Successful use of sex-sorted frozen semen in a commercial OPU-ICSI program

There is increasing interest in the use of sex-sorted semen especially in certain breeds. After artificial insemination (AI) with fresh sex-sorted semen, pregnancy rates between 10% and 60% have been reported (Buchanan. et. al. Theriogenology. 2000; 53:1309-1321; Lindsey. et. al. Equine Veterinary Journal. 2002; 34:128-132; Lindsey. et. al. Animal Reproduction Science. 2005; 85:125-130). With sex-sorted frozen thawed semen the reported pregnancy rates are lower ranging from 0 – 27% (Linsey. et. al. Equine Veterinary Journal. 2002; 34:121:127; Clulow. et. al. Animal Reproduction Science; 2007; 108:287-297; Gibb. et. al. Equine Veterinary Journal. 2016; 49(2): 160-166). Limited studies have shown that although pregnancies can be achieved with ICSI using frozen-thawed sex sorted semen, cleavage rates are low (20% -30%) (Carnevale. et. al. Reproduction, Fertility, Development. 2008. 21(1) 228; Colleoni.et.al. Reproduction Fertility, Development. 2008, 21(1):228-229). Since those reports, further improvements in sex-sorting stallion semen by flow cytometry have been made. The semen from a polo stallion of proven fertility was collected and the raw semen parameters assessed with a phase contrast microscope, NucleoCounter SP-100 (Chemometec, Allerod, Denmark) and IVOS CASA system (Hamilton Thorn IVOS, Massachusetts, USA). The sample was then centrifuged through EquiPure (Nidacon, Mölndal, Sweden), the pellet recovered and extended in media for final sorting by flow cytometry (Cogent, Chester UK). The semen was selected for the X-chromosomeand frozen into 0.25ml straws with a concentration of 5 million sperm per straw. Post thaw results of the semen were 17.1% total motility, 10.1% progressive motility, 26.2% non-viable sperm and purity of 95%. The oocytes from four different mares were fertilized with the sex-sorted semen resulting in 3 embryos. Two embryos were thawed and transferred into suitable recipients. One transfer resulted in a pregnancy due in April 2023. The third embryo has not been transferred yet. The results of ICSI using frozen sex-sorted semen were compared to conventionally frozen semen used for ICSI in 23 commercial OPU-sessions (summary Table 1). There were no significant differences between ICSI with sex sorted semen from one stallion and the controls (χ2 test, P≤0.05). This study shows that frozen-thawed sex-sorted semen can be successfully used for ICSI. However, further improvement of the flow cytometry and cryopreservation protocols are needed to improve the post-thaw quality of semen.