Title

Studies on the Effects of the Induction of Cyclic Ovarian Activityduring Early Postpartum Using GnRH and PGF2α on theSubsequent Reproductive Performance in Dairy Cows( 本文（FULLTEXT) )

Author(s) Carlos, Santiago Amaya Montoya

Report No.(DoctoralDegree) 博士(獣医学) 甲第238号

Issue Date 2007-09-14

Type 博士論文

Version author

URL http://hdl.handle.net/20.500.12099/23183

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Studies on the Effects of the Induction of Cyclic

Ovarian Activity during Early Postpartum Using

GnRH and PGF2α on the Subsequent Reproductive

Performance in Dairy Cows

(乳牛における GnRH と PGF2α製剤を用いた分娩後の

早期卵巣賦活化処置による繁殖成績向上に関する研究)

2007

The United Graduate School of Veterinary Sciences

Gifu University

(Obihiro University of Agriculture and Veterinary Medicine)

Carlos Santiago Amaya Montoya

ii

Contents

Page Chapter 1. General introduction 1

1.1 Actual trends in the fertility of high producing dairy cows 1 1.2 Postpartum resumption of ovarian activity 2 1.3 Relation between postpartum energy status and resumption

of ovarian activity during postpartum 3 1.4 Characteristics of the resumption of luteal activity during the

early postpartum period in dairy cows 5 1.5 Hormonal control of the ovarian activity for the reduction

the postpartum anovulatory interval 6 1.6 General objectives 9

Chapter 2. General Materials and Methods 10 2.1 Animals 10 2.2 Hormonal treatments 10

2.3 Ovarian ultrasonography 11 2.3.1 Frequency and description of examinations 11 2.3.1a Detection of ovulation after treatments 11 2.3.1b Ultrasound technique and evaluation of the

ovarian morphology 11 2.4 Blood collection and hormone determination 14 2.5 Biochemical analysis 16 2.6 Statistical analysis 17 Chapter 3. Induction of Ovulation with GnRH and PGF2α

at Two Different Stages during the Early Postpartum Period in Dairy Cows: Ovarian Response and Changes in Hormone Concentrations 19

3.1 Introduction 19 3.2 Materials and method 21 3.2.1 Animals and hormonal treatment 21 3.2.2 Ovarian ultrasonography 21 3.2.3 Blood collection and determination of hormones 22 3.2.4 Statistical analysis 22 3.3 Results 23

iii

3.3.1 Ovulatory response 23 3.3.2 Follicular dynamics 23 3.3.3 Plasma concentrations of FSH and IGF-1 24 3.3.4 Ovulatory follicle, CL periodicum and induced CL development 24 3.3.5 Plasma concentrations of P4 and E2 26

3.4 Discussion 26 3.5 Summary 31 Chapter 4. Cyclic Ovarian Activity and Fertility Traits of Cycling and

Non-Cycling Dairy Cows Induced to Ovulate with GnRH and PGF2α Treatments 21days postpartum 43

4.1 Introduction 43 4.2 Materials and method 44 4.2A Study 1: Ovulatory and cyclicity response of dairy cows under experimental conditions 44 4.2A.1 Animals 45 4.2A.2 Evaluation of the luteal activity within

21days postpartum 45 4.2A.3 Hormonal treatment 46 4.2A.4 Observation o the ovulatory response 46 4.2A.5 Ovarian cyclicity 46 4.2B Study 2: Ovulation, cyclic activity and fertility responses of dairy cows under commercial conditions 47 4.2B.1 Animals 47 4.2B.2 Evaluation of the luteal activity within 21 days postpartum 48 4.2B.3 Hormonal treatment 48 4.2B.4 Ovarian response 48 4.2B.5 Ovarian cyclicity and assessment of the fertility 49 4.3 Statistical analysis 49 4.4 Results 50 4.4A Study 1 50 4.4A.1 Occurrence of early first ovulation and response to GnRH and PGF2α 50

iv

4.4A.2 Plasma P4 levels by 28 days postpartum in GnRH-PGF2α treated cows 51 4.4A.3 Traits for the commencement of luteal activity 51 4.4A.4 Characteristics of the ovarian cycles 52 4.4B Study 2 52 4.4B.1 Occurrence of ovulation within 21 days postpartum and luteal formation prior to PGF2αtreatment 52 4.4B2 Plasma P4 levels by 28 days postpartum in the GnRH- PGF2αtreated cows 53 4.4B.3 Traits for the commencement of luteal activity 53 4.4B.4 Characteristics of the ovarian cycles 54 4.4B.5 Fertility traits 54 4.5 Discussion 55 4.6 Summary 61 Chapter 5. The Relation between Metabolism and Ovarian Status on the Ovulatory Response in Dairy Cows Treated with GnRH and PGF2αduring Early Postpartum 75

5.1 Introduction 75 5.2 Materials and methods 76 5.2.1 Animals 76 5.2.2 Evaluation of the luteal activity within 21 days postpartum and following the induction of ovulation 77 5.2.3 Hormonal treatment 77 5.2.4 Observation of the ovarian response 78 5.2.5 Blood sampling and steroid hormone analysis 78 5.2.6 Biochemical analysis 79 5.2.7 Statistical analysis 79 5.3 Results 80 5.3.1 Occurrence of ovulation within 21 days postpartum and response to GnRH and PGF2α 80

5.3.2 Ovarian morphology and endocrine status 80 5.3.3 Metabolic status 81 5.4 Discussion 82

v

5.5 Summary 86 Chapter 6. Reproductive Performance of High Producing Early Post_ partum Dairy Cows Induced to Ovulate with GnRH and PGF2α21 Days Postpartum under Commercial Farm Conditions 90 6.1 Introduction 90 6.2 Materials and methods 91 6.2.1 Animals 91 6.2.2 Hormonal treatment 91 6.2.3 Sampling frequency and analysis of P4 92 6.2.4 Evaluation of ovulation within 21 days postpartum and estimate of the ovulatory response prior to PGF2α treatment 92 6.2.5 Biochemical analyses 92 6.2.6 Reproductive management and diagnosis of gestation 93 6.2.7 Statistical analysis 93 6.3 Results 93 6.3.1 Changes in the endocrine status between day 21 and day 28 postpartum 93 6.3.2 Metabolic status 94 6.3.3 Fertility traits 94 6.4 Discussion 96 6.5 Summary 100 Chapter 7. Summary and Conclusions 107 Summary (Japanese) 114 Acknowledgements 118 References 121

1

Chapter 1

General Introduction

1.1 Actual trends in the fertility of high-producing dairy cows

An intense genetic selection to reach higher milk production has been practiced

world wide by the dairy industry. In Japan for example, continuous increases in milk

production have led to a 70% increase in one year production over the last 30 years (64).

Conversely, the fertility of dairy cows has declined. Milk production and reproductive

performance are genetically and phenotypically related (65). However, reproductive

traits have low heritability (55) and a consistent improvement could be seen only after

several years of work by genetic selection.

The early re-establishment of normal ovarian cycles is of paramount

importance because cows need to be bred early to calve at a year interval to make the

dairy industry profitable. However, failure to cycle and display estrus and suboptimal

and irregular estrous cycles are some of the very important identified factors that

depress fertility by lengthening the time to conception (55, 65).

The resumption of ovarian activity early in the postpartum as a pre-requisite for

subsequent cyclicity and early breeding has been rigorously studied, but is not yet

properly understood.

The long term expected in order to see improvement in the fertility by

performing selections through fertility traits have led research to focus on the use of

hormonal treatments to solve the afflicted reproductive performance in the dairy

industry. Some factors specific of the postpartum period in the actual high-producing

dairy cow in relation to the effects of hormonal treatments need further study and will

2

be the focus of this dissertation

1.2 Postpartum resumption of ovarian activity

During late pregnancy as in other physiological states in dairy cows (e.g.

before puberty), the high concentrations of estrogens (from placental origin in cows)

alone or in combination with similarly high concentrations of progesterone (P4) exert a

negative feed back effect on the hypothalamic-hypophysial axis reducing both the

formation and the secretion (amplitude and frequency) of gonadotropins from the

adenohypophysis, namely luteinizing hormone (LH) and follicle-stimulating hormone

(FSH). This phenomenon has been regarded as the main cause for the suppression of

follicular development during late pregnancy and the first 4 to 5 days postpartum (4, 67,

79). Following the regression of the corpus luteum of pregnancy and the decline in the

concentrations of gestational estrogens, the negative feed back effects of steroids on

gonadotropin releasing hormone (GnRH) are removed inducing an increase in FSH

concentrations (4, 78). FSH concentrations peak from 4-5 days postpartum (2, 4). This

increase in FSH is followed by the first postpartum follicular growth that precedes a

process of selection and culminates with the presence of dominant follicles (DF) (9.9

mm~) within 10 days postpartum (4, 44, 85, 88).

Three fates of DFs during the early stages of postpartum have been reported: 1)

ovulation in nearly 50% of the cows (2, 3, 44), 2) continued growth followed by

variable periods of anovulatory follicle turnover that involves atresia (4, 44, 85), and 3)

formation of anovulatory cystic follicles (2, 4, 81, 85, 88).

Failure of DF to ovulate prolongs the postpartum anovulatory interval. The

ovulatory fate of DF is related with an adequate LH pulse frequency necessary for the

3

support of an active estradiol production by DF (4, 14) and with the presence of

sufficient LH receptors in the DF (78). Estradiol exerts positive feedback on the

hypothalamus to stimulate, through GnRH release, an ovulatory surge of LH and FSH

(87). However, the positive feedback stimulation of estradiol during postpartum does

not occur until the presence of its receptors in the hypothalamus and the anterior

pituitary are in enough concentration (67). Concentrations of receptors considerably

increase at day 22 postpartum (67). It has been suggested that a low concentration of

receptors causes the hypothalamic-hypophysial axis to be less sensitive to the positive

feedback effects of E2 before first ovulation (67).

1.3 Relation between postpartum energy status and resumption of ovarian activity

during postpartum

During early postpartum, most dairy cows undergo a period of negative

energy balance (NEB) (41, 98). The main reason for this energetic deficiency is an

inappropriate upkeep of dry mater consumption of cows in relation to their high milk

production (90). To compensate the energy demanded by milk production (high

production of lactose) and the regeneration and function of body tissues (e.g. ovary,

uterine involution), dairy cows mobilize adipose tissue reserves as non-esterified fatty

acids (NEFA) (41, 73). In cattle as in humans, NEFA are taken up mainly by the liver,

and transformed into triglycerides (TG) and secreted as very low density lipoproteins

(VLDL) or are β-oxidized in the mitochondria and peroxisomes (41). However, the

cow’s liver has a decreased potential to release TG as VLDL, explaining the reason why

ruminants are more prone to develop fatty liver syndrome when TG accumulation is

high due to equally high production of NEFA. The incomplete metabolism (through

4

β-oxidation) of NEFA leads terminally to the activation of the neo-glycogenic pathway

that produces an excess formation of metabolites, e.g. ketone bodies, necessary as

alternative energy resources for extra-hepatic tissues. While consistent findings on the

negative effects of high NEFA concentrations on ovarian structures of lactating cows

have been reported (38, 39), the direct relation of this metabolite and the occurrence of

first postpartum ovulation is still equivocal. Some reports show no relation (2, 3, 42),

but others do (44). In contrast, a consistent relation for a delay has been shown for low

glucose (Glu) and high concentrations of the ketone body β-hydroxybutirate (BHB)

(74).

After parturition, the concentrations of growth hormone (GH) increase,

promoting lipolysis and increase in milk production. GH induces the release of

insulin-like growth factor 1 (IGF-1) by binding its own receptors in the liver (54). IGF-1

acts on extra-hepatic tissues including reproductive tissues (54). The IGF synergizes

with gonadotropin hormones to stimulate ovarian function by promoting growth and

steroidogenesis in ovarian cells (23, 54, 78). During the first follicular wave postpartum,

IGF-1 has been proposed as a factor limiting ovulation of the first wave DF. That is,

IGF-1 in concert with increased LH pulsatility enhance estradiol-17β (E2) secretion by

the follicle that finally induces an ovulatory surge of LH (24, 44, 78). However, the

intensity (2, 73, 90, 98), duration (92) and the period postpartum to the negative most

(nadir) energy balance (2, 14) have been related to the delay of the first postpartum

ovulation. The nadir in energy balance has been reported to occur within 2 weeks

postpartum in Holstein cows (2, 73, 92, 98) with follicles developing during this period

experiencing low pulsatile LH support and limited IGF-1 and E2 production (2, 14, 42).

5

1.4 Characteristics of the resumption of luteal activity during the early postpartum

period in dairy cows

It has been widely reported that under a normal recovery of the uterine

condition an early resumption of luteal activity enhances the fertility of dairy cows (17,

43, 86, 92, 93). The length of postpartum anovulation in cows can be accurately and

objectively measured by the determination of P4 alone (52) or in combination with

ultrasound imaging (72). Recent reports in high producing Holstein cows frequently

monitored using P4 concentrations indicated that the postpartum anovulatory interval is

increasing (32, 89).

Studies using transrectal ultrasonography (3, 42, 85, 88) have shown that

ovulation of the first wave DF occurs around 15 days postpartum (range: 12-17 days).

Ovulation within 21 days postpartum has been recently proposed as an index of the

subsequent reproductive performance (43). However, Sakaguchi et al (80) questioned

the benefits of the early start of ovarian cycles in dairy cows.

A short luteal phase (4-7days) occurs in most cows and small ruminants

following the first ovulation postpartum as well as in other physiological conditions that

involve the change from periods of anestrous to cyclicity such as puberty and seasonal

anestrus (24, 33, 34, 49). The reason for the short lifespan of the first CL postpartum

seams to be the lack of previous exposure to P4, as pre-treatment with P4 produce

normal lifespan CL (24, 34). Therefore, it has been suggested that exposure to P4

(endogenous or exogenous) during postpartum is important for the normalization of

postpartum estrous activity (76).

The length of normal ovarian cycles in cows has been defined to last 18 to 24

days (31) and refers to the time in days between two consecutive ovulations. In contrast

6

to the importance of an early resumption of normal ovarian activity, recent reports show

that nearly 50% of high producing dairy cows have a delayed return to normal ovarian

cyclicity (32, 43, 89). The proportion of abnormal cycles increases in cows undergoing

delayed first ovulation (43, 89). Cows having abnormal ovarian cycles show increased

days to conception, more services per conception, lower first service pregnancy rates,

and more interventions due to fertility problems (52, 89).

The genetic selection for milk production and the consequent increased

partitioning of nutrients has been directly related with the incidence of abnormal cycles.

Cows having parameters related with a deep NEB; lower dry matter intake, increased

body weight loss within 21 days postpartum and thereafter, more energy and lower fat

contents in milk, and the highest BHB levels by 21 days postpartum, have one or more

types of abnormal estrous cycles (92).

1.5 Hormonal control of the ovarian activity for the reduction of the postpartum

anovulatory interval.

The process to achieve an improvement in the fertility of the actual

high-producing dairy cows requires the systematic integration of goals focusing on the

improvement of management, nutrition, overall and fertility-related health programs,

and the enhancement of submission, conception and pregnancy rates. The culling rate

due to infertility would be higher for cows that do not show estrus within a 60-day

voluntary waiting period (76). Out of all the possible interventions to improve fertility,

those targeting improvements in the submission, conception and pregnancy by using

hormonal treatments seem to give more predictable results (76). However, according to

the continuous drop in fertility of dairy cows throughout the years and to the several

7

factors affecting reproductive recrudescence during postpartum, further research is

needed to establish and update the physiological and the cost benefits of hormonal

interventions in research and farm level conditions.

The hormonal control of the reproduction in cows requires understanding of

the ovarian function.

In the ovary of normally cycling cows, the development of follicles occurs in

phases that include recruitment of a cohort of small follicles, selection and dominance

of a single follicle (8.5mm~) which suppresses the growth of other follicles in the same

pool, and finally, atresia of the DF when a functional CL is present. In conjunction,

these phases conform what has been termed one “follicular wave” (1, 25, 51). Two or

three follicular waves occur during one estrous cycle in cows (51). Upon luteal

regression or removal of P4, follicular maturation and the associated increase in

estradiol synthesis occur, triggering a LH surge that causes ovulation of a DF (56).

Consecutive treatments with GnRH or any of its analogues and prostaglandin

F2-alpha (PGF2α) have been applied in a 6-7 day interval to control an spontaneous (18,

94) or an induced ovulation (71) of cycling cows. The rationale is the presence of a

mature DF (10mm~) and a PGF2α -responsive CL around 7 days following ovulation.

Earlier studies demonstrated that the release of LH from the pituitary in

response to GnRH treatment is fully restored after 7 to 14 days following parturition (21,

46). Occurrence of ovulation following treatment with GnRH during the anestrous

period has been equivocal, some works report high ovulatory response (16, 28, 58) and

others low (29, 107). The response in ovulation after GnRH depends on the presence of

a fully mature follicle (56).

Some concerns exist on the benefits that an early first ovulation has on the

8

subsequent fertility. In specific, the early exposure to P4 during postpartum may induce

uterine infections by down-regulating the uterine immune system (80). The postpartum

anestrous interval is either prolonged (20) or shortened (5) when ovulation is induced

within the first 2 weeks postpartum by using GnRH analogues alone.

The equivocal information on the benefits in the fertility of an

early-spontaneous or induced first ovulation requires further investigation.

9

General objectives

1. To determine the ovulatory, morphological and the endocrine responses to the

combination of treatments with GnRH and PGF2α during two different stages

postpartum.

2. Study comparatively the estrous activity of spontaneous ovulating cows and cows

treated with a GnRH and PGF2α protocol started on day 21 postpartum under

distinct farming conditions.

3. Study of the relation of the response to the induction of ovulation with GnRH and

PGF2α and the metabolic status during the first 3 weeks postpartum.

4. Determination of the influence of the early treatment using the combination of

GnRH and PGF2α on the different parameters of fertility in high producing dairy

cows managed for commercial purposes.

10

Chapter 2

General Materials and Methods

2.1 Animals

Early postpartum high producing dairy cows (n=195) were studied at different

periods during the years of 2004 though 2006. In chapters 3, 4 and 5, cows were

managed in the Field Center of Animal Science and Agriculture at Obihiro University

of Agriculture and Veterinary Medicine. In chapters 4, 5 and 6, cows managed under

commercial farm conditions in Iwate prefecture and the town of Urahoro in the

Tokachi area of Hokkaido, Japan were also studied. The postpartum period to the

beginning of the experiments ranged from 21 to 37 days; with 21 days postpartum

being the period during which most experiments were carried out. The cows were

housed under several confinement conditions. The feeding system was based on a total

mixed ration (TMR) in most of the cases and the cows had free access to water.

Grazing was allowed in some locations during the summer months. Milking was

performed at least twice daily.

2.2 Hormonal treatment

Treatments were performed intramuscularly with 10μg of a GnRH analogue

(Buserelin acetate: Itorelin®; ASKA Pharmaceutical Co., Ltd. Tokyo, Japan), regarded

as GnRH hereafter, followed 7 days later by the intramuscular treatment with an

analogue of PGF2α that consisted of either 500μg of Cloprostenol (Resipron-C®. ASKA

Pharmaceutical Co., Ltd.) or 5.0 mg of Etiproston tromethamine (Prostavet®. Virbac

S.A., France) regarded as PGF2α hereafter.

11

2.3 Ovarian Ultrasonography

2.3.1 Frequency and description of examinations

In chapter 3, the ovaries of all cows were scanned at 24 h intervals starting

from the day of GnRH treatment and continuing until the detection of a

protocol-synchronized ovulation. In chapter 5, the ovaries in hormone-treated cows

were scanned only on the days of hormone treatments.

2.3.1a- Detection of ovulation following treatments. In order to detect the

occurrence and the time of ovulation of a dominant follicle (DF) responsive to the

GnRH treatment, ultrasonographic observations were performed rectally at 12-h

intervals between the 1st and 2nd day following the initial treatment. Confirmation of

ovulation following treatment with PGF2α was performed by ultrasonic scanning means

at 24-h intervals during five consecutive days.

2.3.1b-Ultrasound technique and evaluation of the ovarian morphology.

All observations were performed using an Aloka SSD-1700 ultrasound scanner

equipped with a 3 cm-7.5-MHz convex array transducer (UST-995-7.5; Aloka Co.).

Each ultrasonographic procedure was carried out under suitable research conditions.

After completely emptying all feces in the rectum, the ovaries were approached in a

gentle manner in order to minimize disruption in their anatomical location within the

abdominal cavity. Ultrasound scanning was systematically carried out in the same

direction at each observation to facilitate the location, count and follow up of all ovarian

structures when the study so demanded. Sequential images saved in the cine memory

were used to select and measure the maximum recorded size. Measurements were done

using the ultrasound internal caliper; and the image print outs together with diagrams of

the location of all detected structures were used as basis for subsequent follow ups.

12

According to their largest diameter, follicles were classified into small (3-5

mm), medium (6-9 mm) and large (≥10 mm) as reported previously (104). The

development and total number of follicles within each class, as well as all spontaneous

(CL periodicum) and treatment-induced corpora lutea (induced CL) were recorded daily

throughout the duration of the synchronization protocol. Pictures of snapped frozen

images and sketches of the location and the number of the several structure types in

each ovary were used to monitor the morphological development throughout the

treatment period.

The new-synchronized ovulatory follicle (OVF) was defined retrospectively as

the largest follicle that ovulated after PGF2α and whose emergence occurred around

GnRH treatment. In order to specify the developmental characteristics of the OVF, its

relation with the growth of the largest subordinate follicle was studied. The subordinate

follicle was defined as one that emerged together with and grew for some time at the

same rate as the OVF while being the second largest follicle present in the ovary.

Based on daily ultrasound observations, follicular deviation (dominance) was

defined as the beginning of the greatest difference in growth rates between the OVF and

the largest subordinate follicle at or before the examination when the second largest

follicle reached its maximum diameter (26). Representative profiles for the different

days to deviation are depicted in Fig.2.1. The development of the induced CL and the

CL periodicum after a GnRH-induced ovulation was analyzed until its limits were not

identifiable.

13

0

5

10

15

20

25

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 9 10 11

Protocol days

GnRH

PGF2α

DEV

DEV

Fig.2.1 Representative graphs in two experimental cows depicting the protocol days at

which morphological deviation (DEV) occurred between the new-synchronized

ovulatory follicles（●）and the second largest (subordinate) anovulatory follicles（○）.

Follicle diameter (mm)

14

2.4 Blood collection and hormone determination

Blood samples were obtained by caudal venipuncture at several intervals

according to the nature of the experiment. Every 24-h sampling intervals were used

when a detailed examination of the changes was required. In order to correlate the

morphology and endocrine changes brought about by the experiment, samples were

obtained just before each scanning and / or hormonal treatment (GnRH or PGF2α). All

samples were obtained using sterile 10-ml vacutainer tubes containing Heparin sodium

(Venoject®) or into plain-tubes (Venojet®. Terumo, Tokyo, Japan) containing 200 μl of

stabilizer solution (0.3M EDTA, 1% acetylsalicylic acid, pH 7.4). Tubes were

immediately chilled in ice water and centrifuged at 4℃ for 20 minutes at 3000 r.p.m.

The obtained plasma was stored at -30℃ until the determination hormones and / or

metabolites were performed.

Progesterone (P4) and estradiol-17β (E2) levels were determined in duplicate by

double antibody enzyme immunoassays (EIA) as previously described for the former

(61) and the latter (101), respectively. P4 extraction from plasma samples was done as

follows: after the addition of 1 ml Diethyl ether to 200 μl plasma aliquots contained into

1.5 ml cryovials, strong shaking was done for 20 minutes using a multi-sample shaker

(MicroMixer E-36 ®, TAITEC. Japan). The plasma and the ether were allowed to

separate into different layers by a 20-min rest at room temperature, followed by freezing

at -30℃ for at least 4 h (range: 4 to 12 h). After freezing, the ether layer was decanted

into 5 ml test tubes and evaporated by warming the tubes in a hot water bath at 50℃ for

15 to 20 min and until the ether smell was not present. Immediately after, 200 μl of

assay buffer (7.12g Na2HPO4 X 2H2O; 8.5g NaCl; 1.0g BSA; pH 7.2) were added to

15

each tube followed by 10-sec vortex cycles within 1 minute. The recovery rate of P4 (2

ng) was 87 %. The P4 level of standards or samples were analyzed in duplicates of 15 μl

after incubation for at least 17 h at 4 ℃ with 100 μl-antibody against progesterone

(OK-1)(1: 200,000) and 100 μl-horse radish peroxidase (1: 50,000). The standard curve

ranged from 0.05 to 50 ng / ml and the ED50 of the assay was 7.56 ng / ml. The intra-

and interassay coefficients of variation were 4.2 and 12.5 % respectively.

E2 extraction from plasma samples was carried out adding 6 ml of Diethyl ether

to 2 ml plasma samples contained into 20-ml scintillation glass vials; followed

immediately by one-hour shaking in a SA-31® shaker (Yamato Scientific CO., LTD.

Tokyo, Japan). Following a 30-min rest at room temperature, samples were frozen at

-30℃ for 12 h. The ether layer was decanted into 10 ml test tubes and evaporated as

described in the extraction procedure for P4. Thereafter, 100 μl of assay buffer were

added and immediately after each tube was vortexed as previously stated. The recovery

rate for E2 was 85 %. Standards and samples were incubated with 100 μl-antibody

against estradiol (AS-A) (1 : 200 000) and 100 μl -horseradish peroxidase (1: 150 000).

The standard curve ranged from 2 to 2000 pg / ml and the ED50 of the assay was 3.3 pg /

ml. The intra- and interassay coefficients of variation were 8.4 and 14.9 % respectively.

Determination of insulin-like growth factor-1 (IGF-1) in plasma was performed

by EIA after extraction of binding proteins by acid-ethanol mixture (87.5 % ethanol and

12.5 % 2N hydrochloric acid) (44). Thirty μl of human IGF-1 standard (Roche,

Indianapolis, USA, 0.39 to 50 ng / ml) dissolved in assay buffer or sample were added

to each well coated with anti-rabbit γ-globulin antiserum. In addition, 100μl of

biotin-labeled hIGF-1 (x 10,000) and rabbit anti-hIGF-1 (x 40,000; NIDDK,

AFP18111298) diluted in assay buffer were distributed in all wells, and then incubated

16

for 72 h at 4 ℃. Finally, colorimetric treatments were carried out. The Intra- and

interassay coefficient of variations were 5.7 and 6.6 %, respectively. The ED50 of the

assay system was 2.5 ng / ml.

Measurement of FSH concentrations from straight plasma was done in

duplicate by double antibody EIA as previously described (100). The standard curve

ranged from 0.18 to 12 ng / ml, and the ED50 of the assay was 1.7 ng / ml. The intra-

and interassay coefficient of variations averaged 8.3 and 14.6%, respectively.

2.5 Biochemical analyses

Blood samples obtained throughout the postpartum and / or prior to hormone

treatments were used to assess the metabolic status of cows. Metabolite measurements

included concentrations of glucose (Glu), non-esterified fatty acids (NEFA),

β-hydroxybutirate (BHB), and aspartate aminotransferase (AST). All metabolites were

measured using a clinical chemistry automated analyzer (TBA-120FR, Toshiba Tokyo,

Japan) (Fig.2.2).

17

Fig.2.2. Clinical chemistry automated analyzer.

2.6 Statistical analysis

The days of GnRH and PGF2α treatments were regarded as d 0 and d 7,

respectively. The data with binomial distribution were analyzed by contingency

chi-square and differences were detected using Fisher’s exact test. All data with linear

distribution, e.g. biochemical traits during postpartum, morphology and endocrine

responses to the GnRH-PGF protocol, were evaluated using repeated measures ANOVA

as reported previously (102). Comparisons of means were carried out using Student’s t

test or ANOVA followed by Tukey-Kramer honestly significant difference test. While

evaluating estrous activity postpartum, comparison of means for the evaluation of the

effect of an early ovulation (spontaneous or induced) on the subsequent estrous activity

was carried out using Dunnett’s test with non-treated cows showing a late first ovulation

18

as the negative control. All calculations were done using the JMP statistical software

(Version 5.1; SAS institute). Differences were considered significant at P < 0.05.

19

Chapter 3

Induction of Ovulation with GnRH and PGF2α at Two Different Stages

during the Early Postpartum Period in Dairy Cows: Ovarian Response

and Changes in Hormone Concentrations.

3.1 Introduction

In Japan, a decline in the reproductive performance of dairy cows has been

noticed (64). First ovulation within 3 weeks postpartum positively affects the fertility by

increasing the number of exposures to P4 before insemination (17, 43, 86). However,

our previous study revealed that as much as 47% of these cows do not show an early

ovulation (43). Since spontaneous ovulation within 3 wk postpartum enhances the

outcomes of fertility, a hormonal treatment able to induce ovulation and posterior

cyclicity by this time would be beneficial for an increase in reproductive performance at

farm level.

The release of sufficient LH from the pituitary in response to exogenous GnRH

is restored after 7-21 days postpartum (21, 28, 46, 58). This surge in LH is followed by

ovulation of large follicles in a greater (16, 28, 58) or a lesser (28, 107) proportion of

cows. Nearly half of the cows ovulate spontaneously by 21 days postpartum (43). This

result indicated that half of the cows already started ovarian cyclicity but the rest did not.

Therefore, the ovulatory response to GnRH may differ between cows that had or not

ovulated spontaneously by 21 days postpartum.

It was early reported that treatment of cows with GnRH alone during the early

postpartum increased the incidence of pre-breeding anestrous (20). This phenomenon

20

was related to an increased rate of uterine infections facilitated by the increased P4

levels. This suggested the need of a luteolisin to cause regression of corpora lutea (CL)

and subsequent estrous activity.

Consecutive treatments with GnRH and PGF2α have been applied in a 6 to 7

–day interval to control ovulation in cycling cows (18, 71). After induction of

ovulation by the GnRH treatment, a new follicular wave emerges, and the posterior

PGF2α treatment induces regression of the CL followed by spontaneous ovulation of

new dominant follicles (18). However in early postpartum dairy cows, the same

hormone regime in a 10- day interval did not allow for a synchronous ovulation

following treatment with PGF2α (5).

In cattle and ewes, the CL that form after a spontaneous or an induced first

ovulations have variable lifespan (34). Therefore, CL derived from the first postpartum

ovulation may show variable response in regression after PGF2α treatment.

In dairy cows, several studies reported the use of GnRH and PGF2α during

early postpartum (5, 20). However, there is little information about the ovarian and the

hormonal changes during the treatment process. FSH is a key factor for the growth of

cohorts of follicles before (2) and after first ovulation (1). IGF-1 and FSH synergize to

favor the selection, and to improve the function of dominant follicles (23). Therefore,

FSH and IGF-1 were examined during the treatment period.

The aim of this study was to determine whether treatments with GnRH and

PGF2α at two different stages during the early postpartum period (on 21 days or around

37 days after calving) can induce ovulation in dairy cows. The follicular dynamics,

development of the CL, as well as the hormonal response in comparison to cycling cows

were studied.

21

3.2 Materials and Methods

3.2.1 Animals and hormonal treatment

Lactating Holstein cows (n=14) managed under free-stall confinement in the

Field Center of Animal Science and Agriculture at Obihiro University of Agriculture

and Veterinary Medicine were used in this experiment. On the first day of treatment (d

0), all cows received a 10 μg-single i.m. injection of GnRH followed 7 days later (d 7)

by a 500 μg-single i.m. injection of PGF2α (Cloprostenol: Resipron-C®. ASKA

Pharmaceutical Co., Ltd.). Animals were equally grouped depending on the days

postpartum at the beginning of the treatment protocol. The first group (n=7; 3

primiparous and 4 multiparous) received the GnRH treatment 21 days postpartum

(GnRH21). The second group (n=7; 3 primiparous and 4 multiparous) received the

GnRH treatment at a mean of 37 days (GnRH37; range: 34-41 days). This study was

carried out from July 2004 through July 2005.

Since luteal activity, as indicated by plasma P4 levels at the beginning of

ovulation synchronization protocols affect the ovarian response (63), GnRH21 group

was divided into two groups based on ultrasound findings and on plasma P4 levels on d

0 as follows; 1) GnRH21-CL, three cows (1 primiparous and 2 multiparous) had an

identifiable-functional CL (CL periodicum)(P4 ≥ 1 ng/ml) and 2) GnRH21-NCL, four

cows (2 primiparous and 2 multiparous) did not have CL (P4 < 1 ng/ml). In contrast, all

cows in GnRH37 had functional CL (GnRH37-CL).

3.2.2 Ovarian ultrasonography

The changes in the morphology of the ovaries were monitored daily using

22

transrectal ultrasonography starting from the day of GnRH treatment (d 0) until the

detection of ovulation after treatment with PGF2α (d 7) as described in Chapter 2. In

order to detect ovulation after GnRH treatment, additional observations were performed

at 12-h intervals during the subsequent 1st and 2nd day. To analyze the changes in

follicular dynamics after GnRH treatment, the observed follicles were classified into

small (3-5 mm), medium (6-9 mm) and large (≥10 mm) sizes as reported previously

(104). The growth of the follicle ovulating after PGF2α was analyzed as described in

Chapter 2.

3.2.3 Blood collection and determination of hormones

Blood samples were obtained by caudal venipuncture at 24 h intervals just

before each scanning and / or hormonal treatment (GnRH or PGF2α) using sterile 10-ml

tubes containing heparin sodium (Venoject®., Terumo. Tokyo, Japan). Tubes were

immediately chilled in ice water and centrifuged at 4℃ for 20 minutes at 3000 rpm. The

obtained plasma was stored at -30℃ until hormone determination. The concentrations

of P4, E2, FSH and IGF-1 were determined by enzyme immunoassays (EIA) following

the procedures described in Chapter 2.

3.2.4 Statistical analysis

The days of GnRH and PGF2α treatments were regarded as d 0 and d 7,

respectively. The data with binomial distribution were analyzed by Fisher’s exact test.

All data with linear distribution was analyzed using the fit model platform of the JMP

statistical software (Version 5.1; SAS institute). Data are presented as mean ± SEM.

Differences between means were compared by Student’s t test. Differences were

23

considered significant at P < 0.05. CL were considered to have undergone regression as

a direct effect of a PGF2α treatment when the P4 levels dropped from ≥ 1 ng / ml at the

time of the PGF2α treatment, to levels < 1 ng / ml within the following 48 hrs.

3.3 Results

3.3.1 Ovulatory response

Neither presence of CL periodicum nor interval postpartum had a significant

effect on the ovulatory response to the treatments. Treatment with GnRH induced

ovulation in all cows of the three groups. The size of dominant follicles ovulated by the

GnRH treatment was significantly larger in GnRH21-NCL (21.2 ± 1.5 mm) than in

GnRH37-CL (15.9 ± 1.4 mm; p

24

and GnRH21-CL. In contrast, GnRH21-NCL had more small follicles than GnRH37-CL

on d 2 (9.5 ± 3.8 vs. 2.4 ± 1.1; p < 0.05), d 3 (7.5 ± 2.1 vs. 2.1 ± 0.6; p < 0.01) and d 4

(8.3 ± 1.6 vs. 2.7 ± 1.1; p < 0.01). The number of medium-size follicles (6-9 mm) did

not differ among the three groups (Fig. 1b). Significant effects of day (p< 0.05) and a

group by day interaction (p< 0.05) were detected for the number of large follicles (≥10

mm). More large follicles were present on d 5 in GnRH21-NCL than in GnRH37-CL

(2.5 ± 0.3 vs. 1.4 ± 0.2; p < 0.05).

3.3.3 Plasma concentrations of FSH and IGF-I

Analysis of FSH concentrations from d 0 to d 4 revealed an effect of group

(p

25

The general characteristics of the development of the ovulatory follicles are

summarized in Table 3.1. The ovulatory follicle seemed to have emerged earlier in

GnRH21-NCL (Table 3.1, Fig.3.4a). The ovulatory follicle was present at the time of

GnRH treatment in 1 out of 4 cows in GnRH21-NCL. By d 1, ovulatory follicles were

detected in all cows in GnRH21-NCL, and only in 1 out of 3 and 1 out of 7 cows in

GnRH21-CL and GnRH37-CL, respectively. By d 2, the ovulatory follicle was detected

in all cows of the three groups. Therefore, the growth of the ovulatory follicle was

analyzed from d 2 to d 7. There were significant effects of group (p

26

presented in Fig. 3.5a and Fig. 3.5c.

Ultrasound images of the changes in the different ovarian structures of

representative cows in GnRH21-NCL, GnRH21-CL and GnRH21-CL are shown in Figs.

3.5-3.7.

3.3.5 Plasma concentrations of P4 and E2

There was a significant effect of group (p

27

consecutive treatments with GnRH and PGF2α at two different stages during early

postpartum. The present results indicate that the treatment with GnRH in the early

postpartum induced ovulation in all cows. Earlier studies demonstrated that the pituitary

release of LH in response to GnRH treatment is fully restored after 7 to 14 days

following parturition (21, 46). At the time of GnRH in the present study, all cows in the

three groups had follicles larger than the size (10 mm) at which dominant follicles

acquire ovulatory capacity in response to LH (83). This may explain the high ovulation

rate obtained in the present study.

Most dairy cows develop a large follicle within 10 days postpartum (2). The

dominant follicle of the first wave ovulates in mean 15 days postpartum (range: 12-16

days) (3, 85, 88). In this study, ovulatory follicles exceeded 8.5 mm in diameter 4 days

after ovulation, and grew approximately 1-2 mm/day. Therefore, the dominant follicle

emerging after an early first postpartum ovulation has the potential to reach or exceed

the 10-mm size by 21 days postpartum. On the other hand, if ovulation of the dominant

follicle of the first wave postpartum fails, it would be substituted by a follicle of the

subsequent wave (2, 88) which becomes dominant by 20 days postpartum (88). Thus,

there seems to be a high possibility of encountering a large follicle when GnRH is

administered 21 days postpartum. This may allow for a good first ovulatory response

around this day.

A longer-lasting follicular recruitment having a cohort with a greater number of

small follicles (3-5mm) was induced in GnRH21-NCL as compared to GnRH37-CL. As

days postpartum increase, the depletion of small size follicles also increases (57). In

addition, the early postpartum period is characterized for the lack of replenishment of

small follicles (19, 57), resulting in a reduced presence of small follicles towards or by

28

35 days postpartum. These findings indicate that the number of small follicles present

for recruitment may be lesser if cows are treated later in the postpartum. Furthermore,

GnRH21-NCL had a higher FSH concentration at the time of GnRH treatment.

Increments in FSH concentrations precede the emergence of follicular waves throughout

the estrous cycle (1). IGF-1 has been reported to have a synergistic effect on follicular

growth together with FSH (23). However, IGF-1 levels did not differ among the groups.

Our results suggest that a larger pool of small follicles coupled with higher FSH

concentrations at the time of GnRH treatment were responsible for making more

gonadotropin pre-stimulated follicles available for recruitment in GnRH21-NCL.

The similar patterns in the dynamics of medium size follicles (6-9 mm) found

in both postpartum stages are in agreement with reports showing no changes in the

dynamics of medium size follicles as days postpartum increase (19, 57). Our findings

also suggest that the dynamics of medium size follicles were similar in all groups

because the 6-9 mm diameter range is transitional for follicles increasing or decreasing

in size.

In the present study the ovarian structures (ovulatory follicle and induced CL)

derived from the GnRH induced ovulation were larger in size in GnRH21-NCL. In dairy

cattle, the size of the CL correlates with the size of the original follicle (84, 96).

However, the size of dominant follicles ovulated by the GnRH treatment differed only

between GnRH21-NCL and GnRH37-CL. This result indicates that the presence of a

functional CL at the onset of the protocol had a stronger effect on the size of the induced

CL.

Progesterone regulates the development of both growing follicles and CL in a

dose dependent manner (13). In addition, high P4 concentrations down regulate LH

29

secretion (6). The hormonal milieu during d 0 to d 5 in GnRH21-NCL was characterized

by mean P4 concentrations (0.9 ng / ml) lower than the subnormal level (2.4 ng/ml)

reported to allow for increases in LH pulse frequency (77). LH plays an important role

during and after the process of follicular selection (26) and supports for the growth of

CL (69). Therefore, it is plausible to consider the involvement of higher gonadotropins

support (mean, basal and / or episodic) under low P4 levels on the enhanced

development of ovarian structures in GnRH21-NCL.

The concentration of E2 during the growth period of ovulatory follicles was

greater in GnRH21-NCL. Estradiol is one of the factors involved in the regulation of

FSH concentrations during the estrous cycle (7). The decrease in FSH levels in

GnRH21-NCL, or the increased concentrations observed in GnRH21-CL might have

been regulated by high and low E2 levels in each group, respectively. However, in

GnRH37-CL, FSH concentrations remained low in the presence of similarly low E2

concentrations, suggesting the involvement of other regulatory factors. Inhibin has been

reported to play a major role in the suppression of FSH levels during the postpartum

period (40). Further research is needed to better understand the role of inhibin on the

regulation of FSH after a hormonally induced ovulation in the early postpartum period.

The rate of regression of CL (induced CL and CL periodicum) in response to

PGF2α was high in all groups regardless of morphological differences. This result

suggests that a fully functional status was achieved by induced CL 7 days after GnRH

treatment in all groups.

Despite differences in the overall size of ovulatory follicles in our study,

morphological dominance was attained in all groups equally and comparably to the size

and time previously reported (26). In addition, the similarities in daily growth and the

30

high ovulatory response following PGF2α in all groups clearly shows that the

development of the ovulatory follicle when the protocol started 21 days postpartum was

not different from that in normal cycling cows.

In conclusion, dairy cows as early as 21 days postpartum are effectively

induced to ovulate by a 7-day GnRH and PGF2α synchronization protocol regardless of

the ovarian cyclicity status. The details of the ovarian and hormonal status herein

presented may provide information to develop the hormonal intervention capable to

reduce the partum–conception interval. Complementary investigation is necessary to

determine the impact of an early induced ovulation on the subsequent estrous activity

and fertility in the dairy cow.

31

3.5 Summary

The aims of this study were 1) to determine whether dairy cows can be induced

to ovulate by the treatment with GnRH followed by PGF2α during the early postpartum

period and 2) to describe their ovarian and hormonal responses according to ovarian

status. Cows were divided in two groups and received 10 μg of buserelin followed by

500 μg of cloprostenol 7 days apart starting from 21 (GnRH21, n=7) or around 37 days

postpartum (GnRH37, n=7). The groups were further classified according to presence

(-CL) or absence (-NCL) of functional corpora lutea (CL) on the day of GnRH

treatment (d 0): GnRH21-NCL (n=4), GnRH21-CL (n=3) and GnRH37-CL (n=7).

Ovarian morphology was monitored and the concentrations of P4, E2, FSH and

insulin-like growth factor 1 (IGF-1) were measured. All cows ovulated after

administration of GnRH. The P4 levels of the GnRH21-NCL group from d 0 to d 5 were

lower than those of the GnRH21-CL (p

32

GnRH21-CL and GnRH37-CL groups, respectively. In conclusion, a 7-day

GnRH-PGF2α synchronization protocol can effectively induce dairy cows to re-start

ovarian activity as early as 21 days postpartum, regardless of the ovarian status.

33

Table 3.1 Time of ovulation of dominant follicles after GnRH treatment and

development parameters of ovulatory follicles. Values are means ± SEM.

GnRH21: enrollment into the protocol on 21 days postpartum. GnRH37: enrollment into the protocol on 37 days postpartum. NCL: Absence of corpus luteum; CL: presence of corpus luteum DF: Follicle of ≥ 10mm in diameter at the time of GnRH treatment. Deviation: Beginning of the greatest difference in growth rates between the two largest follicles.

GnRH21-NCL GnRH21-CL GnRH37-CL (n=) 4 3 7 DF ovulation (hr) 36.0 ± 0.0 36.0 ± 0.0 37.7 ± 1.7 Ovulatory follicle emergence (day) 1.0 ± 0.0 1.8 ± 0.3 2.0 ± 0.2 Size at emergence (mm) 4.8 ± 0.1 5.2 ± 0.5 6.3 ± 0.3

Deviation (day) 1.5 ± 0.5 2.0 ± 0.1 1.3 ± 0.2 Size at deviation (mm) 8.4 ± 0.6 9.3 ± 1.1 9.1 ± 0.5

Growth rate (mm/day) 1.6 ± 0.2 1.4 ± 0.1 1.7 ± 0.2

34

Fig. 3.1 Number of: a) small (3-5 mm), b) medium (6-9 mm) and c) large (≥10 mm)

follicles within the GnRH-PGF2α protocol in early postpartum dairy cows. d 0: day of

GnRH treatment; d 7: day of PGF2α treatment. Experimental groups are classified

according to the presence (-CL) or absence (-NCL) of functional CL at the time of

GnRH treatment: GnRH21-NCL (n=4), GnRH21-CL (n=3), GnRH37-CL (n=7). Data

are shown as mean ± SEM. Values with different letters in the same day differ (p

35

Fig. 3.2 Concentrations of FSH during d 0 to d 4 of the GnRH (d 0) - PGF2α (d 7)

protocol. Classification of experimental groups is described in the legend to Fig. 3.1.

Data are shown as mean ± SEM. Values with different letters in the same day differed

significantly (p

36

Fig. 3.3 Concentrations of IGF-1 during d 0 to d 4 of the GnRH (d 0) - PGF2α (d 7)

protocol. Classification of experimental groups is described in the legend to Fig. 3.1.

Data are shown as mean ± SEM.

80100120140160180200220240

0 1 2 3 4

IGF-

1 (n

g/m

l)

GnRH21-NCL GnRH21-CL GnRH37-CL

Day of protocol

80100120140160180200220240

0 1 2 3 4

IGF-

1 (n

g/m

l)

GnRH21-NCL GnRH21-CL GnRH37-CL

80100120140160180200220240

0 1 2 3 4

IGF-

1 (n

g/m

l)

80100120140160180200220240

0 1 2 3 4

IGF-

1 (n

g/m

l)

GnRH21-NCL GnRH21-CL GnRH37-CL

Day of protocol

37

Day of protocol

E 2(p

g/m

l)P 4

(ng/

ml)

Indu

ced

CL

diam

eter

(mm

)O

vula

tory

folli

cle

diam

eter

(m

m)

(a)

(d)

(c)

(b)

0

5

10

15

20

25

GnRH21-NCL GnRH21-CL GnRH37-CL

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

7

8

9

10

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 7

0

5

10

15

20

25

GnRH21-NCL GnRH21-CL GnRH37-CL

0

5

10

15

20

25

30

35

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 7

Day of protocol

E 2(p

g/m

l)P 4

(ng/

ml)

Indu

ced

CL

diam

eter

(mm

)O

vula

tory

folli

cle

diam

eter

(m

m)

(a)

(d)

(c)

(b)

Day of protocol

E 2(p

g/m

l)P 4

(ng/

ml)

Indu

ced

CL

diam

eter

(mm

)O

vula

tory

folli

cle

diam

eter

(m

m)

(a)

(d)

(c)

(b)

0

5

10

15

20

25

GnRH21-NCL GnRH21-CL GnRH37-CL

0

5

10

15

20

25

GnRH21-NCL GnRH21-CL GnRH37-CL

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

7

8

9

10

0

1

2

3

4

5

6

7

8

9

10

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 70.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 7

0

5

10

15

20

25

GnRH21-NCL GnRH21-CL GnRH37-CL

0

5

10

15

20

25

GnRH21-NCL GnRH21-CL GnRH37-CL

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 70.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 7

Day of protocol

E 2(p

g/m

l)P 4

(ng/

ml)

Indu

ced

CL

diam

eter

(mm

)O

vula

tory

folli

cle

diam

eter

(m

m)

(a)

(d)

(c)

(b)

38

Fig. 3.4 Growth patterns (mean ± SEM) of the ovulatory follicle (a), induced CL (b),

and concentrations of P4 (c) and E2 (d) within the GnRH-PGF2α protocol. d 0: day of

GnRH treatment; d 7: day of PGF2α treatment. Classification of experimental groups is

described in the legend to Fig. 3.1. Overall mean size of the ovulatory follicle and

induced CL in GnRH21-NCL were larger (p

39

Fig.3.5 Changes in diameter of the induced CL and changes in plasma progesterone following the treatment with PGF2α in responsive cows (a, b: GnRH21-NCL, n=3; GnRH21-CL, n=3; and GnRH37-CL, n=7) and in one refractory cow of the GnRH21-NCL group (c, d). Classification of experimental groups is described in the legend to Fig.3.1. Arrows represent the time of treatment with PGF2α. Data are mean ± SEM.

0

5

10

15

20

25

30

35GnRH21-NCL

GnRH21-CL

GnRH37-CL

0

1

2

3

4

5

6

7

8

9

10

7 8 9 10 7 8 9 10

Refractory

Day of protocol

a)

b)

c)

d)

P4 (ng/ml)

Induced CL (mm)

40

DF

indu

ced

CL

OV

F

L2

Fig.

3.5

. U

ltras

ound

imag

es o

f a re

pres

enta

tive

anov

ulat

ory

cow

indu

ced

to o

vula

te fo

llow

ing

the

treat

men

t on

day

21

post

partu

m (G

nRH

21-N

CL)

with

a p

roto

col i

nclu

ding

GnR

H a

nd P

GF 2

α. C

L: c

orpu

s lut

eum

,DF:

dom

inan

t fol

licle

, OV

F:

ovul

ator

y fo

llicl

e, L

2: se

cond

larg

est f

ollic

le. G

radi

ng li

nes:

5m

m.

d 0

d 2

d 3

d 4

d 7

d 1

GnR

H tr

eatm

ent

PGF 2

αtr

eatm

ent

Day

of d

omin

ance

DF

indu

ced

CL

OV

F

L2

Fig.

3.5

. U

ltras

ound

imag

es o

f a re

pres

enta

tive

anov

ulat

ory

cow

indu

ced

to o

vula

te fo

llow

ing

the

treat

men

t on

day

21

post

partu

m (G

nRH

21-N

CL)

with

a p

roto

col i

nclu

ding

GnR

H a

nd P

GF 2

α. C

L: c

orpu

s lut

eum

,DF:

dom

inan

t fol

licle

, OV

F:

ovul

ator

y fo

llicl

e, L

2: se

cond

larg

est f

ollic

le. G

radi

ng li

nes:

5m

m.

d 0

d 2

d 3

d 4

d 7

d 1

GnR

H tr

eatm

ent

PGF 2

αtr

eatm

ent

Day

of d

omin

ance

41

CL

perio

dicu

m

DF

indu

ced

CL

OV

F

L2

Fig.

3.6

. U

ltras

ound

im

ages

in a

re

pres

enta

tive

cycl

ic c

ow

indu

ced

to o

vula

te

follo

win

g th

e tre

atm

ent

with

a p

roto

col i

nclu

ding

G

nRH

and

PGF 2

α

star

ted

on d

ay 2

1 po

stpa

rtum

(GnR

H21

-C

L). C

L: c

orpu

s lut

eum

, D

F: d

omin

ant f

ollic

le,

OV

F: o

vula

tory

folli

cle,

L2

: sec

ond

larg

est

folli

cle.

Gra

ding

line

s:

5mm

.

d 0

d 2

d 3

d 4

d 7

GnR

H tr

eatm

ent

PGF 2

αtr

eatm

ent

Day

of d

omin

ance

CL

perio

dicu

m

DF

indu

ced

CL

OV

F

L2

Fig.

3.6

. U

ltras

ound

im

ages

in a

re

pres

enta

tive

cycl

ic c

ow

indu

ced

to o

vula

te

follo

win

g th

e tre

atm

ent

with

a p

roto

col i

nclu

ding

G

nRH

and

PGF 2

α

star

ted

on d

ay 2

1 po

stpa

rtum

(GnR

H21

-C

L). C

L: c

orpu

s lut

eum

, D

F: d

omin

ant f

ollic

le,

OV

F: o

vula

tory

folli

cle,

L2

: sec

ond

larg

est

folli

cle.

Gra

ding

line

s:

5mm

.

d 0

d 2

d 3

d 4

d 7

GnR

H tr

eatm

ent

PGF 2

αtr

eatm

ent

Day

of d

omin

ance

42

CL

perio

dicu

m

DF

indu

ced

CL

OV

F

L2

Fig.

3.7

. U

ltras

ound

im

ages

in a

re

pres

enta

tive

cycl

ic c

ow

indu

ced

to o

vula

te

follo

win

g th

e tre

atm

ent

with

a p

roto

col i

nclu

ding

G

nRH

and

PGF 2

α

star

ted

arou

nd d

ay 3

7 po

stpa

rtum

(GnR

H37

-C

L). C

L: c

orpu

s lut

eum

, D

F: d

omin

ant f

ollic

le,

OV

F: o

vula

tory

folli

cle,

L2

: sec

ond

larg

est

folli

cle.

Gra

ding

line

s:

5mm

.

d 0

d 2

d 3

d 4

d 7

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43

Chapter 4

Cyclic Ovarian Activity and Fertility Traits in Cycling and

Non-Cycling Dairy Cows Induced to Ovulate with GnRH and PGF2α

Treatments 21 days Postpartum

4.1 Introduction

The consistent relation between an early first ovulation and the subsequent

improvement in fertility in dairy cows has been widely reported (17, 43, 86, 93). An

early ovulation is particularly important because enhances normal estrous activity,

shortens the partum-first service interval and increases conception rate to first service

(43, 93). However, it was previously reported that nearly half of postpartum dairy cows

fail to have an ovulation by 21 days postpartum (43). The only selection of cows on the

basis of increased milk production, as has occurred during the recent years, delays the

postpartum interval to first ovulation (30).

The normality of the estrous cycles that follow the first postpartum ovulation is

important for an early breeding and an early conception during the postpartum period

(89). When compared to normally cycling cows, cows with abnormal cycles

(anovulation and/or prolonged luteal phase) during the pre-breeding period had lower

100-d AI submission, conception and pregnancy rates (89). The proportion of normal

cycles improves in cows ovulating spontaneously within 3 weeks postpartum (43)

During early postpartum, most dairy cows undergo a period of NEB,

resulting in the mobilization of adipose tissue in the form of NEFA as the primary

option to compensate the energy demands of lactation. NEB within 21 days postpartum

44

has been highly correlated with the time to first ovulation (11). The type of energy status

under which the first postpartum wave dominant follicles grows finally rate limits its

estrogen production and thus the capacity to induce LH surge and ovulation (11, 44).

The treatment of dairy cows with a GnRH analogue during the early

postpartum is effective to induce ovulation as (5, 58). However, the benefits of inducing

ovulation only with GnRH in the early postpartum are equivocal. While some studies

show a detrimental effect on fertility due to an early exposure to P4 (20), others show

improvement (5). The additional treatment with PGF2α after GnRH prevents postpartum

anestrous (20). Due to the frequently short lifespan of the first postpartum CL (34) and

to the need for the presence of a large follicle at the time of PGF2α to assure ovulation

(70), the interval in days between the two treatments is important for the presence of

responsive ovarian structures that could allow subsequent cyclicity.

As shown in Chapter 3, enrolling dairy cows as early as 21 days postpartum

into a protocol including consecutive treatments with GnRH and PGF2α in a 7-day

interval is effective to synchronize ovulation and has the potential to activate ovarian

cycles.

Two different studies involving cows managed either under research or under

commercial conditions were conducted to describe the ovarian cyclic activity and the

fertility of dairy cows treated with GnRH and PGF2α by 3 weeks postpartum.

4.2 Materials and Methods

4.2A. Study 1: Ovulatory and cyclicity responses of dairy cows under experimental

conditions

45

4.2A.1 Animals.

Postpartum lactating Holstein cows that calved between June 2004 and March

2006 were used to examine the efficacy and the factors influencing the response to a 7-d

GnRH-PGF2α protocol started on d 21 postpartum. Animals were managed under

free-stall confinement in the Field Center of Animal Science and Agriculture at Obihiro

University of Agriculture and Veterinary Medicine (Obihiro, Japan). Cows were offered

a total mixed ration including grass, corn silage and concentrate. Grazing was also

allowed between the months of May and October and milking was performed twice

daily (0600 and 1700). The 305-day milk yield average was 9,119 kg.

Hormonal treatments with GnRH and PGF2α were given to fifteen cows (GP

group, n=15). During the same period, thirty two non-treated cows (C group, n=32)

were monitored and served as controls. The major guidelines of the experimental design

including sampling frequency, hormonal treatments and evaluated parameters are shown

in Fig. 4.1a.

4.2A.2 Evaluation of luteal activity within 21 days postpartum

To asses whether first ovulation occurred within 21 days postpartum, plasma P4

levels in blood samples collected two to three times weekly until 21 days postpartum

were evaluated. An early first ovulation was identified as to have occurred when a P4

concentration of ≥1 ng/ml was detected within 21 days postpartum as reported

previously (43). In this regard, cows in the GP and C groups were classified as having

(-CL) or not (-NCL) ovulated within 21 days postpartum. Expected groups were:

GP-CL, GP-NCL, C-CL and C-NCL.

46

4.2A.3 Hormonal treatment

Treatment was performed intramuscularly with 10μg of a GnRH analogue

(Buserelin acetate: Itorelin®; ASKA Pharmaceutical Co., Ltd. Tokyo, Japan) 21 days,

followed by 500μg of PGF2α intramuscularly 28 days postpartum (Cloprostenol:

Resipron-C®. ASKA Pharmaceutical Co., Ltd.).

4.2A.4 Observation of the ovulatory response

In the GP group, occurrence of ovulation following GnRH and PGF2α

treatments was monitored using ultrasonography at 12-h intervals from postpartum days

22 to 24, and at 24-h intervals from postpartum days 29 to 34, respectively. Ovulation

was confirmed by the disappearance of dominant follicles (≥8mm) following either

treatment. Any cow failing to ovulate following treatment with GnRH was classified as

“non-synchronized” considering that, in dairy cows, a rather synchronous start of a new

wave of follicular growth and the formation of luteal tissue occur after GnRH-induced

ovulations. This is based on the results shown in Chapter 3 and on previous reports (71).

Cows were identified as to have undergone luteolysis following treatment with

PGF2α when plasma P4 concentrations dropped to < 1 ng/ml after 48-h. Cows in the GP

group were then further identified as having successfully ovulated (+) [GP-CL(+),

GP-NCL(+)] or failed to ovulate (-) [GP-CL(-), GP-NCL(-)] following the treatment

with PGF2α.

4.2A.5 Ovarian cyclicity

The patterns of luteal activity postpartum were monitored based on plasma P4

47

levels. In the GP cows classified as –NCL, blood samples collected daily from 21 until

28 days postpartum were used to identify the day of return to luteal activity. Thereafter,

samples were obtained twice weekly in both GP and C until the determination of a 3rd

ovarian cycle. The day of return to cyclic ovarian activity was defined as the day when a

second rise in P4 ≥1 ng/ml was detected following the demise of a previous luteal phase

(C group), or when a second rise in P4 concentrations ≥1 ng/ml occurred after an

induced demise of CL by the treatment with PGF2α (GP group).

Cows not showing P4 rises ≥1 ng/ml prior to 45 days postpartum were

considered to have delayed first ovulation and were termed as “inactive”.

The length of the estrus cycle following the first postpartum ovulation has been

reported to be of short duration (24). Therefore, comparison of the length of the estrous

cycle between the GP and the C groups was based on the cycle between the 2nd and the

3rd P4 rise in the C group or on the one between the 1st and the 2nd P4 rises following

treatment with PGF2α in the GP group according to their response to the protocol. An

estrous cycle of normal length was defined as the one having a 18-24 day interval

between two consecutive P4 rises ≥1 ng/ml as previously reported (31).

4.2B Study 2: Ovulation, cyclic ovarian activity and fertility responses of dairy

cows under commercial conditions.

4.2B.1 Animals.

Postpartum lactating Holstein cows (n=48) were part of a field trial study

carried out from May through December 2006. Animals were managed under either

stanchion, free or tie stall conditions in four distinct commercial dairy farms in the

48

Tokachi area of Hokkaido (Japan). The feeding system in one of the four herds was

based on a total mixed ration including. In the remaining three herds, the concentrate

feed was offered separate from the other components of the ration. The 305-day milk

yield average for the four herds ranged from 9,391 to 11,597 kg.

As described in study 1, hormonal treatments were given to twenty-five cows

(GP group). During the same period, twenty-three non-treated cows (C group) served as

controls. The major guidelines of the experimental design including sampling frequency,

hormonal treatments and evaluated parameters are shown in Fig.4.1b.

4.2B.2 Evaluation of luteal activity within 21 days postpartum

To monitor the ovarian activity within 21 days postpartum, plasma P4 levels in

blood samples collected once weekly from 1 to 28 days postpartum were evaluated. An

early first ovulation was identified as to have occurred when a P4 concentration of ≥1

ng/ml was detected prior to or by 21 days postpartum. In this regard, cows in the C and

GP groups were classified as having (-CL) or not (-NCL) ovulated within 21 days

postpartum as described in study 1.

4.2B.3 Hormonal treatment

Treatments were performed intramuscularly with 10μg of the GnRH

analogue 21 days postpartum, followed by 5.0 mg of the PGF2α analogue (Etiproston

tromethamine: Prostavet®. Virbac S.A., France) 28 days postpartum.

4.2B.4 Ovarian response

Study 2 was designed to allow the monitoring of the postpartum cyclic activity,

49

the ovulatory response to the treatment, and to evaluate the fertility by causing the less

possible interference in the management of the herds involved. In this regard, no

ultrasound observations of the reproductive organs were performed. Therefore, the

occurrence of ovulation following treatments was evaluated by analyzing individual P4

profiles. Treated cows with P4 levels going from ≥1ng/ml at the time of PGF2α treatment

to levels

50

small number of observations (in frequency and/or number of P4 rises), cows designated

as “non-synchronized” or as “inactive” were removed from analyses for the hormone

concentrations and ovarian cycles. Binomial data were tested by contingency chi-square

and differences were detected using Fisher’s exact test. Evaluation of the effect of the

induction of ovulation on the postpartum cyclic ovarian activity among cows grouped

according to treatment (GP vs. C), cyclic status at the beginning of treatment (-CL vs.

–NCL) and differences in the ovulatory response after the treatment with PGF2α (+ or -)

were tested for differences by the Dunnett’s test using control cows without ovulation

by 21 days postpartum (C-NCL) as the negative control. All calculations were done

using the JMP statistical software (Version 5.1; SAS institute). Differences among

means were considered significant at p

51

between 72 h post GnRH and before PGF2α treatment. Following treatment with PGF2α,

four [GP-CL (+)] out of four (100%) and six [GP-NCL (+)] out of eleven (55%) cows in

the GP-CL and the GP-NCL groups ovulated, respectively. All ovulations were detected

within six days. Three [GP-NCL (-)] out of the five remaining cows of the GP-NCL

group that failed to ovulate after PGF2α treatment were cows that ovulated following

GnRH treatment. The two remaining cows were the same ones classified as

non-synchronized.

4.4A.2 Plasma P4 levels by 28 days postpartum in the GnRH-PGF2α treated cows

Plasma P4 concentration did not differ between the GP-NCL (+) and GP-NCL

(-) groups (Table 4.2). However, both groups tended (p=0.08) to have less plasma P4

compared to the GP-CL (+) group. No corpora lutea were detected in non-synchronized

cows by the time of PGF2α treatment and plasma P4 remained at < 1ng/ml. Two days

following PGF treatment, the GP-NCL (-) group had significantly greater (p

52

cows of the GP-CL (+) group. The number of luteal phases within 60 days postpartum

(traditional end of the voluntary waiting period) tended to be more for C-CL (2.4 ± 0.2,

p=0.08) and GP-NCL (+)(2.6 ± 0.2, p=0.05) when compared to the C-NCL group (1.9

± 0.2). Representative P4 profiles during postpartum in the C and GP (+) cows are

shown in Figure 4.2.

4.4A.4 Characteristics of the ovarian cycles

Based on the data from P4, evaluation of the estrous cycle between the 2nd and

the 3 rd luteal activity showed no difference in the proportion of normal (18-24 days)

or abnormal cycles (short or long) across the groups. However, cows in the GP group

had improved proportions of normal cycles in an overall basis despite the presence of a

spontaneous or an induced ovulation after PGF2α treatment when compared to the C

group (GP: 82 % vs. C: 47 %; p 24 days) tended to be reduced in the GP group when compared to the C

group (1/11, 9 % vs. 13/32, 41 %; respectively. p=0.05).

4.4B Study 2 4.4B.1 Occurrence of ovulation within 21 days postpartum and luteal formation prior to PGF2α treatment

According to plasma P4 levels, 6 out of 23 (26 %) cows in the C group showed

ovulation within 21 days postpartum (C-CL). Likewise, ovulation occurred in 5 out of

25 (20 %) cows in the GP group (GP-CL). The remaining cows in the C (C-NCL: n=17,

74%) and the GP groups (GP-NCL: n=20, 80%) did not have ovulation within 21 days

postpartum. By day 28 postpartum a functional CL was present in 5 out of 5 (100%) and

53

16 out of 20 (80 %) cows in the GP-CL and GP-NCL groups, respectively. Following

treatment with PGF2α, 5 out of 5 (100%) and 5 out of 16 (31.3 %) cows in the GP-CL

and the GP-NCL groups had an induced luteolysis [GP-CL (+) and GP-NCL (+),

respectively]. Luteolysis to PGF2α treatment did not occur in 11 out of the 16 (68.8 %)

cows that had formed a CL by 28 days postpartum in the GP-NCL group [GP-NCL (-)].

4.4B.2 Plasma P4 levels by 28 days postpartum in the GnRH- PGF2α treated cows.

Plasma P4 concentrations prior to PGF2α treatment on day 28 postpartum did

not differ between the GP-NCL (+) and GP-NCL (-) groups (3.7 ± 0.7 and 2.7 ± 0.4

ng/ml, respectively) (Table 4.2). However, P4 levels in GP-CL (+) were significantly

greater than levels in GP-NCL (-) (4.9 ± 1.0 vs. 2.7 ± 0.4 ng/ml, p

54

first luteal activity 30 days postpartum. The number of luteal phases within 60 days

postpartum was more in the C-CL (2.7 ± 0.2, p

55

proportion of cows in the C group than in the GP cows conceived by 150 days

postpartum (65 vs. 36%, respectively; p

56

cows ovulated (in both GP-CL and GP-NCL groups). Similarly, plasma P4 levels ≥

1ng/ml by 28 days postpartum were observed in 80% of the GP-NCL cows and in all

cows of the GP-CL group in study 2. These results are in agreement with the results

presented in Chapter 3, and with those reported by others (5, 16, 28).

Two cows in study 1 failed to ovulate after GnRH treatment, while 4 cows in

study 2 presumably did so based on the absence of luteal activity by 28 days postpartum.

Both GnRH-anovulatory cows in study 1 were nesting follicles larger than the size at

which follicles acquire the capacity to ovulate (10 mm) (data not shown) (83). Failure to

ovulate might have been due to a reduced number of LH receptors (45) or caused by a

reduced capacity of the follicle to bind to LH due to ongoing atresia (35) as reported

previously. The reasons for the ovulatory failure after GnRH treatment in four cows in

study 2 are also uncertain. As in study 1, ovulation failure in study 2 might have been

due to absence of GnRH-responsive follicles. Three of the four cows that failed to

ovulate after the GnRH treatment in study 2 did not have luteal activity for at least 45

days postpartum, and the remaining cow showed its first lutea activity two days after

treatment with PGF2α. Nevertheless, the high rate of ovulation after GnRH treatment on

day 21 and the proportion of cows with a functional CL by day 28 in both studies

further demonstrates, in agreement with the results presented in Chapter 3, that

ovulation and synchronization of a new follicular wave can be effectively induced

despite differences in the ovarian cyclicity status during this period.

In both studies, the rate of ovulation after PGF2α was lower than the rate of

ovulation to GnRH. One reason that limited ovulation after PGF2α was the failure to

ovulate in response to GnRH treatment in the three anestrous cows classified as

non-synchronized in these studies. In a limited number of cows, the absence of

57

ovulation by 72 h following treatment with GnRH as observed in the

ultrasound-monitored group, and by an increase in P4 levels ≥ 1ng/ml 2 to 5 days after

PGF2α treatment in both studies indicated that these cows had ovulations within 4 days

prior to PGF2α treatment. Therefore, non-synchronized cows had CL in the very early

stages of formation at the time of treatment with PGF2α. Since CL in the early stage of

formation are refractory to PGF2α- induced luteolysis (60), the lack of a responsive CL

by 28 days postpartum in these cows was the cause of a failed ovulation after PGF2α.

In the GP-NCL (-) cows that ovulated to GnRH treatment in both studies,

failure of the CL to regress prevented the occurrence of ovulation after PGF2α treatment.

Interestingly, the P4 levels prior to PGF2α treatment in the GP-NCL (-) group were

similar to those in the GP-NCL (+) group, in which luteolysis was successfully induced.

These results indicate that, despite differences in PGF2α-induced regression, the

development and the steroidogenic function of the induced CL in the GP-NCL groups

were similar. However, the high rate of luteolytic failure of cows in the GP-NCL that

had a functional CL by the time of PGF2α treatment under commercial conditions was

unexpected. One probable reason for the degree of unresponsiveness between the two

studies could have been the heterogeneity of conditions (i.e., animals, environmental)

among the four commercial herds. Moreover, pharmacokinetic differences between the

two different PGF2α analogues used in either study can not be discarded as a possible

cause. The responsiveness of newly formed CL to undergo luteolysis may depend on

both the magnitude and the duration of the luteolytic stimuli (70). It is probable that

Cloprostenol, based on its long half life (t1/2= 3 hrs) (70), is more resistant to

metabolism than Etiproston tromethamine (no published information on t1/2).

Tromethamine salts, which are from natural PGF2α origin have very short half-lives (75).

58

A shorter milk withdrawal period is recommended for Etiproston tromethamine (12

hours) (Virbac S.A., France) in comparison to Cloprostenol (24 hours) (ASKA

Pharmaceutical Co., Ltd. Tokyo, Japan), being the main reason for its use in study 2. In

dogs, Etiproston tromethamine has been reported to be less effective than Cloprostenol

for CL regression when administered in a single dose (50).

Interestingly, luteolysis was unanimously synchronized in the GP-CL (+)

groups in both studies. This result is in accordance with reports showing improvement

in the response to hormonal synchronization programs in cows maintaining luteal

activity (62, 63). Probably, the reason for the presence of CL prior to PGF2α and the

uniform luteolytic response in this group was because cows were in the early luteal

phase when the treatment protocol started. The optimum suggested stage for the start of

a GnRH- PGF2α synchronization protocol in cycling cows is the period between days 5

and 10 of the estrous cycle (62)

The treatment with the GnRH- PGF2α protocol to early postpartum dairy cows

reduced the time postpartum to recovery of cyclic ovarian activity in both GP-CL (+)

and GP-NCL (+) groups. Moreover, the mean interval in days from the PGF2α-induced

luteolysis to the beginning of the two consecutive luteal phases (2nd and 3rd P4 rises,

respectively) observed under research conditions was precisely replicated in the field.

These results confirm that ovulation after GnRH and the synchronization and ovulation

of a new follicle after the PGF2α treatment did occur in the GP-CL (+) and the GP-NCL

(+) groups in the commercial dairy herds. Since days 5-9 of the estrous cycle yield the

best synchronization and fertility results after timed appointed breeding (70, 97), the

synchronous start of a luteal phase close to the end of the voluntary waiting period as

herein reported may be a prospective base for non-estrus detection based breeding

59

strategies.

The length of the estrous cycle following the protocol in cows that responded

with ovulation after both treatments was normal in the majority of the cases. Moreover,

estrous cycles of long durations were significantly reduced to the point of being almost

absent in treated cows. In contrast to a previous report in which normal cycles were

reduced when the treatment with GnRH preceded PGF2α 10 days (5), our present

findings may suggest a 7 day period as optimum for the synchronization of ovulations

and the cyclic ovarian activity when similar pharmacological treatments are to be used

during this period.

Based on the number of exposures to P4 within 60 days postpartum, a

significant acceleration of the cyclic ovarian activity occurred under field conditions in

the C-CL group (2.7 luteal phases) as well as in the GP-CL (+) and GP-NCL (+) groups

(2.6 and 2.8 luteal phases, respectively). A similar tendency was observed under

research conditions suggesting that, in this case, the small number of animals was a

limiting factor to obtain comparative results among the studies. Furthermore, in these

treated