The effect of uterine massage and number of embryo flushing attempts on embryo recovery in mares.

The aims of this study were to determine the effect of the embryo flushing technique and the number of flushing attempts performed by operators of different experience on embryo recovery (ER). Ten non-lactating mares were inseminated with the same stallion in six cycles each (n = 60). Embryo flushing (EF) was performed 7-9 days after ovulation by three operators (OP; 20 EF cycles each): OP1 had performed >500 EF before the study, while OP2 and 3 had performed 0 EF. Each EF was performed with 2 flushing attempts (FA) using 1L of ringer's lactate "in-and-out" using two EF techniques: 1) uterine massage (UM): continuous ballottement and massage of the uterus per rectum during ringer lactate recovery, 2) gravity flow (GF): the ringer lactate was allowed to flow back without massaging the uterus. In both groups, 20 IU of oxytocin were administered at the second FA and the ringer lactate was allowed to remain in the uterus for 3 min before recovery. An extra FA was performed in each group using 0.5 L of ringer lactate and uterine massage. More embryos (P < 0.05) per ovulation were recovered in the UM (17/33, 0.51) than in the GF group (8/36, 0.22). For the UM group, 16/17 embryos (94.1 %) were recovered in the first FA, while only one embryo in the second FA (1/17, 5.9 %). In the GF group, 4 embryos were recovered in each FA. No embryo was found in the extra FA in the UM group, while seven additional embryos were found in the GF group (5/7 flushed by OP1; P < 0.05). The overall ER per cycle was 70, 40, and 45 % for OP1, 2 and 3, respectively. In conclusion, highest embryo recovery is achieved in EF performed with UM, with the majority of embryos being flushed in the first FA.

Long-term performance of show-jumping horses and relationship with severity of ataxia and complications associated with myeloencephalopathy caused by equine herpes virus-1.

BACKGROUND: Equine herpesvirus myeloencephalopathy (EHM) has severe impact on the sport horse population. OBJECTIVE: Study the influence of EHM on the likelihood of affected horses to return to their previous performance and investigate the association of clinical variables with prognosis. ANIMALS: Twenty-six horses positive for equine herpesvirus type 1 (EHV-1) were admitted to a veterinary teaching hospital (VTH) during a natural EHM outbreak at an international jumping event. METHODS: Data collected from the VTH, the International Equestrian Federation, and surveys completed by the riders and horse owners were retrospectively analyzed. RESULTS: Horses affected by EHM had 68% chance of returning to exercise, and 52.9% were able to achieve their preoutbreak performance level. Horses with an ataxia grade at admission ≥4/5 had an increased fatality rate (P < .05) and 10% chance of reaching their preoutbreak performance level. None of the horses with both vascular and urinary complications returned to their previous performance level. Finally, horses vaccinated against EHV-1 and those with urinary complications had a 71.4% and 43.7% fatality rate, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Horses affected by EHM were able to return to their previous performance levels, but certain clinical variables were negatively associated with postoutbreak performance. Ataxia grade upon admission and the development of systemic signs of vasculitis and urinary complications were potential poor prognostic indicators in sport horses. Variables linked to fatality included prior vaccination against EHV-1, ataxia grade upon admission, and the development of urinary complications.

Effects of repeated embryo flushing without PGF2α administration on luteal function, percentage of unwanted pregnancy and subsequent fertility in mares.

BACKGROUND: PGF2α is commonly given at the end of embryo flushing (EF) to shorten the interval to the next oestrus and ovulation. OBJECTIVES: To determine the effect of repeated EF on plasma progesterone concentration, percentage of mares with endometritis, unwanted pregnancy and subsequent fertility in mares flushed without the use of PGF2α . STUDY DESIGN: Controlled experiments. METHODS: Nine mares were inseminated in seven consecutive cycles (n = 63), to either perform an EF (n = 54) 7-9 days after ovulation or left pregnant (n = 9). PGF2α was not used to induce oestrus. Ultrasound examination and blood sampling were performed just before the EF and 72 h later to determine changes in progesterone concentration and signs of endometritis. RESULTS: The overall percentage of positive EF/pregnancy was 55.5% (30/54) and 66.7% (6/9), respectively. The likelihood of pregnancy/positive EF in the first three cycles was 55.5% (15/29). This was not different (p > 0.1) from the fertility of the last four cycles (69.4%, 25/36). In five EF cycles (9.3%), mares had signs of endometritis and early luteolysis (progesterone <2 ng/mL) 72 h after EF. The reduction in progesterone concentration by 72 h after EF was greater (p < 0.05) for Day 9 (-2.3 ± 0.7 ng/mL) than Day 7 (-1.0 ± 0.8 ng/mL) or Day 8 (-1.3 ± 1.1 ng/mL) cycles. The progesterone concentration in non-flushed mares did not vary significantly during the sampled period (Day 7-12). There were 5 cycles in which the donor mare remained pregnant after the EF, although four were from a single mare. MAIN LIMITATIONS: The mare population was limited to barren and maiden mares. The cycle order and operator allocation to each EF were not randomised. CONCLUSIONS: EF induces a subtle, but significant reduction in progesterone concentrations compared with non-EF cycles. However, the percentage of mares with EF-induced full luteolysis is low (9.3%). The fertility of mares after repeated EF without administration of PGF2α was unaffected; however, there is a considerable risk of unwanted pregnancy (5/27 = 18.5%) in donors from which an embryo was not recovered.

Seasonal absence of supplementary corpora lutea in pregnant mares and the relationship with pregnancy loss.

Two experiments were conducted in the Northern (UK) and Southern (Brazil) hemispheres to determine the effect of season (month of conception) on the development of supplementary CL (SCL) and the relationship with pregnancy loss. In experiment 1, 199 pregnancies were followed between Day 14 and term, to determine the number of SCL and pregnancy viability (Northern Hemisphere). From the 199 pregnancies, 178 were obtained from inseminations during the breeding season (March-September), while the rest, 21 pregnancies resulted from conceptions in the non-breeding season (October to February). Pregnancies conceived in the breeding season were more likely (P < 0.01) to have at least 1 SCL (75.8 %, 135/178) than pregnancies from the non-breeding season (33 %, 7/21). However, the pregnancy loss between Days 35 and 120 of pregnancy in mares with no SCL was similar (3.5 %, 2/57; P >0.1) than from mares with SCL (1.4 %, 2/142). In Experiment 2 (Southern Hemisphere), three groups of recipients were used based on their ovarian activity at the time of embryo transfer: Anestrus (n = 8), transitional (n = 7) and cyclic (n = 7) recipient mares. While all transitional and cyclic mares developed at least 1 SCL, only 50 % of anestrous recipients (4/8) developed SCL by 120 of gestation. In conclusion, the development of SCL in pregnant mares is influenced by the time of season of conception, therefore it appears to be regulated by the photoperiod and the endogenous seasonal variation in gonadotropin concentrations. Mares with no SCL were not at increased risk of pregnancy loss.

The Timing of the Maternal Recognition of Pregnancy Is Specific to Individual Mares.

The present experiment aimed at determining whether the timing of the maternal recognition of pregnancy (MRP) was specific to individual mares by determining when luteostasis, a failure to return to oestrus, reliably occurred in individuals following embryo reduction. Singleton (n = 150) and synchronous twin pregnancies (n = 9) were reduced in 10 individuals (5-29 reductions/mare) at pre-determined time points within days 10 (n = 20), 11 (n = 65), 12 (n = 47), 13 (n = 12) or 14 (n = 15) of pregnancy. Prior to embryo reduction, the vesicle diameter was measured in 71% (106/150) of the singleton pregnancies. The interovulatory interval (IOI) was recorded on 78 occasions in seven of the mares in either non-pregnant cycles (n = 37) or those in which luteolysis followed embryo reduction (n = 41). The earliest time post-ovulation at which the embryo reduction resulted in luteostasis in an individual was 252 h (mid-Day 10). Consistency in luteostasis following embryo reduction showed individual variation between mares (272-344 h). Binary logistic regression analysis showed an individual mare effect (p < 0.001) and an effect of the interval post-ovulation at which embryo reduction was undertaken (p < 0.001). However, there was no significant effect of vesicle diameter at the time of embryo reduction (p = 0.099), nor a singleton or twin pregnancy (p = 0.993), on the dependent of luteolysis or luteostasis. The median IOI between individual mares varied significantly (p < 0.05) but was not correlated to the timing of MRP. The timing of MRP varied between the mares but was repeatable in each individual. The factors and mechanisms underlying the individuality in the timing of MRP were not determined and warrant further study.

Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling.

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.

The Effect of Treatment with Oestradiol Benzoate on Oestrus Expression and Endometrial Oedema in Anovulatory and Cyclic Mares.

Oestrogens treatment is often used to induce oestrus behaviour in anoestrous mares to aid in the collection of stallion semen and as recipient mares to receive embryos when combined with progesterone. However, there are no studies to describe the effect of dose and individual mare on the intensity and duration of the response, in both anoestrous and cyclic mares. In Experiment 1, 13 anoestrous mares were treated with one of five doses of oestradiol benzoate (OB) (1, 1.5, 2, 3 and 4 mg) per mare in five consecutive treatment periods (n = 65), to determine the response in terms of endometrial oedema and oestrous behaviour. Experiment 2 and 3 used 3 mg of OB in cyclic mares to confirm or deny the presence of an active corpus luteum (CL). There was a dose rate of OB and individual mare effect (p < 0.05) on the intensity and persistence of endometrial oedema and oestrous behaviour. A total of 2 mg OB was enough to induce endometrial oedema and oestrous behaviour within 48 h in most mares. Mares with an active CL did not show endometrial oedema following treatment of 3 mg OB.

In vitro aging of stallion spermatozoa during prolonged storage at 5°C.

Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca2+ ]i and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n = 17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca2+ ]i decreased over time (1 h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca2+ ]i in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1 h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5°C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions.

The effect of dexamethasone and flunixin-meglumine on ovulation, endometrial oedema, and inter-ovulatory interval length in the mare.

The use of flunixin-meglumine (a potent non-steroidal anti-inflammatory drug) during the critical period of intrafollicular prostaglandin production before ovulation (24 and 36 h after hCG treatment) results in a high rate of ovulatory failure and formation of haemorrhagic anovulatory follicles (HAF) in the mare. Dexamethasone is commonly used to prevent persistent mating-induced endometritis in susceptible mares, but the effect on ovulation blockage within the pre-ovulatory critical window of intrafollicular prostaglandins production following hCG administration has not been determined. Six mares were followed during four consecutive cycles in a crossover design; once in oestrus with a follicle of >32 mm in diameter, mares were treated with hCG (Hour 0) and assigned to one of 4 groups randomly: 1) FM, mares received 1.7 mg/kg flunixin-meglumine at Hour 24 and 36; 2) CON, mares received no further treatment. 3) DEX1, mares received 0.1 mg/kg dexamethasone at Hour 24, and 4) DEX2, mares received 0.1 mg/kg dexamethasone at Hour 24 and 36. For all groups, ovulation and HAF rates, endometrial oedema profiles and the inter-ovulatory intervals (IOI) were determined and compared statistically. All CON and DEX mares ovulated normally and did not form any HAF. On the contrary, FM mares developed a HAF in 83% of cycles (P < 0.01). The endometrial oedema score was lower following DEX administration than FM (P < 0.05). The mean IOI was longer (P < 0.05) in DEX1 and DEX2 groups (26.5 and 26 days, respectively) than in CON and FM groups (21.5 and 22 days, respectively). In conclusion, dexamethasone treatment given either once or twice during the critical window of hCG-induced ovulation did not block or delay ovulation, but had a similar ovulation rate than untreated control mares. However, the inter-ovulatory intervals of dexamethasone treated mares was longer than control and FM treated mares. Finally, dexamethasone treatment was more effective in reducing endometrial oedema than FM.

The post-ovulatory rise in progesterone is lower and the persistence of oestrous behaviour longer during the first compared with the second cycle of the breeding season in mares.

Mares are seasonally polyoestrous breeders. Therefore, the first ovulation of the season, following winter anoestrus, is the only cycle in which mares ovulate without the presence of an old CL from the previous cycle. The objective of this study was to compare the length of oestrous behaviour, and plasma progesterone concentrations during the early post-ovulatory period between mares after the first and second ovulation of the breeding season. Overall, 38 mares and 167 oestrous periods were used in the study. From those, 11 mares were used during the first and subsequent oestrous period to measure and compare the post-ovulatory rise in progesterone concentration, whereas all the mares were used to compare the length of the post-ovulatory oestrous behaviour between the first and subsequent cycles of the breeding season. The persistence of the post-ovulatory oestrus was longer (p < .001) following the first ovulation of the year (median of 52 h) compared with the subsequent ovulations (median of 36 h for second and later ovulations groups; n = 38 mares). The progesterone concentration at any of the four 8 h-intervals analysed (28, 36, 76 and 84 h post-ovulation) was lower (p < .01) following the first versus the second ovulation of the year. By 36 h post-ovulation the progesterone concentration of mares at the second ovulation of the year had passed the threshold of 2 ng/ml (2.1 ± 0.33 ng/ml), whereas in the first cycle it was 1.2 ± 0.13 ng/ml. In conclusion, mares had lower progesterone concentrations in their peripheral circulation and longer persistence of oestrous behaviour following the first ovulation of the year compared with the second and subsequent ovulatory periods of the breeding season.

The effect of priming and duration of oestradiol benzoate treatment before progesterone administration on embryo development and survival in anestrous recipient mares.

The effect of three different hormonal protocols to prepare anestrous recipient mares on embryo survival was evaluated. The first group consisted of only progesterone administration (NE) 4 days before embryo transfer, while the recipients from the other two groups received a single administration of 2.5 mg of oestradiol benzoate (SE) 2 days earlier or 8 mg of oestradiol split in increasing doses for 5 consecutive days (LE) ending 3 days before progesterone treatment. The likelihood of recovering an embryo 2 days after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the no oestrus, short and long oestrous groups respectively (p = .09). In conclusion, the presence and duration of oestradiol treatment before progesterone administration tended to influence the embryo survival in anestrous recipients 2 days after transfer. The surviving embryos recovered from the three different groups of recipients did not show any difference in size and morphology.

Artificial Long Estrus Protocols Administered Prior to Progesterone Increase Endometrial Uterocalin Expression in Anestrous Mares.

Uterocalin (P19) is a lipocalin protein that has binding activity for the provisioning of the embryo with lipids, including those essential for morphogenesis and pattern formation. Therefore, it is thought that P19 plays an important role in supporting the survival of the early embryo. Previous research has shown that the endometrium from acyclic mares expressed more P19 following a simulated long estrus: treatment of 8 mg of estradiol benzoate (EB) split over five consecutive days, followed by long-acting progesterone administration (LA-P4) 72 hours later, when compared to a single 2.5 mg EB treatment. The main objective of this study was to evaluate if a new long estrus protocol, with fewer EB administrations, also increases endometrial uterocalin expression. Fifteen mares were assigned to three groups: long estrus 5 administrations (LE5; a total of 8 mg of EB in increasing doses was given over five consecutive days, Day -7 to Day -3); long estrus 2 administrations (LE2; 3 and 5 mg EB were given on Day -7 and Day -4); and short estrus (SE; a single dose of 2.5 mg EB was administered on Day -2). All groups received LA P4 on day 0 (D0). Daily ultrasonography and blood collections were performed to assess uterine edema and estradiol and progesterone plasma concentrations, respectively. Endometrial biopsies were collected on Day 4 to evaluate P19 expression by RT-qPCR. There were no differences in P19 expression between groups LE2 and LE5. However, P19 expression was higher (P < .05) in the LE groups than in the SE group. Both LE protocols produced high uterine edema (score 2-3) for at least 6 days. There was no effect of group (LE2 or LE5) on overall estradiol and progesterone concentrations (P > .1). In conclusion, the two EB administrations protocol is more feasible for preparing acyclic recipient mares at field conditions. However, a positive effect of the LE protocols on embryo survival and viability in anestrous treated mares still needs to be confirmed.

A practical guide to estimate the age of the early CL by ultrasonography in mares examined for the first time to be used as recipients in a commercial embryo transfer program.

The echogenicity of the early CL undergoes obvious changes during the first 5 days of luteal development. This phenomenon could potentially be used to estimate the age of the CL in mares which have not been checked frequently for the diagnosis of ovulation. The objective of this retrospective study was to determine the accuracy of estimating the age of the early CL (Day 0 to Day 4; Day 0 = Day of ovulation) in recipient mares examined for the first time in the breeding season to be used in a commercial embryo transfer (ET) program. The post-transfer pregnancy rate and embryo loss of 28 recipient mares whose CL age was estimated at the first scan of the breeding season (at least 2 months gap from the previous examination) were compared to that of 41 recipients in which the Day of ovulation had been determined by daily examinations and that received an embryo on the respective day. There was no difference (P > 0.1) in pregnancy (85.7 and 85.3%) or early embryonic loss (12.5% and 11.4%) between groups, respectively.

Effect of Timing of Postovulatory Insemination Relative to Human Chorionic Gonadotropin/Buserelin Treatment With 1 Straw of Frozen-Thawed Semen on Mare Fertility.

The reproductive management of mares for frozen semen artificial insemination (AI) can be costly and labor intensive. Predicting the exact time of ovulation can be challenging even when ovulation-inducing drugs are used. The main objective of this retrospective study was to determine whether there was an effect of interval between examinations to detect ovulation on likelihood of pregnancy and early embryonic loss in mares after postovulatory breeding with a single straw of frozen/thawed semen. The second objective was to determine the efficacy of two different drugs (human chorionic gonadotropin vs. buserelin) for timely induction of ovulation. The length of the interval from penultimate check to ovulation had no significant effect on pregnancy or embryo loss rates (4 hours: 34.1% and 13.3% vs. 8 hours: 26.1% and 0% and 16 hours: 34.5% and 10%, respectively) nor did the ovulation-inducing drug used, number of the cycle, or the stallion. In conclusion, there appears to be no advantage of checking mares for ovulation during the late evening and night hours when using a postovulatory AI protocol and ovulation-inducing drugs.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

In vitro-produced horse embryos exhibit a very narrow window of acceptable recipient mare uterine synchrony compared with in vivo-derived embryos.

In recent years, the number of equine invitro-produced embryos (IVP) has increased markedly; as yet, there are few reports on what constitutes an 'ideal' recipient for an IVP embryo. This study retrospectively investigated the effects of recipient mare oestrous cycle characteristics on the likelihood of pregnancy after transfer of IVP (n=264) and invivo-derived embryos (n=262). IVP embryos tolerated only a narrow window of recipient mare 'synchrony', with transfer on Day 4 after ovulation resulting in a higher likelihood of ongoing pregnancy (69%) than transfer on Days 3, 5 or 6 (53.2%, 41.3% and 23.1% respectively; P=0.02). In contrast, Day 8 invivo-derived embryos tolerated a wide range of uterine (a)synchrony, with no difference in pregnancy or pregnancy loss for recipients that ovulated between Day 4 and Day 9 before transfer. However, transferring invivo-derived embryos to recipients that had a longer oestrus preceding transfer resulted in higher Day 12 and ongoing pregnancy rate (P<0.01). This effect was not significant in IVP embryos. In conclusion, Day 6-8 IVP blastocysts survive best after transfer to Day 4 recipient mares; Day 8 invivo-derived embryos survive equally well in Day 4-9 recipients, but do better in mares that have a long preceding oestrus.

Mare and stallion effects on blastocyst production in a commercial equine ovum pick-up-intracytoplasmic sperm injection program.

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.

Effect of the Interval From Prostaglandin F2alpha Treatment to Ovulation on Reproductive Efficiency Rates in a Commercial Equine Embryo Transfer Program.

In the present study, 2.228 cycles of 180 Polo Argentino donor mares from an embryo transfer program in Argentina were examined to evaluate the effects of: (1) Interval from Prostaglandin F2alpha analog treatment to ovulation (ITO) on embryo recovery rate (ERR); (2) ITO on number of embryos per flushing (EPF); (3) ITO on multiple ovulation (MO) rate; (4) ITO from donor mare on pregnancy rate (PR) in recipient mares. Mares were inseminated with fresh semen from 31 fertile stallions in the induced estrus. Embryo flushing was performed 7-8 days postovulation. Following embryo flushing, donor mares were treated with prostaglandin F2alpha analog (cloprostenol 250 µg). The ERR increased along with the ITO (P = .01), with the lowest ERR (30.7%) for mares with an ITO of <4 days, and the highest (78.3%) in mares with an ITO of 10 days. The ITO from the donor mare in which the embryo was recovered did not have a significant effect on PR: ITO <6, 6 to 10, and >10 days were 74.6, 81.4, and 77.3%, respectively. The number of EPF and MO rate increased gradually along with the ITO (P < .05). In conclusion, the results of this study indicated that the ITO is positively correlated with the embryo recovery and the multiple ovulation rate.

The recipient's Day after ovulation and the number of corpora lutea influence the likelihood of pregnancy in mares following transfer of ICSI frozen embryos.

Asynchronous embryo transfer is an excellent tool to investigate how subtle differences in the uterine environment affect embryo development and survival. Progesterone secreted from the corpus luteum following ovulation is one of the main factors responsible for establishing endometrial receptivity for the pre-implantation embryo via complex alterations in the expression of genes involved in the secretion of the histotroph. The objective of this retrospective study was to determine whether the recipient's Day after ovulation and the number of CL at ET influence the pregnancy rates of IVP horse embryos. The study included 650 heterologous frozen ICSI horse embryo transfer cycles and evaluated the pregnancy and ongoing pregnancy rate. The ongoing pregnancy was significantly lower in recipient mares with ET performed 5 and 6 days after ovulation (47.4% and 37.5%, respectively) than in recipients with ET 4 days after ovulation (67.3%). Furthermore, Day 5 recipient mares (Day 0 = Day of ovulation) with 2 corpora lutea (CL) at the time of ET had lower ongoing pregnancy rate (36.1%) than Day 5 recipient mares with 1 CL (51.9%). In contrast, the presence of 2 CL was associated with a higher ongoing pregnancy rate (75.8%) in recipient mares with a less advanced uterine stage at the time of ET (Day 3 and 4 after ovulation), compared to recipients with only 1 CL at ET (62.7%). In conclusion, both the number of days after ovulation and the number of CL recorded in the recipient mare at ET influenced the ongoing clinical pregnancy rate. This study highlights the importance of establishing exactly when progesterone rises above a threshold (relative to the Day of ovulation) when trying to determine the optimal window for transferring an IVF/IVP embryo.

The Effect of Different Flushing Media Used to Aspirate Follicles on the Outcome of a Commercial Ovum Pickup-ICSI Program in Mares.

The in vitro production of embryos by ovum pickup (OPU) and intracytoplasmic sperm injection (ICSI) is gaining popularity among horse breeders and veterinarians. Various collection media are available for flushing follicles during OPU. The objective of this study was to determine whether the type of flushing media used to aspirate follicles and collect oocytes influences the outcome of a commercial equine OPU-ICSI program. Two commercial embryo flushing media (EFM1 and EFM2) supplemented with heparin were compared with a flushing media designed specifically for the collection of oocytes (oocyte flushing media [OFM]) on the outcome of OPU-ICSI parameters in 234 Warmblood mares. The OPU-ICSI performed in mares using one of the EFM1 resulted in a lower (P < .05) blastocyst rate and blastocysts per OPU-ICSI session (11.9 ± 13.2%, 0.88 ± 1.3) than the OFM (19.2 ± 15.2%, 1.24 ± 1.2). Unlike the EFM2 solution, the heparin used to prepare the EFM1 contained preservatives including benzyl alcohol, a component known to alter the oocyte membrane, which might have been responsible for the lower developmental competence of oocytes collected with EFM1. In conclusion, exposure of oocytes (<1.5 hours) to one of the flushing medium tested in this study affected negatively the outcome of the OPU-ICSI commercial program when compared with flushing media designed for collection of equine oocytes. Care should be taken when choosing the components of the flushing media used to collect oocytes. Further research should be carried out to confirm the potential negative effect of the preservatives used in multidose heparin vials.