Comparison of two different synchronization programs in
New Zealand dairy cattle
Drs. V.E.M. Captein
Research project Oct 2010 -Jan 2011
Supervisors: Dr. R. Laven and Dr. P.L.A.M. Vos
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
TABLE OF CONTENT
I
LIST OF ABBREVIATIONS.....................................................................................................4
II
ABSTRACT........................................................................................................................5-6
2.1 ENGLISH ABSTRACT…………………………………………………………………………………………………….5
2.2 DUTCH ABSTRACT……………………………………………………………………………………………………5-6
III
PURPOSE AND LIMITATIONS OF THIS RESEARCH PROJECT..................................................7
IV
INTRODUCTION...............................................................................................................8-9
V
LITERATURE REVIEW....................................................................................................10-13
VI
MATERIALS AND METHODS.........................................................................................14-17
6.1
ANIMALS.....................................................................................................................14
6.2
TREATMENT...........................................................................................................14-15
6.3
ULTRASOUND..............................................................................................................15
6.4
BLOOD SAMPLES...................................................................................................15-16
6.5
ASSAYS........................................................................................................................16
6.6
MILK PRODUCTION.....................................................................................................16
6.7
STATISTICAL ANALYSIS AND DATA HANDLING......................................................16-17
VII
RESULTS.......................................................................................................................187.1
AGE AND BCS..............................................................................................................18
7.2
MILK YIELD..................................................................................................................19
7.3
RESPONSE TO FIRST GNRH....................................................................................19-20
7.3.1 OVULATION RATE..........................................................................................19
7.3.2 SIZE OF PRE-OVULATORY FOLLICLE ...............................................................20
7.4
DF SIZE AT DAY 7.........................................................................................................20
7.5
DF SIZE AT DAY 9.........................................................................................................21
7.6
RESPONSE TO SECOND GNRH................................................................................21-22
7.6.1 OVULATION RATE..........................................................................................21
7.6.2 SIZE OF PRE-OVULATORY FOLLICLE................................................................22
7.6.2.1 RELATION PRE-OVULATORY FOLLICLE SIZE AND MILK YIELD............22
7.7
FOLLICULAR DYNAMICS.........................................................................................22-24
7.7.1 DYNAMICS ALL COWS INCLUDED...................................................................22
7.7.2 DYNAMICS WITHOUT DEVIATED RESPONDERS.............................................23
7.7.3 DYNAMICS DEVIATED RESPONDERS VERSUS NORMAL RESPONDERS...........24
7.8
CL MEASUREMENTS....................................................................................................22
VIII
DISCUSSION.................................................................................................................25-28
8.1
METHODOLOGY.....................................................................................................25-26
8.1.1 SELECTION OF COWS.....................................................................................25
8.1.2 MEASUREMENTS......................................................................................25-26
8.1.3 CATHETERIZATION.........................................................................................26
8.2
RESPONSE TO FIRST GNRH....................................................................................26-27
8.2.1 OVULATION RATE..........................................................................................26
8.2.2 SIZE OF PRE-OVULATORY FOLLICLE..........................................................26-27
8.3
DF SIZE AT DAY 7.........................................................................................................27
8.4
DF SIZE AT DAY 9.........................................................................................................27
8.5
RESPONSE TO SECOND GNRH................................................................................27-28
8.5.1 OVULATION RATE.....................................................................................27-28
8.5.2 SIZE OF PRE-OVULATORY FOLLICLE................................................................28
8.6
FOLLICULAR DYNAMICS..............................................................................................28
IX
SYNCHRONIZATION PROGRAMS IN THE NETHERLANDS…………………………………………………..29
X
CONCLUSION....................................................................................................................30
XI
ACKNOWLEDGEMENTS ....................................................................................................31
XII
REFERENCES......................................................................................................................32
2
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
XIII
ATTACHMENTS
A: DETAILS DEVIATED RESPONDERS
B: RECORDING SHEET DURING ULTRASOUND
3
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
I.
ANCOVA
AI
BCS
CL
DF
EDA
ElISA
EU
E2
FSH
FTAI
GnRH
GPG
GPG + P4
IVABS
LH
Mm
NZ
ODB
PGF2α
P-value
P4
RIA
Rpm
SD
TG
LIST OF ABBREVIATIONS
= Analysis of Covariance
= Artificial Insemination
= Body Condition Score
= Corpus Luteum
= Dominant Follicle
= Exploratory Data Analysis
= Enzyme-Linked Immuno Sorbent Assay
= European Union
= Oestradiol
= follicle stimulating hormone
= Fixed Time Artificial Insemination
= Gonadotrophin-releasing Hormone
= GnRH-Prostaglandin-GnRH program
= GnRH-Prostaglandin-GnRH + Progesterone program
= Institute of Veterinary, Animal and Biomedical Sciences
= Luteinizing Hormone
= millimetre
= New Zealand
= Oestradiol Benzoate
= Prostaglandin F2 alpha
= Probability value
= Progesterone
= Radio Immuno Assay
= Revolutions per minute
= Standard deviation
= Treatment group
4
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
II.
ABSTRACT
The main objective of this research is to study the effect of removing a progesteronereleasing device out of the treatment of a regular GnRH-Prostaglandine-GnRH + P4 program,
on follicular dynamics and oestrus synchronization in post-partum anoestrus cows, in the
New Zealand situation.
Animals included in the experiment were selected from one farm, comprising a total of 22
Friesian dairy cows. Animals used for the experiment were multiparous, were not observed in
heat, had no sign of being cyclic after two ultrasonographic examinations, and had no
palpable evidence of uterine or ovarian abnormalities.
The cows were randomly allocated to receive a treatment following the GPG + P4 program
or to receive a treatment following the same protocol but than without P4.
Ultrasonography was performed from day 0 to 8 once a day, and twice daily on days 9, 10
and 11, to record the presence and diameter of the dominant follicle (DF), and further
follicular dynamics. Blood sampling took place on a daily basis in the coccygeal vein to
measure P4 and E2 concentrations, and on a hourly basis at day 9 of the program, to follow
LH-level dynamics. Fixed time AI was performed on the 10th day, 16 hours after the second
GnRH injection.
No significant difference was found between the two treatment groups concerning the
number of cows with a synchronised ovulation, although a trend showed a better
synchronization after treatment with GPG+P4. The only significant difference (p=0.050)
found in this experiment was the diameter of pre-ovulatory follicles after the first GnRH
administration in the GPG+P4 group were (on average 3 mm) smaller than in the GPG group.
SAMENVATTING
Het belangrijkste doel van dit onderzoek is, om in post-partum anoestrische koeien in de
Nieuw Zeelandse situatie, het effect van het wegnemen van de progesteron-afgevende
spiraal uit het reguliere GnRH-Progesteron-GnRH + P4 synchronisatie programma, op de
folliculaire dynamiek en mate van synchronisatie van oestrus, te bestuderen.
De dieren die deelnamen aan dit experiment zijn geselecteerd op één bedrijf, en bevatte een
totaal van 22 Friesian melkkoeien. De dieren die gebruikt zijn in dit experiment waren
multipaar, nog niet in oestrus waargenomen, hadden geen teken van cycliciteit na twee
echoscopische onderzoeken, en hadden geen palpabele uteriene of ovariele abnormaliteiten.
De koeien zijn willekeurig verdeeld over behandelgroepen volgens het GPG+P4 programma,
of het GPG programma zonder P4.
Echoscopisch onderzoek werd van dag 0 tot 8 dagelijks uitgevoerd, en twee maal daags op
dag 9, 10 en 11, om de aanwezigheid en diameter van het DF, en verdere ontwikkelingen op
het ovarium op te nemen. Bloed afnemen vond dagelijks plaats in de vena coxygea, om P4
en E2 concentraties te meten. Op dag 9 werd er elk uur bloed afgenomen, om het LH-niveau
5
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
te kunnen volgen. Kunstmatige inseminatie werd toegepast op dag 10, 16 uur na de tweede
GnRH injectie.
Er werd geen significant verschil gevonden tussen de twee behandelgroepen wat betreft
aantal koeien met een gesynchroniseerde ovulatie. Er was echter een trend die een betere
synchronisatie aangaf bij de GPG+P4 behandeling. Het enige gevonden significante verschil
was dat de diameter van de pre-ovulatoire follikel na de eerste GnRH injectie in de GPG+P4
groep (gemiddeld 3 mm) kleiner was dan in de GPG groep.
6
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
III.
PURPOSE AND LIMITATIONS OF THIS RESEARCH PROJECT
Within the curriculum of Veterinary Medicine at the University of Utrecht all students have
to accomplish a research project, after finishing their 4th or 5th year.
This final report is based on the research project completed at the Massey Universities’
Institute of Veterinary, Animal and Biomedical Sciences (IVABS), New Zealand, from October
till December 2010.
This research project is part of a long-term PHD project about the comparison of three
different synchronization programs in heifers and post-partum anoestrus cows. (GPG,
GPG+P4, PG+P4)
Supervisors in this project are R. Laven (Massey University) and P.L.A.M. Vos (Utrecht
University), and PhD-student S.K. Sahu.
The main purpose of this research is to study the effect of removing a progesteronereleasing device from the regular used FTAI program (GnRH-Prostaglandine-GnRH + P4), to
reduce costs and labour, on follicular dynamics and oestrus synchronization in post-partum
anoestrus cows, in the New Zealand situation.
This report will finish with a brief extrapolation of the results to possibly apply them in the
Dutch dairy industry to solve comparable reproduction problems, and to find out in what
way synchronization programs will be financially interesting.
As an effect of this research project being part of a larger project, the sample-size is not
sufficient to give significant results in all parameters of the study. The data has to be
extended with data of earlier and following experiments. The power analysis of this
experiment is based on LH-surge dynamics.
Unfortunately, at the moment of writing the report, data for the results of the hormone
concentrations are not yet available from the laboratory. More over, incomplete
information was available on pregnancy rates and therefore not used in the report.
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
IV.
INTRODUCTION
Different production systems ask for different approaches to solve the problems that go
along with the systems. The pasture-based dairy production system in New Zealand (NZ)
brings separate difficulties to a more intensive, confinement-based system like most farms
in the Netherlands. (Bo, 2007)
The main difference is the seasonal breeding system used in NZ. NZ dairy farmers try to
match pasture growth with the increased energy demand at the onset of lactation.
In this seasonal rhythm, cows must give labour to a calf every year. A 365 days calving
interval does not give cows a long anoestrus period. All cows need to conceive within
approximately 80 days after calving.
Oestrus synchronization is an important solution to improve reproductive performance in
cattle. Two main groups ask for more attention during the breeding season: heifers and
post-partum anoestrus cows.
Heifers normally graze separately from the lactating cows and special effort needs to be
made to access them for oestrus detection, and the following artificial insemination (AI).
For this reason many farms let the bull do the job, and the widespread use of AI is still
limited. The use of AI in heifers would be facilitated by an oestrus synchronization program
that allows for a single fixed-time AI to achieve an acceptable pregnancy rate. AI would give
the opportunity to increase the genetic value of the calves and so the replacement herd.
Furthermore, using synchronization programs for heifers makes it possible to let most of the
heifers calve early in the season; with the advantage that these animals can find their own
place in the herd before the cows with higher parity come in, and will have a longer period
post-partum before the breeding-season starts again.
Post-partum anoestrus is a major reproductive problem in the NZ dairy industry. The
percentage of cows in the herd not being detected in oestrus by the start of the breeding
season is on average 20%. As already mentioned above, there is not much time to lose
because of the strict 365 days calving interval. For this reason, synchronization protocols
are used on these cows. (McDougall, 2010a)
Oestrus synchronization is not an approach just from the last couple of years. Many
different strategies, programs and hormones have been part of the research, all over the
world. But still, the ideal synchronization program has not yet been found. The European
Union (EU) ban (2006) on oestradiol-17 β and its related ester derivates as a result of the
concerns regarding consumer’s health, had its effect on NZ where the use of oestradiol
benzoate (ODB) is effectively banned for all veterinary indications. (Laven, 2008)
As a consequence, most synchronization programs, including oestradiol-17 β or one of its
derivates, had to be reviewed and experiments with other hormones were started to test
new designed protocols including GnRH (Gonadotrophin-releasing Hormone) administration
and intravaginal progesterone (P4) -releasing devices.
Several of those new oestrus synchronization regimens had variable but mostly
disappointing results under NZ field conditions, and asks for thorough research to learn
more about the mechanisms underlying why the response to synchronization differs every
time. The response to synchronization of the main groups; heifers and post-partum
8
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
anoestrus cows hold most interest, because of their apparently reduced sensibility for these
programs. (Cavalieri et al., 2006; Laven et al., 2009)
Of these two groups of interest, the post-partum anoestrus cows will be the main subject of
this report. The aim of this research is to improve the understanding, and optimise the
effect of two different synchronization programs in NZ dairy cows.
Concluding the research with a brief extrapolation of these results to Dutch dairy farming, to
see if synchronization programs would be financially interesting, and to check if the results
may be useful in understanding or solving comparable reproduction problems in the
Netherlands.
As already mentioned a substantial amount of research has been published on FTAIsynchronization programs for anoestrus cows, including information on follicular dynamics,
hormone-levels, and others. But the gap in research still is the integration of all these
factors, in the New Zealand situation. Only a few studies focus on the NZ situation, and
mostly they do not include the entire process from anoestrus till pregnancy. (e.g. McDougall,
2010b)
Most NZ researches mainly compare pregnancy rates, while much more happens in
between, which is needed to have a good understanding and to be able to say what really is
the best synchronization program.
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
V. LITERATURE REVIEW
Oestrus synchronization is used for two main purposes. On one hand; to increase the
number of (successfully) inseminated cows in a short period of time, and on the other hand;
to obviate the need for heat detection. These two purposes of oestrus synchronization are,
in NZ dairy herds, represented by the group of post-partum anoestrus cows, and the group
heifers, respectively.
To understand the differences in response between the cows, and to create a
synchronization program as efficient as possible and economically attractive, knowledge of
normal reproductive function and the way these exogenous hormones manipulate this
reproductive function is required.
The main hormones used in NZ Dairy industry are Prostaglandine-F2 Alpha (PGF2α), to
induce luteolysis, and GnRH, P4, and oestradiol, to synchronise follicular development. As
mentioned in the introduction, oestradiol is no longer allowed for any veterinary indication,
so will not be reviewed in this paper. (Laven, 2008)
To understand the effect of the different hormones, an overview of follicular dynamics in
the dairy cow is needed.
Follicular dynamics
Follicles follow a wave-like pattern on the ovary. In on average 2 or 3 follicular waves per
oestrus cycle, the follicular development can be subdivided in 3 stages: growth, deviation
and selection. In each wave a group of about 6 follicles will grow larger than 4-5 millimetre
(mm) in diameter. One of those follicles will be selected to become the dominant follicle
(DF). The DF will start to grow and differentiate, while the other follicles of the same wave
will regress. In normal oestrus cycles, which means without the administration of any
exogenous hormones, this DF will regress as well when it is the first wave( in a two-waveoestrous-cycle), or the first or second wave (in a three-wave-oestrus-cycle). Although these
DF normally would regress, they can ovulate as long as the endocrine conditions are suitable,
which is useful in modern synchronization programs. (Ptaszynska, 2006)
Recruitment of follicular waves
A small rise of follicle stimulating hormone (FSH) makes the follicles start to grow in the
beginning of a follicular wave. The FSH surge reaches a peak when the future DF of the
emerging wave is approximately 4 mm in diameter. (Ginther et al., 1996) Despite the
declining FSH, the follicles continue to grow at an equivalent rate for 2 or 3 days after
emergence. (Ginther et al., 1997)
Selection and lifespan of the DF
It is not clearly understood, for what reason one out of the cohort of follicles recruited after
FSH-rise, is selected to become dominant. It might be selected on its greater capacity of E2
production. (Ginther et al., 2000) While the other subordinate follicles cease to grow
because of the low FSH concentration: E2 and inhibin secretion of the growing follicles
suppress FSH (MacMillan et al., 2003; Peter et al., 2009). The DF continues its growth,
because of its responsiveness (thanks to LH-receptors) to the increasing LH-levels. The FSH
negative feedback decreases when the DF undergoes atresia. The increase in blood FSH
triggers the recruitment phase of the next wave. The LH-pulse pattern is controlling the
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
growth, lifespan and oestrogen activity of the DF. The final LH-surge can induce ovulation of
the DF.
The FSH and LH-patterns are dependent of the GnRH levels. GnRH is a hormone produced
by the hypothalamus, and stimulates the FSH and LH release by the pituitary. (Senger, 2005)
Post-partum anoestrus cows
The post-partum anoestrus cows are the main reason for use of synchronization programs
in the dairy industry in NZ. Anoestrus is a general term to indicate that a cow was not seen
in oestrus, because she was not detected in oestrus (poor detection or weak/absent oestrus
behaviour, termed suboestrus), or because the cow did not come into oestrus (true
anoestrus).
It is true anoestrus which causes most problems with post-partum anoestrus cows in NZ.
There are 4 types of true anoestrus. The cycle can stop at different points as shown in figure
1. (Peter et al., 2009) Type II is the most common way post-partum anoestrus occurs in NZ
dairy cows. (Laven, pc, 2010) This means, the cow does develop a DF in the physiological
way, but in the last stage the DF fails to ovulate and goes in atresia. A new follicular growth
wave can start.
McDougall and Rhodes (1999) found that under NZ management systems, between 10%
and 30% of anoestrus cows have a CL, and following the figure, are type IV-anoestrus cows.
This off course has its effect on the responsiveness on the breeding program. (McDougall et
al., 1999)
(This group of cows is not the subject of the recent study, since the cows in the experiment
will be excluded when they have a CL.)
Type II is the
most common
way post-partum
anoestrus occurs
in NZ dairy cows.
(Laven, personal
communication,
2010)
Figure 1: Schematic representation of types of anoestrus conditions based on ovarian follicular and
luteal dynamics (Peter et al., 2009)
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
Effect of the different hormones used in modern synchronization programs:
Prostaglandin F2 alpha
PGF2α brings about regression of the CL. The regression makes it possible for a new oestrus
cycle to begin, since the P4-production by the CL will be stopped. That will cause the end of
the negative feedback on the hypothalamus, what means, no inhibition of GnRH release
anymore: new ovulations are possible. For this reason PGF2α is a frequently used hormone
in dairy cattle. A limitation of this indication is the absence (most anoestric cows do not
have a CL), or insensitivity of the early corpus luteum (CL) to luteolytic actions of the PGF2α.
The underlying mechanisms of this poor sensitivity are meagre understood, but differential
gene expression in different stages of CL development may account for differences in signal
transduction pathways associated with luteal sensitivity. (Goravanahally, 2009)
In general, the CL will be sensitive for PGF2α between day 6 and 16 of the oestrus cycle (the
period of natural prostaglandin F2a release). Injection of PGF2α within that period will cause
regression of the CL ending the luteal phase and a new follicular phase begins. Although this
is a good start to synchronize oestrus, a single injection is not sufficient. When injecting a
group of cows with unknown stages of the cycle, the following oestrus will not be at the
same moment for all cows. It depends on the stage of the animals’ follicular development
when the animal is treated. Animals with a functional DF are in oestrus within 2-3 days after
injection, while animals at the pre-dominance phase of the wave will require 2-4 days to
form a DF, and hence have a longer and more variable interval to the onset of oestrus.
To give a cow an effective treatment with PGF2α it needs to meet 2 conditions: It needs to
be a cycling cow, and the moment of injection has to be between day 6 and 16 of the cycle,
(since the CL has to be sensitive). To pre-empt the different stages the cows are in, PGF2α is
often used in combination with GnRH-injection. (Ptaszynska, 2006; Amiridis et al., 2000)
GnRH
Administration of GnRH will cause an LH-surge and consequently an ovulation. The first
GnRH injection of a GnRH-Prostaglandin-GnRH -synchronization program (GPG) can be given
at a random stage of the cycle. This injection will cause ovulation or luteinisation of the DF.
If the GnRH is given when a new follicular wave is just started, so no Luteinizing Hormone
(LH) -responsive follicles are yet grown to be ovulated, the GnRH will not cause ovulation or
luteinisation; this should be about 15 % of the cows (Pursley et al., 1995). For these cows,
the follicular wave is not altered by the GnRH administration. Thus by the time of the
second GnRH injection, a DF should be present and is able to react and ovulate as a
response to second GnRH administration.
P4
Progesteron is normally produced by the CL, and is needed for a normal cycle and to
maintain pregnancy. It reduces the GnRH release, thus inhibits new ovulations.
P4 can be administrated by injections, but the labour-effective CIDR (Controlled intravaginal
drug release) is often used as an ‘artificial’ CL in synchronization programs.
GPG: Ov- synchr
Ov-synchr is a commonly used name of the GPG program.
As a result of these three injections, follicular dynamics should be tightly synchronised, and
ovulation occurs 26-32 hours after the second GnRH injection. The ovum is normally fertile
12
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
4-6 hours after ovulation, while frozen semen has got a lifespan of 36-48 hours. Therefore,
an insemination at 17-24 hours after second GnRH injection is expected to give optimal
conception results. (Pieterse, 2008)
GPG vs. GPG + P4
In this research we will compare a GPG program with a GPG+P4 program.
To clarify the expected positive effect of addition of P4 to the treatment, some more details
of the post-partum ovary are required.
The first postpartum ovulation is often accompanied by so called ‘silent heat’, strictly
speaking: no oestrus behaviour. This first ovulation is often not fertile, because of the short
luteal phase that follows. (Webb et al., 1980; Laven, pc, 2010)The addition of P4 in the Ovsynchr program is thought to be effective since it will create a luteal phase of normal length,
and via this mechanism, better conception rates. (Sheffel et al., 1982)
Thanks to new insights the length of addition of P4 in the protocol has been abbreviated to
only 7 days, and so also will shorten the length of the luteal phase, different than Sheffels’
study.
McDougall compared the Ovsynchr program with and without addition of P4, and found
better conception rates in the programs with P4.
Another mechanism found in several studies, is that if the dominant follicle not ovulates
after first GnRH, they will be supported and grown under progesterone-dominance (in the
GPG+P4-program), but will not ovulate until the progesterone-releasing device is taken out
and the second GnRH is given. This results in higher pregnancy rates (55.2 vs 34.7 %) (Yaniz
et al., 2004)
Economical aspects
Given that farmers usually dry off all cows on one predetermined calendar day, cows calving
later in the subsequent season have a shorter lactation length and for that reason will have
less economical value. In the cost-benefit analysis of McDougall (2010b) it was concluded
that Ovsynchr+P4 treatment without any diagnostic procedure was the most cost-effective
option. Via partial budgets and decision trees it became clear that Ovsynchr+P4 treatment
had the highest net benefit.
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
VI. MATERIALS AND METHODS
6.1
Animals:
Post-partum Friesian cows (n=49) were detected for oestrus before the planned start of the
breeding season at one of Massey’s own farms, Massey nr. 4, using tail paint. Massey nr 4 is
a dairy unit, located in the periphery of Massey University. Cows not detected in oestrus by
the start of the breeding season (20th October 2010) were enrolled for the study. At the 28th
of October 2010, these cows were checked using ultrasound. Reproductive tract of all the
enrolled cows were palpated and palpable uterine or ovarian pathology was recorded.
Additionally ultrasonographic examinations of the ovaries were performed and the
presence or absence of CL was defined. Cows with palpable evidence of uterine infections or
adhesions or other pathology of the ovaries or uterus were excluded. 31 cows out of those
49 met the requirements. At the day of starting the experiment (3rd or 5th November 2010),
the ovaries were checked again on absence or presence of CL, using ultrasound. BCS was
also recorded at a 1 to 10 scale. Cows without a CL after both checks were defined
‘anoestric’. After this final selection 22 cows were enrolled and selected for the study. These
22 selected cows were randomly divided in two different treatment groups, each having 11
animals, with respect to days post-partum. The cows were on average 32.4 days postpartum.
6.2
Treatment:
All the enrolled cows received two different treatment regimens as described below in table
1, and visualized in figure 2. The drugs used for the synchronization were OvurelinTM 1ml
i/m (Gonadorelin (= GnRH-effect), 100 µg / ml), OvuprostTM 0.5 ml i/m (Cloprostenol, 250
µg/ml) (= PGF2α effect) and Cue-MateTM (Progesterone 1.56 g) manufactured by Bomac
Laboratories Ltd, Auckland NZ. The cows from both the treatment groups were again
randomly assigned to two groups, and were scheduled two days apart, to manage blood
sampling at day 9.
Table 1: treatment protocol of GPG (n+11) and GPG+P4
Day of 0, AM
experim
ent,
AM/PM
GPG
Inject 1 ml
Ovurelin (100
(n=11)
µg/ml I/M) =
GnRH
GPG + P4 Insert P4 device
+ Inject 1 ml
(n=11)
Ovurelin (100
µg/ml I/M)
= GnRH
7, AM
9, PM
(12.00 AM)
Inject 2 ml Ovuprost Inject 1 ml
(250 µg/ml I/M)
Ovurelin
= PGF2α
(100 µg/ml
I/M) = GnRH
Remove P4 device + -doInject 2 ml Ovuprost
(250 µg/ml I/M)
= PGF2α
10, AM
(8.00 AM)
FTAI (16 – 20
hours
after
second GnRH
injection)
-do-
14
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
Figure 2: Overview of treatment protocol (Laven, 2008)
6.3
Ultrasound:
Ovarian structures were monitored using a real time B-mode ultrasound scanner, Mindray
DP-6600 Vet, equipped with 7.5 MegaHertz, linear endorectal transducer 75L50EAV.
Ultrasonography was performed in the cows once a day from day 0 (= day of treatment) to 8,
to determine ovarian follicular changes, and twice a day on days 9, 10 and 11, to confirm
ovulation. Because the exact time of ovulation after the second GnRH is of more interest
than the time of ovulation after the first GnRH, the twice daily ultrasonography is only done
after the second GnRH. The presence or absence of a DF (> 9mm) and/or CL was recorded
for each cow individually, and mapped during scanning. The ultrasound images were
recorded digitally for detailed examination and measurements with use of the program
Image-J, at the same day of scanning. Diameter of the ovarian structures was estimated by
averaging the following two measurements: at the widest point and at a right angle to the
first measurement. Ovulation was diagnosed when the preovulatory follicle was no longer
present. Cows were examined for pregnancy by transrectal ultrasonography at 32 days after
AI.
6.4
Blood samples:
Blood samples were collected from all cows via coccygeal venopuncture into BD Lithium
Heparin Vacutainers of 10 ml on days 0 to day 14 and on days 16, 18, 21 and 22 for
determination of P4 and E2 concentration.
To measure LH-surge dynamics, the cows were sampled on day 9, the day of second GnRH
treatment, on hourly basis, which means at -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and
14 hours from GnRH treatment, where the sample at 0 hours, is taken right before GnRH
treatment.
To obtain these samples in the most effective way, a jugular catheterisation was done with
the first 5 animals. 18 gauge catheters, with a length of 45 mm and a flow of 100 ml/min,
(Smiths medical, Optiva®) were used, and extended with a tube, which was fixated at the
withers. For the fixation of the catheter, superglue was used. To prevent the catheters from
blocking, the catheters were filled and flushed with heparinised saline, (1 ml heparin in 1
litre saline). Since the catheters were blocked within one hour, blood sampling was
15
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
continued following a repeated scheme: 2x left jugular, 2x right jugular and 2x coccygeal
vein.
Blood samples taken daily were analysed for FSH, E2 and P4 concentrations. The hourly
taken blood samples were used to measure corticosterone and LH-levels. The
corticosterone measurement is part of the assays to exclude an effect of stress (possibly
caused by taking the blood samples) on time of ovulation
Plasma was separated by centrifugation at 2000 revolutions per minute (rpm) for 20 min at
4 °C. The plasma was separated using disposable Pasteur pipettes within 2 hours after
sampling, and were stored in CRYO-tubes in the freezer (- 20 °C) till final assay.
6.5
Assays:
P4, E2, corticosteronee
P4, E2 and corticosterone concentration in the blood plasma will be measured by using
commercially available Radio Immuno Assay (RIA) kit.
FSH and LH
FSH and LH concentrations will be measured using a commercial available Enzyme-Linked
Immuno Sorbent Assay (ELISA).
6.6
Milk Production:
Milk production (in litres per day) of the animals was recorded twice a day. The data was
used to make sure the cows were randomly divided in the treatment groups, so that no
possible found difference was subscribed to treatment, while it was possibly caused by
difference in milk production. Milk yields of the 19 days before start of treatment were used.
6.7
Statistical Analysis and Data Handling
The data obtained was subjected to Descriptive Statistics (Exploratory Data Analysis – EDA)
to obtain the mean and standard deviation (SD) of the variables measured. Various
statistical modelling tools like multiple regression, multivariate analysis and analysis of
covariance (ANCOVA) were used to establish the relationship between different parameters.
A probability value (p-value) of P ≤ 0.05 was considered significant. The software Excel 2007
and Minitab were used for analysis.
The power analysis of this experiment is based on LH-surge dynamics and is illustrated in
table 2.
Table 2: Power analysis of the experiment
Variable
(eg,
Weight)
Means or
Expected
Difference
SD
Type 1
error (ά)
LH
25ng/ml
25
0.05
Type 2
error (β)
Power
Number
of animals
needed
82%
10
per
treatment
group
.
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
The following definitions are used to be able to categorize the cows:
- Responders versus deviated responders after first GnRH injection: To distinguish
responders from deviated responders, ovulations within the 25-72 hour interval after
injection, are assumed to be a normal response to the GnRH, and before or after this
interval it is assumed to be a deviated response since these cows did not ovulate as a
response on the GnRH administration.
- Responders versus deviated responders after second GnRH injection: To distinguish
responders from deviated responders, ovulations within the 13-36 hour interval after
injection, are assumed to be a normal response to the GnRH, and before or after this
interval it is assumed to be a deviated response since these cows did not ovulate as a
response on the GnRH administration.
Measurement method of:
- pre-ovulatory follicle: The size of the pre-ovulatory follicle is based on the measurement of
that DF on the day before ovulation. Only the responders are used in calculating the mean.
- CL: It was difficult to get clear pictures of the CL and the DF at the same ovary. More
pictures were taken to get a good picture of both the structures. But this was not done
consistently, and because the DF was of larger importance in this study, the CL
measurements are not used in this report.
17
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
VII. RESULTS
7.1
Age and BCS
Age and BCS of the cows at the day the experiment started are shown in table 3 and in the
boxplot in figure 3. The multivariate analysis showed that there was no significant difference
among treatment groups for age or BCS (P=0.654): the cows were correctly, randomly
distributed over the TGs.
Table 3: Age (days) and BCS of cows at the start of the experiment.
TG
n
GPG
11
GPG+P4 11
Age in days
BCS (scale 1-10)
1620 ± 536
1520 ± 474
3.8 ± 0.34
3.6 ± 0.32
Values are mean ± SD. No significant difference was found
The boxplot in Figure 3, shows outliers in BCS of the GPG+P4 group. These outliers signify
that the BCS of these cows are aberrant from the minimum and maximum values. This
means that the other data-sets have all data within the minimum and maximum values,
which are showed by the whiskers. The median is showed in the boxplot of age, because the
median in BCS comes together in the border of the boxes.
Boxplot of Age, BCS
1
Age
2600
2
BCS
4.50
2400
4.25
2200
2000
4.00
1800
1600
3.75
1400
1200
3.50
1000
GPG 1
GPG+P4 2
TG
GPG
GPG+P4
Figure 3: Boxplot of age and BCS at time of start of experiment; GPG (n=11), GPG+P4 (n=11)
18
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
milk yield in litres per day
7.2
Milk yield
Figure 4 shows the average daily milk yield per treatment group, 2 weeks prior to the start
of the experiment. No significant difference was observed for the average daily milk yield
between the groups (p=0.31).
36
34
32
30
28
26
24
22
20
18
16
GPG (n=11)
GPG + P4 (n=11)
d- d- d- d- d- d- d- d- d- d- d- d- d- d- d- d- d- d19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2
Days before start of experiment
Figure 4: Average daily milk yield per treatment group before start of experiment, no significant
difference between groups.
7.3 Response to first GnRH
7.3.1 Ovulation rate
After the first GnRH injection 12 out of 22 cows ovulated within the 25-72 hours after
treatment, (6 out of GPG and 6 out of GPG+P4). Out of those 12 cows, 2 cows of the GPG
group ovulated between 25 and 48 hours. The other 10 cows (4 GPG cows, and 6 GPG+P4
cows) ovulated between 49 and 72 hours. (Fig. 5)
In both the treatment groups, 5 cows did not ovulate within the 25-72 hours after treatment,
which is illustrated as ‘deviated response’ in Figure 5. 1 cow ovulated at day -1 (GPG), 1 cow
ovulated at day 0 (GPG), 1 cow ovulated at day 3 (GPG+P4). The other 7 cows did not
ovulate before day 6.
19
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
7
6
5
number of cows
4
GPG (n=11)
GPG + P4 (n=11)
3
2
1
0
25-48 hrs
49-72 hrs
deviated response
Time after first GnRH injection
Figure 5: Distribution cows related to the observed ovulation time, after the first GnRH injection on day 0.
7.3.2 Size of pre-ovulatory follicle
The mean size of the pre-ovulatory follicle per treatment group is shown in table 4. There
was a significant difference between GPG and GPG+P4. (p=0.05) In table 4, n=12, because
only the cows which had a normal response were used in these calculations.
Table 4: Size of pre-ovulatory follicle after first GnRH treatment, n=12
TG
n
Size of pre-ovulatory follicle (mm)
GPG
GPG+P4
6
6
16.2 ± 3.07a
13.2 ± 1.30b
Values are mean ± SD, different superscripts within column are significant different.
7.4
DF size at day 7
Table 5 shows the mean size of the DF at moment of PGF2α treatment. There is no
significant difference found between the two treatment groups, (p=0.90).
Table 5: Size of DF at the moment of PGF2α administration, at day 7
TG
n
Size of DF (mm)
GPG
GPG+P4
11
11
15.3 ± 2.59
15.1 ± 5.81
Values are mean ± SD. No significant difference was found
20
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
7.5
DF size at day 9
Size of DF before second GnRH injection, day 9 am, is shown in table 6.
Table 6: Size of DF before second GnRH administration, at day 9
TG
GPG
GPG+P4
n
11
11
Size of pre-ovulatory follicle (mm)
15.9 ± 3.79
15.9 ± 3.59
Values are mean ± SD. No significant difference was found
7.6
Response to second GnRH
7.6.1 Ovulation rate
As shown in Figure 6, 15 out of 22 cows in total, responded with an ovulation between the
interval of 13-36 hours after second GnRH, of which 7 were GPG cows and 8 were GPG + P4
cows. The interval in which most responders ovulated was even narrower: 13 out of the 15
responders ovulated within the interval of 25 to 36 hours (6 GPG and 7 GPG+P4 cows).
7 out of 22 cows ovulated on different moments or not at all. 1 cow ovulated on day 6
(GPG+P4), 2 cows ovulated on day 7 (both GPG), and 1 cow ovulated on day 8 (GPG). One
cow did not ovulate at all, (GPG), and 2 cows did not ovulate anymore since day 2 (both
GPG+P4).
Figure 6: Ovulation rate after second GnRH, per treatment group. (!! Attention: the time intervals at
x-axis differ from figure 5, since scanning was done twice daily at this point of experiment)
21
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
7.6.2 Size of pre-ovulatory follicle
The mean size of the pre-ovulatory follicle per treatment group is shown in table 7. There
was no significant difference between GPG and GPG+P4, (p=0.45). Only the normal
responders are used in this calculation.
Table 7: Size of pre-ovulatory follicle, after second GnRH, n=15
TG
GPG
GPG+P4
n
7
8
Size of pre-ovulatory follicle (mm)
17.8 ± 2.86
16.4 ± 3.83
Values are mean ± SD. No significant difference was found.
7.6.2.1
Size of pre-ovulatory follicle in relation to milk production:
This study did not show a significant correlation between size of pre-ovulatory follicle and
milk-production. The Pearson correlation was 0.186 within the GPG-group, and 0.391 within
the GPG+P4 group. So a small positive correlation was found.
7.7
Follicular dynamics
7.7.1 Dynamics all cows included
All cows are included for calculating the mean, 11 cows per treatment group. All data are
used until day 10, PM. In figure 7 is the mean of the largest follicle per treatment group per
day shown.
22
diameter of largest follicle (mm)
20
18
16
GPG (n=11)
14
12
GPG + P4
(n=11)
10
8
6
4
2
d0
d1
d2
d3
d4
d5
d6
d7
d8
d9 d9 d10 d10
AM PM AM PM
Day of experiment
Figure 7: Follicular dynamics of all cows, with mean of largest follicle which is present each day, per
treatment group. Vertical lines in pink are pointing out the moments of respectively GnRH,
Prostaglandin and GnRH injection.
22
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
7.7.2 Dynamics without deviated responders
Figure 8 is comparable with figure 7, but now are the cows with a deviated response, so no
ovulation, as a reaction on second GnRH are excluded in this figure. This means cows 234,
422, 576, 578 of GPG, and cows 37, 85, 322 of GPG+P4 are not part of the data set used for
this graph. Also is the DF which is present after ovulation, not counted in the data set
anymore. The data set ends after their ovulation.
diameter of largest follicle (mm)
22
20
18
16
14
GPG (n=7)
12
GPG + P4 (n=8)
10
8
6
4
2
d0
d1
d2
d3
d4
d5
d6
d7
d8
d9 d9 d10 d10
AM PM AM PM
Day of experiment
Figure 8: Follicular dynamics with deviated responders excluded, and data until ovulation. Vertical
lines in pink are pointing out the moments of respectively GnRH, Prostaglandin and GnRH injection.
23
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
7.7.3 Dynamics deviated responders versus normal responders
In Figure 9 is shown how the follicular dynamics of responders versus deviated responders
are. The data set of the responders consists out of mean DF size per day, until the day
before ovulation. For the deviated responders, the whole data set until 10 AM is taken,
unless a cow ovulate, than the data set is taken until day before ovulation, because of the
fact that some cows ovulated around day 7, and other cows did not ovulate at all. Deviated
responders are again cows: 37, 85, 234, 322, 422, 576 and 578.
24
Diameter of largest follicle (mm)
22
20
18
16
Deviated responders (n=7)
14
12
Normal responders (n=15)
10
8
6
4
2
d0
d1
d2
d3
d4
d5
d6
d7
d8
d9
AM
d9
PM
d10
AM
Day of Experiment
Figure 9: Dynamics of largest follicles comparing responders with deviated responders. Vertical
lines in pink are pointing out the moments of respectively GnRH, Prostaglandin and GnRH
treatment.
24
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
VIII.
DISCUSSION
8.1
Methodology:
8.1.1 Selection of cows
The methodology of selecting the post-partum anoestrus cows was questionable since there
were some cows which did not seem to be ‘non-cyclers’. Two cows in the GPG-group
ovulated on day -1 and day 0 of the experiment. These two cows (number 300 and nr 576),
were possibly wrongly enrolled for this study. Missing the CL of the ovulated follicle can be
expected, because at day 0 it still was a corpus haemorrhagicum, which is difficult to
distinguish using ultrasound. But 6 days before the enrolment of these cows, the cows were
checked on CL-presence as well. At that moment, the cows which ovulated on day -1 or 0,
and were probably normal cycling cows, should have had a CL from the previous cycle.
(Senger, 2005) Either these CL’s were missed during ultrasound at the 28th of October, or
these two early ovulations were for these cows their first ovulation after a anoestrus-period
(which means they are cyclers rather than non-cyclers), so there was no CL which could be
diagnosed.
It is possible that misclassification of cows occurred and that some of the cows diagnosed
without a CL did in fact have a CL, and are for this reason not suitable for using in the recent
study. Previous studies have estimated the sensitivity and specificity for detection of a CL
following ultrasonography to be between 85 to 94% and 46 to 78% respectively, using
concentrations of P4 as the gold standard. (Silva et al. 2007; Stevenson et al. 2008). For this
research the sensitivity of the CL detection is important, since as less false negatives as
possible, the fewer cows would have been wrongly enrolled for this study.
8.1.2 Measurements
Ovarian follicular dynamics in synchronised cows with different treatment protocols, in
relation to follicular diameter and P4 and E2 levels in plasma, was the main purpose of this
study.
By the use of ultrasound techniques, follicular dynamics were followed, and pictures were
taken every day of each ovary. This picture was taken to include the largest cross-cut
diameter of the largest follicle on that ovary. Several times, more than one picture was
taken per ovary to try to show the largest cross-cut diameter of the largest follicle and of
the CL. This method still has limitations with respect to the measurement of other ovarian
structures than the largest follicle. Also some pictures are taken from different angles, which
makes it difficult to decide whether it is still the same DF or if another came in place.
Measurement of ovarian structures with the program Image-J is a subjective procedure.
Especially when the pictures are not very clear, this makes it difficult to see the
circumference of the structures. Nevertheless all measurements were performed by the
same person making the variation between measurements as small as possible.
The ultrasound measurements were done by two different operators. There can be a
difference in the way they manipulated the ovary during scanning.
As mentioned before, there were cows which ovulated at day -1 or day 0 of the experiment.
The only reason this was observed is because these cows were in the second group. This
was the group which was scheduled two days apart from the first group of cows. Because
the first group was measured with ultrasonography anyway, the second group was done as
25
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
well, while their treatment was not yet started. At this point the experimental design was
not consistent. It is possible that in the first group there were cows which ovulated just
before start of trial as well. The results would have been more consistent if all cows were
checked by ultrasonography in the days before the trial started.
8.1.3 Catheterisation
The persons catheterizing the cows were inexperienced in doing so. This might be a reason
why the catheters were blocked within one hour. Another factor was that, a shed where the
cows could stay fixated and had ability to eat, for duration of 15 hours, was not available.
Instead of staying fixated, cows had the ability to walk around in a small area to eat and
drink.
8.2
Response to first GnRH:
8.2.1 Ovulation rate
After first GnRH treatment, 55% of the cows responded with an ovulation. This percentage
is lower compared to other results of studies. Vasconcelos (1999) found a 64% ovulation
rate after first GnRH.
Vasconcelos (1999) showed that response to the first GnRH is influenced by the stage of the
oestrus cycle at initiation of the synchronization program. An explanation for the lower
percentage in the current study could be that more cows were in an unfavourable stage of
their cycle, meaning before Day 5 or after Day 12 of the oestrus cycle.
Cows with DFs not able to respond to LH because of their lack of LH-receptors will not
ovulate. 15% of the cows should be in this stage. Another explanation is that the cows in the
current study were post-partum anoestric, while the cows which Vasconcelos used were
already seen in standing heat, before included in the study.
The synchronization of ovulation is better in the GPG+P4 group than in the GPG group. All
responders in GPG+P4 ovulated in the 49-72 hour-interval after treatment, while GPG also
had 2 cows ovulating at day 1.
Another factor: Not all cows were really anoestric because of their ovulation at day minus 1
or day 0.
8.2.2 Size of pre-ovulatory follicle
In the recent study the size of the pre-ovulatory follicle after first GnRH significantly differed
between the two treatment groups. The GPG group had a mean pre-ovulatory follicle which
was 3 mm larger than the GPG+P4 group had.
A biological explanation for giving significantly larger pre-ovulatory follicles after first GnRH
is because of the P4-device in GPG+P4-group. This inhibits the release of FSH and LH after,
and so does not stimulate the DF for growing larger. The lack of this P4-device in GPG group,
could cause the larger pre-ovulatory follicles. (Senger, 2005)
The reason for this significant difference could also be partly subscribed to the stage of cycle
at moment of starting treatment, and serum progesterone concentration at PGF2α.
Vasconcelos et al. (1999), found a significant difference between pre-ovulatory follicle and
these parameters. Another factor can be the moment of ovulation during the day. The size
of the pre-ovulatory follicle after first GnRH is based on the follicle at the day before the
26
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
follicle is gone. That means that there is a range of 24 hours in which the ovulation can
occur, and will declare a part of the variation between the treatment groups.
8.3
DF size at day 7
The SD in GPG+P4 group is quite big. This is a result of one of the cows in this group which
ovulated at day 6. The follicle which was biggest at day 7 was as a result of the recent
ovulation, only 7 mm. This is the main reason for the big SD. Compared to another study
(Kim et al., 2005), the recent study had larger DFs at the moment of PGF2α injection. Kim et
al. found a mean DF size of 12.1 ± 0.6, in cows treated with a GPG+P4 protocol. Since this
protocol is quite comparable to the protocol used in the recent study, no different reason
for this difference is to blame, other than that it is a Korean situation, with Holstein cows,
and other persons doing the measurements.
8.4
DF size at day 9
The overall mean sizes of DFs before second GnRH injection are comparable to a study of
Stevenson 2006, where diameters ranged between 14.2-15.5 mm in GPG group, and
between 15.5-16.4 in the GPG+P4 group, in non-cycling cows. Although, in the recent study,
there is no significant difference found between the treatment groups, unlike the findings of
Stevenson. Stevenson found that cows treated with a P4-device had significantly larger
follicles. This could be an effect of the sample-size: recent study 22 cows, versus 144 cows
(in the non-cycling-group) in Stevensons study. Another factor which might affect this, is the
earlier discussed issue (see: ‘methodology: selection of cows’) that maybe not all cows in
the recent study were true anoestric. Stevenson writes that CIDR-treated cows had larger
follicles: ‘except in cycling cows having an active CL (high P4) before PGF2α injection’.
(Stevenson et al., 2006)
Compared to findings of Kim et al. (2005), DF sizes are quite similar as well. Kim found a 15.4
± 0.5 mm as mean size of DFs at day 9, in a GPG+P4 protocol.
8.5
Response to second GnRH
8.5.1 Ovulation rate
68 % of the cows reacted with an ovulation within 13-36 hours after second GnRH. This
percentage is disappointing when compared to Vasconselos (1999), who found a 87%
ovulation rate. This was, again, in only cycling cows, so the ovulation rate might be higher
because of that.
Atkins found a slightly lower ovulation-rate: 65%, but this was found in beef-heifers, so may
not be the best representative group. (Atkins et al., 2008) Looking at findings of Kim (2005),
the results of the recent study were disappointing, since Kim found a 100 % synchronization
of ovulation, 20 out of 20 cows ovulated within 40 hours after GnRH treatment in a GPG+P4protocol. This situation might be not completely comparable with NZ situation since this was
measured in Korea-dairy farms, and were not specifically in post-partum anoestrus. (Kim et
al., 2005)
That 13 of the 15 ovulating cows ovulate between 25 and 36 hours after second GnRH
treatment, is comparable with the results of the study of Peters et al, 2002, where the
synchronization of ovulation after 2nd GnRH occurs within 26-32 hours after synchronization.
(Peters et al., 2002)
27
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
The P4 device might contribute to the fact that in the GPG+P4 group, only one out of 4 cows
ovulated at day 6, 7 or 8. The progesterone dominance from the P4-device, will block the
possibility of ovulation for the DFs at that moment. For the GPG group, no Progesterone
dominance will be available when no CL was formed, so there is a higher possibility of
ovulating before the set time. 3 cows out of the GPG group ovulated before the wished
interval.
8.5.2 Size of pre-ovulatory follicle:
The mean sizes of pre-ovulatory follicles after second GnRH, are larger when compared to
other studies. The fact that in this recent study, no significant correlation between milk
production and size of pre-ovulatory follicle could be due to the fact that the power of this
study was not big enough to show this. The correlation that was found, was positive, which
is in line with Vasconcelos study (1999). He found a significant correlation which meant that
each 1 lb (0.45 kg) (http://www.convertunits.com/from/lb/to/kg ) increase in milk
production was a 0.032 mm increase in follicular size. The reason for increased follicular size
with increased milk production is not clear but is likely to be related to increases in LH pulse
frequency. (Vasconcelos et al., 1999)
Lopes also found the positive correlation: Cows producing more milk, develop larger follicles,
but with lower circulating oestradiol. There high metabolic rate influences will influence
their endocrine status. (Lopes et al., 2004)
8.6
Follicular dynamics
As to see in Figure 6 and 7, there are no big differences in follicular dynamics, when the
both treatment groups are compared. The drop in diameter from day 10 AM and PM is
partial a result of some of the cows that already ovulated at day 10, their new biggest
follicle instead of the ovulated follicle makes the average drop. But as to see in Figure 8,
there is still a little drop at day 10 AM, while the data of the ovaries which already ovulated
are not part of the data set anymore. This means that before the DF ovulates, a small drop
in diameter appears. At the start of the follicular wave, there is a trend that GPG has got
smaller DFs on the ovaries of the responding cows.This is conflicting with the finding that
pre-ovulatory follicles were significantly bigger in the GPG group. A contributing factor to
this difference is that the cows used in these dynamics are the responders on second GnRH,
while in calculating the pre-ovulatory size after first GnRH, only the responders after first
GnRH are used. (Figure 8).
Figure 9 points out the difference in follicular growth in responders versus deviated
responders. The deviated responders show less follicular growth.
28
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
IX. SYNCHRONIZATION PROGRAMS IN THE NETHERLANDS
The confinement based dairy industry in the Netherlands is difficult to compare with the
New Zealand situation. The main reason why in NZ oestrus synchronization is used is the
seasonal breeding system, and so matching the pasture growth with the increased energy
demand at onset of lactation. This reason is not relevant in the Dutch situation.
But other factors in the Dutch system have resulted in a growing number of dairy farms
interested in applying synchronization programs on their farms.
For many years, farmers use hormone treatments on individual ‘problem’ cows, to get them
oestric and pregnant again. On an average Dutch dairy farm, 15% of the cows will be treated
with some fertility-hormones to improve their chances on pregnancy. This includes the cows
with already a too long calving interval, too many failed inseminations or anoestric cows.
But to use these hormones as a preventive tool, before the calving interval becomes too big,
and/or the milk yield drops too much in a further stadium of lactation, and to use these
hormones on a structural basis, like the synchronization programs in NZ, does not have a
long history in the Netherlands yet.
But for larger farms, with at least more than 100 cows, synchronization of ovulation on (a
part of) the herd, could give benefits.
- Oestrus synchronization can be used as a management tool to be able to regulate and
spread the workload. Working via protocols can be more efficient.
- Some field examples show better pregnancy rates as a result of synchronization
- Calving interval will be smaller, and so will be the time the milk yield drops, which gives as
a result higher milk production.
- Especially for farms using automatic milking systems (robots), it is important to have a herd
of approximately the same size, whole year round. Using these tight synchronization
programs can make this possible.
On the other hand, these synchronization protocols cost money. And it is only possible to
give good results if the farmer is a good planner.
And maybe the biggest problem in The Netherlands would be: the image of the dairy
industry. Consumers do not like the idea of using hormones in animals used for food
production. Hormones are often confused with the growth hormones used in USA’s meat
industry. The fertility hormones are not dangerous, but it is difficult to convince the
consumers of dairy products of this.
For some Dutch farms, ovulation synchronization is a solution, and will find more and more
application. But for earlier mentioned reasons, most (smaller) farms will only use fertility
hormones
for
the
individual
‘problem’
cow.
(https://www.partners-inreproduction.nl/Content/Artikel%20Melkvee%20Magazine.pdf )
29
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
X. CONCLUSION
Concluding, these results have shown a trend in a better synchronization of ovulation after
treatment with GPG+P4. The only significant difference (p=0.050) found in this experiment
was the diameter of pre-ovulatory follicles after first GnRH administration in the GPG+P4
group were (on average 3 mm) smaller than in the GPG group. However, it must be
acknowledged that the number of cows in this experiment was too small to gain more
significant results. In order to obtain a more reliable view of the effects of the treatment,
more research should be carried out on this subject.
Oestrus synchronization in the Dutch dairy industry is not very common yet. But for the
larger farms, oestrus synchronization can be beneficial especially used as a management
tool; being able to spread the workload, and in reducing the calving interval to optimize milk
production.
30
Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
XI. ACKNOWLEDGEMENTS
I would like to thank:
-
-
-
-
Richard Laven for giving me the opportunity to carry out my research at the
Massey University, for supervising this project, for teaching me a lot about the
New Zealand way of farming and for taking me to all those other farms to
practise my veterinary skills
Peter Vos for giving me feedback during my stay in New Zealand, and especially
for giving comments and improvements to finalize my report.
Santosh Kumar for giving me the opportunity of working together on his PhD
project and giving me a lot of opportunities to think how the experiment would
work the best.
Conrad, for managing that the cows were ready, at all moments we needed
them for ultrasonography and/or blood sampling.
Dorien Eppink for helping me to get started with the project and continuing her
work, sharing frustrations, learning me about statistics, and staying motivated
in a room without fresh air but only computers with a lot of pictures.
Nicole, Irene and Marrit for helping out in the weekend of hourly blood
sampling; it would not have been possible without their help.
My kiwi-veterinary flatmates for teaching me that veterinary students have the
same spirit in each country and for heaps of fun!
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
XII. REFERENCES
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approaches and treatments of anoestrous dairy cows. New Zealand Veterinary Journal 58(2), (2010b
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hormone, PGF2α and P4. Journal of Dairy Science Vol. 93 No. 5, (2010a) pp. 1944 – 1959
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cows by manual palpation, transrectal ultrasonography and plasma progesterone
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(2006) pp. 1-114
 Pursley JR, Mee MO, Wiltbank MC, Synchronization of ovulation in dairy cows using
PGF2α and GnRH. Theriogenology 44, (1995) pp. 915-923
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 Stevenson JS, Tenhouse DE, Krisher RL, Lamb GC, Larson JE, Dahlen CR, Pursley JR,
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Comparison of two different synchronization programs in New Zealand dairy cattle - Drs. V.E.M. Captein
XIII: ATTACHMENTS
ATTACHMENT A: Details different responders
Tables 8 and 9 show that 4 cows are consistent deviated responders: the other cows had a
deviated response after either 1st GnRH r 2nd GnRH injection. 9 out of 22 cows (41%)
ovulated after first and after second GnRH treatment within the taken intervals.
Table 8: Comparison between individual cows per treatment group (TG): Bold and Italian cow numbers were
consistent deviated responders.
Deviated responders 1st GNRH Deviated
GnRH
85
37
216
85
234
234
300
322
310
422
422
576
432
578
responders
2nd
527
576
604
Table 9: deviated responders with explanation of response, again consistent deviated responders in bold
and Italian.
Cow number
37
85
216
234
300
310
322
422
432
527
576
578
604
Deviated response
Ovulation at day2, no observed ovulation anymore
Ovulation at day 6, no observed ovulation anymore
Ovulation at day 3, ovulation at day 10 PM
No ovulations
Ovulation at Day 0 and day 10 AM
Only ovulation at day 10 PM
Only ovulation at day 2
Only ovulation at day 7
Only ovulation at day 10 PM
Only ovulation at day 10 PM
Ovulation at day -1 and day 8
Ovulation at day 1 and 7
Only ovulation at day 10 AM
35
ATTACHMENT B: recording sheet during ultrasound
Cow No…………………………… …..Treatment Group …………………………………………………BCS………………………………….
Day/Date
Follicular Dynamics/ Ovarian Picture
CL
-2
-1
0
1
2
Left Ovary
DF
Picture
CL
Right Ovary
DF
Remarks/
Ovulation
Picture