Successful use of sexed stallion semen for in vitro embryo production; a preliminary study

The use of sex-sorted semen to obtain offspring of a desired sex in commercial livestock breeding programs has been in a state of rapid growth as sorting technology has evolved (Neculai-Valeanu and Ariton, Animals 2021, 11, 1182-1194, 2021). In horses, sex-sorted semen has resulted in successful pregnancies using low-dose or hysteroscopic artificial insemination, albeit with decreased pregnancy rates (Clulow et al, Animal Reproduction Science 108: 287–297, 2008; Gibb et al, Eq Vet J 49:160–166, 2017). Even though sex-sorted semen has been used to produce viable in vitro produced (IVP) embryos, the cleavage and blastocyst rates remain low (Colleoni et al, Reproduction, Fertility and Development 21(1): 228-229, 2008). Using current sperm sorting technology (ST Inc, Navasota, TX), we investigated fertilization, cleavage, and embryo development rates after ICSI using a time-lapse imaging system. To sex sort the semen, the ejaculate was diluted in 1:1 in a casein/cholesterol-based semen extender (BotuSemen Gold) supplemented with 60ug/ml of Hoechst 33342. The diluted semen was centrifuged through a colloid suspension (EquiPure) for 25 min at 300g using 3mL of EquiPure per billion sperm. After centrifugation, the sperm pellet was split into two aliquots: NON-SEXED, resuspended in BotuCrio, cooled and frozen in 0.5 ml straws at ∼ 100 million/ml, and SEXED, resuspended in a proprietarychemically-defined medium (EquiShip, ST Genetics), and transported to the sorting facility at ∼20°C. At the laboratory, the sperm were passed through the SexedULTRA™ Genesis III flow cytometer. After approximately 40 million total events X- and Y-bearing sperm were recovered, diluted in BotuCrio at a concentration of 2 million/ml, loaded into 0.5 ml straws and frozen. Oocytes were obtained from mares using transvaginal ultrasound-guided aspiration, shipped/held for 24h, followed by in vitro maturation. The matured MII oocytes from each aspiration were randomly split and injected with SEXED and NONSEXED sperm. A total of 56 oocytes were fertilized. We observed 24/29 and 23/27 cleaved oocytes, resulting in 11 and 12 blastocysts in SEXED and NONSEXED groups, respectively. No significant difference was detected in the cleavage and blastocyst rates among the groups. Embryo kinetic analysis revealed a slightly shorter duration from injection to blastocyst formation in the SEXED versus NONSEXED groups (183h and 188h, respectively). Higher number of embryos are required to compare the morpho-kinetics of male and female embryos at each developmental stage. This is a preliminary report, showing the successful use of frozen sexed semen in a commercial in vitro embryo production setting and further studies are ongoing.