Intracytoplasmic sperm injection of in vitro-matured equine oocytes.

This research explored the effectiveness of Intracytoplasmic Sperm Injection (ICSI) on horse oocytes matured in a laboratory setting. The study found that the procedure can result in fertilization and early embryo development, but the rate of cell division was not substantial.

Experiment Process

• The team of researchers used horse oocytes (eggs) that were aspirated from ovaries obtained from a slaughterhouse. These oocytes were matured in an in vitro environment for 36 hours at a temperature of 38 degrees Celsius.
• The process of intracytoplasmic sperm injection (ICSI) was performed using thawed stallion semen. The sperm was purified using a technique known as “swimup” in a medium containing human serum albumin.

After ICSI

• Post sperm-injection, the oocytes were further either cultured in vitro for a period of 10, 20, or 72 hours; transferred to the oviducts of pseudopregnant mice; or transferred to a mare that had been synchronized, after an initial bout of in vitro culture.
• These transferred ova were retrieved after a period of 72 hours. All ova were then fixed, stained, and processed for both light and transmission electron microscopy examinations.

Observations and Results

• After 10 hours of injection, they observed the formation of a single pronucleus in 2 out of the 12 presumptive zygotes along with abundant cortical granules in the subplasmalemmal region, an area just beneath the cell membrane.
• However, 20 hours post-injection, they observed 2 pronuclei in 6 of the 12 injected oocytes, a fertilization rate of 50%. Almost all cortical granules had been released by this stage.
• The cleavage rate, a significant stage in early embryonic development signifying successful fertilization and the onset of development, in vitro was 16% after 72 hours in culture. The embryos developed up to the 6 to 8 cell stage.
• Despite the successful induction of fertilization and early embryonic development in vitro, the in vivo cleavage rate was low, with only one out of ten recovered ova having divided to the 2-cell stage.

Conclusion

• In conclusion, the researchers found that intracytoplasmic sperm injection of equine oocytes resulted in successful fertilization, including pronucleus formation and cortical granule release, indicating the activation of the oocytes.
• However, these promising signs were not followed by substantial cleavage of the zygotes, indicating that further improvements in the technique may be necessary to increase the success rates of ICSI in equines.