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 addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

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.

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.