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Factors influencing oocyte recovery and in-vitro production of equine embryos in a commercial OPU/ICSI program

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Effect of maternal age on estrogen production in mid to
late pregnancy in the mare
M.A. Alonso
, F.J. Affonso
, M.G. Meirelles
, R.C. Simas
, D.F.
, V.V. Hernandes
, M. Nichi
, M.N. Eberlin
, C.B.
Department of Animal Reproduction, College of Veterinary
Medicine and Animal Science, University of Sao Paulo, SP, Brazil;
Thomson Mass Spectrometry Laboratory, University of Campinas,
SP, Brazil
*Corresponding author:
The fetoplacental unit is responsible for estrogen production
from day 50 onward, which reaches a peak between days 150
and 270, and then declines gradually to baseline at the time of
parturition. Estrogen levels can be utilized as an index of fetal
well-being and placental function. Factors that can affect steroid
production are parity, breed, photoperiod, pregnancy length and
the individual [Palme R, et al. Reprod Domest Anim
2001;36:273e7]. Few studies focusing on the endocrinology,
specically estrogen metabolism, of equine pregnancy during
mid to late gestation are found in the literature. The aim of the
present study was to evaluate the effect of maternal age on
estrogen production (estradiol 17
eES, equilin eEQ and
equilenin - EL) in pregnant mares. Thirty-six pregnant mares
were divided according to age [Group1: 4-8 years (n¼14);
Group2: 9-12 years (n¼13); Group3: 13 years (n¼9)]. Plasma
was collected weekly starting at twenty-six weeks prepartum
until parturition. Standards ES-E8875, EQ-E8126 and EL-33693
) were used for estrogen measurements by liq-
uid chromatography coupled with tandem mass spectrometry
Data were analyzed by ANOVA (GLM Procedure of SAS) followed
by the Tukey's test and t-test was used to evaluate the effect of
week. Transformations were required due to non-normal dis-
tribution. Medians of the non-transformed data were therefore
used. Signicance was considered when p<0.05. Data are pre-
sented as pg/ml
. ES concentrations were higher in Group2 (6.47)
compared to Group3 (4.64), but neither group differed sig-
nicantly from Group1 (5.53). EQ differed statistically between all
three groups; Group2 (439.73) had the highest concentration,
whereas Group3 had the lowest (107.25). EL behaved differently;
Group2 (2606.18) had the highest concentrations, but the other
two groups did not differ statistically (1528.31; 977.76, Group1
and 3 respectively). There was a signicant effect of week on EQ
and EL, but not on ES concentrations. The highest value for EQ and
EL was seen 14 weeks prior to parturition. EQ started to rise
statistically between 23 and 19 weeks and to decrease between 6
and 2 weeks. EL increased between weeks 26 and 24 prepartum,
then decreased between 6 and 1 weeks. The Group1 exhibited
lower estrogen concentrations than Group2 for all estrogens
measured, possibly reecting uterine immaturity in younger
mares [Wilsher S, Allen, WR. Equine Vet J 20 03;35:476-83].
Group2 mares likely have a greater area of fetomaternal contact,
resulting in larger foals with larger gonads which can produce
greater amounts of DHEA to be aromatized by the placenta [Palme
R, et al. Reprod Domest Anim 2001;36:273e7]. These ndings are
in agreement with Dr Allen [Allen, WR, et al. Endocrinol
2002;172:237-46], who suggested that the size/capacity of the
chorioallantois and fetal gonads correlates with the level of
estrogen synthesis. These results show a relationship between the
uterus, placenta and fetus in estrogen synthesis and secretion,
and help dene the normal range of estrogens during normal
pregnancy and parturition.
Key Words: estradiol 17
, equilin, equilenin, mare
Financial Support FAPESP 2012.08929-6. Equine Reproduction
Center TOK eBiritiba Mirim, Brazil.
Factors inuencing oocyte recovery and in-vitro
production of equine embryos in a commercial OPU/
ICSI program
A. Claes
, C. Galli
, S. Colleoni
, D. Necchi
, G. Lazzari
C. Deelen
, M. Beitsma
, T. Stout
Department of Equine Sciences, Faculty of Veterinary Medicine,
Utrecht University, Utrecht, the Netherlands;
Avantea, Laboratory
of Reproductive Technologies, Cremona, Italy;
Fondazione Avantea;
Dept. of Veterinary Medical Sciences, University of Bologna,
Bologna, Italy
*Corresponding author:
In-vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and
Intracytoplasmic Sperm Injection (ICSI) is becoming increasingly
popular in the equine industry because it offers several advan-
tages over conventional embryo transfer. In particular, multiple
offspring can be produced from sub- and infertile mares and
stallions, or from a single straw of frozen semen. Furthermore,
oocyte recovery can be performed as an out-patient procedure,
year round without hormonal manipulation, which makes it
appealing for the owners/riders of competition mares. Never-
theless, these advanced reproductive techniques are complex and
therefore relatively expensive, while little is known about factors
that inuence the success of equine IVEP. The objectives of this
study were to retrospectively identify factors that inuence
oocyte recovery and the success of IVEP. A total of 159 Warmblood
mares were presented once (n¼105 mares) or more times (n¼54
mares, range: 1-10 times) for OPU/ICSI and the following data
were recorded: time of year (season), mare age, reproductive and
athletic performance history and antral follicle count (AFC: total
number of follicles >4mm). Mares were classied as fertile, sub-
fertile or infertile, and the primary cause of reduced fertility or
infertility was recorded, if known. Ovum Pick-Up was performed
at Utrecht University and recovered oocytes were shipped over-
night at 20-22
C to Avantea where in-vitro maturation, ICSI and
embryo culture was performed as described by Galli et al. [Galli C Theriogenology 2014;81: 138e151]. Linear and logistic
regression was used to assess the potential inuence of donor
mare age, reproductive history, athletic performance history, AFC
and season on oocyte recovery and the success of IVEP. Overall,
252 OPU sessions were performed, yielding a mean of 12.8
oocytes per OPU with an average oocyte recovery success of 54%.
Oocyte recovery percentage was inuenced by donor mare age
(P¼0.03) and season (P¼0.04); oocyte recovery rates decreased
with mare age and were higher during the spring than autumn.
The total number of OPUs that resulted in one or more in-vitro
produced embryos was 147 (58%) with a mean of 1.8 embryos per
successful OPU and a range of 1-8 embr yos. Successful production
of an embryo was inuenced by AFC (P¼0.0004) and the repro-
ductive history of the donor mare (P¼0.04), whereas none of
season (P¼0.9), donor mare age (P¼0.4), or athletic performance
(P¼0.3) had an impact on the production of an embryo. The odds
of producing an IVP embryo increased as AFC increased. Fur-
thermore, infertile mares were 4 to 6 times less likely to produce
an IVP embryo than sub-fertile or fertile mares, which did not
differ from each other. Moreover, mares infertile due to severe
uterine abnormalities (predominately chronic intractable uterine
uid or infection) were 13 times less likely to produce an embryo
than fertile mares. To conclude, AFC and the reproductive history
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e8468
of the donor mare are important factors in predicting the success
of equine IVEP. In particular, equine IVEP is less successful in
chronically infertile mares, and reduced success is even more
pronounced in infertile mares with chronic severe uterine
Potential to reduce negative effects of exercise stress on
number of pregnancies in a commercial embryo
transfer program
M. Pinto
, M. Miragaya
, P. Burns
, R. Douglas
, D. Neild
atedra de Teriogenología, Facultad de Ciencias Veterinarias,
Instituto de Investigaci
on y Tecnología en Reproducci
on Animal,
Universidad de Buenos Aires, Chorroarín 280, 1427 Ciudad
onoma de Buenos Aires, Argentina;
Biorelease Technologies LLC,
1222 Richmond Rd, Lexington, Kentucky 40502, USA
*Corresponding author:
Exercise stress has a negative impact on embryo transfer ef-
ciency. For example, a 34% embryo recovery rate [Sertich P. J Am
Vet Med Assoc 1989; 195(7):940-944]; [Mortensen JC et al, Anim
Reprod Sci 2009; 110:237-244], 43% incidence of poor quality
embryos [Smith RL et al, J Anim Sci 2012; 90:3770-3777] and a
36% pregnancy rate post transfer [Allen WR In: Havemeyer
Foundation Monograph Series Nº1; 1999, p. 63-65]. have been
reported. Administration of nonsteroidal anti-inammatory
drugs (NSAIDs) may inhibit the inammatory response produced
after non-surgical embryo transfer. In addition, progesterone is
administered to some recipient mares to improve uterine con-
ditions prior to the transfer and continued after the transfer to
ensure plasma progesterone levels are compatible with preg-
nancy. The aim of this study was to evaluate embryo recovery
rates using BioRelease Deslorelin vs hCG and to increase post-
transfer pregnancy rates by jointly administering BioRelease
progesterone and a NSAID (unixin or meloxicam) to recipents.
Seventeen upper-level show jumping mares stabled and in daily
training, were used as donors. To induce ovulation, 1 mg IM
BioRelease Deslorelin (BioRelease Technologies, Lexington, Ken-
tucky, USA) was injected in treated cycles (n¼66), or 2500 IU hCG
IV (Ovusyn
, Syntex, Buenos Aires, Argentina) was given in con-
trol cycles (n ¼79) when a 35mm + follicle was present. Articial
insemination with extended fresh semen (500 x 10
motile sperm) was carried out in both groups immediately after
injecting the ovulation induction agent. Day 8 embryos were
recovered and non-surgically transferred using a speculum and a
cervical traction forceps. Recipient mares (n¼73) were randomly
assigned to one of three groups: Group A received a single
injection of 1.5 g IM BioRelease progesterone (Progesterone LA
300, BioRelease Technologies, USA) and 3 IV injections of 0.5 g of
unixin meglumine (Flunix
Deltavet, Argentina), one the day of
the transfer and one on the next two successive days. Group B
received 1.5 g IM BioRelease progesterone and a single dose of 1.5
g IM BioRelease Meloxicam (Meloxicam LA, BioRelease Tech-
nologies, USA) at the moment of embryo transfer. Group C did not
receive any treatment. Pregnancy diagnosis was carried out 7
days post transfer. Results were analyzed using comparisons of
proportions. More embryos were recovered per cycle (13%
increase) when donor mares in training for show jumping com-
petition were induced to ovulate with BioRelease deslorelin
(60.6%; 40/66) than with hCG (46.8%; 37/79). While both recipient
groups given NSAIDs in combination with BioRelease progester-
one numerically had higher pregnancy rates (A: 70.8%; 17/24 and
B: 75 %; 15/20) compared to non-treated control recipients (47.1%;
33/70), pregnancy rates were signicantly higher only in recipi-
ents given LA Meloxicam treatment at the time of transfer. The LA
Meloxicam is released over a 72 hour period making it more
practical to use as it requires a single IM injection versus the 3 IV
unixin meglumine injections. Thus, to minimize the effects of
exercise stress on ET efciency, a combination of BioRelease
deslorelin to induce ovulation in donors and BioRelease proges-
terone and LA Meloxicam in recipients at the time of transfer may
offer an interesting alternative for improving results in commer-
cial ET programs.
Key Words: Embryo transfer, exercise stress, NSAID, progester-
one, deslorelin, hCG
Can pregnancy survival after embryo transfer be
predicted based on early ultrasound examinations in
the recipient mare?
J.K. Morrissey
, R.A. Ferris
, D. Trundell
, K. Loncar
, D. Scoeld
, P.M. McCue
Equine Reproduction Laboratory, Department of Clinical Sciences,
Colorado State University, Fort Collins, CO, USA 80521
*Corresponding author:
Embryo transfer is a common procedure in the equine breeding
industry. Ultrasound examinations are performed on recipient
mares 4 to 9 days after transfer to determine pregnancy status.
The goals of this study were to compare the size of embryonic
vesicles at days 11, 12, 14, and 16 for embryos that survived to
25 days versus pregnancies that did not survive to 25 days. A
retrospective study was performed using reproduction records
at the Equine Reproduction Laboratory, Colorado State Uni-
versity. Donor mares were bred with cooled or frozen semen.
Embryo collection procedures were performed either 7.5 or 8
days after ovulation. Transrectal ultrasound pregnancy exami-
nations were performed on the recipient mares 11, 12, 14, 16 and
25 days (embryo age). Pregnancy survival was determined for
recipient mares up to day 25 of pregnancy. As a comparison,
embryonic vesicle diameter at day 14 of pregnancy was also
determined for mares bred to carry their own pregnancy. Stat-
istical comparisons were made using a paired t-test with sig-
nicance considered to be p <0.05. All data are presented as the
mean ±SEM. Embryos collected on day 8 from mares bred with
frozen semen (349.5 ±12.6
m; n¼89) were smaller (p<0.05)
than embryos collected from mares bred with cooled semen
(587.3 ±75.3
m; n¼20). An overall pregnancy rate of 80.8 %
was achieved following transfer of 313 embryos. Pregnancy
survival from day 11 to day 25 (embryo age) was evaluated in
169 recipient mares. Embryonic vesicles from mares bred with
frozen semen were signicantly smaller in size at days 11, 12,
and 14 days (3.3 ±0.4, 5.4 ±0.3, and 12.7 ±0.8 mm, respec-
tively) compared to those from mares bred with cooled semen
(5.6 ±0.4, 8.3 ±0.4, and 15.4 ±0.5 mm, respectively). However,
the difference in size was not signicant at the 16-day ultra-
sound exam (20.6 ±1.3 vs 23.2 ±0.7 mm, respectively;
p¼0.108). There was no difference (p <0.05) in embryonic
vesicle diameter at day 14 of pregnancy between mares bred to
carry their own pregnancy (16.5 ±0.4 mm; n¼27 mares) and
pregnant recipient mares (16.3 ±0.7 mm; n¼116 mares) for
pregnancies that survived to 25 days. Pregnancies that survived
to the 25-day examination had signicantly larger embryonic
vesicles at 12, 14 and 16 days of embryo age (8.7 ±0.3, 16.5 ±
0.4, and 24.5 ±0.5 mm, respectively) compared to those that did
not survive (5.7 ±0.7,10.5 ±1.2 and 16.6 ±2.0 mm, respec-
tively). In summary, ultrasound examination at days 12, 14 and
(Equal contribution)
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84 69
... Morphocytometric analysis was performed using the MIP software through an inverted microscope (Bertero et al., 2017), taking into study the thickness of the zona pellucida and cumulus expansion after maturation, oocyte diameter, presence and size of the first polar body. Morphocytometric measurements were performed and complemented by morphological examination; we allowed the reinstatement of oocytes into four quality classes according to oocyte diameter, expanded cumulus size, pellucida membrane thickness and the length and width of the first polar body (Claes et al.,2016). ...
... During the maturation period (in vivo and in vitro) of equine oocytes, the cells from the cumulus oophorus expand. In in vivo case, after induction of ovulation, 83% and 100% of the oocytes have an expanded cumulus following 12 h and 35 h post maturation, respectively, (Claes et al.,2016). After in vitro culture, most oocytes have an expanded cumulus depending on the culture medium. ...
... The maturation rate of compact oocytes selected under such conditions ranges from 14 to 25% and 66 to 78% for expanded oocytes (Hinrichs and Schmidt, 2000). In contrast, other studies have reported up to 85% of collected oocytes as having compact cumuli (Choi et al., 2003;Claes et al.,2016), this percentage is commonly reported as over 50%. Many of the above factors are interrelated, e.g., the difference in the proportion of compact oocytes and their maturation rate observed among studies may be due not only to classification criteria but also to the follicles selected for the collection, as both the proportions of oocytes which are compact and the maturation rate of compact oocytes increase with follicle size (Scott et al., 2001;Iwata et al., 2004). ...
Full-text available
In the current study, 930 equine oocytes were collected, 765 (82.25%) of which were considered viable for in vitro maturation. All selected oocytes were matured for 27 h at 38.50 0 C in an atmosphere of 5% CO2 in humidified air. 25 mm hepes-buffered TCM-199 supplemented with 2 mm sodium pyruvate, 1mm l-glutamine, penicillin (75 mg/ml), streptomycin (50 mg/ml) and 10% fetal calf serum was used for in vitro maturation. Following maturation, matured COCs were examined to investigate for first polar body formation. The morphocytometric assessment was performed using the motic image plus (MIP) software with an inverted microscope. Morphocytometric examination results showed highly significant differences (p<0.001) among groups of matured oocytes (zona pellucida thickness; <13 µm, cumulus oophorus thickness; < 10 µm and oocytes diameter; < 100 µm), where the rates were 63.39%, 33.59% and 30.58%, respectively. The results of morphocytometric evaluation based on zona pellucida thickness, cumulus oophorus thickness and oocyte diameter of the total 765 cultured oocytes showed that 197 oocytes (25.75%) were classified as excellent mature, 108 oocytes (14.11%) as mature good, 203 oocytes (26.53%) as immature and 257 oocytes (33.59%) were considered as degenerated. It is concluded thatthe oocytes rates differed according to the parameters (cumulus oophorus, oocytes diameter, zona pellucida).
... This methodology provides a solution for subfertility in both mares and stallions [1,4] and might lead to a more efficient and economically profitable use of fertile mares. The advantages include the necessity for only a small amount of scarce and expensive semen [2,5], the production of embryos at any moment of the year, without compromising sport engagements, and the possibility to determine the gender of the embryo before cryopreservation and transfer [1]. Individuality of the mare and the number of retrieved oocytes come across as the two main parameters influencing a successful outcome of OPU-ICSI [5,6], though male factors have to be considered too [7]. ...
... The advantages include the necessity for only a small amount of scarce and expensive semen [2,5], the production of embryos at any moment of the year, without compromising sport engagements, and the possibility to determine the gender of the embryo before cryopreservation and transfer [1]. Individuality of the mare and the number of retrieved oocytes come across as the two main parameters influencing a successful outcome of OPU-ICSI [5,6], though male factors have to be considered too [7]. ...
... Therefore, establishment of a predictive model for OPU-ICSI outcome in mares would represent a useful tool to support client counselling and management of expectations. The success of equine in vitro embryo production is significantly influenced by many factors such as the identity of the donor stallions and mares, the antral follicle count, the number of oocytes recovered, and notably the clinician and laboratory expertise [5,6]. The oocyte developmental capacity appears to be less affected by the sperm donor than by the oocyte donor, leading to the hypothesis that a factor intrinsic to the mares could be used as a marker for success [6]. ...
Full-text available
Anti-Müllerian hormone (AMH) reflects the population of growing follicles and has been related to mammalian fertility. In the horse, clinical application of ovum pick-up and intracytoplasmic sperm injection (OPU-ICSI) is increasing, but results depend largely on the individuality of the mare. The aim of this study was to assess AMH as a predictor for the OPU-ICSI outcome in horses. Therefore, 103 mares with a total follicle count above 10 were included in a commercial OPU-ICSI session and serum AMH was determined using ELISA. Overall, the AMH level was significantly correlated with the number of aspirated follicles and the number of recovered oocytes (p < 0.001). Mares with a high AMH level (≥2.5 µg/L) yielded significantly greater numbers of follicles (22.9 ± 1.2), oocytes (13.5 ± 0.8), and blastocysts (2.1 ± 0.4) per OPU-ICSI session compared to mares with medium (1.5–2.5 µg/L) or low AMH levels (<1.5 µg/L), but no significant differences in blastocyst rates were observed. Yet, AMH levels were variable and 58% of the mares with low AMH also produced an embryo. In conclusion, measurement of serum AMH can be used to identify mares with higher chances of producing multiple in vitro embryos, but not as an independent predictor of successful OPU-ICSI in horses.
... In the horse, in vitro embryo production (IVEP) has become a commercial reality in the past decade and equine reproduction centres now increasingly offer ovum pick-up (OPU) followed by intracytoplasmic sperm injection (ICSI) to overcome fertility problems in mares and stallions, to increase the offspring of a single frozen sperm straw and to plan and increase the propagation of valuable female genetics (Claes et al. 2016;Maserati and Mutto 2016;Morris 2018). An important complement to clinical ICSI would be the ability to cryopreserve oocytes, which would improve overall flexibility. ...
Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters.
The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.
In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yielding an average 13.8 immature oocytes, which were shipped overnight to a specialized ICSI laboratory (Avantea). One or more blastocysts (range: 0–13 blastocysts) were produced from 78% of procedures with a mean of 2.12 blastocysts per session; the likelihood of pregnancy after transfer of a cryopreserved thawed IVP blastocysts in 2021 (n = 781) was 77.7%. Several donor mare, recipient mare, stallion and embryonic factors influence the likelihood of producing an in vitro blastocyst or achieving pregnancy. Approximately 60% of the transferred IVP blastocysts yield a foal; moreover, neither gestation length nor the health of foals is noticeably influenced by IVEP. On the other hand, a skewed sex ratio towards colts is apparent among IVEP foals resulting from day 7 but not day 8 embryos, suggesting that male embryos develop more rapidly in vitro. Although serious complications after OPU are uncommon, owners should be aware of their existence, because some complications can be life-threating.
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Research focused on female gamete vitrification has increased attention to develop a reliable cryopreservation method to preserve immature equine oocytes. Despite the intensive implementation of biotechnological procedures for horse breeding, vitrification of immature equine cumulus-oocyte complexes (COCs) remain to be clearly elucidated. We aimed to determine the relative transcript level of target genes Bone morphogenetic protein 15 (BMP15); Bcl-2-associated X protein (BAX); and Caspase 3 (CASP3) in equine COCs prior to and after vitrification. Ovarian follicles were aspirated from ovaries collected from an abattoir. A total of 240 COCs were collected and distributed into vitrified COCs (VIT, n=120) and non-vitrified (Non-VIT, n=120) groups. Then, COCs were preserved and relative transcript expressions of BMP15, BAX, CASP3 were measured and normalized against GAPDH performed by qRT-PCR. In addition, 38 COCs were evaluated to assess chromatin configuration of germinal vesicle stage prior and after vitrification by exposure to 10 μg/ml of bisbenzimide. A difference was observed in the COCs’ mRNA level of abundance for the BAX gene between the VIT (2.05 ± 0.47) and (0.85 ± 0.08) Non-VIT groups. There was no difference in mRNA relative transcript level of CASP3 and BMP15 in Non-VIT (0.63 ± 0.20 and 1.55 ± 0.73, respectively) compared to VIT (0.64 ± 0.01 and 2.84 ± 2.20, respectively) equine COCs. All COCs where considered at immature stage of development even though COCs in Non-VIT group showed higher condensed chromatin configuration compared to VIT (100% vs 60.7%, respectively). We demonstrate that BMP15 and CASP3 are detected in VIT and Non-VIT immature COCs. In conclusion, BAX is expressed highly in vitrified immature equine COCs and indicates that activation of apoptosis signaling cascades in cells exposed to vitrification.
Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry due to the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through OPU (Ovum Pick Up). After collection the oocytes can be fertilized by ICSI using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without loss of viability and that increased in vitro maturation time can double the number of embryos produced is fueling the uptake of the OPU technique by several clinics that are shipping oocytes of their client’s mares to specialized ICSI laboratories for embryo production and freezing. In this paper we present a retrospective analysis of 10 years of work at Avantea with a special focus on the last 3 years. Based on our data an average production of 1.7-2 embryos per OPU-ICSI procedure can be obtained from Warmblood donor mares with a pregnancy rate of 70 % and a foaling rate in excess of 50 %. OPU-ICSI offers the added value of freezing embryos that allows the development of embryo commercialization worldwide to the benefit of top horse breeders who are endorsing this technology as never before.
The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient day after ovulation at transfer. The odds ratio for foaling was ~0.63 for transfer of Day 8 (46%) compared with Day 7 (56%) IVP blastocysts. The highest likelihood of pregnancy (72%) and foaling (60%) was observed when IVP blastocysts were transferred to recipient mares on Day 4 after ovulation. Finally, the sex (colt:filly) ratio was higher after transfer of Day 7 (71%:29%) than Day 8 (54%:46%) IVP blastocysts, suggesting that the speed of embryo development is sex dependent. In conclusion, the speed of invitro embryo development in our IVEP system affects the likelihood of foaling and the sex of the foal.
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This study retrospectively examined the degree to which success within a commercial ovum pick-up (OPU)-intracytoplasmic sperm injection (ICSI) program varied between individual mares and stallions. Over 2 years, 552 OPU sessions were performed on 323 privately owned warmblood mares. For mares that yielded at least one blastocyst during the first OPU-ICSI cycle, there was a 77% likelihood of success during subsequent attempts; conversely, when the first cycle yielded no blastocyst, the likelihood of failure (no embryo) in subsequent cycles was 62%. In mares subjected to four or more OPU sessions, the mean percentage of blastocysts per injected oocyte was 20.5% (range 1.4-46.7%), whereas the mean number of blastocysts per OPU-ICSI session was 1.67 (0.2-4.2). Age did not differ significantly between mares that yielded good or poor results. The number of recovered oocytes per OPU was positively associated with the likelihood of success (P<0.001). Although there were considerable between-stallion differences, most stallions (14/16) clustered between 15.6% and 26.8% blastocysts per injected oocyte, and the number of blastocysts per OPU (mean 1.4; range 0.2-2.2) was less variable than among mares. In conclusion, although both mare and stallion affect the success of OPU-ICSI, mare identity and the number of oocytes recovered appear to be the most reliable predictors of success.
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