Sofie Peere

Background: While fast in vitro embryo development has been associated with improved outcomes, the relationships of slow in vitro embryo development and vitrified versus fresh in vitro produced (IVP) embryo transfer to foaling percentage and foal sex are less explored. Objectives: To determine the relationships of (1) day of blastocyst formation (D7–11 after intracytoplasmic sperm injection (ICSI)) and (2) vitrification and warming method (one‐ and three‐step) versus fresh transfer with pregnancy, early pregnancy loss (EPL), foaling percentage and foal sex. Study Design: Retrospective clinical study. Methods: Blastocysts ( n = 201) were collected on days 7–11 after ICSI of in vitro matured oocytes from Warmblood mares and transferred either fresh or vitrified‐warmed into recipient mares on day 4 after ovulation. Pregnancy, EPL, foaling percentage, and foal sex were compared using multivariable generalised linear mixed‐effects logistic regression models. Results: Day of blastocyst formation was significantly associated with pregnancy outcome at D14 ( p = 0.048), D42 ( p = 0.046) and foaling percentage ( p < 0.001). The odds of foaling decreased with developmental age, with significantly lower odds after D11 blastocyst transfer (OR = 0.0045, 95% CI 0.00034–0.061; p < 0.0001). Slow embryo development was associated with a significantly higher proportion of female offspring. The odds of obtaining a filly versus a colt were 5.6‐fold higher for D10 blastocysts than D7 (95% CI 1.14–25.0; p = 0.034). Transfer of fresh versus vitrified‐warmed blastocysts, using a one‐ or three‐step protocol, did not significantly affect pregnancy or foaling outcome. Main Limitations: Small sample size per day of blastocyst formation. Conclusions: Slow in vitro embryo development is associated with decreased pregnancy and foaling outcomes following transfer of D11 IVP blastocysts. Transfer of D10 IVP blastocysts yields acceptable foaling percentages and is associated with a higher proportion of female offspring. Vitrification is effective for preserving equine embryos and allows the use of a simplified one step warming protocol.

Background: While fast embryo development has been associated with improved ICSI outcomes, the effects of slow embryo development (D9-11 post ICSI) and of vitrification vs. fresh IVP transfer on foaling rates and the foal’s sex are less explored. Objectives: To determine: 1) the effect of day of embryo development (D7-11) and 2) of vitrification and warming assessing two methods: direct warming in isotonic commercial embryo holding medium (one step) or serial dilution warming (three step) vs. fresh IVP embryos, on pregnancy, early pregnancy loss, foaling rates and the foal’s sex Study design: Retrospective clinical study Methods: Blastocysts (n=201) were collected on D7-11 after ICSI of in vitro matured (IVM) oocytes from Warmblood mares (n=22) and transferred either fresh or vitrified-warmed into recipient mares on D4 post-ovulation. Pregnancy, early pregnancy loss, foaling rates and the foal’s sex were compared by RStudio statistics. Results: The day of blastocyst development did not affect initial pregnancy rates, but transfer of D11 IVP embryos resulted in significantly higher early pregnancy loss (D7,-8,-9,-10 and -11; 0%, 5.7%, 11.1%, 20.0% and 83.3%, resp., p < 0.001) and lower foaling rates (D7,-8,-9,-10 and 11; 88.2%, 64%, 60.8%, 50% and 10%, resp., p < 0.0001). The foal’s sex showed a skewed sex ratio towards female in D10 (78.9%:21.1%) vs. D7 (35.7%:64.3%; P = 0.02), D8 (50%:50%; P = 0.05) and D9 (48.4%:51.6%) IVP embryos. Transfer of fresh vs. vitrified-warmed (one-and three step protocol) embryos did not significantly affect pregnancy nor foaling rates. Main limitations: Small sample size per day of blastocyst development Conclusions: Slow in vitro embryo development negatively affects foaling rates after transfer of D11 IVP embryos. Transfer of D10 IVP embryos yields acceptable foaling rates and favors female offspring. Vitrification provides a safe method to preserve equine embryos with the flexibility to use a one-step warming protocol.

Transfer of in vitro-produced (IVP) equine embryos is associated with a higher incidence of monozygotic multiple pregnancies, but this remains a rarity. The outcome of monozygotic twin gestation is poor, and good management techniques are lacking. In addition, hydrops conditions are exceptional in horses, with hydroallantois occurring more frequently than hydramnion. Most hydrops conditions will lead to compromise or death of the foal and mare. A 6-year-old multiparous standardbred recipient mare, 332 days pregnant, diagnosed with a monozygotic twin and hydrops with a dead co-twin, gave birth with an assisted vaginal delivery. The dead foal, previously determined at 9 months by ultrasound, turned out to have the dimensions and hair corresponding to an 8-month-old foal. The other foal was born alive and considered healthy and normal-sized for its age. Both mare and foal were discharged from the clinic one week after parturition. This report is the first to describe the birth of a healthy foal born from a monozygotic twin pregnancy in the presence of hydrops, following transfer of a single IVP equine embryo. In addition, it is rare to find a non-macerated, non-mummified dead co-twin after fetal death at 8 months together with a healthy full-term foal.

Twin gestation in the mare is undesirable and can have disastrous consequences. As in many cases, the key to success in twin management lies in a thorough follow‐up and accurate recording of clinical findings in the pre‐breeding examination. A pregnancy diagnosis in the mobility phase is imperative for a good outcome in the event of twin reduction. If a twin gestation is not diagnosed during this early pregnancy stage, several other procedures exist for managing post‐fixation twins (>16 days) with varying degrees of success. Most twin pregnancies are the result of multiple ovulations (dizygotic twins). However, monozygotic twins are also sporadically diagnosed, due to the increasing number of transferred in vitro produced equine embryos. In these cases, the most optimal treatment strategy still needs to be determined. This review provides an overview of the various twin reduction techniques described with the expected prognosis as well as of some less reported techniques with their results. In addition, physiological events and the reduction techniques are demonstrated to the user in virtual 3‐dimensional illustrations.

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.