Equine in vitro fertilization with frozen-thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes.

The research investigates the viability of using frozen-thawed horse sperm in in-vitro fertilization (IVF) and shows the process brings about changes conducive to fertilization, resulting in successful fertilization at a shorter incubation period.

Methods and Procedures

• The scientists previously found success in horse IVF using fresh sperm that had been left to incubate for a lengthy period of 22 hours before being combined with oocytes (egg cells).
• In this study, the potential of performing equine IVF with frozen-thawed sperm was evaluated.
• A special apparatus known as a sperm separation device (SSD) was employed to select sperm. This method led to significantly improved fertilization rates compared to the swim-up or colloid centrifugation selection techniques.
• Several pre-incubation durations, including 15 minutes, 3 hours, 6 hours, and 9 hours, were used in the experiment.

Results and Observations

• The highest fertilization rate of 73.3% came from sperm that had pre-incubated for 9 hours. This defied expectations, as the rate eclipsed those found in sperm left to incubate for shorter and longer timeframes. Notably, the 9-hour pre-incubation rate was statistically superior to those garnered from 15-minute and 3-hour spans.
• Diverse freezing extenders yielded differing fertilization rates, with a notable disparity recorded (45.9% vs. 85.5%).
• Using this technique, blastocysts (early-stage embryos) were produced from frozen-thawed semen from three stallions. Of the 9 resulting blastocysts transferred to mares, 7 developed into embryonic vesicles.
• A rise in the incidence of anti-protein tyrosine phosphorylation staining of the total sperm tail was observed throughout the pre-incubation.
• Sperm cells with and without said staining in the tail were found to bind to the oocyte cumulus following their joint incubation.

Conclusion

• The study concluded that frozen-thawed equine sperm could effectively fertilize oocytes after a reduced pre-incubation window of 9 hours, implying the freezing and thawing induced capacitation-related (capacity for fertilization) changes.
• The researchers also found pre-incubation of sperm may affect how frozen-thawed sperm acquire the ability to fertilize, a mechanism that might differ from that of fresh sperm.