Developmental capacity of equine oocytes matured and cultured in equine trophoblast-conditioned media.

The research explores the most effective culture media for the in vitro maturation of equine oocytes and in vitro culture of zygotes. Several media was used, and the results highlighted that, although certain types resulted in higher cumulus expansion, there were no significant differences in fertilization, cleavage rates, and development stages among treatments.

Research Methodology

• The research involved using Cumulus-oocyte complexes (COCs) obtained from ovaries derived from a slaughterhouse.
• COCs were washed and cultured in various media: TCM-199, F10-DMEM, or c-F10-DMEM.
• c-F10-DMEM was created by conditioning F10-DMEM medium with the culture of an equine trophoblast monolayer for 3 or 4 days.
• All media included follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and 10% fetal calf serum (FCS).
• After 28 to 30 hours, the expansion of cumulus cells was scored from 0 (no expansion) to 4 (fully expanded).
• Oocytes with a visible first polar body were chosen for subsequent procedures and cumulus cells were removed using enzyme hyaluronidase.
• About one-third of the zona pellucida, the membrane surrounding the oocyte, was cut using a fragment of a razor blade, a process called partial zona removal.

Fertilization and Result Analysis

• Fertilization involved washing fresh stallion semen in BGM3, a modified Tyrode’s medium, and capacitating with c-AMP and ionomycin, followed by the addition of sperm to the oocytes at a concentration of 10(6) spermatozoa/mL.
• After 20 hours, some presumptive zygotes were stained for visualization while the rest were cultured further in 100% TCM-DMEM conditioned medium.
• Cumulus expansion was found to be higher in F10-DMEM and c-F10-DMEM, as compared to the TCM-199 control. However, the formation rates of the polar body showed no significant difference among the treatments.
• Fertilization rates of oocytes matured in the three different media also did not show significant disparity.
• Similarly, the rates of cleavage, development to morula, and development to blastocyst stages, did not differ significantly among the treatments.
• Embryos derived from the process were transferred to recipient mares, but unfortunately, none resulted in a continued pregnancy.

Conclusion

• This study effectively scrutinized different culture media for the in vitro maturation of equine oocytes and the culture of zygotes.
• Despite the differences in cumulus expansion across media, there were no significant variations in terms of polar body formation, fertilization rates, cleavage rates, and development stages.