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.

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.

In vitro 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 in vitro-produced (IVP) and in vivo-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 in vivo-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 in vitro 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.

Background Monozygotic multiple pregnancy is rare in horses, but may be more common after transfer of an in vitro produced (IVP) embryo. Objectives To determine the occurrence, incidence, characteristics and outcome of monozygotic siblings arising from in vivo and IVP equine embryos. Study design Retrospective case series. Methods A total of 496 fresh in vivo and 410 frozen‐thawed IVP blastocysts, produced by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes from Warmblood mares, were transferred into recipient mares. The likelihoods of pregnancy and multiple pregnancy were calculated, and the clinical features and outcome of any multiple pregnancy were recorded. Results The likelihood of pregnancy after transfer of a single IVP or in vivo embryo was 62% (254/410) and 83% (413/496), respectively. The incidence of multiple pregnancy was 1.6% (4/254) and 0% (0/413) for IVP and in vivo blastocysts, respectively. More specifically, three IVP blastocysts yielded twin embryo propers/foetuses, and one IVP conceptus developed three distinct embryonic bodies. Interestingly, only one embryonic vesicle was detected at all ultrasonographic examinations prior to embryo proper development. Multiple embryonic bodies only became apparent at later scans to check for an embryo proper and heartbeat, or when the recipient mare aborted. Two twin pregnancies aborted spontaneously at 3 and 9 months, respectively, while the heart beat was lost from all three embryos in the triplet pregnancy before day 35 of gestation. Twin reduction by per rectum compression of one fetal thorax was attempted at day 50 of gestation in the fourth case; however, both foetuses were lost. Main limitations Small number of cases. Conclusions In vitro embryo production resulted in a higher incidence of multiple monozygotic pregnancy, which could only be diagnosed after development of the embryo proper and is likely to result in pregnancy loss later in gestation if left untreated.

The success of in vitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of in vitro 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 in vitro-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 in vitro 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 in vitro embryo development in our IVEP system affects the likelihood of foaling and the sex of the foal.