I

 

REPRODUCTIVE TRAITS AND FREEZABILITY OF SEMEN OF RAMS FROM TWO DESERT SHEEP 

ECOTYPES IN THE SUDAN. 

BY

Babiker Abdel Atti Elsharif 

B.V.Sc., M.V.Sc. University of Khartoum.

 

                   

A Thesis

Submitted in fulfillment of the requirements of 

The University of Khartoum for the Degree of Ph.D. 

 

Supervised by

Dr. Sharaf  A. Makawi 

B.V.Sc., Ph.D. University of Khartoum. 

  

 

 

Department of Reproduction and Obstetrics,

Faculty of Veterinary Medicine.       

 January, 2010.                           

              

II

 

 

 

Dedications To my father …………….....

To my mother……………..

To my wife……………..…

To my sons and daughter

With endless love

 

III

Table of Contents 

Dedication .............................................................................................................. II

Table of contents ................................................................................................. III

List of tables ...................................................................................................... VIII

List of figures.......................................................................................................IX

Acknowledgement. ............................................................................................ X

English Abstract ............................................................................................... XII

Arabic Abstract ................................................................................................ XV

Introduction ...................................................................................................XV1I

Chapter one: .............................................................................................

Review of literature: ..............................................................................1

1-Reproduction in sheep...........................................................................2

1-1 Libido and mating behaviour in rams ...............................................2

1-2 Semen . ..............................................................................................5

1-3 Semen characteristics. .......................................................................6

1-3-1 Colour and consistency of semen. ................................................7

1-3-2 Volume of ejaculate. ......................................................................8

1-3-3 Concentration of spermatozoa .......................................................8

1-3-4 Sperm motility. ..............................................................................9

1-3-5 Sperm cell morphology .................................................................9

1-4 Factors affecting semen production. .............................................11

1-4-1 Age of the ram. ............................................................................11

1-4-2 Nutrition. ......................................................................................11

1-4-3 Testes size. ...................................................................................14

IV

1-4-4 Environment ................................................................................17

1-4-4-1 Temperature. ............................................................................18

1-4-4-2 Photoperiod ..............................................................................19

1-4-5 Frequency of semen collection. ...................................................20

1-4-6 Breed ............................................................................................21

1-5 Seasonal effect on reproduction ......................................................22

1-6 Semen collection. ............................................................................24

1-6-1 Collection from the vagina after mating ......................................24

1-6-2Artificial vagina. ...........................................................................24

1-6-3 Electro-ejaculation. ......................................................................25

1-7 Semen dilution. ...............................................................................26

1-8 Semen freezing ................................................................................32

1-9 Cryopreservation of spermatozoa. ..................................................33

1-10 Thawing of frozen semen . ..........................................................35

1-11 Oestrous synchronization. .............................................................36

1-12 Insemination of ewes ....................................................................40

1-12-1 Vaginal insemination. ................................................................42

1-12-2 Cervical insemination. ..............................................................42

1-12-3 Laparoscopic insemination. ......................................................45

1-13 Fertility following AI. ...................................................................46

Chapter two: ............................................................................................

2- Materials and methods: ..................................................................51

2-1 Site of study. ...................................................................................51

2-2 Latitude and climate. .......................................................................51

V

2-3 Animals. ..........................................................................................51

2-3-1 Housing ........................................................................................52

2-3-2 Feeding . ......................................................................................52

2-3-3 Body weight recording. ...............................................................53

2-3-4 Scrotal circumference measurements. .........................................53

2-4 Reaction time. .................................................................................53

2-5 Semen collection. ............................................................................53

2-6 Semen evaluation ............................................................................55

2-6-1 Appearance of ejaculate. ............................................................55

2-6-2 Volume of ejaculate ....................................................................55

2-6-3 Colour and consistency of semen. ..............................................55

2-6-4 Sperm motility. ...........................................................................56

2-6-4-1 Mass activity . ..........................................................................56

2-6-4-2 Individual motility. ...................................................................57

2-6-5 Sperm cell concentration . ..........................................................57

2-6-6 Sperm cell morphology. .............................................................59

2-6-6-1 Wet preparation. .......................................................................59

2-6-6-2 Dry preparation ........................................................................59

2-6-7 Live-dead spermatozoa. .............................................................59

2-7 Extender preparation. ...................................................................60

2-8 Semen dilution. .............................................................................60

2-9 Semen cooling. ...............................................................................61

2-10 Semen packing. .............................................................................61

2-11 Semen freezing. .............................................................................62

VI

2.12 Pre- and post-freezing rejected samples ………………………....62

2-13 Thawing of frozen semen. ...........................................................62

2-14 Oestrous synchronization. .............................................................62

2-15 Insemination of ewes. ..................................................................63

2-16 Statistical analysis .........................................................................66

2-17 Conception rate…………………………………………………..64

2-18 Experimental design……………………………………………..64

2-19 Statistical analysis………………………………………………..65

Chapter three ...........................................................................................

3-Results: ..............................................................................................68

3-1 Semen characteristics ......................................................................68

3-1-1 Colour and consistency of ejaculate. ...........................................68

3-1-2 Ejaculate volume. ........................................................................69

3-1-3 Mass activity. ...............................................................................70

3-1-4 Individual motility .......................................................................71

3-1-5 Sperm cell concentration. ............................................................72

3-1-6 live sperm count. .........................................................................72

3-1-7 Sperm cell abnormalities. ...........................................................73

3-2 Live body weight. ........................................................................74

3-3 Scrotal circumference. .................................................................75

3-4 Relationship between body weight, scrotal circumference, semen

volume and sperm cell concentration.....................................................76

3.5 Reaction time. ................................................................................77

3-6 Freezability of semen. ..................................................................79

VII

3-7 Conception rate. ...........................................................................81

Chapter four: ...........................................................................................

Discussion: ............................................................................................84

4-1 Semen characteristics. .....................................................................85

4-1-1 Colour and consistency of ejaculate ............................................85

4-1-2 Ejaculate volume .........................................................................85

4-1-3 Mass activity. ...............................................................................87

4-1-4 Individual motility. ......................................................................87

4-1-5 Sperm cell concentration. ............................................................88

4-1-6 Live Sperm count. ........................................................................89

4-1-7 Sperm cell abnormalities. ............................................................90

4-2 Scrotal circumference. ....................................................................92

4-3 Reaction time. .................................................................................93

4-4 Freezability of spermatozoa. ...........................................................94

4-5 Rejection rate. ..................................................................................95

4-6 Fertility following AI. .....................................................................96

4-7 Conclusions and recommendations. ...............................................98

5- References. .....................................................................................100

 

 

VIII

List of Tables 

1-............................................................................................... N

umber and percentage of ejaculates with different colours and

consistency of Hamari and Kabashi rams..................................68

2- Number and percentage of watery ejaculates in different seasons

of the year of Hamari and Kabashi rams………………………69

3- Means and standard errors of semen traits in different breed….70

4- Means and standard errors of semen traits in different seasons.71

5- Means and standard errors of sperm cell abnormalities in

different breed………………………………………………….74

6- Means and standard errors of sperm cell abnormalities in

different seasons……………………………………………….75

7-............................................................................................... E

ffect of season and breed on scrotal circumference and body

weight..........................................................................................79

8-............................................................................................... P

erson`s correlation coefficient between body weight, scrotal

circumference, semen volume and sperm cell concentration from

Hamari rams................................................................................80

9-............................................................................................... P

erson`s correlation coefficient between body weight, scrotal

circumference, semen volume and sperm cell concentration from

Kabashi rams...............................................................................80

         10‐The reaction time of Hamari and Kabashi rams in different

seasons of the years ....................................................................81

11-The effect of breed and season on pre- and post-freezing rejection

IX

batches and post-thaw motility of frozen semen……………...…82

12-Fertility parameters of frozen-thawed semen obtained from

              Hamari and Kabashi rams ........................................................82

List of figures  

1- The effect of season on sperm abnormalities of Hamari rams…………………………………………………………….76

2- The effect of season on sperm abnormalities of Kabashi rams…………………………………………………………….77

3- The effect of season on pre- and post-rejection sample rate of Hamari rams…………………………………………….76

4- The effect of season on pre- and post-rejection sample rate of Kabashi rams………………………...………………….76

5- The effect of breed on pre-, post and total percentage of rejected samples ……………………………………………….76

Appendix:

1-Climatic conditions in Khartoum during the experimental period..141

2-Semen characteristics Anova table…………………..…………….142

3-Sperm cell abnormalities Anova table…………………..…………143

X

Acknowledgements

I wish to express my sincere gratitude to Dr. Sharaf A. Makawi,

Department of Reproduction and Obstetrics, Faculty of Veterinary

Medicine, University of Khartoum, for his keen supervision, deep

interest, invaluable advice and guidance during this work, to whom I am

very grateful.

My sincere acknowledgement is also due to Dr. Faisal Hassan

Ibrahim, the former executive manager of LADCO (Live-stock and

Agricultural Development Company) whose financial support,

enthusiasm and close co-operation have made this investigation

possible.

I am most grateful to Dr. Adil Salim Al Garai, Faculty of

Veterinary Medicine, University of Khartoum, Dr. Khalid Alssabayil,

King Saud University, Qasim, Agriculture and Veterinary Collage,

K.S.A., Mr. Mohamed Zein Elsharif, Dr.Suhair Sayed, for their fruitful

assistance, for providing me with many scientific papers.

For expert technical assistance I wish to acknowledge my

indebtedness to my colleagues at the National Animal Breeding and AI

XI

Centre, Kuku, Dr. Mohamed Saad, Dr. Salih Graham, Miss. Somaya

Mohamed Adam and Miss. Husna Hussain.

I would like to express my sincere gratitude to Mr. Taha Yasin,

for his assistance for the application of the oestrous synchronization and

AI programme in ewes, and I am most grateful for his friendship.

My thanks are also due to Mr. Emam, Mr. Abdalla Dago and

Mr. Abu Amna Osman, for their care after the animals during the course

of this study.

Also I wish to thank Dr. Mohamed Khair Abdella, Faculty of

Animal Production, University of Khartoum, for his help with statistical

analysis and results of this experiment.

My thanks also due to Meteorology Department and Mahmoud

El Hassan, for providing me with meteorological data.

Last, but not the least, I wish to sincerely thank my family, my

wife, my sons, Ayman, Ahmed, Mohanad and Awap and my daughter

Shemookh. They know how to create a comfortable studying

environment for me.

XII

Abstract

The objective of this experiment was to study the influence of breed

and season on sexual performance and semen characteristics of two

Sudan Desert sheep ecotypes. Freezability and fertility of frozen semen,

collected from the two breeds, was also investigated. The rams were

2 - 4 years old at the start of the experiment with initial average live

body weights of 73.60 and 61.20 kg for Hamari and Kabashi rams,

respectively. All animals were maintained under uniform conditions of

feeding and management. A total of 293 semen ejaculates were

collected by means of an artificial vagina from six Hamari and three

Kabashi rams. Data of Hamari and Kabashi rams showed that the over-

all means for seminal attributes were 1.31 and 1.23 ml for semen

volume, 2.73 and 2.37 scores for mass motility, 68.11% and 67.91% for

individual motility, 2.83 and 2.95(x109) sperm/ml for sperm cell

concentration, 76.90% and 84.73% for live spermatozoa. The breed had

a significant effect on live cell percentage and mid-piece abnormalities.

The summer exerted a significant effect on all semen characteristics and

sperm cell abnormalities except semen volume. Reaction time was

XIII

16.14 for Hamari and 20.10 seconds for Kabashi rams. Breed and

winter season exerted significant effect on RT. Mean values of SC were

33.12 and 31.38 cm for Hamari and Kabashi rams, respectively. Breed

exerted a significant effect on SC but the season of the year did not.

The ram semen was diluted with extenders containing glucose,

egg yolk and glycerol and buffered with Tris-citrate. The post-thaw

progressive motility were 56.75% and 56.19% and the incidence of total

rejected batches of semen before and after freezing were 46.15% and

38.79% for Hamari and Kabashi rams, respectively. Breed and season

had no significant effect on post-thaw motility of frozen semen, but

there was a significant difference (P< 0.05) between the two breeds and

summer season in the rate of rejected semen after freezing (15.38% vs.

6.35%); better results were obtained with Kabashi breed. One hundred

and fourteen ewes, aged 2-3 years, were given vaginal progestagen

impregenated sponges for 14 days and were injected with 500 i.u PMSG

on day of sponge removal for oestrous synchronization. All females

were inseminated in the cervix with 0.5 ml frozen-thawed semen, 55

hours after sponge removal. There was no significant difference in

pregnancy rate after AI and in the lambing rates and prolificacy between

Hamari and Kabashi breeds.

XIV

The present study showed that the two breeds are continuous

breeding animals and they are capable of producing semen of good

quality all the year round. The semen collected was suitable for freezing

and the results obtained after cervical insemination will positively

contribute to intensive sheep production.

XV

 

المستخلص

على النشاط الجنسي وخواص السائل مواسمال هدفت التجربة لدراسة تأثير الساللة و

شملت الدراسة اختبار قابلية تجميد السائل . المنوي لساللتين من الضأن الصحراوي السوداني

سنوات  4- 2آان عمر الخراف. المنوي ومن ثم خصوبة النطف المجمدة لتلك الساللتين

آل الخراف . آجم للحمري والكباشي على التوالي61.2 و 73.6وزنها عند بداية الدراسة و

خراف حمرية 6قذفة منوية من 293 ت جمع. تمت تغذيتها ورعايتها تحت ظروف مماثلة

مل لحجم 1.23و 1.31 آان متوسط خواص القذفات المنويةت النتائج أنوضحأ .آباشية 3و

2.83، للحرآة الفردية % 68.11 و %67.91، درجة للحرآة الجماعية 2.73و 2.37 ،القذفة

. للحيامن الحية%84.73 و 76.90%، لعدد الحيوانات المنوية في المليلتر)109x 2.95 (و

آما أثر . أثرت الساللة تأثيرا معنويا على نسبة الحيامن الحية وعيوب عنق الحيوانات المنوية

على آل تشوهات الحيوانات المنوية وخصائص السائل المنوى فصل الصيف تأثيرا معنويا

ثانية 20.10 و 16.14 للحمرىوجد أن متوسط التفاعل الجنسي . ماعدا حجم القذفة

33.12متوسط محيط المناسل بلغ . تأثيرا معنويا على زمن التفاعل الجنسى لهالشتاء.لكباشيل

ة لها تأثيرا معنويا على هذه الصفة أما فصول وآان السالل. للكباشىسم 31.38وللحمرى

.السنة فلم يكن لها تأثير معنوى

صفار البيض والجلسرول، تم تخفيف السائل المنوى فى مخففات تتكون من الجلكوز

اإلذابة و التجميدمتوسط حرآة الحيوانات المنوية بعدبلغ . السترات-ومعادلتها بالترس

للحمري %38.79و %46.15 ات المبعدة قبل وبعد التجميد العيننسبةو%. 56.19 و56.75%

لم يكن للساللة وفصول السنة تأثير معنوى على حرآة الحيوانات .والكباشي على التوالي

XVI

البيانات توضح وجود فروق معنوية بين الساللتين وفصل الصيف المنوية بعد اإلذابة ولكن

ل على نتائج جيده مع الحصو )%6.35 مقابل 15.38(في نسبة العينات المبعدة بعد التجميد

اسفنجات مهبلية مشبعة بهرمون سنوات 2 - 3نعجة في عمر 114 أعطيت.لساللة الكباشي

وحدة عالمية من مصل الفرس الحامل 500يوما ثم حقنت 14 البروجستيرون لمدة

PMSG) ( 0.5 عنق الرحم ب فىلقحت آل النعاج . عند ازالة االسفنجة وذلك إلحداث الشبق

ا فرقم يكن هناكل. ساعة من ازالة االسفنجة المهبلية55 مذاب بعد -مل سائل منوى مجمد

.ين الحمري والكباشيب والخصوبة نسبة الوالداتو في نسبة الحمل بعد التلقيح امعنوي

و وانتاج سائل منوي ذمستمرالتناسل القدرة على الامتين لهل أن السال الدراسة أوضحت

والنتائج المتحصل عليها من التلقيح في .وامكانية تجميده جيده على مدار العام مواصفات

. لتوليد األغنامواعدة تشجع فى تطبيقها على نطاق واسعأعطت نتائج عنق الرحم

XVII

Introduction:

The sheep population in the Sudan was estimated to be 51.067

million heads, with an annual increasing rate of one percent (Ministry of

Animal Resources: 2008).

Mcleory (1961) classified the sheep of Sudan into eight distinct

ecotypes according to locality, tribe and origin. Of these ecotypes, the

Sudan Desert sheep constitutes 65 percent of the sheep population in the

country. These animals are normally found in the desert and semi-desert

areas of the Sudan (annual rain-fall ranging between 75 and 300

mm).The desert sheep dominate the export market of live sheep and

mutton. However, Hamari and Kabashi sub-types are dominant over the

other Desert sub-types and collectively the Desert sheep play an

important role in the national economy either through satisfying the

local need or through their export revenue of hard currency (cited

Babiker et al.,1988).

Most of sheep population, in the Sudan, is raised under the

traditional nomadic system of management. Thus they are exposed to a

wide range of adverse climatic and nutritional stresses in addition to

XVIII

endemic diseases. Official and private sectors exert efforts to shift

towards intensive modern system were limited and short-lived.

In any breeding programmes, the use of superior sires could have

a direct effect on the reproductive and productive traits of the resultant

progeny, and this could satisfy both the producer and consumer interest.

Breeding soundness examination is the first step in management of rams

for optimum breeding performance. Selection of the most fertile ram

may not only improve the lamb crop but also allows for a large ewe to

ram ratio and thus reduces the cost of keeping many rams.

One of the keys to successful artificial insemination (AI)

programmes is to know when to inseminate the female. There are

various methods of oestrous synchronization which have been used

successfully in sheep. Utilization of progestagins and follicular

stimulants (eCG) for controlling the oestrous cycle and for inducing

out-of-season breeding has been commercially available for many years.

When oestrus synchronization is employed, extra rams are required to

mate the ewes over a very short period, AI will reduce the number of

rams needed and facilitate a faster insemination.

The progress made in cryopreservation of ram semen has opened

the possibility for conservation and utilizing of frozen semen of elite

rams in sheep improvement programme. Though, AI with frozen semen

XIX

in sheep has not been well spread in the world, probably because of the

intensive nature of sheep breeding and relatively poor fertility results

obtained when frozen semen is used for cervical insemination

(Söderquist, 1999).

However, the knowledge gained in studying the reproductive

pattern of rams is useful when deciding the best period to breed ewes

with high quality semen or the best periods to harvest ram semen meant

for freezing for AI purposes.

Previous studies on the measurement of the reproductive

capacity, seasonality and fertility of Sudan Desert sheep have been

carried by many investigators (Galil and Galil, 1982; Alsayed, 1996;

Suhair, 1997; Manahil, 1999).

Introduction of modern techniques such as oestrous

synchronization and AI with fresh diluted semen in the Sudan sheep is

recent and confined to research institutes (Alsayed, 1996; Manahil,

1999; El Mubark, 2001; Alsayed, 2001). However, wide-scale adoption

of AI using fresh or frozen semen and monitoring of the success rate of

this procedure remained to be investigated.

The basis of production of ram semen in the laboratory is to train

the ram to ejaculate in an artificial vagina. Then the semen is collected,

XX

grossly and microscopically evaluated. Then the semen is process,

frozen and stored until insemination.

The objective of the study:

(1) To study the influence of the breed and season on semen quality

and sexual performance.

(2) To assess the relationship between semen volume, sperm cell

concentration, body weight and scrotal circumference

measurement of the two breeds.

(3) To compare the freezability of the semen of the two breeds in

different seasons of the year.

(4) To study the conception rate in the oestrous synchronized ewes

inseminated with the frozen semen obtained from the two breeds.

It is hoped that the production of frozen semen and the

understanding of the seasonal impacts on the reproductive performance

of the Hamari and Kabashi breeds would assist to formulate a rational

breeding programme for genetic improvement at the appropriate time of

the year and at different localities using semen from the same proven

sire. This will offer significant opportunities to reach maximum

exploitation of this superior genetic material through this technique

which will be introduced for the first time in the Sudan.

XXI

1  

 

 

 

 

 

 

 

 

 

Literature Review

 

 

 

 

 

 

 

 

2  

1. Reproduction in sheep:

1.1 Libido and mating behaviour in rams:

The term libido is commonly used to describe sex-drive in entire

male animals (Fraser, 1974). Libido is primarily dependent upon

androgenic steroid hormones, which allow mating, and aggressive

behaviour to occur, as well as maintaining the function of all parts of the

male reproductive system (Arthur et al., 1998). The most obvious

expression of libido is during the copulatory behaviour which can only be

expressed post-puberally. However, the actions of copulation may be

mimicked by pre-puberal males and by females while under oestrogenic

influence (Chenoweth, 1981). Ram exhibit libido by behavioural

components such as nosing the female perineum, nudging, flicking of

tongue, striking out with a fore-limb and low pitched bleats (Nabil et al.,

2006). Although these activities may all is described as sexual behaviour

the following definition can be used (Chenoweth, 1981):

Libido: the willingness and eagerness of a male animal to mount and

attempt service of a female.

Mating behaviour: The behaviour of the male animal in the periods

immediately before, during and after service.

Sexual performance tests have been used as a tool to predict ram

performance (Kilgour, 1993; Stellflug et al., 2006). Libido and mating

3  

ability are important traits which can affect production significantly

(Chenoweth, 1981). In intensive breeding programmes a single ram can

breed 8–10 ewes daily, or well over 100 ewes in the course of a single

oestrous cycle (Chenoweth, 1981). In some trials measuring the

reproductive capacity of rams, great variations have been reported

between individuals (Hulet et al., 1962). However, rams tend to distribute

their services among receptive ewes through the choice of ewes with

which they have not mated before or with which they have mated less

frequently (Chenoweth, 1981). In closely, confined groups of ewes,

dominant rams inseminate the majority of ewes and may prevent other

rams from breeding (Hulet et al., 1975).

Different methods have been employed to assess sex-drive in rams

including reaction time to service, the number of services within a limited

time period and libido score (Chenoweth, 1981). Changing the stimulus

female for second ejaculation had no effect on the semen characteristics

or the inter-ejaculation interval. However, this increased the number of

mounts needed to achieve a second ejaculation, while preventing physical

contact with the stimulus animal after the first ejaculation reduced the

number of mounts (Lezama et al., 2003).

Yearling rams engaged in more leg-kicking bouts and showed

higher mounting frequency than two years Awassi rams (Kridli and Said,

4  

1999). Sexual performance of rams that differ in age (maturation) and

sexual experience, when they have one or two relatively brief exposures

to oestrous ewes can bring the sexual performance of virgin rams up to

the levels comparable to that of experienced males (Price et al., 1991).

However, exposing sexually naive rams to oestrous ewes before the

breeding season may be necessary to improve their sexual performance

(Kridli and Said, 1999). Trejo et al. (1990) reported that, breed of ram

had no significant effect on the interval from contact with a female to

ejaculation (reaction time), the interval between the first and second

ejaculation (recuperation time), the total number of services per ram in 30

minutes or the number of mounts per ejaculation. Lezama et al. (2001)

found that, spreading semen from a different ram in the vulva of the

stimulus animal was more efficient than spreading semen of the same

male or the absence of semen, in shortening the inter-ejaculatory interval,

but no relationship was found between this effect and semen

characteristics.

It is well known in temperate zones that ram sexual activity is

subjected to seasonal variations and the intensities varies from breed to

breed. These variations can affect all the components of reproductive

function particularly semen quality and its fertilizing ability (Salamon

and Robinson, 1962; Smyth and Gordon, 1977; Dufour et al., 1984;

5  

Melpomeni et al., 2004). However, rams have been reported to show

more sexual activity in autumn, while in another study it was observed

that some decline occurred in the libido of Targhee rams during the

spring (Chenoweth, 1981). However, Mickelsen et al. (1981) suggested

that, if the sex-drive were normal in some breeds of rams, perhaps twice a

year breeding would be realistic, if fertility of ewes were normal. Ibrahim

(1997) found that, the mean reaction time for local and cross-bred rams

(Local x Chios) raised in United Arab Emirates were reported to have an

average RT of 43.7 seconds which affected mainly by season.

Furthermore, he showed that, the reaction time was shorter (20 seconds)

in summer and longer (72 seconds) in autumn, indicating that the high

temperature in summer did not decrease sexual activity of the rams.

It has been reported that rams fed on a high protein supplement

displayed a more intense sex-drive than rams fed on a low protein

supplement (Chenoweth, 1981).

1.2 Semen:

Semen is the normal discharge of the reproductive organs

ejaculated by the male. It contains sperm cells and seminal plasma. The

seminal plasma is a mixture of fluids secreted mainly by the vesicular

glands, with lesser contribution from the testes, epididymides, deferent

ducts and the accessory glands (Herman et al., 1994). The seminal plasma

6  

of rams is clear or opaque fluid, usually isotonic, neutral fluid with a pH

close to 7.0 (Evans and Maxwell, 1987). However, prolonged exposure of

spermatozoa to seminal plasma has been found to have an adverse effect

on sperm function. The loss of sperm heterogeneity and membrane

integrity during treatment and that the freezing-thawing process, were

less apparent when seminal plasma had been removed from semen

samples before freezing (Ollero et al., 1997). Therefore, the separation of

mammalian spermatozoa from seminal plasma is a practice routinely used

in the laboratory and in assisted reproductive technology application

(Perez et al., 2001). In contrast, the seminal plasma components

prevented and reverted cold-shock damage on sperm membrane and

improved the viability and fertility of frozen-thawed sperm (Jobim et al.,

2005). Moreover, the seminal plasma proteins that bound to the sperm

surface may be responsible for sperm membrane stabilization

(Dominguez et al., 2008).

1.3 Semen characteristics:

The routine semen evaluation for artificial insemination application

relies on assessing a number of parameters such as sperm motility,

concentration and morphology for prediction of fertility in the male

(Correa and Zavos, 1994). The hypo-osmotic swelling test (HOS-test) is

used to evaluate the sperm functional status (integrity) and permeability

7  

(Moussa, 1999 and Zeidan et al., 2006).The development of computer-

assisted sperm analysis (CASA) allows an objective assessment of

different cell characteristics: motion, velocity and morphology (Sancho et

al., 1998 and Verstegen et al., 2002).

1.3.1 Colour and consistency of semen:

The normal colour of semen is milky–white or pale cream (Evans

and Maxwell, 1987). Some rams can have a very yellowish colour of a

normal creamy ejaculate; this is normal for those rams which have high

levels of riboflavin in the ejaculate and which is a harmless pigment

(Nabil et al., 2006). Mature ram during breeding season would have a

thick, creamy consistency semen samples. A sub-normal ram may

produce a sample looks like cloudy water or thin milk (David, 1994). Any

abnormal colour, odour or foreign material such as hair, faecal particles,

bedding and dirt indicate poor hygienic quality (Nabil et al., 2006). The

presence of blood or pus flakes may indicate affection in reproductive

tract (Nabil et al., 2006). Dabas et al. (1997) reported that, most of the

semen samples collected from six Potanwadi rams, aged 2-4 years, during

the pre-breeding, breeding and post-breeding seasons, showed creamy

thick (48%) or creamy white and thick (42%) consistency. The normal

semen colour of Sudan Desert sheep was found to be creamy-white

varying from slightly thick to very thick (Galil and Galil, 1982).

8  

1.3.2 Volume of ejaculate:

Normal volume of ejaculate in adult rams (1.5 years of age or

older) is of 0.8 to 1.2 millilitre (ml), with a mean of one ml (Foote, 1974;

Gomes, 1977). However, Herman et al.(1994) reported that, the average

volume of ram ejaculate was about 1.0 to 1.5 ml. Dabas et al. (1997)

found that the ejaculate of Patanwadi rams averaged 0.96+0.03 ml. The

Sudan Desert sheep showed an average of 0.99 ml with a range of 0.82 to

1.22 ml for the ejaculate volume (Galil and Galil, 1982). Moreover, the

average ejaculate volume that was reported by Alsayed (1996) in the

same breed was 1.37 ml, while that found by Manahil (1999) averaged

1.62 ml. Chiboka (1980) found that, in the African dwarf rams , there was

a significant week seasonal effect on the volume of semen during the dry

period but not in the rainy season.

1.3.3 Concentration of spermatozoa:

Good quality ram semen contains 3.5 to 6.0 billion spermatozoa

per ml (Evans and Maxwell, 1987). However, Herman et al. (1994)

reported that, the average ram ejaculate contains from 1 to 3 billion

sperm. Cameron et al.(1987) found that, the mean number of

spermatozoa ejaculated by an individual ram varied from 140 million to

1050 million, following depletion of the epididymal reserves of

spermatozoa. Galil and Galil (1982) found that, the sperm count of the

9  

Sudan Desert sheep was 2.452 billion per ml with a range of 1.85 to 3.4

billion. However, Alsayed (1996) reported that the average concentration

of spermatozoa was 2.5 billion per ml while that reported by Manahil

(1999) was 3.4 billion per ml for the same breed.

1.3.4 Sperm motility:

The normal percentage of motile spermatozoa is around 75 percent

with a range between 60 and 80 (Gomes, 1977). In the Sudan Desert

sheep, the sperm mass motility score was found to be 4.12 with a range of

3.19 to 4.8 scores and individual motility was 79.5% with a range of 63%

to 89% (Galil and Galil, 1982). Manahil (1999) reported that, the mass

motility score of Sudan Desert sheep ranged between 4 and 5 (average

4.3 + 0.5). The individual motility of Sudan Desert sheep ranged between

75–90% (average 83.9+5.3%).

1.3.5 Sperm cell morphology:

The evaluation of the incidence of morphological abnormal

spermatozoa in an ejaculate is important. Only sperm cells with intact

acrosome and normal structure are fertilizes the ovum. The normal

mature ram spermatozoa head is approximately 8 micrometer (µ m) long,

4 µ m wide and 1µ m thick. The mid-piece is approximately 14 µ m long

and the tail is 40 to 45 µ m long. The entire sperm measures

approximately 65 µ m (Sorensen, 1979).

10  

Under electronic microscope, the sperm head has a regular oval

shape with a pronounced apical ridge, a nuclear ring and two

protuberances on the lateral rim. The acrosome consisted of dark and

light zones, and the tail tapered off posterior to Jensen’s ring (Massanyi,

1989).

Many studies have shown that the initial ejaculate contains a

greater number of abnormal cells; these abnormalities consist, for the

most part, of head malformations and proximal cytoplasmic droplets,

which indicate incomplete spermatogenic activity and incomplete

epididymal function (Skinner and Rowson, 1968). The percentage of

abnormal spermatozoa showed larger variations between semen

collections and between different sires (Colas et al., 1990). The presence

of immature forms of sperms may indicate that a ram is being

overworked (David, 1994). Dabas et al. (1997) found that, the average

abnormal spermatozoa of Patanwadi rams were 7.9+0.01 percent.

However, Galil and Galil (1982) observed that, the abnormal

spermatozoa of the Sudan Desert sheep was 3.5 percent with range of

2.40 to 4.99.

11  

1.4 Factors affecting semen production:

The main factors which may affect the quality and quantity of

semen include; age of the ram, nutrition, testes size, environment,

frequency of semen collection and breed.

1.4.1 Age of the ram:

In sheep as in most other species, the quality and quantity of semen

is strongly influenced by age of the animal (Demrici, 1993; Rege et al.,

2000). The age at the first appearance of spermatozoa in ejaculates in

three breeds of rams (Chios, Serres and Karaguniki) averaged 20.1+0.31,

20.4+0.37 and 20.6+54 weeks and body weight averaged 36.4+0.56,

36.5+0.7 and 34.9±0.99 kg respectively (Alexopoulos et al.,1991).

However, the semen volume, sperm motility score and sperm

concentration increased with increasing age, while the percentage of dead

and morphologically abnormal spermatozoa decreased (Alexopoulos et

al., 1991).

1.4.2 Nutrition:

The influence of nutrition on reproduction is mediated via the

effects of dietary constituent on the hypothalamic–pituitary gonadal axis

in both ewes and rams (Brown, 1994). The extensive supplementation of

the ram-lamb during intra-uterine and post-natal life could result in higher

testicular growth and sperm production when they become adults (Bielli

12  

et al., 2000). However, over-feeding of young rams could be detrimental

to their fertility (Fourie et al., 2004). The morphology of the seasonal

testis regression of Corridale rams is not easily reverted by the improved

feeding, so the enhancement in testis development when pre and post-

pubertal rams grazed improved pasture was short-lived. The tendency to

increase Sertoli cells numbers elicited by treating animals with improved

pasture plus grain supplementation from foetal to pre-pubertal life might

result in larger-lived consequences to spermatogenic capacity (Bielli,

1999).

Changes in the nutrition of mature rams lead to profound responses

in testicular size and therefore the rate of production of spermatozoa.

These effects are largely due to changes in the size of the testes and

seminiferous tubules and in the efficiency of spermatogenesis (Fernandez

et al., 1993; Martin and Walkden-Brown, 1995).

In mature rams, however, there is a delay of six to seven weeks in

the response of spermatozoa numbers to diet, reflecting the time it takes

for the development of spherical spermatoid in the germinal epithelium to

fully mature spermatozoa in the distal caudal epididymides (Robinson,

1996). The effect of nutrition has been demonstrated by results which

have shown that daily sperm production tends to decline as the level of

energy and protein in the diet declines. The rate of sperm production per

13  

gram of testis was approximately 40% greater in Merino rams maintained

on a high, rather than low plane of nutrition (Cameron et al., 1988).

Similarly, Oldham et al. (1978) achieved rates of spermatozoa production

of 13.6 billion versus 7.5 billion per day, through providing different

levels of nutrition. Martin and Walkden-Brown (1995) and Martin et al.

(1999) concluded that, nutritional effect can be divided into short-term

(10–20 days) effect that mainly act on the neuroendocrine system

(Stimulates Gn RH/LH pulse frequency) controlling testicular activity

and long-term (several months) effect that act on testicular growth and

sperm production. The increasing level of feeding by 30% of total

nutrients above the recommended level or 38% of the total protein,

increased semen volume per ejaculate and improved semen quality

(Khachirov, 1984). The addition of lysine at a rate of 4.36% and

methionine at a rate of 3.9 to 4.0% of the weight of crude protein, to the

diet of ram during the breeding season increased ejaculate volume by

17.8%, sperm concentration by 28.5%, sperm resistance by 17.1% and

fertilizing ability by 4.0% (Zpydnev and Orekhov, 1989).

Supplementation of the diet with 18–36 gm sun flower oil daily,

increased semen quality and quantity, motility increased by 11.3–12.4%

and conception rate using frozen semen was 5.6–10.7% higher for

supplemented rams (Davidenko et al., 1990). Rams given diet containing

12% cotton-seed meal did not differ from that of the control group in

14  

their body weight, semen volume, semen motility and concentration of

spermatozoa, but the percentage of abnormal spermatozoa was higher in

the cotton-seed fed rams (17%) than the controls. The histological

examination of the testes showed that rams fed cotton-seed meal had

larger lumen diameter, fewer number of cell layers and lower wall

thickness in seminiferous tubules, and smaller size of Sertoli and Leydig

cells than the control rams (Arshami and Ruttle, 1989). In rams fed diets

containing gossypol, ejaculated sperms appear normal under light

microscopy, but the integrity of the membrane of sperm cells may be

damaged due to the extensive damage of germinal epithelium (Randel et

al., 1992).

The effect of vitamin E and selenium added to the ration was

studied by Gokcen et al. (1990); they found that, the semen

characteristics and acrosomal integrity were significantly better in

supplemented animals than the controls. Tenlibaeva (1991) observed that,

the addition of vitamins A and E to the diet has a positive effect on sperm

production and on the fertility level of ewes.

1.4.3 Testes size:

Testes are the primary organs of reproduction in the male. They are

two in number, and are carried outside the body wall in the scrotum

(Herman et al., 1994). There are no significant differences between the

15  

weight and volume of the left and right testes and epididymides (Colyer,

1971; Martinez et al., 1994). The testes have at least two functions:

(1) Production of spermatozoa (male germ cells).

(2) Production of endocrine substances or male hormones

(particularly testosterone) that markedly affect the

development and behaviour of the male (Herman et al.,

1994).

Testis size has been regarded as a trait of choice in male (Matos and

Thomas, 1992). Scrotal circumference has been suggested as an indicator

of seminal performance and male libido as well as the age at puberty. The

testicular development in young rams was found more closely associated

with body weight than with its age (Matos and Thomas, 1992). Master

(1988) suggested that, the young ram to be used for breeding should have

scrotal circumference more than 30 centimetres (cm) and that value of

35 cm is the normal for two years old rams. However, the scrotal volume

decreased by 18–26% in rams mated for 4–6 weeks, with only part of the

decrease being accounted for by the 4–12% decrease in live weight

(Knight et al., 1987).

In many studies testicular measurements have been evaluated and

related to some seminal parameters, usually sperm concentration and

sperm motility (Ruttle et al., 1982; Gipson et al., 1985). Testicular

16  

circumference is directly related to sperm production and rams with

larger testes produce more sperm (Langford et al., 1998). However, the

ejaculate volume was found to be significantly correlated with testis size

(Vijil et al., 1987; Demirci, 1993). Rege et al. (2000) found that , genetic

correlations of scrotal circumference with semen volume (0.55±0.11),

mass motility (0.62±0.20), individual motility (0.54±0.12), concentration

(0.25±0.04) and proportion of abnormal spermatozoa (-0.75±0.24) in 12-

month for Menz and Horro old rams. The selection based on this trait,

have appreciable favourable correlation response in semen quality and

spermatozoa production. Follicular stimulating hormone (FSH) levels, at

all ages, were correlated negatively with scrotal size; these findings

indicate that FSH levels in ram lambs may be useful for predicting adult

testis function (Langford et al., 1998). Colas et al. (1990) concluded that,

lambs could inherit seasonal variation in testicular diameter.

In 3.5 to 4 years, Pelibuey rams, the daily sperm cell production

was found to be 27.5+1.5 million sperm cells per gram of testicular

parenchyma and 5094+366 million sperm cells per testis. Epididymal

sperm reserve accounted for 28.1+1.3 billion spermatozoa per organ

(Martinez et al., 1994). However, Cardoso and Queiroz (1988) reported

that, in Brazilian hairy rams, the mean values obtained for sperm

production were 22.8+8.9 million sperm cells per gram of testis

17  

parenchyma and 2.2+1.3 billion sperm-cells per testis per day. The

efficiency of spermatogenesis and degeneration of different

spermatogenic cells under normal conditions of the environment in rams

was 47.58 percent, since one spermatogonium produces 30-45 spermatids

against the expected number of 64 (Bilaspuri and Guraya, 1986). The

spermatogenic cycle was estimated to take 42.28 days from the first

spermotogonial mitosis of A1 spermatogonia to the liberation of

spermatozoa (Cardoso and Queiroz, 1988).

However, clinical examination of genital organs seems to offer a

reliable source of information about rams, and may be considered

adequate in sheep breeding (Weirzbowski and Kareta, 1993). The

consistency of testis is described as firm and elastic. Although there are

grades within these dimensions, (firm and elastic) deviations from this

including both soft and flabby and firm and dull, are also used. Any

enlargements of the epididymis should receive due attention (Gallway,

1966).

1.4.4 Environment:

Photoperiod and temperature are two of the most important

environmental factors influencing reproduction of sheep. Seasonal

variation in sexual activity among small ruminants depend on latitude and

day length. Melatonin secreted during the night by the pineal gland

18  

enables the animal to determine the length of the day. This hormone can

be used to advance mating season of sheep and increase its fecundity. The

use of artificial photoperiodic variations and melatonin eliminates or

alters seasonality of sperm production (Chemineau, 1993).

1.4.4.1 Temperature:

The effect of high temperature on the reproductive performance of

ram is well established. The exposure of the animal to hot temperature

results in a change in the morphological profile of the semen (Zamba,

1971; Marai et al., 2007). Under heat stress, libido, semen production and

fertilizing ability of males are significantly reduced. However, in females,

heat stress reduces the oestrous period and level of fecundity and

increases early embryonic mortality (Chemineau, 1993). In sub-tropical

conditions, the environmental temperature significantly affect sperm

motility, sperm concentration and sperm number per ejaculate (Daader et

al., 1987). Howarth (1969) suggested that the deleterious effect of

elevated temperature on sperm quality is due to damage sustained during

the late stages of spermatogenesis and would further attest to the apparent

resistance of epididymal spermatozoa to the effect of heat. The resistance

of epididymal spermatozoa to the effects of heat could be due to their

lower metabolic rate compared to dividing cells. Ibrahim (1997) observed

that, the best semen quality of local rams in United Arab Emirates was

19  

obtained in winter, but the semen collected in other seasons is also judged

to be of satisfactory quality. Galil and Galil (1982) concluded that, Sudan

Desert sheep appeared to be able to breed through-out the year and their

spermatogenesis was not seriously affected by high environmental

temperature.

1.4.4.2 Photoperiod:

The hypothesis proposes that melatonin secreted by the pineal

gland acts at separate sites in the brain and pituitary gland to regulate

gonodotrophin and prolactin secretion, and these responses mediate the

effects of photoperiod on the time and characteristics of the seasonal

reproductive cycle. The pituitary gland is the proposed site of action of

melatonin for the control of prolactin secretion (Lincoln, 1999). The

seasonal cycle of reproduction is a result of a complex interaction of at

least four distinct mechanisms (Pelletier, 1996):

• Light, which stimulates gonadotrophic activity mainly under

conditions of natural or artificial decreasing of day length?

• The intensity of gonadal steroid negative feed-back, which

varies depending on the day length.

• The existence of an inherent annual pattern of reproduction.

• The existence of photosensitive phase during which light

stimulates gonadotrophic activity.

20  

Thus gonadotrophins and gonadal steroid hormones act in concert with

prolactin to determine the functional state of the reproductive axis. This

interaction potentially affects the functional state of the gonads, accessory

sex glands, secondary sex characteristics and the expression of sexual and

aggressive behaviour during sexual cycle in seasonally breeding

mammals (Lincoln, 1999).

Further studies, showed that, light regime with alternating 1-2

month periods of increasing and decreasing day length stimulates the

gonadotrophins in alternate months and maintains permanent sexual

activity (Malpaux et al., 1995). With this regime, plasma testosterone

never reaches the levels which are considered inhibitory at the end of the

breeding season (Pelletier, 1996). This alternating light regime has

maintained the quantity and quality of semen at levels normally observed

during the breeding season (Pelletier, 1996). Although, the treatment with

melatonin may be considered as an alternative to photo-stimulation for

the preparation of rams for out-of-season breeding (Hanif and Williams,

1991).

1.4.5 Frequency of semen collection:

Frequent ejaculation (4, 5 or 8 times daily) for 14-36 days duration

produced a decrease in semen volume by 25–53%, sperm motility by 19-

36% and sperm concentration by 19–55% (Thwaites, 1995). Semen of the

21  

second ejaculate was significantly less in volume, greater in motility,

closer to neutrality and less per cell concentration when two separate

successive ejaculates were obtained, however, characteristic of the

second ejaculate fell within the normal range of high quality semen

(Ibrahim, 1997).

1.4.6 Breed:

Breed differences in semen quality and quantity was reported in

many studies. Langford et al. (1998) found that, Canadian rams produced

more sperm per ejaculate than Finnish Landrace (7.0 billion compared to

3.3 billion). Fernandez et al. (1993) observed that, the Corriedale rams

had lower semen production and sperm concentration and higher

incidence of sperm abnormalities than rams of other breeds (Polwarth,

Merilin and Merino). However, Vivanco (1989) studied the influence of

breed and reproductive characters of rams under high altitude conditions.

He found that, Criollos tended to have better semen quality than

Corriedales and Junins at first ejaculation and at all ages. Abdel-Rahman

et al .(2000) reported that the Somalian ( Barabari ) and Sudan (Sawakni)

breeds showed reduction in sperm concentration, percentage of live

spermatozoa, highest percentage of individual motility and the highest

concentration of sodium (Na) chloride (Cl) and inorganic phosphorus (P)

in the whole semen, seminal plasma and spermatozoa, while superior

22  

sperm density, percentage live spermatozoa and seminal concentration of

potassium (K) and calcium (Ca) were evidenced in the Najdi, Naemi and

to lesser extent, in Merino breeds. However, no breed difference was

observed in semen quality between local Emirate breed and its cross with

Chios breed (Ibrahim, 1997).

1.5 Seasonal effect on reproduction:

Reproduction in sheep is a complex mix of environmental factors

and hormonal responses in ewe and ram. Several studies have indicated

that semen production is influenced by seasonal changes (Colas and

Brice, 1976; Jennings, 1976; Loubser and Van Niekerk, 1983; Mandiki et

al., 1998). Dufour et al. (1984) showed that, the seasonal variation can

affect all the components of the reproductive traits, being lowest during

the summer and highest during the fall. Significant differences between

the breeding and non-breeding seasons were obtained from the blood.

The activities of the acid phosphatase, aspartate and alanine, amino-

transferase and concentration of total protein and sialic acid, all were due

to a significant decrease in values during breeding season (Karcheva and

Baulov, 1989). Sheep from temperate latitudes display seasonal variation

in their reproductive performance which is controlled by the annual

fluctuation in the day length (Malpaux et al., 1995). There is a seasonal

variation in the dimension of the reproductive organs of the animals and

23  

that during the breeding seasons, testicular size and weight are intimately

correlated (Nwoha, 1996). The seasonal variation in testis size has been

recorded, but rams appeared to be sexually active when introduced to

oestrous ewes out of season (Dyrmundsson et al., 1989; Melpomeni et

al.2004). The magnitude of seasonal variation in semen quality was not

sufficient to prevent rams from being used for breeding throughout the

year (Gündogan, 2006). Berg (1999) reported that, in rams there were no

total seasonal limitation of the sexual function, but the gonadal activity is

somewhat reduced in the period from early spring to next autumn. Dabas

et al. (1997) reported that, season had a significant effect on mass activity

of semen, pH, sperm concentration and percentage of dead and abnormal

spermatozoa of Patanwadi rams. In sub-tropical conditions,

environmental temperature significantly affects sperm motility, sperm

concentration and sperm number per ejaculate (Daader et al., 1987). In

West African dwarf rams, semen volume, sperm cell concentration did

not differ between dry and wet seasons, whereas, percentage of

progressive motility, total abnormalities and live sperm in dry season

were different from those in the rainy season which showed better quality

in these criteria (Chiboka, 1980). Seasonal variation in testicular

morphology between Corriedale rams subjected to different feeding

levels was observed. These findings suggested that nutritional factor

contributed, at least partly, to the differences in variation observed (Bielli

24  

et al., 1997). The season exerts a significant influence on semen

freezability in Leccese ram, with the best performance occurring in

summer and autumn period, corresponding to the breeding season in

temperate zones (D´Alessandro, 2003).

1.6 Semen collection:

Semen may be obtained by one of three methods:

(1) Collection from the vagina after mating.

(2) Artificial vagina.

(3) Electrical stimulation (Electro-ejaculation).

1.6.1 Collection from the vagina after mating:

The collection of semen from the vagina of a recently bred ewe is

possible by use of a syringe with soft nozzle. However, such semen is

small in quantity, mixed with mucus, sloughed cells, debris and micro-

organisms. This procedure is no longer in practice (Herman et al., 1994).

1.6.2 Artificial vagina (AV):

The AV is the best method for collecting natural ejaculate. Its also

allows evaluation of sperm quality, potency (sperm delivery to the

vagina) and libido of the animal. Moreover, when the AV is used for

semen collection, rutting behaviour of rams appeared to be normal

(Sackman and Schone, 1990). The AV provides suitable temperature,

pressure and lubrication (Evans and Maxwell, 1987). The AV consists of

25  

a medium–hard rubber casing about 20 centimetres (cm) in length and

about 4 cm in diameter, with a soft rubber lining, about 25 cm in length

placed in the casing and each end folded over the edges and held in place

by rubber bands (Herman et al., 1994).

The temperature of the AV at the time of collection should be

between 41° and 44°C. If the temperature drops below 40°C or goes

above 45°C, it may inhibit ejaculation. The required pressure in the AV

may vary from one ram to another, but with a little experience, an

operator will soon determine the proper pressure (Herman et al., 1994).

As the ram mounted the teaser, the penile sheath is grasped by the

operator and the penis is directed into the AV. The collector must be alert

because the ram moves very quickly and makes a rapid single thrusting

motion (Sorensen, 1979).

1.6.3 Electro-ejaculation:

Electrical stimulation works successfully in sheep and it is similar

to the procedure described for the bull. Small rectal probe for rams are

commercially available for this purpose. Semen obtained by electrical

stimulation is usually few in sperm number and thinner in consistency

than that obtained with an AV (Herman et al., 1994). In addition,

electrical ejaculation resulted in a larger volume of semen, higher pH,

lower concentration and gross motility (Matter and Voglmayer, 1962).

26  

However, there was no difference in fertility found between ewes bred

with semen collected by AV or electro-ejaculation (Hackett and

Wolynetz, 1982).

The rectal probe is pressed forward and downwards against the

floor of the pelvis by an assistant and short stimuli (3-8 seconds) are

applied at 15-20 seconds intervals. After 3-5 bouts of stimulation,

accessory glands secretion will flow, followed by semen (Evans and

Maxwell, 1987). If the penis is not extended, it may be necessary to push

the sheath and prepuce back a long the shaft to expose the penis and the

glans before semen collection (Sorensen, 1979). Ejaculation (usually

without erection) generally takes place in the rest phase between

stimulations and it may be necessary to milk the sheath to retrieve the

sample (David, 1994).

However, about 20 semen collections a week can be taken from a

ram during the breeding season, but only four or five a week out of

season (David, 1994).

1.7 Semen dilution:

Semen extenders provide some nutrients for the metabolic process

of sperms, protect against cold shock, buffer the effects of toxic and

acidic substance produced by sperm metabolism and aid in maintaining

the proper osmotic pressure and mineral balance (Herman et al., 1994).

27  

Semen extenders usually contain a buffer such as sodium citrate

dehydrate or Tris (Tri- hydroxy methyl amino methane); egg yolk or

milk, which contains macromolecules that provide protection against cold

shock; simple sugars, such as glucose and fructose, that serve as a source

of energy for sperm; glycerol which is added as a cryoprotectant to

reduce the intercellular formation of ice crystals and solute effects during

the freeze-thaw process and antibiotics such as gentamycin, tylosin which

inhibit a variety of micro-organisms found in semen (Herman et al.,

1994). The addition of glycerol at 5˚C results in better sperm survival

than addition at 30˚C, although the difference is only small (Fisher and

Fairfull, 1989). It is due to ability of glycerol to pass through the

membrane at 30˚C. Cell membranes are less permeable to glycerol at 4˚C

so it has a less toxic effect (Critser et al., 1988). Lipid-binding proteins

present in seminal plasma (sp proteins) induce cholesterol and

phospholipids removal of sperm membrane which is detrimental to sperm

preservation. Low-density lipoproteins present in egg yolk interact with

lipid-bind proteins which minimize the cholesterol and phospholipids

removal. Skimmed milk, which is devoid of lipoprotein, also protects

sperms during storage by milk casein (Annick and Puttaswamy, 2006).

High egg-yolk concentrations reduce the post-thaw viability of ejaculated

spermatozoa in several species including rams (Watson and Martin,

28  

1975). In addition, egg-yolk reduces the acrosome integrity of goat

spermatozoa (Abo agla and Terada, 2004).

Three extenders were used for processing of ram semen (Gil,

1999); each of them has two fractions for two-step extension

methodology, the first fraction without glycerol and the second with 14%

glycerol, thus resulting in a final concentration of 7% (v/v).

These extenders were prepared as follows:

(i) Powdered skim milk extender (A):

this extender consisted of reconstituted non fatty milk powder

(11% w/v) heated to 95°C for 10 minutes and then cooled to room

temperature. It is prepared into two fractions.

Fraction A-1 was prepared by adding egg yolk (5% v/v) to the

reconstituted milk and antibiotics (0.03 g penicillin and 0.04 g

streptomycin/100 ml.).

Fraction A-2 was prepared by adding egg yolk (5% v/v), glycerol

(14% v/v), 224 mM of fructose and antibiotics as described above.

29  

(ii) Clarified powder skimmed milk extender (B):

It was prepared exactly like extender (A), but it was, somewhat,

clarified by centrifugation (3310g at 5°C for 20 min.). Centrifugation was

repeated with the supernatant.

Fraction B-2 was centrifuged before adding glycerol (14% v/v).

Only supernatant between the overlying lipoid and the compact pellet at

the bottom was used.

(iii) Tris–egg yolk-glycerol extender (C):

It consisted of 263 mM Tris, 85 mM citric acid, 73 mM fructose

and 20% (v/v) egg yolk.

Fraction C-1 consisted of extender C clarified by centrifugation (as

described for extender B).

Fraction C-2 consisted of C-1 plus glycerol (14% v/v). The

antibiotic mixture used in this extender is the same as that used in

extenders A and B.

Furthermore, Evans and Maxwell (1987) tabulated four different

formulae of Tris-citric acid-fructose-egg yolk-glycerol extenders for one

– step dilution methodology. One part of semen added to 1, 2, 3 or 4 parts

of extender according to the consistency of semen samples. Although,

the sperm viability was higher for semen processed in the clarified milk–

30  

based extender compared to both milk-based extenders as well as the

Tris-citric acid fructose based extender (Söderquist, 1999). Significantly a

higher percentage of motile, progressive and membrane-intact

spermatozoa were recorded for milk than for the Tris extenders (Yániz et

al., 2008).

The current use of ingredients of animal origin, such as egg yolk,

milk and bovine serum albumin in semen extenders presents a risk of

microbial contamination, and has led to the search for alternatives. Such

an extender is commercially available for bull semen (Bioexcell®, IMV,

L´Aigle, France ) and it has previously been tested in vitro for freezing

ram semen, with satisfactory results (Gil et al., 2003). A soybean lecithin-

based extender (AndroMed®, Minitüb, Germany) is a Tris-based buffer

containing phospholipids, citric acid, sugars, antioxidant and glycerol,

and has been utilized successfully for cryopreservation of bovine (Aires

et al.,2003) and ovine semen (Fukui et al.,2008). Gil et al. (2003) found

that, the subjective motility was slightly higher in Bioexcell extender than

in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38

vs. 37.7%; NS), and the percentage of incapacitated spermatozoa

(28.5vs.26.3%; NS). Furthermore, the results obtained indicate that when

freezing ram semen, Bioexcell containing 6.4% glycerol may be used as

31  

an alternative extender to the conventional milk extender containing 5%

egg yolk (Gil et al., 2003).

The diluents have a quadratic effect on motility when increasing

glucose concentration in the diluents with a maximum of 55 mM. At this

sugar concentration, motility was higher for diluents containing glucose

and fructose than for containing lactose, sucrose or trehalose (Molinia et

al., 1994).

Semen processing protocols when centrifugation was included,

percentages of motile spermatozoa, sperm with intact membrane as well

as incapacitated spermatozoa were higher compared to those processed

without centrifugation, but the situation was opposite when the sperm

numbers were calculated (Söderquist, 1999). The protocol with adjusted

extension of semen yields higher numbers of viable spermatozoa per A1

dose compared to the protocol involving semen centrifugation (Gil,

1999). However, semen dilution in different ratios (v/v) had no effect,

since the overall post-thaw spermatozoa viability was highly dependant

on the freezing method and the cryoprotectant used (Pontbriand et al.,

1989). Moreover, Colas (1975) reported that, ram semen diluted to a

constant concentration rather than volume to volume achieved superior

results.

32  

1.8 Semen freezing:

Polge et al. (1949) were the first to report a protective effect of

glycerol, which in combination with low rates of cooling, resulted in high

survivals after freezing and thawing of spermatozoa from several species.

The challenge to cells during freezing is not their ability to endure storage

at very low temperatures; rather it is the lethal effect of an intermediate

zone of temperature (-15° to -60 °C) that the cell must traverse twice,

once during cooling and once during thawing (Mazur, 1985).

The goal of freezing semen is to decrease the temperature from 5°

to -196°C in gradual steps to avoid damaging the delicate sperm cells.

This can be done by utilizing cryogenic nitrogen vapour, dry ice or

alcohol bath (Herman et al., 1994). The temperature variation involved in

freezing and thawing of semen, however, inevitably reduces the

proportion of motile spermatozoa and causes ultra-structural, biochemical

and functional damage (Söderquist, 1999). Healey (1969) reported that,

the ram frozen semen examined in the electron microscope, showed

consistent damage to the outer spermatozoal membrane and acrosome

complex, the damage ranged from slight swelling of the acrosome to total

removal of cytoplasmic regions. The initial freezing temperature has a

significant effect on spermatozoa motility and velocity following post-

thawing , were the best motile spermatozoa achieved at – 125°C freezing

33  

temperature (Bag et al.,2002). However, the freezing in small dimensions

straws resulted in a very quick drop of temperature, leading to

intracellular crystallization of water, this may cause less damage to the

cells during the temperature drop than the extreme dehydration associated

with slow freezing (Berg, 1999). Pontbriand et al. (1989) cited that,

cooling rates of 6 to 24 °C per minute and 10 to 100 °C per minute have

been reported as acceptable, demonstrating that ram spermatozoa tolerate

a wide range of cooling velocities. Kaya et al. (2001) concluded that,

melatonin administration to sperm donors improved freezability of ram

semen collected from these rams and reduced enzyme leakage through

sperm cells during cryopreservation. Although the manual freezing of

straws in a liquid nitrogen vapour 5 cm above the liquid nitrogen in

freezing tank was comparable to that of automatic temperature controlled

liquid nitrogen freezing chambers, the later may provide more reliable

results when cryopreserving semen from rams under field conditions

(Pontbriand et al., 1989).

1.9 Cryopreservation of spermatozoa:

Sperm survival after freezing-thawing is dependent on freezing

technique, cryopreservation, diluents composition, glycerol

concentration, dilution rate, cooling, equilibration interval and thawing

method (Graham et al., 1978; Bearden and Fuquay, 1984).

34  

Cryopreservation of spermatozoa for many species leads to series of

alterations resulting in a marked decrease in their fertility (Parks and

Graham, 1992). Freezing and thawing lead to a decrease in both

membrane integrity and acrosome integrity.

It is suggested that the structural integrity of ram spermatozoa is largely

un-affected after cryopreservation, and that damage to plasma membrane

is primarily responsible for the low fertility of frozen–thawed

spermatozoa (Valcarcel et al., 1996). The processing and storage of ram

semen lead to reduced sperm motility and disruption of membrane

integrity. It is generally assumed that these changes are detrimental, and

are associated with reduced fertilizing ability after cervical insemination.

However, recent evidence suggests that, while many spermatozoa remain

motile after storage, the membranes of the motile spermatozoa are

destabilized to the point where they may not survive further ageing in the

female reproductive tract after cervical insemination (Maxwell and

Watson, 1996).These membrane changes are similar to the capacitation of

acrosome reaction of spermatozoa (Maxwell and Watson, 1996;

Chatterjee et al., 2001). Thus, stored spermatozoa may require less

capacitation time in the female reproductive tract, and may readily

fertilize oocytes if placed in their immediate vicinity, as with in vitro

fertilization, tubal or intra-uterine insemination. It is argued that some of

these changes can be prevented or reversed using antioxidants in order to

35  

improve fertility following cervical insemination (Maxwell and Watson,

1996; Mara et al., 2005; Bucak et al., 2008; Atessahin et al., 2008). Colas

(1975) reported that, ram semen cryopreserved in straws resulted in

fertility higher than that of semen frozen through pelleting on dry ice.

Even when semen was stored for eight hours in a refrigerator, conception

rates were significantly lower with refrigerated semen than with fresh

semen (Cordova et al., 1989).

1.10 Thawing of frozen semen:

According to general recommendations, thawing of ram semen

should be carried-out quickly and at a relatively high temperature.

However, if thawing is not fast enough, the ice crystals formed will

increase in size before the melting point is reached, thus injuring the

spermatozoal membranes and other cellular structures (Berg, 1999).

Moreover, thawing straws in a water–bath at a higher velocity

(60°C for 8 seconds) had no effect on spermatozoal motility, progressive

status ratings, or acrosomal integrity when compared to a lower rate

(37°C for 20seconds) (Pontbriand et al.,1989). Söderquist (1999)

concluded that sperm can be thawed at 50°C for 9 seconds instead of

70°C for 5 seconds without further reducing sperm motility or membrane

integrity. Thus lower thaw temperature would facilitate a wide spread use

of frozen–thawed ram semen under farm conditions.

36  

Melatonin implantation to ram in the breeding season improved

post-thaw sperm viability and intact acrosome rates without influencing

the motility rate, that the post-thaw alkaline phosphates release through

sperm cells was significantly lower in the melatonin-treated group in

comparison with untreated controls (Kaya et al, 2001). However,

individual ejaculates of ram semen can only be considered suitable for

storage and use for insemination if the percentage of forward moving

spermatozoa is not less than 40% upon thawing and 30% after 5– 6 hours

of incubation (Evans and Maxwell, 1987).

1.11 Oestrous synchronization:

Sheep are seasonally polyoestrus; their normal breeding season

depends upon breed and geographic location. The physiological basis of

the breeding season seems to be induced by a rise in the production of

melatonin by the pineal body during the reduction in the day light (Berg,

1999). Ewes are able to monitor changes in the daily photoperiod by the

circadian secretion of melatonin from the pineal gland. Melatonin output

is regulated by photoperiod and elevated concentrations are found in

blood only during the hours of darkness (O`Callaghan, 1994; Rosa and

Bryant, 2003). The length of oestrus cycle is 16-17 days (Monika, 2006).

The oestrous period lasts approximately two days; ovulation takes place

in the second half of the oestrus, 30-36 hours after the onset of the heat

(Sorensen, 1979; Taljaard et al., 1991). Ewes in oestrus do not exhibit

37  

sufficiently clear symptoms of their state and it is necessary to use teaser

males for identification (Chemineau et al., 1991). The time in oestrus can,

to a certain extent, be assessed by examination of the cervical mucus

when the speculum is inserted into the vagina in connection with

insemination. In the first 12 hours of the heat the mucus is absolutely

clear, in the next 12 hours increasingly opaque, in the following 12 hours

white or yellow-white and very viscous, and in the last 12 hours smoke

grey and more dry and solid (Berg, 1999 ).

Levels of plasma progesterone at oestrus ranged from 0.1 to 0.5

ng/ml and during the luteal phase from 3 to 6 ng/ml. Levels found during

seasonal anoestrus were within the range of those observed at oestrus. In

animals which ovulated, the plasma progesterone concentration either

remained basal or rose to a lower level <2 ng/ml than that found during

the luteal phase of the cycle (Haresign et al., 1975).

When Targhee and Fin x Targhee ewes were subjected to different

photoperiods, the percentage of exposed ewes lambing was lowest for

natural day length (37%) and highest for 8 light: 16 hours of darkness

(81%). Considering percentage lambing in combination with interval to

lambing the 8L: 16 D treatment proved to be the most effective (Slyter et

al., 1986).

The treatment of anoestrous ewes with medroxy-progesterone

acetate (MAP) priming increased the ovulation rate by increasing the

38  

number of follicles that responded to PMSG (Leyva et al., 1998a). FGA

sponges (fluorogestone acetate), containing 30 mg are recommended for

use in ewes in seasonal anoestrus and 40 mg for ewe lambs and ewes in

the breeding season. MAP sponges contain 60 mg and can be used for all

purposes (Chemineau et al., 1991). Moreover, progestagen treatment is

administered for 10 – 12 days in the non-breeding season or 12 – 14 days

during the breeding season in the ewe (Chemineau et al., 1991). Though,

the treatment with MAP sponges does not adequately synchronize oestrus

and ovulation among cyclic ewes due to the difference to follicular

patterns that results, depending on the stage of the cycle at the time of

sponge insertion (Leyva et al., 1998b). An intramuscular injection of

PMSG at the end of progestagen treatment (at sponge removal) in the

ewe, increases follicular growth, the duration of oestrus, ovulation rate

and advances the onset of oestrus (Chemineau et al., 1991). The use of

progestagen sponges plus PMSG for the induction of out-of-season

breeding in ewes is associated with a number of recognized short-

comings. A possible reason for variability in conception rates is the high

incidence of complete embryonic loss or due to fertilization failure in

ewes that are induced to super-ovulate after treatment with a dose of

PMSG (Haresign, 1992). Langford et al. (1983) found that, ewes

inseminated at 55 and 60 hours after sponge's removal, conception rates

(in June and October) were 82 and 87% with PMSG and 18 and 48%

39  

without PMSG respectively. The corresponding lambing rates were 60

and 74 with PMSG and 10 and 26% without PMSG.

Furthermore, melatonin (Regulin) can advance the onset of the

breeding season of commercial sheep flocks. This treatment appears to

overcome a number of the short-comings of progestagen sponges plus

PMSG (Haresign, 1992). Treatment with melatonin implant (18 mg) 35

days before inserting progestagen-impregnated sponges (14 days) and

PMSG (500 iu, on the day of sponge removal) showed AI results

significantly higher in lambing rate compared to ewes not implanted with

melatonin (60.4 vs. 32.6%) (Laliotis et al.,1998).

Pre-treatment of seasonally anoestrous ewes with PMSG (750 iu)

or oestradiol benzoate (50 µ g) 24 or 7 hours respectively before a single

injection of synthetic LH-RH (150 µ g) significantly increased the release

of LH compared to that after injection of LH-RH alone (Haresign and

Lamming, 1978).

Fukui and Roberts (1981) found that, the injection of 24 mg PGF2α

(prostaglandin) between day 6 and 12 of the oestrous cycle resulted in a

higher proportion of ewes exhibiting oestrus (92.9%) within 5 days of

treatment as compared to the other two doses (16.6 and 66.6% for 8 and

16 mg PGF2α, respectively).They concluded that the occurrence of

ovulation depended on the dose of PGF2α rather than the stage of breeding

season.

40  

Chemineau et al. (1991) concluded that, timing of AI after oestrous

synchronization with a source of progesterone and PMSG, the optimal

timing for a single insemination (semen either liquid state or in deep-

frozen state) is 55±1 hours after sponge removal. In two inseminations,

they can be performed at 50–60 hours after sponge removal. In ewe

lambs, in which oestrus appears earlier than in adult ewes, it is necessary

to inseminate at 50±1 hour after sponge removal of the implant. Manahil

(1999) reported that, the treatment of Sudan Desert sheep with

progestagen intra-vaginal sponges accompanied by PMSG has a merit

resulting in significantly higher pregnancy rate to first insemination than

luprostiol or progestagen alone.

1.12 Insemination of ewes:

The ewe has a miniature cervix structured like that of the cow. The

circular folds are much more distinct and many in number (six or seven).

The intra-vaginal portion extended further and lies closer to the floor than

in the cow (Sorensen, 1979). The gross anatomy of ovine cervix is

described with particular reference to the positioning of annular folds.

The second fold is eccentric to the other concentric folds and presents a

physical barrier to the passage of a straight instrument, a phenomenon

that has prevented the use of deep cervical or uterine artificial

insemination (Thomas and Homer, 1981). Naqvi et al. (2005) found that,

the average number of funnel shaped folds in the cervix of Malpura and

41  

Kheri ewe lambs and adult ewes were 3.2±0.19 and 3.4±0.22. However,

the second and third-folds from the os were observed to be eccentric in

both ewe lambs and adult ewes. The cervix measures 1 cm (0.4 inch) in

diameter and 5 cm (2 inches) long (Sorensen, 1979). However, Souza et

al. (1994) found that, in 134 Polwarth and 138 Corriedale ewes, cervix

length averaged was 5.9 and 6.7 cm respectively, cervix diameter was 0.8

cm in both breeds and a cervical flap was found in 52.2% of the cervixes

examined.

Therefore, in sheep, most AI is performed with diluted fresh semen

while only a few ewes are inseminated with frozen semen (Colas, 1983).

Alsayed (1996) found that, AI in Sudan Desert sheep with undiluted

semen gave 90% conception rate, while diluted semen gave 57.1% and

hand mated controls were 91.6%.

However, deep-frozen ram semen is not easy to use, mainly

because of three factors: fertility of deep-frozen semen is about 20

percent lower than that of fresh semen; the required number of

spermatozoa appears to be higher than fresh semen and the technique is

more expensive and more complicated (Colas, 1975). Since insemination

at an optimal time in the heat is essential to achieve an acceptable fertility

results, it is of utmost importance to be able to preserve and store the

semen under optimal conditions (Söderquist, 1999). There are three

methods of semen deposition in sheep:

42  

(i) Vaginal insemination.

(ii) Cervical insemination.

(iii) Laparoscopic insemination.

1.12.1 Vaginal insemination:

Vaginal insemination consists of deposition of semen into the

anterior vagina without an attempt to locate the cervix (Evans and

Maxwell, 1987). The poorest results are obtained when semen is left in

the vagina close to the cervix, the so called-shot in the dark method.

Much improved conception rates result from depositing semen about 1

cm into the folds of the cervix (David, 1994). Liquid ram semen diluted

in a milk–based extender and vaginally inseminated once in natural heat,

with a semen dose of 150 million spermatozoa, gave acceptable fertility

results and is to be recommended as the method of choice in Norway

(Heiko et al., 2003).

1.12.2 Cervical insemination:

Cervical insemination involves deposition of semen to a depth of

up to 3 cm into the cervix. If, as in some animals, the cervix will permit

further passage of the pipette, the method can be adopted to become a

non-surgical intra-uterine insemination (Evans and Maxwell, 1987).

Cervical insemination in sheep using frozen semen has not progressed

due to low conception rates (Salamon and Maxwell, 1995). Naqvi et al.

(1998) found that mid-cervical and trans-cervical insemination with

43  

frozen semen resulted in similar lambing rates 28.5 and 22.7 percent

respectively, but no lambing was achieved on os-cervical insemination.

For cervical insemination the dose can vary from 0.5 to 1.0 ml and should

contain from 150 to 200 million viable sperms (Herman et al., 1994).

However, Berg (1999) reported that, the volume that can be deposited in

the cervical canal of the ewe is normally limited about 0.2 ml and this

insemination method necessitates a minimum number of spermatozoa of

approximately 200 million and the semen dose contains at least 50%

normal motile spermatozoa after thawing. Smith et al. (1995) found that,

with trans-cervical insemination method conception results obtained were

higher than partial penetration (52%vs.30%). However, the full

penetration rates with chilled semen produced no better results than

cervical insemination (Smith et al., 1995); while partial penetration was

lower (full 68%, partial 58%). However, Westhuysen et al. (1981) found

that, the number of lambs born by female treated with MAP and PMSG

(300 IU) was significantly higher after double than single insemination in

12 and 24 hours after oestrus was observed. Hair sheep ewes were

synchronized using PGF2α and bred by transcervical insemination with

frozen–thawed semen after 48 hours after the second injection; the

conception rate was (8.7%), while in a subsequent trial, ewes were

synchronized with controlled internal drug release (CIDR) devices

containing 300 mg progesterone for 12 days and inseminated 48 hours

44  

after CIDR removal , resulting in conception rate of 52.9% (Godfrey et

al., 1999).

The Guelph system for trans-cervical AI (GST-AI), as described by

Halbert et al. (1990 a ) and Halbert et al . (1990 b), provides a viable non-

surgical alternative to laparoscopic AI. The Guelph system allows for

consistent penetration of the offset rings of the ewe`s cervix, thus

permitting the semen to be dispelled directly into the body of the uterus.

In many instances, conception rates using this technique have compared

favourably with those achieved using laparoscopic AI (Cunningham and

Deborah, 1994 ). The efficiency (time, labour, equipment and drugs) of

this technique makes the Guelph system comparable to the laparoscopic

method. The equipment is less expensive and time , labour and materials

are not required to prepare the ewe for surgery and no anaesthesia or

sedation is necessary (Naqvi et al.,1997; Anel et al.,2006). However,

Cambell et al. (1996) found that, varying degrees of damage occurs to

the cervical lining over the length of the cervix when the trans-cervical AI

needle penetrated by use of GST-AI technique. Exogenous oxytocin aids

in the transcervical passage of an AI gun into the uterus of ewes, and it

may be an effective adjunct to sheep AI procedures (Sayre and Lewis,

1996). Recently, Ivanka et al.(2009) found that, the treatment with

Cervdil® (East Kilbride, Scotland), a prostaglandin E2 (PGE2) releasing

45  

vaginal insert used for induction of cervical ripening and labour in

women, facilitated transcervical semen deposition in anestrous ewes and

has the promise of a technique to improve transcervical AI in sheep.

It is recommended that, trans-cervical insemination be performed

approximately 42 hours after progestagen pessary removal or

approximately 18 hours after the onset of oestrus (Husein et al1996).

Pregnancy rate of 81 percent have been reported by Halbert et al.(1990 b)

using the Guelph system for trans-cervical insemination. However, using

frozen–thawed semen, the trans-cervical insemination yielded lambing

rates of 50.7 percent for ewes bred during the breeding season (Buckrell

et al., 1994). The difference in pregnancy rate between Suffolk and

Belclare ewes following cervical AI with frozen–thawed semen was due

to sperms traversing the cervix and uterus in a higher proportion of

Belclare than Suffolk ewes, leading to a higher incidence of fertilization

(Fair et al., 2005).

1.12.3 Laparoscopic insemination:

Laparoscopic insemination by-passes the cervix and deposits the

semen directly into the uterine horns. It is a minimally-invasive, minor

surgical procedure. With intrauterine AI, a thousand ewes could be

inseminated seasonally with fresh semen from one ram. Using

intrauterine AI and frozen semen, as many as 25000 ewes could be

46  

annually inseminated with semen from one highly fertile ram (David,

1994). Lambing rates to AI using frozen semen were found to be 62 and

67 percent for laparoscopic and trans-cervical insemination respectively.

Showing that, laparoscopic AI was confirmed as the most suitable

technique for insemination using frozen semen in sheep breeding

programme (Cappai et al., 1998). Laparoscopic AI method requires less

semen (20 million sperm). However, this method requires skill and

special equipment (Herman et al., 1994). However, Smith et al. (1995)

reported that, with frozen semen there were no significant differences

between trans-cervical and laparoscopic AI (64.1 vs. 66.7%,

respectively).

1.13 Fertility following AI:

Fertility, the proportion of ewes lambing of all those exposed to the

ram during defined period (usually expressed as a percentage) varies with

breed, season, age, nutritional status, breeding management and farm

conditions (Monika, 2006). Assessment of ram fertility on basis of semen

characteristics was the objective of several studies and was described and

evaluated by Cameron and Fairnie (1984). No single test accurately

predicts fertility of a semen sample. However, the examination of various

semen characteristics can determine fertility potential (Nabil et al., 2006).

It is well known that a correlation exists between certain semen

47  

characteristics, such as motility and sperm morphology, and field fertility

results for both fresh and frozen semen (Elliot, 1978).

Many studies have shown that within the same breed, there exists a

wide variation in fertility between rams (Mickelesen et al.,1981).

However, Donovan et al.(2004) found that, pregnancy rate was

significantly influenced by breed of ewe and inseminator; these results

suggest that ewe breed may be a critical determinant of the potential for

exploitation of cervical insemination of frozen-thawed semen in sheep

breeding programmes. Langford and Marcus (1982) found that, the

number of ewes lambing after insemination with 200 million sperms were

similar to those bred after natural service at progestagen induced oestrus.

However, when less than or equal to 100 million spermatozoa were

inseminated, fertility fell markedly and the number of lambs per ewe

inseminated decreased (Langford and Marcus, 1982). The total number of

spermatozoa (80 vs. 200 million) inseminated to Karakul ewes in 12 and

22 hours after the onset of the oestrus did not affect the conception rate ,

but it was lower when spermatozoa where in higher volume of fluid (0.2

vs. 0.5 ml) (Roux and Le-Roux , 1976).

Furthermore, Hackett and Wolynetz (1982) observed that, fertility

was 53% for ewes bred by natural mating, 34% for ewes receiving PMSG

and bred by AI and 9% for those not receiving PMSG and bred by AI.

48  

Fertility is very high in all Norwegian breed, about 92.5 percent after AI

with fresh diluted semen, this partially due to the favourable

environmental conditions and low lactation (Berg, 1999).

The fertility results after AI with frozen semen remains

unacceptably low (Graham et al., 1978). In Sweden, Söderquist (1999)

reported that, the average overall fertility with frozen-thawed semen in

synchronized ewes was 39.7%. Pontbriand et al. (1989) suggested that

the poor fertility rates after using frozen-thawed ram semen, likely,

results not only from reduced sperm motility, but also from ultra-

structural damage to sperm integrity. No difference in fertility of frozen –

thawed semen adjusted to the final concentration before packing (200

million sperm) with centrifugation or without centrifugation (30.8% vs.

29.7%) and cervically inseminated during oestrus (Gil et al. 2002). Trials

have been conducted on AI using fresh and frozen semen, but the

conception rate with frozen semen has been considerably lower than

fresh semen, mostly in the range of 30- 60 percent, whereas, for fresh

semen it was 70 percent (Dyrmundsson et al., 1989). Frozen semen from

19 individual rams has been evaluated for fertility, using bovine oocytes

in vitro fertilization procedure. The results showed that, there was a

significant difference between rams ranging from 8.0+2.0 to 89.9+0.4 and

a significant difference between ejaculates from the same ram ranging

49  

from 61.5+13.0 to 87.5+3.2 (Smith and Murray, 1996). The collection of

semen in the fall, facilitates successful freezing of semen and AI in the

spring and this could be useful to determine whether the low fertility in

the out of season mating is due to the ram, the ewe, or both (Mickelsen et

al., 1981). Heiko et al.(2004) concluded that, the superior fertility results

achieved for minitubes compared to ministraws when cervical

insemination with 200 million sperm have been carefully evaluated in

relation to the possible application of a more rational semen production

and simplified semen handling at AI, when using mini straws thawed at

35° C for 12 seconds. The biological effects of the inorganic constituents

(Na, Cl, Ca, K and inorganic P), together with magnesium (Mg), in the

semen on semen quality should be considered in the interpretation of the

results obtained in the fertility evaluation of the various ram breeds

(Abdel Rahman et al., 2000).

50  

 

Materials and methods

 

 

 

 

 

 

51  

2- Materials and Methods:

2.1 Site of study:

This experiment was conducted from April 1999 to March 2000 at

the National Animal Breeding and Artificial Insemination Centre (Kuku,

Khartoum North) where rams were kept and semen collection, evaluation

and processing were carried-out. Insemination of ewes was applied in six

private farms in Khartoum state.

2.2 Latitude and climate:

In Sudan the year is divided into three seasons, winter (November-

February), summer (March–June) and autumn (July–October). In

Khartoum (15° 36 N, 32` E) the summer is dry and very hot (maximum

temperature between 38° and 44°C). Climatic data of temperature,

relative humidity and rainfall during the period of investigation were

collected from the Meteorology Department (Appendex 1).

2.3 Animals:

Six selected Hamari and three selected Kabashi breeding rams

(both are ecotypes of Desert sheep) were used in this study. Rams were

two to four years of age at the beginning of this experiment. Rams were

maintained under uniform conditions of feeding and management. During

the whole period of study, it was observed that, all rams from the two

breeds were free of palpable abnormalities or clinical diseases.

52  

2.3.1 Housing:

Each of the rams was housed in an individual shaded pen (2 x 1.6

meters) with enough space for free movement.

2.3.2 Feeding:

Rams were fed with pea-nut hay (ad libitum). Each ram was

supplied daily with 0.5 kg commercial concentrate (Al Gandol Feed) with

free access to water and mineral licks (10 kg).

The chemical analysis of concentrate feed on dry matter basis was

as follows:

Metabolic energy 11481.39 M.J.

Crude protein 16.5 %

Crude fibres 10 %

Plus different vitamins and minerals needed by the animal.

The mineral blocks composed of (per Kg):

Sodium 37.1 % Zinc 2.5 mg

Magnesium 3.5 mg Iodine 38 mg

Iron 3.0 mg Cobalt 35 mg

Manganese 625 mg Selenium 13 mg

53  

2.3.3 Body weight recording:

The rams were weighed once monthly and body weight was

recorded using a weighing scale.

2.3.4 Scrotal measurements:

Scrotal circumference (SC) and testicular length measurements

were taken while the ram was in the standing position. The testicles were

pushed into the bottom of the scrotum and measured around the widest

point of the testicles with a flexible measuring nylon tape (Mickelsen et

al., 1981).

2.4 Reaction time (RT):

Sexual drive of each ram was estimated by measuring the reaction

time which denotes the interval between the first contacts with the teaser

ewe until ejaculation is completed (Ibrahim, 1997).

2.5 Semen collection:

Rams were subjected to a training period of two weeks to ejaculate

into an artificial vagina (AV) after mounting anoestrous ewe which was

restrained and tied to a post or held by an assistant.

Semen was collected from all rams by means of AV (IMV model

for ovine). This was continued for one year covering the three seasons

(Summer, autumn and winter).

54  

A total of 293 first ejaculates of semen were collected during the

whole period of the study, of which 71 and 22 ejaculates were collected

during the summer, 42 and 24 ejaculates were collected during the

autumn and 85 and 49 ejaculates were collected during the winter from

Hamari and Kabashi rams respectively.

A separate AV was used for one collection only. The AV case was

half-filled with warm water (48-50°C) through a valve fitted in one side.

One end of the inner liner was lightly lubricated with sterile Vaseline to a

depth of three centimetres. The AV was inflated with air through the

valve, this exerts some pressure in the AV cavity but allows easy

penetration of the penis. The temperature of the AV just before semen

collection was around 42-44°C and checked by insertion of a clean

thermometer.

The teaser ewe was secured and tied to a post or held by an

assistant. The operator took a crouching or kneeling position at the right

side of the teaser, and held the AV in the right hand along the flank, with

the open end facing towards the ram and down-wards at an angle of 45°.

The valve of AV was directed down-wards to avoid contact with the ram.

When the ram mounted, the erected penis was directed into the

open end of the AV, the left hand was used to grasp gently the sheath and

deflect the penis into the open end of the AV.

55  

A vigorous up-ward and forward thrust indicated the occurrence of

ejaculation.

Immediately after collection, the AV was turned up while keeping

collecting glass up-right, the pressure was released from the inner liner by

opening the valve for water and air to escape.

2.6 Semen evaluation :

All procedures of semen evaluations were carried-out at the semen

laboratory, immediately after collection as described by Rao (1971).

2.6.1 Appearance of ejaculate:

Appearance of semen was estimated directly from the collection

tube for contaminants such as hair, dirt, faeces, blood and urine.

2.6.2 Volume of ejaculate:

The volume of the ejaculate was measured directly from the

graduated collecting glass tube in millilitres (ml) and to the nearest 0.1 ml

2.6.3 Colour and consistency of semen:

Colour and consistency of semen were assessed by visual

observation directly in the collecting glass tube.

56  

2.6.4 Sperm motility:

2.6.4.1 Mass activity (Gross motility):

Semen mass activity was estimated by observing the degree of

wave motion and general activity of spermatozoa. Mass activity was

estimated by examining a drop of fresh semen on a pre-warmed slide

(37°C) under light microscopy at magnification (10 x objective). The

mass activity is graded from zero to 5 scores according to intensity of

wave motion as described by Evans and Maxwell (1987). The basis of

this method includes:

0 = (Dead) all spermatozoa are motionless

1 = (Very poor) very few spermatozoa (about 10%) are

active, with weak movement only.

2 = (Poor) no waves are formed, but some movement of

spermatozoa are visible . Only 20-40% of sperm cells are

active, and their motility is poor.

3 = (Fair) only small , slow moving waves. Individual

spermatozoa can be observed . 45-65% of sperm cells

are active .

4 = (Good) vigorous movement, but the waves are not so

rapid as for score 5. About 70-85% of sperm cells are active.

57  

5 = (Very good) dense, very rapidly moving waves. Individual

Sperm cells can not be observed. 90% or more of the

spermatozoa are active.

2.6.4.2 Individual motility:

Individual motility of sperm was estimated by examining a drop of

diluted semen with physiological saline solution (0.9% Na Cl) on a pre-

warmed slide (37°C). One drop of semen and two drops of normal saline

were mixed together and covered with a cover-slip. Under a phase

contrast microscope fitted with a hot stage maintained at 37°C and at

magnification (400 x), the individual motility was estimated at a scale

ranging from zero to 100 according to the percentage of sperms moving

straight forward over the field of the microscope. Only sperm that have

forward or progressive movements are included, but sperm with

backward, vibrating or circling movements are not included (Evans and

Maxwell, 1987).

2.6.5 Sperm cell concentration:

The concentration of spermatozoa was counted by the aid of a

haemocytometer according to the method described by Evans and

Maxwell (1987). An improved Neübauer chamber was used. With the red

blood sampler pipette (that has the red agitator in the bulb) a sample of

raw semen was sucked up to the 0.5 mark. The distilled water was drawn

58  

up to the 101 mark of the pipette (made 1/200 dilution with distilled

water). The pipette was agitated by holding it between the thumb and the

index finger and was shaking to ensure dilution of semen sample. Four to

five (4–5) drops were discarded from the end of the pipette to obtain

properly diluted semen from the bulb. The diluted semen with the tip of

the pipette was allowed to run under the cover–glass of the

haemocytometer, then the spermatozoa were allowed to settle for 5–6

minutes before counting. Five large squares, one at each corner and the

one in the centre were counted. Spermatozoa heads lied on the top and

right lines between the squares were included in the counting of each

large or small square.

The spermatozoa in the large squares (5 x 16 = 80 small squares)

were counted to determine the sperm concentration by applying the

following formula:

The number of sperm counted over 80 small squares x 5 (because

only one-fifth of the total small squares were counted) x 10,000 (to

convert the number of sperm counted in the 0.1 cu mm volume to a

millilitre) x the dilution factor.

Number of sperms per millilitre =

No. of sperms counted x 5 x 200 x10 x 1000

= No. of sperms counted x 107

59  

2.6.6 Sperm cell morphology:

2.6.6.1 Wet preparation:

The semen sample was fixed in buffer formal saline in a ratio of

1:10 immediately after collection. A drop of diluted semen was placed on

a microscope slide under cover-slip. The preparation was left for a couple

of minutes to allow sperm cells to settle down. Under phase-contrast

microscopy at magnification of (400x), two hundred sperms were counted

for abnormalities, loose heads, proximal cytoplasmic droplets, mid-piece

and tail abnormalities. The results were expressed as a percentage for

each abnormality in different ejaculates (Rao, 1971).

2.6.6.2 Dry preparation:

Sperm head and acrosome abnormalities were evaluated on air-

dried eosin-nigrosin stained slide. Two hundred spermatozoa were

examined on each slide under oil immersion lens at magnification of (400

x) and incidence of each category was expressed as a percentage for head

and acrosome abnormalities in each ejaculate (Evans and Maxwell,

1987).

2.6.7 Live-dead spermatozoa:

Eosin-nigrosin was used for routine examination of live-dead

spermatozoa. A small drop of semen was mixed well with two drops of

60  

the stain on a pre-warmed slide (37°C). The drops were allowed to reach

body temperature before mixing. Thin smear was prepared from the

mixture. Under high power magnification (400x), two hundred

spermatozoa were examined for live and dead cells. Sperm heads or at

least two thirds of the head stained red, were considered as dead

spermatozoa. The result was expressed as a percentage for live-dead

sperms in each ejaculate (Rao, 1971).

2.7 Extender preparation:

The extender was prepared a day before collection. Tris-based

diluent used in this study was composed of a solution of 3.634 gram (gm)

Tri-hydroxy methyl amino methane (Tris), 0.5 (gm) glucose, 1.99 (gm)

citric acid monohydrate, 15 ml egg yolk, 5 ml glycerol, 100000 i.u

penicillin, 100 mg streptomycin and distilled water added to the level of

100 ml (Evans and Maxwell, 1987).

2.8 Semen dilution:

Dilution of fresh semen was done within the first 10–15 minutes

following collection and evaluation. The ejaculate was placed in a water

bath at 37°C and the diluent was at the same temperature when mixed

with the semen. Depending on a dilution rate (1 semen: 4 diluent), only

good samples of semen were diluted. A clean glass pipette was filled with

required amount of extender, and slowly added to the semen in one–step

61  

extension methodology. Then the diluted semen was mixed gently and

was re-examined for sperm motility (Evans and Maxwell, 1987).

2.9 Semen cooling:

The diluted semen in the flask was carried in a portable water bath

at 32°C. The water level should be higher than that of the diluted semen

inside the flask. Then the water-bath with diluted semen in the flask was

placed inside the upper part of a cold cabinet at 5°C. When temperature

of diluted semen reached 20°C, ice-cubes were added to the water-bath

every five minutes to maintain an even drop in temperature to 5°C.

However, the temperature of diluted semen was reduced form 32°C to

5°C within 45 to 60 minutes, depending on the volume of diluted semen.

Then the diluted semen was left inside the cold cabinet for another one

hour before packing.

2.10 Semen Packing:

Medium size (0.5 ml) IMV plastic straws were filled with the

diluted semen by suction. Each straw contained 200x106 spermatozoa.

The air space at the end of the straws was created by a plastic comb

before sealing with the polyvinyl alcohol powder to prevent straws crack

during freezing. The plugged straws were immersed for two hours in a

water–bath at 5°C to allow equilibration of sperm with diluent

components and to let the plugs to get harder and the excess powder on

62  

the end of the straws fall to the bottom of the bath. Then the straws were

carefully dried with a pre-cold linen towel inside the cold cabinet.

2.11 semen Freezing:

The filled straws were placed horizontally on a cold rack at 5°C

and then lowered into liquid nitrogen vapour LN2 (-80°C) 3-4 centimetres

above the surface of liquid nitrogen in a styrofoam container for 8

minutes. Then the frozen straws were plunged in liquid nitrogen (-196°C)

and then transferred to a storage container.

2.12 Pre- and post-freezing rejected samples:

Diluted semen that showed less than 60% progressive motility after

cooling to 5°C and just before freezing was discarded and considered as a

pre-rejected sample and that frozen semen gave less than 50 percent post-

thawing motility was discarded and considered as a post-rejected sample.

2.13 Thawing of frozen semen:

From each frozen semen batch, two straws, which had been frozen

and stored in liquid nitrogen for a minimum of 24 h, were thawed by

immersion in warmed water at 37°C for 20 seconds (Evans and Maxwell,

1987). Immediately after thawing of semen, the examination of motility

was done on a pre-warmed slide (37°C) with a cover-slip, under phase

contrast microscope. Individual ejaculates of ram semen can only be

considered suitable for storage and use for insemination when the

63  

percentage of forward moving spermatozoa was not less than 50 percent

post- thawing (Berg, 1999).

2.14 Oestrous synchronization:

Ewes were treated with progestagen impregnated intra-vaginal

sponges (Chrono-gest®sponges- Intervet) for 14 days and were injected

with 500 i.u PMSG (pregnant mare serum gonadotrophin - Folligon®-

Intervet) intra-muscular at the time of sponge's removal.

2.15 Insemination of ewes:

A total of 114 first inseminations were performed. Of these, 63

inseminations were done with Kabashi and 51 with Hamari frozen-

thawed semen. All females were inseminated in the cervix with 0.5 ml

frozen- thawed semen at 55 hour after sponge removal. The ewes were

presented for cervical insemination by an assistant lifting the hind

quarters of the ewe over, while the front legs remained standing on the

ground. The steps for cervical insemination were as follows:

(1) A speculum was introduced carefully in the vagina of the ewe (10–

13 cm) and located the cervix.

(2) The amount of the mucus observed in the vagina was drained when

it was necessary.

64  

(3) The insemination syringe was introduced as far as possible into the

cervix. Twisting motion but with no force with the syringe was

used.

(4) Then the plunger of the AI syringe was pressed to expel the semen

while the syringe was drawn slightly (Evans and Maxwell, 1987).

2.16 Conception rate:

The pregnancy was detected by palpation after three months of

insemination. The reproductive parameters used in this study were

fertility and prolificacy. The fertility is the proportion of ewes lambing of

all those artificially inseminated, whereas, the prolificacy is the number

of lambs born per lambing ewe (usually are expressed as a percentage).

2.17 Experimental design:

• Experiment (1) was design to study the influence of

season and breed on reaction time (RT); semen

characteristic, semen volume, sperm motility, sperm cell

concentration, sperm cell abnormalities and live sperm

cells of semen collecte from 6 Hamari and 3 Kabashi

rams. The relationship between ejaculate volume, sperm

cell concentration, body weight and scrotal

circumference were included. The methodology was

described in materials and methods.

65  

• Experiment (2) was design to study the influence of

breed and seasons on freezability of semen obtained

from the two breeds, consisted of the post-thaw

evaluation of frozen ejaculates. Two random straws from

each batch were used to assess the progressive motility

of semen as described in materials and methods.

• Experiment (3) to study the conception rate of oestrous

synchronized ewes (114) in different 6 farms

inseminated with frozen semen obtained from the two

breeds according to the protocol shown in materials and

methods.

2.18 Statistical analysis:

Data were treated and analysed statistically using SPSS 10.1

Package (Statistical Products and Service Solutions for windows) (SPSS

inc., 1999). Physiological and morphological results of semen, post

thawing motility and reaction time were represented as mean+standard

error of the mean. The data were subjected to analysis of variance using

the GLM (General linear Model) and mixed model procedure. The

statistical model included the effect of breed (2), season (3) and

breed*season interaction.

Yijk = µ + βi + Sj + ( βS)ij + eijk

66  

Where:

Yijk = Trait measure on individual kth ram.

µ = Over all mean.

βi = The effect of the ith breed.

i = 1,2.

Sj = Effect of the jth season.

j = 1,3.

(βS)ij = interaction between breedi and seasonj .

eijk = Random error term associated with single records.

The testing of differences between means was done using Scheffe`s

multiple range test.

Correlation analysis using Pearson correlation procedure of SPSS

was used to assess the relationship between body weight, scrotal

circumference, semen volume and sperm cell concentration.

The significance of the difference between the two breeds in

pregnancy rate (number of pregnant ewes/number of ewe inseminated

X100%) was tested using the chi-square test. Prolificacy (number of

lambs born/number of ewes lambed) in the two breeds was compared

using the Student`s t- test. Values of p<0.05 was chosen as an indication

of significance.

67  

 

 

 

Results

 

 

 

 

 

 

68  

 

3-Results:

3-1 Semen characteristics:

3-1-1 Colour and consistency of ejaculate:

The colour and consistency of most semen ejaculates were white-

creamy (70.20 and 58.95%), white-milky (22.22 and 31.58%) and watery

(7.58 and 9.47%) for Hamari and Kabashi rams respectively (Table 1).

Statistical analysis showed that, breed had no effect on semen colour and

consistency, but winter exerted a significant effect on this character. The

watery ejaculates increased during summer and decreased during winter

(12.73 and 18.18 vs. 3.53 and 4.08%) for Hamari and Kabashi rams

respectively (Table 2).

Table (1) Number and percentage of ejaculates with different colour

and consistency of Hamari and Kabashi rams (mean±S.E).

Parameter Hamari Kabashi

White-creamy 139 (70.2%) 56 (58.95%)

White-milky 44 (22.22%) 30 (31.58%)

Watery 15 (7.58%) 9 (9.47%)

69  

Table (2) Number and percentage of watery ejaculates in

different seasons of the year of Hamari and Kabashi rams

Season Hamari Kabashi

Summer 9 (12.68)a 4 (18.18)a

Autumn 3 (7.1)a 3 (12.5)a

Winter 3 (3.53)b 2 (4.08)b

a, b, different superscripts in the same column indicate significant differences (P<0.05).

3-1-2 Ejaculate volume :

The results in table (3) indicated that the mean ejaculate volume for

Hamari and Kabashi rams were 1.31±0.04 and 1.23±0.0.06 ml

respectively. The highest ejaculate volumes (2.5 and 2.3 ml) were

collected during autumn and the lowest ejaculate volumes (0.5 and 0.6

ml) were collected during summer from Hamari and Kabashi rams

respectively. No difference in the mean ejaculate volume was found in

this study between both breeds and seasons. Breed and season of the year

did not exert any significant effect on this semen trait (Table 3&4).

70  

Table (3): Means and standard errors of semen traits in different breeds (M±S.E.)

Parameters Hamari Kabashi

Ejaculate volume (ml) 1.31±0.04 1.23±0.06

Mass motility (1-5 scores) 2.73±0.06 2.37±0.08

Motility ( % ) 68.11±0.79 67.91±1.10

Sperm concentration (x 106/ml) 2.83±0.06 2.95±0.08

Live sperm ( % ) 76.90±1.25a 84.73±1.93b

Total abnormalities( % ) 4.26±1.01 4.47±1.18

a, b, different superscripts in the same row indicate significant differences ( P<0.05).

3-1-3 Mass activity:

As in tables (3 and 4) mean score for mass activity were

2.73±0.06 and 2.37±0.08 for Hamari and kabashi rams respectively.

The highest mean mass activity were recorded during winter

(3.16±0.07 and 3.47±0.10), while the lowest mean scores were

recorded during summer (2.48±0.08 and 2.32±0.15) for Hamari and

Kabashi breed-type respectively. Statistical analysis revealed no

breed effect on mass activity but winter season had a highly

significant effect (P< 0.01) on this trait. The interaction term between

breeds and seasons was significant. This indicates that the ranking of

the two breeds varied among seasons.

71  

Table (4): Means and standard errors of semen traits in different seasons (M±S.E.)

Summer Autumn Winter Parameter

Hamari Kabashi Hamari Kabashi Hamari Kabashi

Ejaculate volume(ml )

1.33±0.06 1.18±0.10 1.36±0.09 1.29±0.10 1.23±0.05 1.21±0.07

Mass motility (1-5 scores)

2.48±0.08a 2.32±0.15a 2.56±0.13a 2.42±0.15a 3.16±0.07b 3.47±0.10b

Motility (% )

60.78±1.19a 60.46±2.14a 71.88±1.78b 71.04±2.05b 71.68±1.03b 72.25±1.44b

Sperm concentration

(x106/ml)

2.39±0.09a 2.52±0.15a 2.88±0.13b 3.15± 0.15b 3.21± 0.07b 3.18±0.10b

Live sperm (%)

74.20±2.22a 82.80±3.84a 77.58±1.98b 85.25±3.04b 78.93±2.30b 86.13±3.04b

Total abnormalities

( % )

5.16±1.20a 5.40±1.51a 3.97±1.02b 4.23±1.12b 3.68±0.84b 3.78±0.97b

a,b, different superscripts in the same row indicate significant differences ( P< 0.05).

3-1-4 Individual motility:

Results furnished in tables (3 and 4) showed that the average

individual motility were 68.11±0.79 and 67.91±1.10 percent for Hamari

and kabashi breeds respectively .The highest mean motility were found to

be 71.88±1.78 and 72.25±1.44 percent during autumn and winter,

whereas, the lowest mean motility were 60.78±1.19 and 60.46±2.14

72  

percent during summer for Hamari and Kabashi breeds respectively.

Summer season showed a highly significant effect (P< 0.01) on

individual motility but breed had no such effect.

3-1-5 Sperm cell concentration:

Results of spermatozoa count presented in tables (3 and 4) showed

that the average sperm cell concentration per ml for Hamari and Kabashi

rams semen were 2.83±0.06 and 2.95±0.08(x109) respectively. The

highest mean sperm concentrations were counted during winter

(3.21±0.07 and 3.18±0.10 billion spermatozoa/ml) while the lowest mean

concentrations were counted during summer (2.39±0.09 and 2.52±0.15

billion spermatozoa/ml) for Hamari and Kabashi breeds respectively.

Statistical analysis showed that breed and summer season exerted a

highly significant effect (P< 0.01) on this trait.

3-1-6 Live sperm count:

The mean live sperm cell percentage in this study was

found to be 76.90±1.25 and 84.73±1.93 percent for Hamari and

Kabashi rams respectively (Table 3). The average live sperm cell

percentage was higher during autumn and winter (78.93± 2.30 and

85.25±3.04 percent) and the lowest values were observed during

summer (74.20±2.22 and 82.80±3.84 percent) for Hamari and

Kabashi rams respectively. Breed and summer season exerted a

73  

highly significant effect (P< 0.01) on this character. The interaction

term between breeds and seasons was highly significant. This

indicates that the ranking of the two breeds varied among seasons

(Table 4).

3-1-7 Sperm cell abnormalities:

The results in tables (4, 5 & 6) showed that the mean percentage

for mid-piece, tail, head, acrosome abnormalities, loose heads and

total abnormalities were 1.91±0.14 and 1.52±0.16; 5.42±0.21 and

5.95± 0.31; 6.38±0.21 and 6.65±0.32; 1.73±0.12 and 1.64± 0.18;

5.88±0.19 and 6.57±0.29; 4.26±1.01 and 4.47±1.18 for Hamari and

Kabashi rams respectively. The incidences of mid-piece and tail

abnormalities were significantly affected by summer season (P< 0.05)

where, head abnormalities and loose heads were highly affected by

summer season (p<0.01). Breed affected significantly (p<0.05) mid-

piece abnormalities. There was a significant difference between

summer and autumn on acrosome abnormalities. However, winter

was intermediate and it was not significantly different from either

summer or autumn. The mean percentage of total abnormalities were

higher (5.16±1.20 and 5.40±1.51) in summer than winter (3.68±0.84

and 3.78±0.97) for Hamari and Kabashi breeds respectively.

74  

3-2 Live body weight:

The initial average of live body weights at the start of the

experiment were 71.60±1.91 and 60.50±2.50 kg, whereas, the

final weights at the end of the experiment were 81.78±2.06 and

71.13±2.73 kg for Hamari and Kabashi rams respectively. Season

of the year did not exert a significant effect on live body weight.

Table (5): Means and standard errors of sperm cell abnormalities in different breeds (M±S.E.)

Parameters Hamari Kabashi

Mid-piece

Abnormalities

1.91±0.14a 1.52±0.16b

Tail

Abnormalities

5.42±0.21 5.95±0.31

Head

Abnormalities

6.38±0.21 6.65±0.32

Acrosome

Abnormalities

1.73±0.12 1.64±0.18

Loose heads

5.88±0.19 6.57±0.29

a,b, different superscripts in the same row indicate significant differences ( P< 0.05).

75  

3-3 Scrotal circumference:

The average scrotal circumference measurements in this study

were found to be 33.12±0.21 and 31.38±0.31 cm for Hamari and

Kabashi rams respectively (table 7). Breed showed a highly

significant effect (p<0.01) on scrotal circumference measurements

but the season of the year did not. The interaction term between

breeds and seasons was significant. This indicates that the ranking of

the two breeds varied among seasons.

Table (6): Means and standard errors of sperm cell abnormalities in different seasons (M±S.E.)

Summer Autumn Winter Parameters

Hamari Kabashi Hamari Kabashi Hamari Kabashi

Mid-piece

Abnormalities

2.33±0.18a 1.80±0.31a 1.77±0.17b 1.38±0.25b 1.64±0.19b 1.38±0.25b

Tail

Abnormalities

6.4±0.37a 6.60±0.62a 4.71±0.34b 6.00±0.49b 5.14±0.37b 5.25±0.49b

Head

Abnormalities

7.80±0.37a 8.20±0.65a 6.41±0.35b 5.75±0.51b 4.93±0.39b 6.00±0.51b

Acrosome

Abnormalities

2.20±0.21a 1.80±0.36a 1.35±0.20b 1.63±0.29b 1.64±0.22ab 1.50±0.29ab

Loose Heads

7.07±0.34a 8.60±0.59a 5.59±0.32b 6.38±0.46b 5.07±0.35b 4.75±0.46b

a ,b, different superscripts in the same row indicate significant differences ( P< 0.05)

76  

3.4 Relationship between body weight, scrotal circumference

semen volme and sperm cell concentration:

The correlations of body weight with scrotal circumference were

found to be 0.31 (weak) and 0.70 (strong) and with semen volume were

0.90 (strong) and 0.28 (weak) and with sperm cell concentration were

0.11 (weak) and 0.01 (weak) for Hamari and Kabashi rams respectively

(table 8 & 9).

(Figure 1) The effect of season on sperm abnormalities of Hamari rams.

77  

(Figure 2) The effect of season on sperm abnormalities of Kabashi rams.

3.5 Reaction time (RT):

Results illustrated in table (10) showed that the average RTs were

16.41±0.46 and 20.10±1.10 seconds for Hamari and Kabashi rams

respectively. The shortest RTs were recorded in summer and autumn

(14 seconds) in both breed-types, whereas, the longest RTs were

recorded in winter (45 seconds) for Hamari and (35 seconds) for

Kabashi rams. The breed and winter season exerted a highly significant

effect on RT (P< 0.05).

78  

(Figure 3) The effect of season on pre- and post rejection sample rate of Hamari rams.

(Figure 4) The effect of season on pre- and post rejection sample rate of Kabashi.

79  

3.6 Freezability of spermatozoa :

The mean values of freezability of sperms were expressed in terms

of percent post-thaw motility. In the present study the values obtained

were 56.75±5.95 and 56.19±5.32 percent (table 11). Regarding sperm

freezability, neither the breed nor the season was affecting the trait

significantly. The rejected diluted ejaculates of semen before and after

freezing were 30.77%, 32.44% and 15.38%, 6.35% for Hamari and

Kabashi rams respectively. Breed and winter season exerted significant

effect on post-freezing rejection rate (P< 0.05) in both breeds with better

results obtained of Kabashi rams semen.

Table (7):Effect of season and breed on scrotal circumference (Sc) (cm) and body weight (B.wt) (Kg) (mean±S.E)

Breed

Hamari Kabashi

Parameters

B.wt. Sc. B.wt. Sc.

Summer

73.60±1.99 32.63±0.36N.S 61.20±3.45 31.00±0.63N.S

Autumn

75.05±1.87 33.45±0.32N.S 63.00±2.73 31.38±0.50N.S

Winter

81.78±2.06 33.29±0.37N.S 71.13±2.73 31.75±0.50N.S

over-all mean

76.82±1.14 33.12±0.21 65.11±1.73 31.38±0.31

Significance difference at p< 0.05

80  

Table (8): Person`s correlation coefficient between body weight (B.wt), scrotal circumference (Sc), semen volume (SV) and sperm cell concentration (SCC)

from Hamari rams.

 

Correlation significance * P< 0.05 ** P< 0.01

Table (9): Person’s correlation coefficient between body weight (B.wt), scrotal circumference (Sc), semen volume(SV) and sperm cell concentration (SCC) from

Kabashi rams.

Parameters

B.wt.

SC

SV

SCC

B.wt

1

SC

0.70∗

1

SV

0.28

0.38

1

SCC

0.01

0.01

0.08

1

Correlation significance * P< 0.05

Parameters

B.wt. Sc Sv Scc

B.wt.

1

Sc

0.31

1

Sv

0.90*

0.18

1

Scc

0.11

0.54**

0.28

1

81  

Table (10): The reaction time ( RT ) of Hamari and Kabashi rams in different seasons of the year ( mean±S.E)

Season

Hamari

Kabashi

Summer

18.33±0.80a

24.80±1.39a

Autumn

17.47±0.75a

19.13±1.10a

Winter

13.43±0.83b

16.38±1.10b

over-all mean

16.41±0.46

20.10±1.10

a ,b ,c, different superscripts in the same column indicate significant differences ( P< 0.05).

3.7 Conception rate:

During the pregnancy period there was only one case of early

abortion but no incidence of stillbirth or mortality of pregnant ewes or

any other physical abnormalities were detected.

As shown in table (12), the pregnancy rate was (35.3% and 41.3%) and

lambing rate was (35.3% and 39.7%). Chi-square test of the cross-

tabulated variables indicated no significant differences between the

various subclasses. Prolificacy (1.38 and 1.36) was not significantly

different between two breeds.

 

 

 

 

 

82  

Table (11): The effect of breed and season on the pre- and post-freezing rejection batches and post-motilityof frozen semen.

Pre-freezing rejection (%)

Post-freezing rejection (%)

Post-thawing motility (%)

Season

Hamari Kabashi Hamari Kabashi Hamari Kabashi Summer

32.60a 31.00a 24.57a 19.00a 56.96±

6.48 59.00±

5.83 Autumn

33.33a 50.00a 5.56a 00.00b 55.00±

6.32 55.45±

4.98 Winter

21.43b 16.67b 9.29a 00.00b 57.22±

4.15 55.00±

4.47 over-all mean

30.77 32.44 15.38 6.35 56.75± 5.95

56.19± 5.32

a ,b , different superscripts in the same column indicate significant differences ( P< 0.05).

Table (12): Fertility parameters of frozen-thawed semen obtained from Hamari and Kabashi rams.

Ram breed

Parameters

Kabashi

Hamari

63

51

No. of ewes inseminated

26

(41.3%)N.S

18

(35.3%)N.S

Pregnancy rate (%)

25

(39.7%)N.S

18

(35.3%)N.S

Lambing rate (%)

34

(1.36±0.5)N.S

25

(1.38±0.5)N.S

Prolificacy(Mean±S.E)

Significance difference at p< 0.05

83  

(Figure 5) The effect of breed on pre-, post- and total percentage of rejected samples.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

84  

 

 

 

 

 

 

 

 

 

 

 

 

Discussion  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

85  

 

4-1 Semen characteristics:

4-1-1 Colour and consistency of ejaculate:

Records of the colour and consistency of the semen samples

observed in the present study indicated that 70.20, 58.95 percent were

white-creamy, 22.22, 31.58 percent white-milky and 7.58 and 9.47

percent watery for Hamari and Kabashi rams respectively. This result

agreed with other results in literature for Sudan Desert sheep (Galil and

Galil, 1982; Alsayed, 1996; Manahil, 1999, Alsayed,2001) and Dabas et

al. (1997) in Patan-wadi rams. The watery ejaculates increased during

summer and decreased during winter (12.68 and 18.18 vs. 3.53 and

4.08% for Hamari and Kabashi rams respectively) and this was due to the

high ambient temperature during summer which affect all the component

of the reproductive function, particularly the quality of semen (Colas,

1983).

4-1-2 Ejaculate volume:

The average volumes of ejaculate obtained in the present study

were 1.31±0.04 and 1.23±0.06 ml for Hamari and Kabashi rams

respectively. They conformed well to those obtained by Alsayed (1996,

2001) in the Sudan Desert sheep. Similarly Daader et al. (1987) reported

1.26±0.01 ml with Rahmani x Finnish Landrace rams in sub-tropical

86  

conditions, Williams et al. (1990) reported 1.20 ml with Suffolk rams,

Sackmann and Schone (1990) reported 1.20 ml with Mouflon rams,

Salhab et al. (2003) reported 1.20 ± 0.5 ml with Awassi ram and Kafi et

al. (2004) reported 1.2±0.3 ml with Karakul rams. Slightly higher values

were recorded (1.62 ml) by Manahil (1999) in Sudan Desert sheep.

Lower values of ejaculate volumes were reported by various authors,

Chiboka (1980) with West African Dwarf rams, Galil and Galil (1982)

with Sudan Desert rams , Loubser and Van Niekerk (1983) with Angora

rams, Kalous and Samkova (1991) with Sumava rams, Demirci (1993)

with Awassi rams, Zhang et al. (1995) with Xinjiang Fine wool rams,

Ibrahim (1997) with Local Emirate and Local x Chios rams, Dabas et al.

(1997) with Patan-wadi rams, Rege et al. (2000) with Horro and Menz

rams and Lezama et al. (2003) with Katahdin yearling rams. The non-

significant influence of breed and season of the year on the ejaculate

volume is in agreement with findings of many authors (Vijil, 1987; with

Manchega rams, Daader et al.,1987; with Rahmani x Finnish Landrace

rams and Fernandez, 1993; with Corriedale, Polwarth, Merilin and

Merino rams) and is in disagreement with findings of Brewer et al.

(1995) with Dorsal x Romney rams, Gündogan and Demirci (2003) and

Gündogan (2006); who reported significant dependency of volume of ram

ejaculate on season of the year. Generally semen volumes in both breeds

were within the range of ram semen volume reported in the literature.

87  

4-1-3 Mass activity:

The average values obtained for mass activity in the present

study were 2.73±0.06 and 2.37±0.08 for Hamari and Kabashi rams

respectively. These values were lower than those reported by other

authors (Loubser and Van Niekerk, 1983, in Angora rams; Alsayed,

1996, Manahil, 1999, Alsayed, 2001, in Sudan Desert sheep; Kafi et

al., 2004, in Karakul rams). Similarly significant effect of season on

mass activity of spermatozoa was observed by Dufour et al. (1984) in

DLS and Suffolk rams; Vijil (1987) in Manchega rams; Fernandez

(1993) in Corriedale, Polwarth, Merilin and Merino rams; Dabas et

al.(1997) in Patan-wadi rams; Alsayed (2001) in Sudan Desert sheep

and Kafi et al. (2004) in Karakul rams. The interaction term between

breeds and seasons was significant. This indicates that the ranking of

the two breeds varied among seasons.

4-1-4 Individual motility:

The average values of individual motility in this experiment

were 68.11±0.79 and 67.91±1.10 percent for Hamari and Kabashi rams

respectively. These were lower than those values reported by Daader et

al. (1987) in Rahmani x Finnish Landrace rams, Demirci (1993) in

Awassi rams, Zhang et al. (1995) in Xinjiang Fine Wool rams, Manahil

(1999), Alsayed (2001) in Sudan Desert sheep and Salhab et al .(2003) in

88  

Awassi rams. However, they were higher than those values recorded in

West African Dwarf rams (Chiboka, 1980), Danube Fine Wool and Dairy

rams (Karcheva and Baulov, 1989) and Sumava rams (Kalous and

Samkova, 1991). The individual motility was highly significantly (p<

0.01) affected by summer season. Similarly significant effect of season on

individual motility were reported by many authors (Dufour et al.,1984,

Vijil, 1987, Daader et al., 1987, Sackmann and Schone ,1990, Alsayed,

2001, Gündogan and Demirci ,2003 and Gündogan, 2006).

4-1-5 Sperm cell concentration:

The average values of sperm cell count per ml in the present

study were 2.83±0.06 and 2.95±0.08 billion spermatozoa for Hamari and

Kabashi rams respectively. They were similar to those results in the

literature of Sudan Desert sheep (Galil and Galil, 1982; Alsayed, 1996

and 2001; and Dabas et al., 1997). Sperm cell concentration in this study

were relatively lower than those recorded by Demirci (1993); Ibrahim

(1997); Manahil (1999) and Salhab et al. (2003) who reported 3.94, 5.10,

3.38 and 4.0 billion sperm per ml in Awassi , Emirate Local and Emirate

Local x Chios, Sudan Desert sheep and Awassi rams respectively. Lower

values were recorded by Chiboka (1980) with West African Dwarf rams

and Loubser and Van Niekerk (1983) with Angora rams. The present

investigation revealed highly significant effect (P< 0.01) of summer

89  

season on sperm cell concentration. This is in accordance with the

findings of many authors (Vijil, 1987; Daader, 1987; Fernandez, 1993;

Brewer et al., 1995; Alsayed, 2001; Gündogan and Demirci, 2003;

Gündogan, 2006) who reported significant dependency of sperm cell

concentration on the seasonal influences and not on the semen volume.

This indicated that the effect of season on testicular function is greater

than that exerted on accessory gland function (Salah et al. 1992). In the

present results, it can be considered that, the semen concentration in both

breeds fell within the range of good semen quality even in the hot

summer (Evans and Maxwell, 1987).

4-1-6 Live sperm count:

The mean values of live sperm cell percentage in this study

were 76.90±1.25 and 84.73±1.93 percent for Hamari and Kabashi

rams respectively. They were in line with those reported by Galil and

Galil (1982) in Sudan Desert sheep and Daader et al. (1987) in

Rahmani x Finnish Landrace rams. Higher values were recorded by

Dabas et al. (1997) in Patan-wadi rams, Manahil (1999) and Alsayed

(2001) in Sudan Desert sheep. Lower values were reported in

different breeds (Chiboka, 1980, in West African Dwarf rams;

Loubser and Van Niekerk, 1983, in Angora rams). There was a

highly seasonal effect (summer) on live sperm cells percentage in

90  

rams of the two breeds under the present study. This is in agreement

with many authors (Chiboka, 1980; Vijil, 1987; Daader et al., 1987;

Fernandez, 1993; Dabas et al., 1997 and Alsayed, 2001). The

interaction term between breeds and seasons was significant. This

indicates that the ranking of the two breeds varied among seasons.

The percentage of the live spermatozoa in the present study (over

70%) was within the acceptable level needed for good fertility

(Gomes, 1977) even in the hot season. The increase in the percentage

of dead spermatozoa during summer agreed with the previous reports

with Sudan Desert sheep (Galil and Galil, 1982; Alsayed, 2001).

4-1-7 Sperm cells abnormalities:

The mean values of sperm cell abnormalities in this experiment

were 4.26±1.01 and 4.47±1.18 percent for Hamari and Kabashi rams

respectively. These values were in good agreement with the findings

reported by Dabas et al. (1997) in Patan-wadi rams and Manahil (1999)

in Sudan Desert sheep. Higher values were reported by many

investigators (Chiboka, 1980, in West African Dwarf rams; Loubser and

Van Niekerk, 1983, in Angora rams; Daader et al., 1987, in Rahmani x

Finnish Landrace rams; Kalous and Samkova, 1991, in Sumava rams).

Though, lower value was recorded by Demirci (1993) in Awassi rams.

The analysis of data revealed significant differences (P< 0.05) in the total

91  

percentage of abnormal spermatozoa in the different seasons. This is in

agreement with many authors (Daader et al., 1987; Vijil, 1987,

Fernandez, 1993 and Gündogan, 2006). The incidence of head and tail

abnormalities were in accordance with the results recorded by Demirci

(1993) in Awassi rams. The incidence of mid-piece abnormalities in the

present study was in favour of those reported by Alsayed (2001) in Sudan

Desert sheep. The present study indicated that the incidence of loose

heads in both breed-types were higher than those of Alsayed (2001) in

Sudan Desert sheep. Statistical analysis showed a significant summer

effect (P< 0.05) on mid-piece, tail, acrosome abnormalities and highly

significant (p<0.01) on head abnormalities and incidence of loose heads

in both breeds. The best results were recorded during autumn and winter

compared to summer. However, the high ambient temperature during

summer season could be a direct cause in the elevation of sperm

abnormalities where temporary epididymal or accessory dysfunction

could occur (Fayemi and Adegbite, 1982). However, all the rams under

investigation had average spermatozoal abnormalities within the

recommended limits of good quality semen, as determined by Evans and

Maxwell (1987), even during the hot summer.

92  

4-2 Scrotal circumference (Sc):

The mean values for Sc in the present study were

33.12±0.21 and 31.38±0.31 cm for Hamari and Kabashi rams

respectively. These values were similar to those obtained by Master

(1988) in Dohne Merino rams (32.2 cm), Demirci (1993) in Awassi

rams (30.75cm), Zhang et al. (1995) in Xinjiang Fine Wool rams

(33.34 cm) and Kafi et al.(2004) in Karakul rams (32.6). The

measurements of Sc of Hamari and Kabashi rams used in the present

experiment fell within the normal size of breeding rams (Master,

1988). In this study Sc was medium correlated with sperm

concentration (0.54) in Hamari rams and highly correlated with body

weight (0.70) in Kabashi rams. Similarly Trejo et al. (1990) have

reported 0.76 correlation coefficient for Sc with body weight in

Suffolk, Romney, Rambouillet, Pelibuey and Criollo rams. Weak

correlations of Sc with semen volume in the present study of 0.18 and

0.38 for Hamari and Kabashi rams respectively. These values were

not comparable to the values reported in the literature of 0.63 for

Manchega rams (Vijil, 1983); 0.98 for Awassi rams (Demirci, 1993)

and 0.84 for Xinjiang Fine wool rams (Zhang et al., 1995) for

correlations of Sc with semen volume. According to Mickelsen et al.

(1981), Downey et al. (1984), Dyrmundsson et al. (1989), Sackmann

93  

and Schone (1990), Nwoha (1996), Gastel et al., (1995), Gündogan

and Demirci (2003); Melpomeni et al.(2004) and Gündogan (2006)

who reported significant seasonal variations on the testes volume . In

this study no significant difference was found between Sc

measurements in the two breeds in different seasons of the year. The

explanation for that may be that the rams in our study were physically

mature and they were effectively monitored not to fluctuate in their

body weight during the study and were kept indoors all year round

taking the same feeding levels and/or due to the in-significant

extreme temperature fluctuation and day light differences in the

experimental area. This is in agreement with Cameron et al. (1988),

Fernandez (1993), Gastel et al. (1995) and Bielli et al. (1997) who

suggested that the differences existed in seasonal variations in

testicular dimensions in adult rams due to the differences in feeding

levels. The interaction term between breeds and seasons was

significant. This indicates that the ranking of the two breeds varied

among seasons.

4-3 Reaction time (RT):

The mean values for the reaction time to the first mount in the

present study were 16.41±0.46 and 20.10±1.10 seconds for Hamari and

Kabashi rams respectively. They were lower than the mean values

94  

reported by Zuzanna (1984) with rams (70.9 seconds, ranging from 30 to

166.7 seconds), Ibrahim (1997) with Emirates Local and Local x Chios

breeds raised under sub-tropical conditions (43.7 seconds) and Lezama

(2003) with Katahdin yearling rams (73 seconds). Regarding RT, the

breed had no significant effect but the winter exerted a highly significant

effect (P< 0.01). This is conforming to the findings of Trejo et al. (1990)

with Suffolk, Romney, Rambouillet, Pelibuey and Criollo rams raised on

the high plateau in Mexico. The shortest RT recorded was in summer and

autumn (14 seconds) in both breed-types, whereas, the longest time

recorded was in winter (45 seconds for Hamari rams and 35 seconds for

Kabashi rams). These values signify that the high ambient temperature of

summer did not completely depress the sexual activity of the tested rams.

This is in agreement with the results reported by Lindsay (1969) with

Merino rams, Ibrahim (1997) with Local Emirates and Local x Chios

rams and Gündogan and Demirci (2003) in two Turkish breeds. However,

there was a seasonal effect on RT but not to the extent of complete

depression as in temperate breeds and this implies that there was no

photoperiodic effect on this trait.

4-4 Freezability of spermatozoa:

Semen is usually evaluated both before and after freezing with the

aim of identifying sires and/or ejaculates with satisfactory semen quality.

95  

Semen ejaculates are assessed in terms of sperm concentration, sperm

motility and morphology, whereas motility post-thawing is the only

parameter used to evaluate freezing procedures (Rodriguez and Larsson,

1998). The average percentage of post-thawing sperm motility in the

present study was 56.75±5.95 and 56.19±5.32 percent for Hamari and

Kabashi rams respectively. The results in this experiment showed that the

post-thawing motility levels were higher than those found by Loubser and

Van Niekerk (1983) with Angora rams (46.62%). However, our results

were lower to those found by other authors (Söderquist et al., 1997 and

Gil, 1999) in temperate breeds. The difference may be due to the

protocols and techniques used for semen dilution and freezing

(centrifugation). Similarly non-significant effect of breed and season

were found between Hamari and Kabashi breeds in post-thawing motility

(Loubser and Van Niekerk, 1983). Though, the results obtained in the

present study satisfy the criterion described for suitable frozen semen

needed for cervical insemination in both breed (Evans and Maxwell,

1987; Berg, 1999).

4-5 Rejection rate:

The incidence of rejected samples of semen before and after

freezing in the present study were 30.77, 32.44 and 15.38, 6.35 percent

for Hamari and Kabashi rams respectively. The data showed a significant

96  

difference (P< 0.05) between the two breeds and between seasons of the

year in the rate of rejected semen samples after freezing, with better

results for Kabashi breeds (15.38 vs. 6.35). This could mean that Kabashi

rams spermatozoa were more resistant to low temperature during freezing

than that of Hamari rams. Similar results reported by Vlachos and

Tsakalof (1965) that the semen of Friesian rams was more sensitive to

low temperature during freezing than that of Chios. The significant

influence of winter season on semen freezability is in agreement with

finding of D´Alessandro and Martemucci (2003) with Leccese rams and

this is may be due to the seasonal variations in quantity and quality of

seminal plasma proteins (SP), and their differential protective effect

against cold-shock (La Falci et al.,2002; Dominguez et al.,2008). Autumn

and winter seasons were the most favourable periods to harvest high

quality semen for freezing and AI purposes.

4.6 Fertility following AI:

The pregnancy rates following oestrous synchronization and AI

using frozen-thawed semen achieved in the present study were 35.3% and

41.3% for Hamari and Kabashi rams. The results were comparable to

those reported by other authors (Dyrmundsson et al., 1989; Molinia et al.,

1996; Cappai et al., 1998; Naqvi et al., 1998; Sördequist, 1999; Sánchez-

Partida et al., 1999; Anel et al., 2003; Fair et al., 2005). Conventional

97  

cervical insemination in ewes with frozen-thawed semen has not been

applied fully on sheep industry due to disappointing results (fertility 10 –

30%) (Windsor et al., 1994, Salamon and Maxwell, 1995, Maxwell and

Watson, 1996, Monika, 2006). Ewe breed has been shown to have a

major effect on pregnancy rate following cervical AI using frozen-thawed

semen. Conception rate values of 8, 28, 44, and 77% were reported for

Suffolk, Texel, Belclare and Finnish landrace breeds respectively

(Donovan et al., 1999). In Norway higher fertility result in Dala breed

61.4 and Spæl breed 71% were reported (Berg, 1999). The possible

reason for this difference is strongly dependant on breed and age of the

ewes and this is attributable to the morphometric parameters, such as the

external diameter and number of cervical folds, type of cervical orifice

and mucus secretion or depth of penetration of the cervix AI (Fukui and

Roborts, 1978; Berg, 1999; Donovan et al.2004). However, the lower

fertilizing rate following cervical AI must therefore be at least partially

due to impeded sperm transport through the genital tract (Salamon and

Maxwell, 1995). Although, laparoscopic AI is the only method that

ensures intrauterine application of frozen-thawed semen with satisfactory

fertility in sheep under field conditions (Salamon and Maxwell, 2000;

Fukui et al., 2007), laparoscopic AI has problems related to complexity,

high equipment costs and the need of trained technicians (Naqvi et al.,

1997; Anel et al.,2006). The higher fertility results that were obtained

98  

after cervical insemination with fresh semen compared to frozen-thawed

semen in Sudan Desert sheep (Alsayed, 1996, 2001; El Mubark,2001;

Makawi and Manahil,2007), could be due to the reduced viability of

frozen spermatozoa resulting in low numbers of viable or undamaged

spermatozoa reaching the fertilization site (Monika, 2006).

4-7 Conclusions and Recommendations:

(1) Measurement of scrotal circumference is the useful test for

identifying rams that are suitable for breeding and artificial

insemination purposes.

(2) The data obtained clearly showed that the two breeds are

continuous breeding animals as they were capable of

producing semen of good quality all the year round.

However, seasonal fluctuation in semen characteristics were

observed but was not sharp as in temperate breeds.

(3) The semen collected from the two breeds was suitable for

freezing and recovery rate after thawing was within the

acceptable limits needed for cervical insemination.

(4) Semen of best quality was obtained during autumn and

winter which are the suitable periods to harvest semen for

freezing and AI purposes.

99  

(5) The results obtained provided a new approach to the

cryopreservation of ram semen, and could positively

contribute to intensive sheep production and gene

conservation.

(6) Although conception rate achieved following cervical

insemination using frozen-thawed semen is relatively

low, but still it is an important objective of establishing

a cost-effective and widely applicable AI procedure for

acceptable pregnancy rates in sheep.

100  

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Appendix (1): Climatic conditions in Khartoum during the experimental period.

Temperature (C˚) Month

Maximum Minimum Average

Humidity

(%)

Rainfall

mm

April 99 40.8 25.0 32.9 15 0.0

May 42.8 27.8 35.3 21 0.0

June 42.4 27.5 35.0 23 0.0

July 39.7 25.8 32.8 37 1.4

August 39.5 25.8 32.7 43 8.5

September 39.5 25.4 32.5 42 15.3

October 37.9 25.8 30.9 30 34.8

November 35.6 20.6 28.1 23 0.0

December 31.1 16.2 23.7 30 0.0

January2000 30.2 14.7 22.5 27 0.0

February 32.8 16.2 24.5 19 0.0

March 37.6 21.1 29.3 15 0.0

142  

Appendix (2) Seminal characteristic Anova table

Source Variables df Mean square F Sig.

Season Semen volume

Mass activity

Individual motility

Sperm count

Live cells

2

2

2

2

2

.203

42.115

3091.889

12.064

1810.025

.875

47.719

30.654

23.075

29.223

.006

.250

.176

.139

.169

Breed Semen volume

Mass activity

Individual motility

Sperm count

Live cells

1

1

1

1

1

.344

.000

2.150

.746

646.478

1.481

.000

.021

1.427

10.437

.005

.000

.000

.005

.035

Season*breed Semen volume

Mass activity

Individual motility

Sperm count

Live cells

2

2

2

2

2

.100

1.701

10.690

.471

400.158

.432

3.367

.106

.900

6.461

.003

.023

.001

.006

.043

Error Semen volume

Mass activity

Individual motility

Sperm count

Live cells

287

287

287

287

287

.232

.505

100.864

.523

17776.481

143  

Appendix (3) Sperm cell abnormalities Anova table

Source Variables df Mean square F Sig.

Season Abnormal mid-piece

Abnormal tails

Abnormal heads

Abnormal acrosome

Loose heads

2

2

2

2

2

1.551

8.397

29.188

1.264

37.154

3.138

4.358

13.937

1.919

21.652

.050

.017

.000

.156

.000

Breed Abnormal mid-piece

Abnormal tails

Abnormal heads

Abnormal acrosome

Loose heads

1

1

1

1

1

2.192

3.963

1.013

.113

6.544

4.435

2.057

.484

.172

3.814

.039

.157

.489

.680

.055

Season*breed Abnormal mid-piece

Abnormal tails

Abnormal heads

Abnormal acrosome

Loose heads

2

2

2

2

2

.076

2.227

4.019

.534

3.958

.154

1.156

1.919

.810

2.307

.857

.322

.155

.449

.108

Error Abnormal mid-piece

Abnormal tails

Abnormal heads

Abnormal acrosome

Loose heads

61

61

61

61

61

.494

1.927

2.094

.659

1.716