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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
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.
3.7
. U
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ound
im
ages
in a
re
pres
enta
tive
cycl
ic c
ow
indu
ced
to o
vula
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, D
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: sec
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Gra
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5mm
.
d 0
d 2
d 3
d 4
d 7
GnR
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PGF 2
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ent
Day
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ance
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