Incubation of Frozen-Thawed Semen Under Capacitating Conditions Supports Successful In Vitro Fertilization and...

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.

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.

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.

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).

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.