Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection.

The study examines how different chemicals affect the fertilization of horse oocytes (egg cells) when used in a process called intracytoplasmic sperm injection. Found to be most beneficial was a chemical called thimerosal, which increased activation of the oocytes and promoted normal fertilization.

Research Method

• The researchers collected cumulus-oocyte complexes (a structure composed of an oocyte, its surrounding cumulus cells, and the attached mural granulosa cells) from horse ovaries sourced from an abattoir.
• These complexes were then matured in vitro (in a controlled, artificial environment) for around 40-44 hours within a medium called TCM199.
• Once matured to chemically active metaphase II stage, each oocyte is injected with a single, immobilized sperm from a stallion.
• Post-injection, the researchers exposed the complexes to one of four treatments, or left untreated as a control. The treatments were: 10 micromol ionomycin l(-1) for 10 minutes, 7% ethanol for 10 minutes; 100 micromol thimerosal l(-1) for 10 minutes; 250 micromol inositol 1,4, 5-triphosphate l(-1), and lastly, no treatment (control).

Results and Assessments

• Following a further 18-20 hours of culture after treatment, the researchers assessed whether the oocytes had activated and progressed to the second anaphase-telophase stage and formed a female pronucleus.
• The results indicated that thimerosal was the most effective activation agent, with 79% of treated oocytes successfully activating, significantly higher than other treatments.
• The following proportions of oocytes cleaved to the two-cell stage (one of the early stages of embryo development) approximately 24-30 hours post sperm injection within each group: 35% for ionomycin treatment, 26% for ethanol, and 48% for thimerosal.
• No such cell cleavage occurred among the control group or those treated with inositol 1,4, 5-triphosphate.
• Indications of successful fertilization were observed in 29% of ionomycin-treated, 40% of ethanol-treated, and 64% of thimerosal-treated oocytes.

Conclusion

• This study demonstrates that equine oocytes can be successfully “activated” with chemical stimulants such as ethanol, ionomycin, thimerosal, and inositol 1,4,5-triphosphate, with thimerosal showing the highest success rates.
• Furthermore, these results indicate that thimerosal is particularly effective in both promoting oocyte activation and encouraging normal equine oocyte fertilization.
• Importantly, this study suggests that chemical activation may also stimulate parthenogenetic cleavage of oocytes (development of an egg into an organism without fertilization) without there being a need for changes in the sperm head; however, further research is likely needed to fully confirm and understand this outcome.