EFFECT OF CLIMATE ON THE SUCCESS OF AN
EQUINE EMBRYO TRANSFER PROGRAM
T.V.A.F. van Delft
2008-2009
Doña Pilar Embriones
Lincoln, Provincia de Buenos Aires, Argentina
Dr. Fernando L. Riera
Faculty of Veterinary Medicine
Utrecht University
Prof. Dr. T.A.E. Stout
Index
Summary
1. Introduction
1.1 Climate effects on reproduction
1.2 Heat stress
1.3 Climate conditions and Humidex
1.4 Embryo transfer program at Doña Pilar Embriones
2. Research objectives
3. Materials and methods
4. Results
4.1 Humidex and Reproduction data per week
4.2 Humidex versus Successful Embryo Flushes
4.3 Humidex versus Embryo quality
4.4 Humidex versus Pregnancy rate at day 14
4.5 Humidex versus Pregnancy rate at day 40
5. Discussion
6. Conclusions
References
Summary
High ambient temperatures and humidity are known to have a negative effect on
reproductive success in some domestic animal species in tropical and subtropical
countries. Research in species other than the horse has shown that long periods of
high temperatures and/or high relative humidity lead to heat stress which in turn
depresses reproductive performance. For the purposes of investigation, heat stress is
frequently defined using a formula in which relative humidity and temperature are
used to calculate the so-called ‘humidex’ value. The effects of heat stress in the horse
have, however, not been extensively studied and it is not clear what humidex values
for how long constitute heat stress. In this study, the effect of heat stress on various
aspects of reproductive performance was examined by investigating correlations
between climatic variables and success rates of a commercial equine embryo transfer
program in Argentina during the breeding season of 2007-2008.
A total of 2193 embryo flushes were performed, of which 1426 (65%) yielded
an embryo. The transfer of these 1426 embryos into recipient mares resulted in 1062
pregnancies (74.5%) on day 14 which had been reduced to 938 (65.8%) by day 40
representing a day14-40 early pregnancy loss rate of 11.7%. There was a negative
correlation between the humidex value and the likelihood of a successful embryo
flush. An increase of 1 in the humidex was associated with a decrease of 0.52 % in the
success of embryo recovery. Although embryo quality decreased towards the end of
the season, climatic conditions appeared to have only a marginal influence on this
parameter and did not influence the likelihood of pregnancy on either days 14 or 40.
Indeed, if anything the pregnancy rates on days 14 and day 40 seemed to increase
with rising humidex. Although it is not possible to definitively ascribe the decrease in
the success of embryo recovery to climate conditions, this study does provide
evidence of a possible negative influence of ‘heat stress’ on embryo production but
not quality of or day 7-40 survival in the horse.
1. Introduction
Embryo transfer (ET) in horses was first described in 1972 by Oguri and Tsutsumi
(1). Initially, however, some breed registries either would not accept, or imposed
limitations on the registration of foals born by ET, such that the development of
equine ET was slower than in cattle (2). ET in horses has been used commercially
since the 1980s, and was first performed in polo ponies in Argentina in 1989 (3,4).
The Argentinean Polo Pony Breeders Association sets no limits on the number of
foals registered annually and, since its introduction, ET has become increasingly
popular in the polo industry. During the 2007-2008 ET season approximately 4000
pregnancies were produced by ET in Argentina (2). However, Argentinean
veterinarians who practice ET have anecdotally reported that the results of both
embryo flushes and transfers appear to decrease significantly during the hot summer
months of January and February. There is to date, however, no definitive proof that
climate has an effect on reproductive success in the horse. Nevertheless, in 2006 the
results at several European ET centers were surprisingly poor and this also appeared
to be connected to an unusually hot summer. Although numbers were too low to make
any significant conclusions, different aspects of the ET were suboptimal, including the
percentage of flushes that yielded embryos, embryonic development at recovery,
lower pregnancy rates after transfer and higher rates of subsequent embryonic death
(5).
Whether the climate truly influences the success of equine embryo transfer, and to
what degree, appears to warrant further examination. The purpose of this project was
to find out whether climate influences the success of embryo collection and/or
transfer. This analysis was performed using data from a large ET program at Doña
Pilar Embriones in Argentina.
1.1 Effects of climate on reproduction
High ambient temperature and humidity have long been known to negatively influence
reproductive success in domestic animals in tropical and subtropical countries (6).
Research in species other than the horse has shown that periods of high temperature
and humidity cause heat stress and that this negatively affects reproductive
performance. In female mammals, the most obvious consequence is reduced likelihood
of pregnancy. In male mammals, reduced sperm production and quality have been
reported (7). Heat stress can compromise compromise various aspects of reproductive
success including the expression of estrus behavior, the rate of follicular development,
oocyte competence, and embryonic development (7). Heat stress has also been shown
to alter the endocrine status, the luteolytic mechanism, early embryonic development
and fetal growth (8).
In dairy cattle, thermal stress during the first 7 days of embryonic development has
been reported to increase the incidence of retarded embryos and embryos graded as fair
to poor rather than good quality (9,10). The incidence of unfertilized ova was also
significantly increased in heat stressed cows (11). A Hungarian research project on
Holstein cows showed that the average number of corpora lutea diminished during an
El Niño summer (with an ambient temperature increase of 6ºC), compared to a normal
summer. Additionally, the average number of embryos recovered and the quality of
those embryos appeared to be reduced (12). Research on rats has shown that heat stress
diminishes the expression of gonadotropin receptors on the granulosa cells of growing
follicles. This results in a decrease in estrogenic activity by these follicles and an
increased sensitivity of the granulosa cells for apoptosis (13). A similar defect may
explain the decrease in the number of ovulations and corpora lutea during periods of
excessive heat.
Research on sows has demonstrated that in addition to a negative effect of heat stress
on the vitality of embryos during the first days after conception, early embryonic
development is also affected. The number of sows pregnant at day thirty was reduced
by heat stress (14) while both conception rates and litter size are reduced when gilts are
exposed to elevated ambient temperature during days 0 to 16 after mating (15).
In cattle, it has been reported that heat stress can influence the endometrium’s ability to
produce and secrete prostaglandin, which can lead to premature luteolysis and embryo
loss. Most embryo loss due to early embryonic death occurs before day 42 in heat
stressed cattle (16).
1.2 Heat stress
Heat stress is defined as the acute condition which occurs when the body produces or
absorbs more heat than it can dissipate. It is usually caused by prolonged exposure to
high temperatures. Each individual possesses thermo-regulating mechanisms that are
used to adapt to changes in environmental temperature. In the case of heat stress,
these heat-regulating mechanisms become overwhelmed and are unable to effectively
deal with the heat, such that the body temperature continues to climb and resulting in
hyperthermia (17). Factors that contribute to heat stress include physical activity,
body condition of the individual, and the surrounding temperature and relative
humidity. Different species, breeds and individuals show differences in the ability to
cope with environmental changes in temperature and humidity (18).
1.3 Climate conditions and Humidex
Climate encompasses the temperature, humidity, atmospheric pressure, wind, rainfall,
atmospheric particle count and numerous other meteorological elements in a given
region over long periods of time. The standard averaging period is 30 years (19). The
climate in Argentina, as in Europe, consists of 4 seasons. Since Argentina is located in
the southern hemisphere, seasons are reversed. Spring starts on the 21st of September,
summer on the 21st of December and Autumn on the 21st of March. The hottest period
in the province of Buenos Aires is January when temperatures often rise above 30°C,
with an average relative humidity that stays above 60% (Fig 1).
Figure 1: Climate charts (Temperature and Humidity) for Argentina during a typical year.
Climatic factors that may influence the degree of heat stress include environmental
temperature and relative humidity. Therefore, heat stress is frequently defined using a
temperature-humidity formula, the humidex value. The humidex reflects the
combined effect of heat and humidity.
Humidex = T + 5/9 * (e-10)
where: e = vapour pressure (6.112*10^(7.5*T/(237.7+T))*H/100)
T= air temperature (degrees Celsius)
H= humidity (%)
Fig 2: Humidex formula using temperature in °C and relative humidity in %
The current formula for determining the humidex was developed by Masterton and
Richardson of Canada's Atmospheric Environment Service in 1979 (20). Various
reports have suggested that the humidex is most accurate for temperatures higher than
20°C.
1.4 Embryo transfer program at Doña Pilar Embriones
At Doña Pilar Embriones reproduction center, over 1500 embryo transfers are
performed per year. The majority of these ETs are performed on polo pony mares
(Thoroughbred or Thoroughbred-crosses). To achieve this large number of ETs, it is
common to examine 300 mares by rectal palpation and ultrasonography on an average
day during the peak of the breeding season. In such a day approximately 30-50 donor
mares are also artificially inseminated with semen collected from 10-20 stallions, and
approximately 12-20 embryo flushes are performed. Stallions, donor mares and
recipient mares are all kept on the same property (2).
Doña Pilar Embriones is located in Lincoln, a village in the province of Buenos Aires.
The center extends over 600 hectares, where all the horses are housed. The facility
can basically be divided into 3 units. Donor mares are located at two units, is the
stallions are also housed at the main donor mare unit. All the reproduction work,
including inseminations and uterine flushes, are performed at the two donor mare
facilities. The third location is used to monitor recipient mares. Most ETs take place at
this location, because most non-pregnant recipients are housed on pasture close to this
facility. Embryos are transported from the donor facilities to the recipient facility and
are transferred non-surgically within 1- 5 hours after collection. Any other ETs take
place at the main facility. In this case, the recipients required are transported from the
recipient facility to the main facility where they stay on good pasture until after ET
has taken place. Transportation of mares is by horse truck. Since stress can influence
pregnancy results, stress is minimized. Therefore, mares are kept at the same location
and in the same herd as long as possible, and only transported when really necessary.
Selection and management of the stallions
The stallions are all privately owned. Some have had a successful career on the
racetrack or polo field, while others are offspring from a promising bloodline with
excellent quality traits. Stallions that arrive at the facility have been checked for EIA.
They get vaccinated against strangles and are checked for EVA. Stallions are housed
at a separate outdoor area where each stallion has its own paddock. They are fed
alfalfa hay and concentrates. Water is provided ad lib. The stallions are dewormed
every 2 to 3 months.
Selection and management of donor mares
ET is an expensive way to produce offspring and, therefore, predominantly used for
mares of superior quality. Donor mares range from 5 to 18 years of age and weigh
between 400 and 500 kg. A lot of these mares still compete during the Argentinean
polo season in the spring. When the polo season has finished, they move to an ET
farm. During the peak of the season in 2007-2008 Doña Pilar housed approximately
390 donor mares. Mares arriving at the center had been tested for EIA and underwent
a general physical examination, were dewormed, vaccinated (tetanus, influenza and an
autologous vaccine against strangles), and their health and fertility records are
reviewed. Next the mares undergo a breeding soundness examination that includes
evaluation of the external genitalia, rectal palpation and ultrasonography. The cervix
is evaluated by direct digital palpation and if necessary an endometrial swab or
endometrial biopsy is performed. If a fertility problem is apparent, this is treated
before the mare starts on the ET program (2).
Donor mares are housed on pasture in herds of 10- 25. Herds stay together during the
entire season and mares are not mixed between herds; this decreases stress and
injuries due to social behavior. While they are pasture-managed, the mares are also
fed alfalfa rolls and receive concentrates once or twice a day. Water is available ad
lib. The mares get dewormed every 8 to 12 weeks. Mares that have lost too much
weight at the end of the summer are put together on a large pasture where they receive
extra alfalfa and soya leaves to gain weight.
Selection and management of recipient mares
Good selection, quality, and management of recipient mares is the most important
factors in a successful ET program. It is preferred to use recipient mares that have had
a foal earlier in their life because this shows that the mare is able to rear a foal.
1.
2.
3.
4.
5.
6.
7.
good health and body condition
easy to handle and halter broken
body size similar to that of the donor mare
4 to 10 years of age
sound breeding condition
good estrus display
regular cyclicity
Fig 3: Requirements which good recipient mares should meet; Riera (2)
Dona Pilar owns approximately 3000 recipient mares. This includes the recipients that
have given birth to an ET foal in the spring and thus can not be used during the
following season. Recipients are all cross-bred mares and weigh between 400 and 600
kg. They are vaccinated and dewormed exactly as described for the donor mares.
Recipients are identified using freeze brands and records with respect to age, health
status and reproductive history. The breeding soundness examination is the same as in
donor mares (2).
Recipient mares are kept in large herds of about 50 to 100 mares. They are kept on
pasture and are fed extra hay and alfalfa rolls. At the end of the summer, they are also
given corn and soya leaves to prevent loss of body condition. Water is provided ad lib.
Recipients that have undergone ET and pregnant recipients are put on the best
pastures. All the mares are checked daily in the field to see if they are healthy. Sick or
injured mares are isolated in a paddock and treated.
Synchronization of mares
To transfer an embryo from a donor mare to a recipient it is essential that the estrus
cycles of the mares are synchronous. Because many recipient mares are available at
this facility, a donor can often be matched with a recipient that has ovulated
spontaneously at a similar time. Recipient mares that have ovulated 1 day before to 4
days after the donor are considered acceptable recipients. This means that at the time
of ET (donor day 8) the recipients are between day 4 and day 9 post-ovulation. It is
important that P4 levels in the recipient are high enough for the uterus and cervix to
be well prepared at the time of ET. Research has shown that the chances of pregnancy
are lower if recipients that ovulated 1 day prior to the donor mare’s ovulation are
used. However, no significant differences were found for recipients ovulating 0, +24
and +48 hours (58%, 62% and 62% respectively) after the donor (3). The method used
to synchronize donors and recipients depends largely on the number of recipients
available. If there is a large number of recipients, administration of a PGF2α analogue
to 1.5 recipients per donor, 1 to 2 days after the administration of PGF2α to the donor
mare (mostly given to the donor immediately after the previous embryo flush). If the
number of recipients is small, hCG can be used to ‘fine-tune’ the synchronization
protocol. 1500 IU of hCG is commonly administered intravenously to 1-1.5 recipients
with a follicle >35 mm in diameter at the time a donor mare ovulates. Ovulation of the
recipient is expected 36 to 48 hours after hCG administration.
Semen collection, treatment and artificial insemination
Donor mares are artificially inseminated with fresh extended semen. Two different
type of artificial vaginas are used, the Missouri and the Brasileira (Bioteck Botucatu),
where the latter resembles a Hannover artificial vagina.
After semen collection, the filled whirl pack goes to a semen laboratory where it is
filtered. Next the amount of semen is estimated and the color checked. Extender
(35°C) is added to the ejaculate to at least a 1:1 dilution (depending on the number of
mares to be inseminated). The concentration, the percentage of motile sperms and the
percentage of progressively motile sperm are estimated using a microscope. At
regular intervals, the concentration of the semen is determined. Once the semen is
prepared for use it is stored in a container at 20°C. Insemination takes place within 2
hours after semen collection.
Mares that are inseminated and ovulate 24 to 48 hours post-insemination are not
rebred if the semen was of good quality (2). Mares that didn’t ovulate within 48h are
inseminated a second time. Occasionally mares are inseminated after ovulation has
been detected, in this case the mares still have to show good signs of heat and the
cervix still needs to be open. If mares show signs of post-insemination endometritis
they receive uterine lavages in combination with Oxytocin injections for up to 3 days
post-ovulation.
Embryo recovery (flushing)
Donor mares are flushed between days 6.5 and 9 post-ovulation. Day 6.5 is used when
embryos need to be vitrified or when young mares are used. Uterine flushes on day 9
are applied to older mares because embryo development is often slower. The most
common time of flushing is day 8. In case of a double ovulation half of the time
between the two ovulations plus 8 days is used to determine the moment of flushing.
During the procedure, the perineal area is cleaned, and mares are sedated with 50 to
100 mg of Xylazine. The use of acepromazine is considered to be contraindicated
because it relaxes the uterus and makes flushing more difficult. If the strains during
per rectum massage of the uterus, 100 to 120 mg of Buscopan is administered IV to
relax the rectum. The most important aspect of flushing is to maintain a constant flow
of fluid leaving the uterus. This flow will reduce the risk that the embryo sticks
between folds of the uterus. After flushing, the filter where the embryo can be found,
is transferred to a laboratory where searching and washing are performed. After
embryo flushing, mares are treated with a luteolytic dose of a PGF2α analogue to
induce oestrus. If a follicle >35mm is already present on one of the ovaries, the
PGF2α analogue treatment is delayed.
Embryo handling
Once the filter is in the laboratory, the contents are reduced to about 20 ml. This fluid
is swirled carefully and poured into a Petri dish and the filter is rinsed to make sure
the embryo has not been left behind. The Petri dish is swirled so that the embryo
moves to the centre, which makes it easier to find. The Petri dish is put under a
stereomicroscope to search for the embryo. Most embryos can be seen with the naked
eye after being found with the microscope. The stereomicroscope is however required
to evaluate size and quality.
Once the embryo has been found it is washed using VIGRO holding solution. VIGRO
is put in a small petri dish and 3 or 4 drops of VIGRO are put in the lid of the Petri
dish for washing purposes. The embryo is washed by sequential transfer between the
drops using a sterile straw or a tomcat catheter connected to a 2ml syringe. Small
morulas and early blastocysts are washed using a 0.25 ml straw. Larger embryos are
washed using straws with a diameter of 0.5 ml. After washing, the embryo is
transferred to the small petri dish containing Vigro where its quality and size can be
graded. Next it is ready to be loaded into a tranfer pipette.
Embryo development, quality and size
The stage of embryo development at recovery depends primarily on the time of
flushing (days after ovulation) but is also affected by factors such as mare age. Most
embryos recovered are blastocysts and expanded blastocysts but occasionally a
morula or an early blastocyst is recovered. Embryos are generally classified for
quality on a scale of 1 to 4 depending on morphologic characteristics (21). At Doña
Pilar, they use their own grading system. Embryos are graded from 1 to 3, grade 1
representing good-excellent quality, grade 2 for lower quality and grade 3 for poor
quality.
A normal embryo (upto the expanded blastocyst phase) is round and consists of a ball
of cells surrounded by the zona pellucida. During blastocyst formation, the so-called
‘blastocyst capsule’ is formed between the tropgectoderm and zona pellucida (25).
Next the embryo starts expanding and hatches out of the zona pellucida. This phase is
called the expanded blastocyst phase. Embryo size is determined subjectively to be
sure that the materials used to transfer the embryo are suitable for the embryo’s size.
Morula
Blastocyst before hatching
Expanded blastocyst after hatching
Fig 4: Equine embryos at different stages of embryonic development
Loading the embryo
Depending on the size, morulas and blastocysts can be transferred with a 0.25 ml or a
0.5 ml straw and transfer pipette. Larger embryos need to be transferred using a
reversed 0.5 cm transfer pipette. The embryo is aspirated into a straw between 2
columns of air and two columns of medium. The loaded straw is put into a transfer
pipette and pushed toward the tip. The whole process of finding the embryo and
loading the pipette takes about 15 minutes. Embryos are held in the loaded transfer
pipettes at room temperature for a maximum of 3 hours before being transferred into
recipient mares.
Embryo transfer
Before ET, the recipients are palpated per rectum to determine whether the cervix and
uterus are in an appropriate condition to receive the embryo and maintain a
pregnancy. The cervix needs to be well closed and the uterus must have good tone. If
the recipient’s reproductive tract is suitable, the perineal area is washed and dried.
Next the charged ET pipette is manipulated via the vagina through the cervix and into
the right uterine horn where the embryo is deposited. After ET, the recipients are
treated with 1.5 g of long acting progesterone (P4) once. The quality of the ET and the
quality of the recipient’s cervix are classified from 1 to 3 according to the ease of
transfer and tone of the cervix.
2. Research Objectives
There is scientific evidence for an influence of high ambient temperature and
humidity on reproductive success in domestic animals other than horses. The aim of
this study was to determine whether heat stress influences the success of equine
embryo transfer. The parameters which we examined were:
-
Percentage of flushes that yielded an embryo
-
Quality of recovered embryos
-
Pregnancy rate (in %) after transfer to recipient mares at day 14 post-donor
ovulation
-
Pregnancy rate (in %) at day 40 post-donor ovulation
Hypothesis: an increase of temperature and humidity during the summer months will
result in a decrease in the percentage of successful embryo transfers compared to
spring and fall.
3. Materials and methods
To examine the relationship between climate and the results of the ET program,
meterological data for the center and results from the ET program were collected.
Starting on September 1st 2007, the daily temperature and relative humidity were
checked and registered using the website http://www.smn.gov.ar. This website,
Servicio Meteorológico Nacional, is connected to the Department of Defence and is
considered the best national service for meteorological information. The data used
was from from Junín, a village 30 kilometers northeast to the reproduction center. The
data on maximum temperature and relative humidity were collected retrospectively at
the end of the season and used to calculate the daily humidex. The humidex was then
averaged over weekly periods and these values were used to examine the influence of
climate on reproductive success.
Doña Pilar Embriones is a commercial stud and reproduction center, and it is of
critical importance that all the work is done in a precise and timely fashion. The
various steps of the ET program are performed by a team of veterinarians and
technicians, with each responsible for specific aspects of the program. This decreases
the effect of the human factor on results, although everybody working on the ET
program must be considered a variable capable of influencing the results.
The reproduction data used for the study was that from Doña Pilar Embriones during
the season 2007-2008. However, because many mares were still in the transitional
phase at the beginning of the season and only few ETs were accomplished at this
time, the data used for analysis was that collected from Monday October 1st 2007.
Similarly, since the number of embryo recoveries decreased dramatically at the end of
the breeding season when many mares enter anestrus the last date included in analysis
was April 13th 2008.
Reproduction data was registered on a purpose designed data sheet (fig 5). Using this
sheet, the following indices were calculated:
-
Successful Embryo Flushes (SEF) per week; A flush was considered successful
when 1 or more embryos were recovered. SEF was calculated as a percentage of
the total number of flushes during that week (w).
-
Quality of gained embryos recovered; The percentages of recovered embryos
scored as grade 1, grade 2 or grade 3 was calculated for each week.
-
Pregnancy rate (%) at day 14 post donor ovulation; The percentage of
recipients to which an embryo was transferred and the were found to be
pregnant on day 14 was calculated. Only ETs to recipients that ovulated (as
opposed acyclic mares treated with progesterone) were included.
-
Pregnancy rate (%) at day 40 post donor ovulation; The percentage of mares
that had received an embryo and were still pregnant at day 40 was also
calculated per week. Again only ETs to recipients that had ovulated were used.
# Flushing Date
Flushing =
date ET
Date
Date last
Ovulation AI
Embryo
quality
recipient
mare
natural
ovulation
or P4
Pregnant
day 1420
1
2
Fig 5: Data sheet used to register needed reproduction information
Pregnant day
40-60
At the beginning of the season, and particularly at the end of the season, a proportion
of naturally ovulated recipient mares are continued on progesterone supplementation
after ET. At the end of the season, a number of recipients have stopped cycling but
can be used for ET if they receive P4 supplementation starting 2 days after the donor
mare has ovulated. After ET P4 supplementation is continued since there will be no
primary CL present. It was decided to exclude P4 treated recipient mares from this
research because the altered hormonal situation might affect pregnancy and/or
pregnancy loss rates.
The average time of embryo recovery was 8 days post-ovulation. If climate influenced
this step in the ET program this should occur during the period just prior to embryo
recovery. Thus, Successful Embryo Flush (SEF) and embryo quality during the week
(w) would be influenced by the climate during that week (w) but also by the climate
during preceding weeks (w-1) and (w-2).
At Dona Pilar, pregnancy examination is not restricted to a precise day after ovulation
of the donor mare or after ET. The first pregnancy check takes place between days 14
and 20 post ovulation and the second check between days 40 and 60. A recipient
diagnosed as being pregnant on day 18 must also have been pregnant on day 14. Thus
it was valid to consider days 14 and 40 as the times at which mares were diagnosed
pregnant or not.
The period during which the climate could have influenced the pregnancy percentage
on day 14 could include the time between ET and pregnancy diagnosis, but could also
be influenced by the 2-3 weeks prior to embryo recovery (i.e. via effects on embryo
development and quality). Pregnancy rate at day 14 for the ETs from week (w) were
considered to be influenced by the climate during weeks (w), (w +1) and (w +2).
The period during which the climate can have influenced the pregnancy percentage on
day 40 must have been the time after the ET until the moment of the second pregnancy
diagnosis. Pregnancy percentage at day 40 of the ETs from week (w) were considered to
be influenced by the climate during weeks (w), (w +1), (w +2), (w +3), (w +4), and
(w +5).
4. RESULTS
4.1 Humidex and Reproduction data per week
The average humidex was calculated per calendar week. As figure 6 shows, the
average humidex rose until the first week of January, and decreased gradually
thereafter.
Average hum idex per calender week
50,0
45,0
40,0
humidex
35,0
30,0
25,0
20,0
15,0
10,0
5,0
w
w
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ee
k
3
ee 6
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w 51
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5
w 2
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w 1
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w 15
ee
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ee
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17
0,0
Calender weeks (w)
Figure 6: Average humidex per calendar week
Figure 7 shows the results of embryo flushing per calendar week. The best results
were recorded during calendar week 1, this week was also the week with the highest
average humidex. During the period from week 40 to week 15, a total of 2193 embryo
flushes was performed of which 1426 were successful. This equates to 65% over the
entire period. A visible reduction in the % SEF was seen in week 51, during weeks 2
to 4 and during weeks 7 to 9.
% Successful Embryo Flushes (SEF) per calender week
80,0
70,0
60,0
50,0
SEF (%) 40,0
30,0
20,0
10,0
0,0
40
42
44
46
48
50
52
2
4
6
8
10
12
Calender weeks (w)
Figure 7: Successful Embryo Flushes per calendar week
14
Embryo quality was registered in two different ways. First, the percentages of grade 1,
grade 2 and grade 3 embryos were calculated per week (w) as shown in figure 8a.
However, because most embryos were grade 1, grade 2 and grade 3 embryos were
subsequently grouped together, as shown in figure 8b.
Embryo Quality (% ) per week
100%
80%
60%
Q3
Q2
40%
Q1
20%
0%
40 42 44 46 48 50 52
2
4
6
8
10 12 14
Cale nde r we e ks (w)
Fig 8a: Embryo quality (3 grades) per week
Embryo quality appeared to be depressed during week 50 and during weeks 2 to 12.
Embryo Quality (% ) per week
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Q2 & Q3
Q1
40 42 44 46 48 50 52
2
4
6
8 10 12 14
Cale nde r we e ks (w)
Fig 8b: Embryo quality (2 grades) per week
Between weeks 40 and 15, 1426 embryos were transferred to recipient mares. At day
14, the cumulative pregnancy percentage was 74.5% and at day 40 it was 65.8%.
The following two figures (figs 9 and 10) show the pregnancy rates at day 14 and 40
for each calendar week (w). A marked decline in success for both indices was noted at
the end of the season.
Pregnancy rate at day 14
90,0
80,0
70,0
60,0
50,0
Pre gnant (%)
40,0
30,0
20,0
10,0
0,0
40 42 44 46 48 50 52
2
4
6
8
10 12 14
Cale nde r we e k (w)
Fig 9: Pregnancy percentage at day 14 of the recovered and transferred embryos during week
(w)
Pregnancy rate at day 40
90,0
80,0
70,0
60,0
Pregnant (%)
50,0
40,0
30,0
20,0
10,0
0,0
40 42
44 46
48 50
52
2
4
6
8
10 12
14
Calender week (w)
Fig 10: Pregnancy percentage at day 40 of the recovered and transferred embryos during week
(w)
4.2 Humidex versus Embryo flushes
The following figures (fig 11a-c) show how climate conditions during week (w), week
(w-1) and week (w-2) influenced the embryo recovery rate during week (w).
Humidex vs Successful Embryo Flushes
SEF (w)
90,0
80,0
70,0
60,0
50,0
40,0
y = -0,1976x + 73,464
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Hum (w)
a
Humidex (w-1) vs Successful Embryo Flushes
SEF (w)
90,0
80,0
70,0
60,0
y = -0,4291x + 81,717
50,0
40,0
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Hum (w-1)
b
Humidex (w-2) vs Successful Embryo Flushes
SEF (w)
90,0
80,0
70,0
60,0
y = -0,5245x + 84,851
50,0
40,0
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Hum (w-2)
c
Fig 11a-c: The influence of Climate on the success of embryo recovery
The scatter plots in the figures above indicate a negative correlation between climatic
conditions (Humidex) and the success of embryo recovery (SEF). The above figures
also show that SEF in a given week was primarily influenced by climatic conditions 1
- 2 weeks prior to the uterine flush. The formula for the regression line in figure 11c
indicates that an increase of 1 in the Humidex resulted in a 0.52 % decreased in the
likelihood of embryo recovery.
4.3 Humidex versus Embryo quality
Figures 12a to 12c show how climate during weeks (w), (w-1) and (w-2) influenced
embryo quality rate during week (w).
Q1
Hum (w) vs Embryo Quality 1 (w)
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
y = -0,0416x + 94,696
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex(w)
a
Q1
Hum (w-1) vs Embryo Quality 1 (w)
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
y = 0,0449x + 91,6
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex (w-1)
b
Q1
Hum (w-2) vs Embryo Quality 1 (w)
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
y = -0,0822x + 96,352
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex (w-2)
c
Fig 12a-c: The influence of Climate conditions on Embryo Quality
The figures indicate no significant influence of climate during the 2 weeks prior to the
uterine flush or during the week of the uterine flush on embryo quality.
4.4 Humidex versus Pregnancy rate on day 14
The following figures (figs 13a-c) show how climatic conditions during weeks (w), (w
+1) and (w +2) influenced the pregnancy rate at day 14 for embryos recovered and
transferred during week (w).
Pregnancy day14 (%)
Hum(w) vs P14(w)
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
y = -0,1285x + 78,725
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex (w)
a
Pregnancy day 14 (%)
Hum(w+1) vs P14(w)
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
y = -0,0445x + 75,717
0,0
10,0
20,0
30,0
40,0
50,0
Humide x (w+1)
b
Pregnancy day 14 (%)
Hum(w+2) vs P14(w)
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
y = 0,1656x + 68,083
0,0
10,0
20,0
30,0
40,0
50,0
Humide x (w+2)
c
Fig 13a-c: The influence of climate conditions on pregnancy rates on day 14
There was no evidence of an effect of climatic conditions during and soon after ET on
the pregnancy results on day 14.
4.5 Humidex versus Pregnancy rate on day 40
The following figures (fig 14a-f) show how pregnancy rate on day 40 is influenced by
the climatic conditions during the weeks after embryo recovery and ET during week
(w).
Pregnancy day40 (%)
Hum(w) vs P40(w)
90,0
80,0
70,0
60,0
y = 0,0867x + 59,586
50,0
40,0
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex (w)
a
Hum(w+1) vs P40(w)
Pregnancy d40 (%)
90,0
80,0
70,0
60,0
y = 0,4186x + 47,639
50,0
40,0
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex (w+1)
b
Hum (w+2) vs P40(w)
pregnancy d40 (%)
90,0
80,0
70,0
60,0
50,0
40,0
y = 0,8947x + 30,101
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex (w+2)
c
Hum(w+3) vs P40(w)
Pregnancy d40 (%)
90,0
80,0
70,0
60,0
50,0
40,0
y = 0,8659x + 31,856
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex (w+3)
d
Hum(w+4) vs P40(w)
Pregnancy d40 (%)
90,0
80,0
70,0
60,0
50,0
40,0
y = 0,9424x + 29,983
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex(w+4)
e
Hum(w+5) vs P40(w)
Pregnancy d40 (%)
90,0
80,0
70,0
60,0
50,0
40,0
y = 0,7392x + 38,345
30,0
20,0
10,0
0,0
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
Humidex(w+5)
f
Fig 14a-f: The influence of climate conditions on pregnancy rates on day 40
The correlation between climatic conditions and the day 40 pregnancy results were
relatively low.
5. Discussion
During the summer of 2007-2007, the climatic were not as extreme as in previous
years. Temperature and humidity levels did not reach the very high levels expected.
This may in part explain why the effects of climate were not marked.
The average percentage of successful embryo flushes during this season was 65 %
which is consistent with reports of normal embryo recovery rates ranging between
65% and 75% (2). Since more than one embryo can be recovered after a multiple
ovulation, the total number of embryos recovered is generally somewhat higher. For
many years, the veterinarians at Doña Pilar have seen an increase in embryo recovery
rates from September reaching a peak in November. In Argentina, most mares begin
to cycle after seasonal (winter) anestrus in September or even October. The results of
embryo recovery during the first cycle of the season have shown to be lower than in
subsequent cycles, suggesting that the first ovulation of the year may be less fertile
(26).
The findings of this study suggest that heat stress could play a role in the success of
embryo recovery. Better embryo recovery rates were recorded during the first part of
the season, from week 40 to 1 than between weeks 2 and 15. Moreover, the embryo
recovery rate declined as the humidex inceased. In this respect, it was not only the
humidex during the week of the flush, but also the climate during the two weeks prior
to a flush that influenced results. The results for embryo quality showed a decrease
from week 2 until the end of the season. However, the humidex had only a negligible
negative influence on embryo quality. Nevertheless, any negative effects heat stress
appeared to impact in the before embryo recovery, i.e. during the period of final
follicular development, ovulation fertilization and initial development in the oviduct
and very early period in the uterus. Similar effects have been described in other
species (9, 11, 12).
The reduced embryo quality during week 50 and reduced embryo recovery during
week 51 could therefore have resulted form the sharp increase in humidex during
weeks 47 to 49. Similarly, reduced emrbyo quality and recovery in January could
have, in part, been related to increased in humidex during weeks 52 and 1.
By contrast, the reductions in embryo recovery during weeks 7 to 9 were thought to
be more related to managemental and technical problems since they were
accompanied post-breeding endometritis were seen next to the lower results in
embryo recovery. In this respect, it is clear that climate is not the only factor that can
affect the success of embryo recovery and/or embryo quality. Although these results
suggest that heat stress does influence embryo recovery and quality, other forms of
stress may also have a role. Other forms of stress that have been suggested to
negatively influence reproductive success include management, nutrition and social
stresses. The influence of these stressors may also vary over the course of the year
with social stresses being maximal at the start of the season as animals arrive and are
made into groups. The number of donor mares at the center increases markedly in
December and January because competing mares arrive at the end of polo season.
Nutritional stress is also likely to peak in the summer when pasture quality reaches a
nadir.
The fact that success of embryo recovery and the quality of the embryos drops during
the second part of the season can in part explained by the quality of the donor mares.
Donor mares that were successful in producing the desired number of pregnancies
have left the program whereas the less fertile mares remain.
The average pregnancy rate at day 14 post-donor ovulation was 74.5 %. At day 40
post-donor ovulation the average pregnancy percentage was 65.8 %. These results are
similar to those described in the literature (3,27). The pregnancy rates at day 14 postdonor ovulation do not vary much until the end of the summer, when they decrease
noticeable. A similar pattern emerges for day 40 pregnancy rates which begin to
decline after approximately week 7. However, there was o obvious effect of post-ET
humidex values on pregnancy rates at either day 14 or 40. Indeed, a higher humidex
index appeared to be associated with better pregnancy results.
One of the reasons for the decline in pregnancy rates at the end of the season is almost
certainly depletion of the recipient pool and, in particular, loss of the best recipients
which are generally the first to be used and made pregnant. In addition, introduction
of new recipients during the summer may lead to social stress and affect subsequent
results. The newly arrived recipients are either mares used in the previous year from
which the foal has been weaned or newly purchased animals to enlarge the total herd.
As the summer progresses, pasture quality decreases and the recipient mares lose
body condition, which may affect their ability to maintain a pregnancy. All of the
factors may negatively influence the success of the ET program.
In this study, climate conditions post-ET did not affect the pregnancy rates on day 14
or 40 post-donor ovulation. One reason that pregnancy rates did not decrease much
during the summer months could be because the recipients were put on better pasture
after the transfer. This may decrease any nutritional stress.
6. Conclusions
This retrospective study has shown that embryo recovery is negatively influenced by
increased temperature and humidity, but that effects on embryo quality are
insignificant. It appears that pregnancy rates after ET are not influenced by post ET
climatic conditions. It is therefore concluded that any influence of heat stress on
reproductive success is concentrated during the early stages of the reproductive
process, which include follicular development, oocyte development and early
embryonic development. This can result in failure to recover an embryo, or possibly
an embryo of lower quality and reduced developmental competence. Although the
results suggest that pregnancy rates are not negatively affected by humidex, it is
possible that humidex levels reached in the summer in which the study was performed
were insufficient to induce true heat stress.
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