Klaartje Broothaers, Daniel Angel‐Velez, Fabiola Le Gaffric Molto, Mohamed Hedia, Tine De Coster, Jan Govaere, Ann Van Soom, Björn Menten, Katrien Smits
BACKGROUND: In 2022, a repeatable protocol for in vitro fertilization (IVF) using fresh semen was established in horses. This facilitated successful capacitation of equine semen allowing to explore novel applications. OBJECTIVES: We aimed to extend this technique to IVF with frozen-thawed semen and intracytoplasmic sperm injection (ICSI), and determine the outcome parameters such as blastocyst production and euploidy rates. MATERIALS AND METHODS: A total of 221 oocytes were subjected to either IVF with frozen-thawed semen, ICSI with frozen-thawed semen incubated under capacitating conditions (ICSI cap) or control ICSI with washed frozen-thawed semen. Cleavage and blastocyst rates were assessed and compared across the three groups using one-way ANOVA. Shallow whole genome sequencing was performed on embryos obtained from IVF and ICSI cap. RESULTS: We established a repeatable protocol for IVF with frozen-thawed semen resulting in higher blastocyst rates per collected oocyte (22.4%) when compared to control ICSI (16.4%) (p = 0.048). Furthermore, the use of semen incubated under capacitating conditions for ICSI resulted in higher blastocyst rates than washed sperm, with 69.0% versus 50.0% blastocysts per cleaved embryo (p = 0.03) and 27.8% versus 16.4% blastocysts per collected oocyte (p = 0.04), respectively. It also yielded higher blastocyst rates per cleaved embryo than IVF, with 69.0% versus 45.9% (p = 0.04). The average day of blastocyst formation was not different between the three groups (p = 0.73). Shallow whole genome sequencing revealed no differences in aneuploidy rates between IVF (1/17) and ICSI cap (0/18) (p = 0.49). CONCLUSION: The incubation of sperm under capacitating conditions for use in ICSI or IVF with frozen-thawed semen may represent a novel method to improve the clinical efficiency of equine IVP embryos, without affecting aneuploidy rates.