index is partially determined by fertility data of a bull’s daughters. However, bull selection directly based on scrotal circumference has been demonstrated to be even more effective. Moreover, high circumference bulls produce higher fertility offspring, both male and female, and scrotal circumference based bull selection will consequently lead to higher fertile animals. As it is generally accepted that fertility in the HF breed is declining, selection for highly productive albeit better fertile animals should be considered worldwide. Since bull scrotal circumference is a highly heritable trait, this seems the ultimate tool for this. For the BB breed, selecting for larger scrota will probably be less efficient as in other breeds since extremely large scrota are apparently non existent in the BB breed and testicular hypoplasia appears to be breed specific. Crossbreeding BB cows to highly fertile bulls of other breeds might offer opportunities to by-pass this deadlock.

213

SAMENVATTING

Vruchtbaarheid is de ‘conditio sine qua non’om melk- en vleesproductie te genereren bij runderen. Stieren spelen een centrale rol in de voortplanting bij runderen, aangezien zij over het algmeen veel koeien moeten dekken. In België zijn de 2 belangrijkste runderrassen het Belgisch Witblauwe (BWB) vleesvee en het Holstein Friesian (HF) melkvee. Bij BWB fokveestapels die natuurlijk dekkende stieren gebruiken worden nogal eens ontgoochelende drachtigheidsresultaten bekomen. De initiële doelstelling van deze studie was de rol van de BWB stieren in deze problematiek op te helderen. Hierbij werd het internationaal erkende ‘onderzoek van de reproductiegeschiktheid’ (ORG) bij BWB stieren gebruikt en dit werd vergeleken met de resultaten bij HF stieren. Dit onderzoek is eigenlijk een vruchtbaarheidsscreening van stieren, met als doel die dekstieren te selecteren die kenmerken bezitten die gewenst zijn in het kader van een goede vruchtbaarheid en die stieren te weren die kenmerken bezitten die onverzoenbaar zijn met een goede vruchtbaarheid.

Aangezien een dergelijk onderzoek in België slechts zelden wordt uitgevoerd, beschrijven we in hoofdstuk 1 hoe dit dient uitgevoerd te worden alsook hoe de resultaten moeten geïnterpreteerd worden. Een ORG bestaat uit een onderzoek van de algemene gezondheid, een onderzoek van het geslachtsstelsel waarbij zowel uitwendige als inwendige geslachtsorganen beoordeeld worden (en waarbij ook een scrotum omtrekmeting verricht wordt), en een onderzoek van de spermakwaliteit. Daarnaast moet ook de deklust (libido) en de kunde om koeien te dekken (dekbekwaamheid) nagegaan worden om de reproductiegeschiktheid van een stier volledig en juist in te schatten.

Aangezien ORG resultaten van BWB stieren zeer schaars zijn, werden in de eerste originele studie (hoofdstuk 2) jonge qua vruchtbaarheid nog ongeselecteerde BWB en HF stieren, die aangeschaft waren door centra voor kunsmatige inseminatie (KI), aan het ORG onderworpen. Het is eigenlijk vreemd dat een dergelijk onderzoek zo zelden wordt uitgevoerd bij BWB stieren, aangezien er meerdere indicaties zijn, zoals een beperkte scrotale omvang en verschillende erfelijke afwijkingen van het beenwerk, die frekwent voorkomen bij BWB stieren die de vruchtbaarheid negatief kunnen beïnvloeden. Er bleek een duidelijk verschil te bestaan tussen het BWB en HF ras betreffende de reproductiegeschiktheid, waarbij 93.7% van de jonge BWB stieren niet slaagde voor het onderzoek. Bij de HF stieren slaagde 59.3% niet. Binnen het BWB ras was er een leeftijdseffect merkbaar, waarbij de betere resultaten bij oudere stieren verklaard konden worden doordat alleen de best vruchtbare stieren tot op latere leeftijd aangehouden werden. De belangrijkste redenen waarom zoveel stieren niet slaagden voor het ORG waren een ondermaatse sperma morfologie en een te geringe scrotale omtrek.

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Beduidend meer BWB stieren bleken met betrekking tot deze punten een onvoldoende te scoren in vergelijking met HF stieren (82.8 versus 56.0% en 43.8 versus 17.6% onvoldoende stieren van het BWB en HF ras, respectievelijk voor sperma morfologie en scrotale omtrek). Aangaande het eerste punt van de libido, de reactietijd, werden noch rasverschillen noch leeftijdsverschillen binnen het BWB ras waargenomen, terwijl bij het tweede onderdeel van de libido, het enthousiasme van opstijgen, gevonden werd dat dit bij oudere stieren afnam, waarschijnlijk tengevolge van hun grotere ervaring. Er waren echter geen rasverschillen aangaande de libido. Samenvattend kan besloten worden dat er wel degelijk problemen voorkomen bij BWB stieren die een negatieve invloed uitoefenen op de vruchtbaarheid en die de soms ontgoochelende drachtigheidsresultaten van natuurlijk dekkende BWB stieren kunnen verklaren.

Omdat bij zoveel BWB stieren het ORG een negatief resultaat opleverde wegens een ondermaatse spermakwaliteit en aangezien er weinig gegevens betreffende de spermakwaliteit van BWB stieren voorhanden zijn, werd in hoofdstuk 3A aan de hand van een klassiek spermonderzoek de sperma kwaliteit van BWB stieren vergeleken met die van HF stieren, en dit voor verschillende leeftijdsklassen. Volume en concentratie, en dientengevolge ook de hoeveelheid geproduceerde zaadcellen bleken voornamelijk afhankelijk te zijn van de leeftijd. Massa-, totale- en rechtlijnige motiliteit, alsook het percentage levende en normale spermacellen waren significant lager in het BWB sperma. Primaire afwijkingen, waarvan kerncysten, middenstukdefecten en cytoplasma druppels het grootste aandeel vormden, waren beduidend talrijker aanwezig in BWB sperma. Dit rasverschil was voornamelijk te wijten aan het voorkomen van veel verschillende primaire afwijkingen in geringe aantallen en minder aan het voorkomen van grote aantallen van slechts enkele specifieke afwijkingen. Bijgevolg kon worden verondersteld dat stoornissen in de spermiogenese aan de basis lagen van het povere BWB sperma. Aangezien dergelijke sperma afwijkingen significant meer voorkwamen in BWB ejaculaten dan in HF ejaculaten zou het sperma van BWB stieren een genetisch bepaalde hogere gevoeligheid kunnen hebben voor stressfactoren vanuit de omgeving die de spermiogenese verstoren. De kleine scrota die typerend zijn voor het ingeteelde BWB ras, en die een belangrijke reden waren van afkeuring tijdens het ORG, zouden hierbij een rol kunnen spelen.

Het hierboven beschreven klassiek sperma onderzoek bevat echter een aantal subjectieve componenten. Aangezien er objectievere methoden voorhanden zijn om de sperma motiliteit te beoordelen werd de sperma motiliteit ook op semi-geautomatiseerde (hoofdstuk 3B1) en gecomputeriseerde (hoofdstuk 3B2) wijze herbekeken.

In hoofdstuk 3B1 werd nagegaan of de ‘Sperm Quality Analyzer IIC’ (SQA-IIC) geschikt is om stierensperma te beoordelen. Aan de hand van een fotometrische cel registreert dit toestel wijzigingen in optische densiteit die ontstaan als gevolg van bewegende deeltjes in

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een spermastaal en zet deze waarneming om in een numerieke waarde, namelijk de sperma motiliteitsindex, die gebaseerd is op de concentratie, de motiliteit en de morfologie van de spermacellen. De metingen bleken goed herhaalbaar te zijn, maar onze resultaten lieten zien dat de metingen dienen te geschieden bij een concentratie van om en bij de 50 x 106

spermatozoa/ml om enigszins betrouwbaar te zijn, aangezien het maximale meetbereik van het toestel overschreden wordt bij hogere concentraties. En zelfs bij deze optimale concentratie bleek het toesel niet in staat om spermastalen met kleine kwaliteitsverschillen te onderscheiden. Dit is waarschijnlijk het gevolg van het feit dat meerdere parameters bijdragen tot de sperma motiliteitsindex, en een ondermaatse kwaliteit van 1 parameter (vb. motiliteit) kan dan gecompenseerd worden door een goede andere samenstellende parameter (vb. concentratie), wat het moeilijk maakt om accuraat de kwaliteit van verschillende spermastalen te rangschikken. Bovendien bleek dat de voorspelling van de vruchtbaarheid (56 dagen non-return rate) van een spermastaal aan de hand van de sperma motiliteitsindex onbetrouwbaar was. Daarom werd geconcludeerd dat het toestel niet bruikbaar was om kleine motiliteitsverschillen tussen ejaculaten van de 2 bestudeerde rassen efficient te kunnen detecteren.

Als alternatief werd in hoofdstuk 3B2 de spermamotiliteit van beide rassen aan de hand van de Hamilton Thorne CEROS versie 12.2c computer geassisteerde sperma analyse (CASA) objectief beoordeeld en vergeleken. Hierbij werd het rasverschil dat vastgesteld was met de subjectieve, lichtmicroscopische motiliteitsbeoordeling bevestigd. De percentages totaal en rechtlijnig beweeglijke spermacellen waren hoger in HF ejaculaten in vergelijking met BWB ejaculaten, wat het hogere aandeel levende spermcellen in HF ejaculaten weerspiegelt. Bovendien werd een minder efficiente manier van voortbewegen aangetoond bij BWB spermacellen, tengevolge van een lagere staartslagfrekwentie (wat een verminderde voortstuwing laat vermoeden) alsook een verder zijdelings uitwijken van de kop (wat meer wrijvingsweerstand zal teweeg brengen). Dit is waarschijnlijk verantwoordelijk voor de tragere en minder rechtlijnige manier van voortbewegen van BWB spermacellen. Rasverschillen in de afmetingen van de spermakoppen alsook de bewezen aanwezigheid van meer kopafwijkingen (die misschien de kopafmetingen beïnvloeden) bij BWB spermacellen zijn waarschijnlijk mede verantwoordelijk voor de vastgestelde verschillen in beweeglijkheid, aangezien kleine geometrische verschillen in de vorm van de spermacellen grote hydrodynamische wijzigingen kunnen teweeg brengen en dientengevolge de snelheid kunnen afremmen. Daarnaast kunnen ook staartafwijkingen en cytoplasma druppels de snelheid negatief beïnvloeden aangezien ze de voortbeweging kunnen hinderen. Tussen de leeftijdsgroepen binnen het BWB ras werden geen verschillen in spermacelbeweeglijkheid waargenomen. Tussen de in Belgïe en Nederland onderzochte KI stierpopulaties, repectievelijk BWB en HF, werden rasverschillen aangetoond maar deze stieren waren echter geselecteerd voor vruchtbaarheid. In een ongeselecteerde groep stieren van beide rassen

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werden numeriek gelijkaardige verschillen aangetoond maar deze waren meestal niet significant. Desalniettemin laten onze resultaten vermoeden dat een ‘genetische component’ verantwoordelijk is voor de waargenomen rasverschillen in de beweeglijkheid van de spermacellen. Of deze ‘genetische component’ alleen te wijten is aan de verschillen in spermacelmorfologie, of ook afhangt van andere niet aan de morfologie gerelateerde factoren is niet duidelijk.

Bovenvermelde resultaten tonen aan dat kleine scrota en een minder goede spermakwaliteit frekwent voorkomen bij BWB stieren. Het hoge aandeel stieren binnen het BWB ras met zowel een klein scrotum als een ondermaatse spermakwaliteit laat vermoeden dat aangeboren testiculaire hypoplasie of verworven testiculaire degeneratie algemeen voorkomen binnen het ras.

Daarom werd nagegaan (hoofdstuk 4) of testiculaire degeneratie de onderliggende reden was voor de kleine scrota en de verminderde spermakwaliteit. Van BWB en HF KI stieren die geslacht moesten worden, werd kort voor ze geslacht werden een spermastaal genomen en werd na het slachten de testiculaire histologie beoordeeld. Uit het onderzoek van de genomen stalen kwam naar voren dat er bij BWB stieren vaker sprake was van testiculaire degeneratie en dat deze degeneratie was gecorreleerd met het aanwezig zijn van primaire afwijkingen van de spermacellen. Bovendien waren zowel de spermakwaliteitsparameters als de mate van degeneratie gecorreleerd met de relatieve hoeveelheid collageenvezels in het bindweefsel van de testikels. Er was significant meer collageen aanwezig in de tesikels van BWB stieren in vergelijking met die van HF stieren. Het is goed mogelijk dat dit hoger bindweefselaandeel in BWB testikels de bloedvoorziening bemoeilijkt, wat kan resulteren in hypoxie van het testisweefsel en vervolgens in het optreden van degeneratieve veranderingen die uiteindelijk leiden tot een veminderde spermkwaliteit. Dit fenomeen kan beschouwd worden als een milde vorm van testiculaire hypoplasie, waardoor het BWB ras een hoge gevoeligheid heeft voor het ontstaan van testiculaire degeneratie. Rekening houdend met de voorgeschiedenis van de onderzochte stieren, met de meest voorkomende sperma defecten en met het kleine scrotale huidoppervlak waardoor de warmteafgifte is verminderd, lijkt hitte stress de belangrijkste uitlokkende factor voor het ontstaan van degeneratie in de hypoplastische BWB testikels te zijn.

Daarom verdient het aanbeveling om bij BWB stieren op scrotumgrootte te selecteren. Tot op heden worden BWB stieren alleen op hun lineaire classificatie geselecteerd. Dit verschilt van de selectie van HF stieren, waar vruchtbaarheid onrechtstreeks tweemaal in rekening wordt gebracht: de eerste maal bij de sperma beoordeling tijdens het KI toelatingsonderzoek, en de tweede keer tijdens het nakomelingenonderzoek aangezien de vruchtbaarheidsdata van de dochters van een stier mede de stierindex bepalen. Stierselectie gebaseerd op de scrotale omtrek zelf blijkt echter het meest efficïent. Stieren met een grote

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scrotum omtrek fokken namelijk vruchtbaarder stieren en vaarzen. Stierselectie gebaseerd op scrotale omtrek resulteert dus in een vruchtbaarder ras. Aangezien algemeen erkend wordt dat ook de vruchtbaarheid van het HF ras achteruit gaat, is het sterk aangewezen om ook bij HF stieren extra aandacht te schenken aan de vruchtbaarheid. Doordat scrotale omtrek een gemakkelijk overerfbaar kenmerk is, is dit het meest aangewezen stierselectiecriterium.

Binnen het BWB ras zal de selectie op basis van scrotale omtrek waarschijnlijk weinig resultaat opleveren in vergelijking met andere rassen, omdat echt grote scrotumomtrekken niet voorkomen bij BWB stieren en testiculaire hypoplasie wel eens een raskenmerk zou kunnen zijn. Daarom moet het kruisen van BWB koeien met zeer vruchtbare stieren van andere rassen in overweging worden genomen om het probleem van de onvoldoende vruchtbaarheid van BWB stieren het hoofd te bieden.

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DANKWOORD

« The oxen are slow, but the earth is patient »… . Uiteindelijk is ook mijn doctoraat af geraakt. En hoewel een doctoraatsthesis eindigt met een ‘one man show’ is de aanloop naar dit hoogtepunt meestal niet zonder slag of stoot verlopen. Bij deze zou ik diegenen willen bedanken die bijgedragen hebben aan dit proefschrift. En die lijst is lang ... .

Vooreerst wil ik mijn beide promotoren, Prof. Dr. Ann Van Soom en Prof. Dr. Geert Opsomer bedanken voor hun constructieve bijdragen aan dit doctoraat.

Ann, jij was mijn wetenschappelijke ‘rots in de branding’ die mij ervan weerhield te verzuipen, zeker tijdens de eindsprint. Je enerzijds zeer kordate maar terzelfdertijd warme aanpak van alle vragen die tijdens mijn onderzoek rezen waren voor mij een hart onder de riem. Bij jou kon ik altijd terecht en je ‘vrouwelijke aanpak’ van mijn wetenschappelijke problemen was voor mij zeer geruststellend. Voor mij ben je de Cora Kemperman van de veterinaire wetenschap. Ik bewonder de schijnbaar speelse manier waarop je een zware job en je gezinsleven combineert en dat maakt jou, als facultair feministisch geweten, tot een toonbeeld voor alle jonge moeders op onze vakgroep (en zelfs daarbuiten). Het antwoord op jouw levensvraag is dus zonder meer ‘ja’!

Geert, jij was de startmotor van dit proefschrift aangezien jij met de idee op de proppen kwam om de vruchtbaarheid van Witblauwe stieren eens onder de loupe te bekijken (zo klein zijn die testikels dus!). De daaruit voortvloeiende lawine aan gegevens leidde, eens de rook om ons hoofd was verdwenen, tot dit proefschrift. Urenlange, onooglijk vroege autoritten en maandenlang turen door een microscoop, waren ‘part of the job’, maar jij wist als geen ander het droge, soms stoffige wetenschappelijke werk op te fleuren met grappen en grollen. ‘Professor Opsomerski’, ik zie er echt naar uit om nog eens gezamelijk een pint te pakken met een handvol (buurt)collega’s of samen nog eens een congresdiner (manu militari) af te mogen sluiten... .

Prof. Dr. de Kruif, alhoewel je als co-promotor het recht had neutraal langs de zijlijn te kuieren, was je steeds de eerste die mijn (lange) artikels nagelezen had. Je uiterst positieve instelling en nooit aflatende enthousiasme annex werklust, en je gave om nooit ‘the big picture’ uit het oog te verliezen werkten bij mij meer dan inspirerend. Jouw ‘waar gehakt

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wordt vallen spaanders’ mentaliteit, en het vertrouwen en de vrijheid die je iedereen op de dienst schenkt verklaren grotendeels de hoge productiviteit van onze vakgroep.

Ook Prof Dr Luc Duchateau wil ik expliciet bedanken. Luc, jij was de biodiesel die mijn proefschrift weer vlot trok toen dit dreigde te verzanden in wat ik achteraf als mijn ‘wetenschappelijke Middeleeuwen’ beschouw. Je vulde gretig mijn statistische leegte en het klikte meteen. Je vermogen om je direct in te leven in een voor jou overigens vreemd onderzoek (testikelmetingen bij Witblauwe koeien?!?) en direct mee te redeneren hoe de vraagstelling accurater kan gebeuren maakt jou facultair onmisbaar. Je brede kijk maakt jou zonder enige twijfel de grootste facultaire aanwinst van de laatste jaren! Ik wens je zeer veel succes in je verdere carrière en hoop samen nog veel Witblauwen te fokken in Afrika . En ook mijn fietsje is ondertussen afgestoft, dus waar wachten we nog op... ?

Dr. Moyaert, Ignace, jij bent degene die mij bij mijn eerste passen op Waalse KI bodem bijstond. De ‘inside information’ die je mij hierbij meegaf was dusdanig waardevol dat ik mij in Mons, en na de IBR calamiteit ook in Ciney, razendsnel thuis voelde. Zonder jou was mijn stierenverhaal waarschijnlijk nooit geworden wat het nu is. Hopelijk kunnen we in Lotenhulle nog lang en veel samenwerken aan een beter Witblauw... .

Dr. Dernelle, je voudrais vous remercier pour la confiance que vous m’avez faite et pour la liberté avec laquelle j’ai toujours pu faire ma recherche à Mons, Wavre, et à Ciney. Rien n’était jamais trop, et tout ce que je voulais faire était possible. Les nombreuses discussions sur l’élevage d’un certain taureau ou d’un autre m’ont vraiment plu. L’honnêteté avec laquelle vous avez lu mes articles est admirable. Eric, merci pour tout!

Prof. Dr. Rodriguez-Martinez, Heriberto, I would like to thank you for my three inspiring periods at Uppsala. After the andrology course, I arrived in Ghent with thousand and one ideas to continue my Phd research. After my second period in Uppsala, I realised the relativity of semen analysis and the difficulty of correct interpretation of semen defects. My third stay at your department opened a whole new histological perspective that finally led to my ultimate conclusion on Belgian Blue bull fertility. Thanks for everything.

I hope you will be able to solve the problems you are encountering now. I definitely hope to meet you again soon. Please give my warmest regards to Annika and Karin for all their time and energy explaining me the formol saline method, as well as to Lennart.

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Ook Prof Dr. Wim Van den Broeck wil ik bedanken voor het ter beschikking stellen van mankracht, materiaal, en know-how om mijn stalen op de meest vervelende momenten te analyseren.

Prof. Dr. Alex Van Zeveren, Prof. Dr. D. Maes en alle leden van mijn begeleidings- en examencommissie wil ik uitdrukkelijk bedanken voor hun monnikenwerk. Mijn manuscripten, waarbij niet op een tabelletje meer of minder gekeken wordt, en waarbij iedere tabel - eufemistisch gesteld - rijkelijk gevuld is, zijn niet altijd even vlot verteerbaar. Het conscientieus nalezen van mijn doctoraat is dan ook geen sinecure. Bij deze wil ik jullie danken voor jullie engelengeduld.

Geen onderzoek zonder onderzoeksmateriaal, in mijn geval stieren, teelballen en sperma. De KI verenigingen die dit mogelijk maakten, KI Haliba, L’Association Wallonne de l’Elevage / Belgian Blue Group, en HG / CR-Delta verdienen dan ook een dikke ‘merci’. Eric, Christophe, Patrice, Ana, Lisette, Adrie, Jaap en Michiel: bedankt! Maar laat mij ook zeker de mensen in het lab en de stierverzorgers niet vergeten, want dat waren uiteindelijk degenen die het mij naar mijn zin maakten en uren met mij samen doorbrachten om de zoveelste stier te onderzoeken. Marc, Marie-Madeleine, Betsie, Stoffer, Carla, Jan, Trijnie, Gérard, Ralph, Fréderic, Ludovic, Pierre, Jean-François, Sylvain Cauchy, Sylvain Crahay, José, Jean-Marc, Grégory, Joseph, Christophe, Olivier , Arie (die er werkelijk alles voor deed om mij thuis te laten voelen in Harfsen), Berend, Bertus, Alfons, Henk, Peter, Jan-Willemse, Ad, Peter en Wouter: van ganser harte bedankt. Jan R, bedankt voor de mooie foto’s die dienden als kers op de taart. Ik wil heel concreet ook HG bedanken voor hun bijdrage in de drukkosten.

Een welgemeend dankwoord is ook op zijn plaats voor meerdere mensen binnen onze vakgroep, mijn ‘diergeneeskundige nest’ de afgelopen 9 jaar.

Prof Marc Verdonck, ‘éminence grise’, jij was degene die mij met de hulp van Dominiek binnenloodsde op de vakgroep, en ik ben jullie beiden hiervoor dankbaar.Toen ik laatstejaars student was, was jij, waarschijnlijk zonder het te beseffen, door je accurate klinische aanpak reeds mijn ( en waarschijnlijk menig medestudent zijn) veterinaire ‘godfather’ Je ‘no nonsense’ aanpak van alle problemen die zich stellen maken jou een bewonderenswaardig mens en ik vind dan ook dat de faculteit sedert jouw emeritaat een stuk armer is geworden. Daar waar Professor de Kruif onze dienst steeds als een echte manager leidde, heb ik jou altijd als ‘vader van de dienst’ beschouwd. Je luisterend oor wanneer ‘jouw dissident’ een klein of kleiner voor hem onoverkomelijk probleem had, je ongelooflijke relativiteitszin, je pragmatische instelling, en je gave om complexe situaties snel te ontleden

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en hierop dan gepast te anticiperen blijf ik met open mond bewonderen. Voor mij ben jij de enige facultaire levende legende!

Mijn absolute dank gaat ook naar alle collega’s die mee de dag-, nacht-, en weekenddiensten verzorg(d)en op de Buitenpraktijk. Alle onderzoek, hoe fundamenteel ook, dient ooit ergens zijn toepassing in de praktijk te vinden, zoniet is het maar onderzoek voor het onderzoek, en dus niets meer dan vicieuze ijdelheid. Vandaar dat ik uitdrukkelijk diegenen wil lauweren die het dagdagelijks moeten ‘doen’. Niettegenstaande de vele discussiepunten en verschillende meningen in onze ‘duiventil’ heb ik er altijd graag gewerkt. Het feit dat je altijd wel op iemand kan terugvallen zorgt voor een nestwarmte die op weinig andere vakgroepen binnen de faculteit voorkomt. Houden zo!

Jef, ik bewonder de manier waarop jij na zoveel jaren in het veld nog steeds een wetenschappelijke onderbouw noodzakelijk acht bij iedere diagnose. Samen met Marcel zijn jullie de mentoren van vele jonge, soms onbesuisde maar steeds enthousiaste, pas afgestudeerde dierenartsen die ieder jaar weer komen binnenwaaien. ‘Keep up the good work’!

Bart, niettegenstaande het feit dat ik een stuk(je) ouder ben dan jij ben jij het type dierenarts waar ik naar opkijk: handig, practisch ingesteld maar terzelfdertijd zeer belezen, en nooit te beroerd om tot het uiterste te gaan voor het zoveelste kalf dat het waarschijnlijk niet zal halen. Petje af! Jij bent zonder meer één van dé peilers van onze vakgroep, zowel in de Buitenpraktijk als in het laboratoriumwerk. Het ga je goed.

Maar natuurlijk wil ik ook alle andere mensen binnen onze groep bedanken voor de aangename sfeer en het ‘jonge gedachtengoed’. Het werken met jongere collega’s is zonder twijfel de beste remedie tegen verouderen en conservatisme. Iris (zeker jij om zoveel vrijdagen voor mij te verlichten), Mirjan, Stefaan en Hans, nogmaals bedankt om bij te springen in mijn diensten toen ik de race tegen de klok dreigde te verliezen.

Kristof en Catharina, jullie wil ik bedanken om mee nacht en ontij te trotseren om s’morgens om 7 uur aan de andere kant van het land of zelfs in Nederland een paar staaltjes te gaan nemen van de ballen van één of andere stier op weg naar de eeuwige graasvelden. Kristof, bedankt voor de tergend lange bindweefselanalyses op de dienst morfologie.

Tom R, alhoewel jou stille aanpak en mijn luidruchtige stijl mijlenver uiteen liggen konden we het wonderwel vinden en klikte het meteen. Als er binnen onze vakgroep iemand was die begreep waarover ik mij druk maakte en waarmee ik echt in detail over mijn

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onderzoek kon brainstormen en discussiëren, dan was jij het wel. Je bijdrage tot dit doctoraat is daarmee veel groter dan jezelf denkt. Zonder jou was mijn doctoraat ongetwijfeld een artikel armer, en de ruggegraat doorheen mijn verhaal is deels jouw verdienste. Bedankt hiervoor. Je bent een getalenteerd wetenschapper en een absolute verrijking voor onze vakgroep. Alhoewel ik niet echt weg ga zal ik je zeker missen!

Ook Mirjan, Leen, Jo en Katrien ben ik dank verschuldigd om mij telkens voor te laten gaan op de fluorescentiemicroscoop om alsnog mijn deadlines te halen.

En dan wil ik ook de mensen achter de schermen zeker niet vergeten. Hun bijdrage wordt nimmer genoeg geprezen en terwijl de wetenschapper met de roem gaat lopen zijn zij veelal degenen die de weg naar die roem geëffend hebben.

Daarom wil ik zeker Joke explixiet bedanken. Je was, samen met Griet, steeds bereid om mijn media te bereiden, terwijl je meestal slechts heel kort op voorhand wist wanneer dat zou moeten gebeuren en dit dus moest inpassen in je reeds overvolle agenda. En ook Janina, verdient een dankuwel om tijdens haar te korte stage in ons labo zo gretig te helpen en herhaaldelijk de schunnige praat in de slachthuizen te trotseren.

Lobke, Liliane en Bart van de dienst morfologie, jullie wil ik zeker ook niet vergeten, want jullie zijn uren bezig geweest met de voorbereiding van fixatief en het klaarmaken van mijn stalen voor histologie. Ook tijdens de weekends waren jullie, en zeker Lobke, steeds bereid om dit voor mij te doen, waarvoor mijn welgemeende dank.

Ook Ria wil ik bedanken om als een echte regie-assistente mijn agenda te volgen, zodat ik niet voor de zoveelste keer Bram vergat te halen op school of Tim vergat bij de onthaalmoeder omdat ik weer eens te geconcentreerd door een microscoop zat te turen.

Leila, bedankt om mijn veelvuldige uitstappen naar KI centra en slachthuizen in de SAP gereguleerde goede banen te leiden, zodat ik steeds verzekerd was en er zelf niet teveel diesel moest inpompen.

Nadine, ook jou moet ik in de bloemen zetten. Als er mij iemand gecoacht heeft doorheen alle hard labeur (met de uitnodigingen als absolute climax) en mij steeds op het rechte spoor zette als ik weer een zijstraatje dreigde in te slaan, dan ben jij het wel. Je nimmer aflatende interesse in mijn kroost was voor mij dikwijls een aangename verpozing in mijn bijwijlen chaotische onderzoeksgeest.

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En tot slot Roger. Zonder jou had ik het dus echt niet gehaald. De eindspurt in de verzengende hitte begin juni om mijn reeds weerbarstige word documenten, overladen met gigantische tabellen toch maar keurig in een pdf formaat te duwen en dan de headers en footers ook nog te laten kloppen heb ik volledig in jouw schoenen geschoven. Met veel zweetgenoegen en met koortsachtige ijver heb jij deze onbegonnen taak op twee dagen geklaard. Chapeau! Ik hoop dat je er geen kater aan over gehouden hebt. Ook de allerlaatste loodjes heb jij helpen dragen. Binnenkort neem je afscheid van onze vakgroep maar ik denk (en hoop eigenlijk stiekem) dat je dit niet gauw zult vergeten. Bedankt!

Ook een aantal buitenstaanders hielpen mee mijn kar duwen, Dirk Lips, jij niet in het minst. De motiverende gesprekken en je realiteitszin en logische benadering van het fenomeen doctoreren hielden mij met beide voetjes op de grond. Ik hoop dat onze samenwerking op weg naar een beter Witblauw een weergaloos succes wordt.

Marc en Andrea, en Marnix en Veronique, bedankt voor de vele hulp bij de kleine en grotere problemen die zich voordoen in de Tuybenslos 2. De opvang van de kinderen en de verzorging van de dieren als ik er weer eens niet ben is jullie nooit teveel. Het is fijn buren te hebben waarop je altijd kunt terugvallen. Bedankt!

Ook Marcel Christiaenen, Dirk Van Thielen en Walter Van Hofstraeten wil ik bedanken voor het vertrouwen dat zij in mij gesteld hebben voor de toekomst. Ik hoop echt op een productieve samenwerking en nu het doctorale zwaard van Damocles boven mijn hoofd verdwenen is zie ik de runderproblemen met enthousiasme tegemoet.

En last but not least, mijn familie. Mijn ouders wil ik oprecht bedanken voor de kansen die zij voor mij en mijn zussen geschapen hebben om verder te studeren in de richtingen die we wilden. Terwijl ma ons extreem probeerde te verwennen als we thuis waren tijdens de examens was pa degene die ons met peptalk op de been hield. Bedankt daarvoor. Ook mijn beide zussen en schoonbroers wil ik bedanken voor hun interesse in ‘datgeen waar ik nu weer mee bezig was’. Bedankt Lieve om pa bij te brengen dat je een doctoraat niet gauw even op een weekend in elkaar flanst.

Ook mijn schoonouders wil ik heel expliciet vermelden, aangezien zij echt meegeleefd hebben met mijn kleine overwinningen en tegenslagen op weg naar mijn uiteindelijke verdediging. Jacques, jouw nimmer aflatende interesse in mijn werk, mijn onderzoek, en mijn dromen, terwijl je zelf veel grotere demonen moest trotseren dan gelijk wie zal ik nooit ofte nimmer vergeten.

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En tenslotte, mijn allerdierbaarsten: Boke, beer Bram en Timmie, het mooiste wat mij ooit overkomen is. Wat en waar zou ik zijn zonder jullie? Dit doctoraat heeft jullie zeker zoveel opofferingen gekost als voor mijzelf. De vele weken dat ik gewoon niet thuiskwam omdat ik in Nederland boven de stieren bleef slapen, de troosteloze weekends dat ik nog maar eens van s’morgens vroeg tot s’avonds laat achter een microscoop ging zitten en jullie aan jullie lot overliet, en de spanning die ik ook meebracht naar huis toen ik mijn deadlines dreigde te overschreiden. Geduldig bleven jullie wachten tot dit alles overwaaide, en nu is het eindelijk zover. De boomhut die ik de kinderen beloofde moet nu maar eens in de steigers, en ook de kaders raken nu zeker wel aan de muur, chou. En ja Brammie, ik zal vanaf nu mijn GSM afzetten in het weekend en de boeren vertellen dat ik kinderen heb. Begin de weekends al maar vol te boeken!

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CURRICULUM VITAE

Geert (Gerard Cornelius) Hoflack werd geboren op 6 augustus 1969 te Ieper. Na het behalen van het diploma van het hoger secundair onderwijs aan het Klein Seminarie te Roeselare (laureaat Wetenschappelijke A), begon hij in 1987 met de studie Diergeneeskunde aan de Universiteit Gent. Hij behaalde in 1993 het diploma van Doctor in de Diergeneeskunde met onderscheiding.

Na een aantal dierenartsen te hebben vervangen tijdens de vakantieperiode werkte hij gedurende 4 jaar als grote huisdierenpracticus in de provincie Vlaams-Brabant, waar zijn grote liefde voor het Belgisch Witblauwe ras ontstaan is. In 1997 trad hij in dienst bij de vakgroep Voortplanting, Verloskunde en Bedrijfsdiergeneeskunde van de Faculteit Diergeneeskunde, initieel als wetenschappelijk medewerker, later als assistent. In 2001 werd het onderzoek naar de vruchtbaarheid van Witblauwe stieren aangevat, wat uiteindelijk leidde tot dit proefschrift. In het najaar van 2003 werd het diploma Gerechtelijk Expert behaald aan de faculteit Rechtsgeleerdheid van de Universiteit Gent en in 2006 behaalde hij het getuigschrift van de Doctoraatsopleiding in de Diergeneeskundige Wetenschappen. Tijdens de ganse periode aan hogervermelde vakgroep was hij werkzaam in de ambulatorische kliniek van de Buitenpraktijk waar hij mee de dag-, nacht- en weekenddiensten verzorgde en de probleembedrijven vleesvee voor zijn rekening nam.

Geert Hoflack is auteur of mede-auteur van 23 wetenschappelijke publicaties in internationale en nationale tijdschriften en hij nam actief deel aan meerdere internationale congressen. In België en Nederland was hij een veelgevraagd spreker op rundveestudiedagen.

Geert Hoflack huwde in 1995 met Ingeborgh Polis en is de fiere vader van 2 zonen, Bram (05-08-1998) en Tim (21-01-2004).

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BIBLIOGRAPHY

Publications in international journals:

G. Hoflack, D. Maes, B. Mateusen, M. Verdonck, A. de Kruif. 2001. Efficacy of Tilmicosin Phosphate (Pulmotil premix) in Feed for the Treatment of a Clinical Outbreak of Actinobacillus pleuropneumoniae infection in Growing-Finishing Pigs. Journal of Veterinary Medicine, series B; 48, 655 - 664.

B. Mateusen, D. Maes, G. Hoflack, M. Verdonck, A. de Kruif. 2001. A comparative study of the preventive use of tilmicosin phosphate (Pulmotil premix) and Mycoplasma hyopneumoniae vaccination in a pig herd with chronic respiratory disease. Journal of Veterinary Medicine, series B; 48, 733 – 741.

G. Opsomer, H. De Bosschere, G. Vanroose, G. Hoflack, S. De Vliegher, A. de Kruif. 2001. An aggressive angiomyxoma in a cow. The Veterinary Record; 149, 594-595

T. Rijsselaere, A. Van Soom,G. Hoflack, D. Maes, A. de Kruif. 2004. Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyser. Theriogenology; 62, 1292 – 1306.

G. Hoflack, T. Rijsselaere, D. Maes, J. Dewulf, G. Opsomer, A. De Kruif, A. Van Soom. 2005. Validation and Usefulness of the Sperm Quality Analyzer (SQA II-C) for Bull Semen Analysis. Reproduction in Domestic Animals; 40 (3), 237 – 244.

G. Hoflack, A. Van Soom, D. Maes, A. de Kruif, G. Opsomer, L Duchateau. 2006. Breeding soundness and libido examination of Belgian Blue and Holstein Friesian artificial insemination bulls in Belgium and The Netherlands. Theriogenology; 66 (2), 207 – 216.

IGF Goovaerts , GG Hoflack, A Van Soom, J Dewulf, M Nichi, A de Kruif, PEJ Bols. 2006. Evaluation of epididymal semen quality using the Hamilton-Thorne analyser indicates variation between the two caudae epididymides of the same bull. Theriogenology; 66 (2), 323 - 330.

G. Hoflack, G. Opsomer, T. Rijsselaere, A. Van Soom, D. Maes, A. de Kruif, L. Duchateau. Comparison of computer assisted sperm motility analysis parameters in semen from Belgian Blue and Holstein Friesian bulls. Reproduction in Domestic Animals: In Press.

Bibliography

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G. Hoflack, G. Opsomer, A. Van Soom, D. Maes, A. de Kruif, L. Duchateau. Comparison of sperm quality of Belgian Blue and Holstein Friesian bulls. Theriogenology: In Press.

T. Rijsselaere, D. Maes, G. Hoflack, A. de Kruif, A. Van Soom. Effect of body weight, age and breeding history on canine sperm quality parameters measured by the Hamilton-Thorne Analyser. Reproduction in Domestic Animals: In Press.

G. Hoflack, W. Van den Broeck, D. Maes, K. Van Damme, G. Opsomer, L. Duchateau, A. de Kruif, H. Rodriguez-Martinez, A. Van Soom. Testicular dysfunction is responsible for low sperm quality in Belgian Blue bulls. Submitted.

Publications in national journals:

G. Opsomer, D. Beeckman, Y. Scheirlynck, G. Hoflack, A. de Kruif. 1999. Endometritis bij het rund: een blijvend dilemma voor de practicus? Vlaams Diergeneeskundig Tijdschrift; 68, 120-130.

S. De Vliegher, G. Opsomer, G. Hoflack, H. De Bosschere, P. Deprez, A. de Kruif. 2000. Een thymus lymphoma bij een vaars. 2000. Vlaams Diergeneeskundig tijdschrift; 69, 44-46.

G. Hoflack, J. Laureyns, A. Tallieu, A. de Kruif. 2001. Acute sterfte bij kalveren. Vlaams Diergeneeskundig Tijdschrift; 70, 131-137.

G. Hoflack, B. Mateusen, D. Maes, A. Van Soom, M. Verdonck, A. de Kruif. 2001. Embryotransplantatie bij het varken. Vlaams Diergeneeskundig Tijdschrift; 70, 344-354.

G. Hoflack, B. Mateusen, A. Van Soom, D. Maes, M. Verdonck, A. de Kruif. 2001. In vitro fertilisatie bij het varken. Vlaams Diergeneeskundig Tijdschrift; 70, 354-361.

G. Opsomer, S. De Vliegher, J. Laureyns, G. Hoflack, D. Beeckman, A. De Kruif. 2001. Verhoogd coligetal in de tankmelk door Klebsiella oxytoca mastitis. Vlaams Diergeneeskundig Tijdschrift; 70, 50-53.

G. Opsomer, H. De Bosschere, G. Vanroose, G. Hoflack, S. De Vliegher, A. de Kruif. 2001. Een agressief angiomyxoma bij een koe. Vlaams Diergeneeskundig Tijdschrift; 70, 138-141.

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S. De Vliegher, J. Laureyns, G. Hoflack, D. Beeckman, G. Opsomer, A. de Kruif. 2001. Een geval van een fataal verlopende pleuritis veroorzaakt door een slokdarmdivertikel bij een vaars. Vlaams Diergeneeskundig Tijdschrift; 70, 405-407.

G. Hoflack, J. Dewulf, S. De Vliegher, A. de Kruif. 2002. Geslachtsdeterminatie van de foetus bij koeien met behulp van het ovatec/trac toestel. Vlaams Diergeneeskundig Tijdschrift; 71, 74 – 80.

G. Hoflack en A. De Kruif. 2003. Enkele aspecten van de diergeneeskundige begeleiding van bedrijven met Belgisch Witblauwe zoogkoeien. Vlaams Diergeneeskundig Tijdschrift; 72, 243 – 255.

D. Beeckman, S. De Vliegher, G. Hoflack, G. Opsomer, A. de Kruif . 2003. Subklinische Pseudomonas Aeruginosa mastitis op een melkveebedrijf. Vlaams Diergeneeskundig Tijdschrift; 72, 108 – 111.

G. Hoflack, J. Laureyns, A. de Kruif. 2004. Het afscheuren van de navelstreng bij kalveren. Vlaams Diergeneeskundig Tijdschrift; 73, 53-57.

G. Hoflack, G. Opsomer, D. Maes, A. de Kruif, A. Van Soom. 2006. Assessing bull fertility: the breeding soundness evaluation. The Flemish Veterinary Journal; 75, 216 – 227.

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