First attempts for vitrification of immature oocytes in donkey (Equus asinus): Comparison of two vitrification methods.

This study looks at the preservation of immature donkey oocytes using cryopreservation in order to contribute to genetic banks for endangered donkey breeds. Two vitrification methods were compared in this study, one using INRA-Freeze™ medium and the other using TCM199-Hepes supplemented with bovine serum albumin.

Research Context

• With poaching, habitat loss, and competition for food resources, many wild donkey breeds are facing severe threats. Additionally, the rise of mechanization in agriculture and transport put domestic donkey breeds at risk of extinction. To combat this, creating genetic banks for these endangered donkey breeds is of pressing importance.
• Cryopreservation, a process that preserves cells or whole tissues by cooling them to sub-zero temperatures, could be an effective tool in storing female genetics. Specifically, the preservation of immature jennies oocytes is considered in this study.

Methodology

• The aim of this research was to determine the conditions required for the vitrification of immature donkey oocytes, using equine oocytes as a control.
• Researchers collected asine and equine immature cumulus-oocyte complexes through transvaginal ultrasound-guided follicular aspiration. They then flushed these to obtain oocytes surrounded only by corona radiata.
• These oocytes were then exposed to increasing concentrations of dimethyl sulfoxide, ethylene glycol, and sucrose as cryoprotectants. Vitrification was performed either in a Solution of INRA-Freeze™ medium or TCM199-Hepes supplemented with bovine serum albumin.
• Then, the oocytes were warmed in decreasing concentrations of sucrose and processed for in vitro maturation

Results

• The researchers found a recovery rate of 48% for jennies oocytes and 42% for mares oocytes. This translates to an average of 4.8 oocytes per female for jennies and 3.5 oocytes per female for mares.
• It was discovered that none of the jennies re-warmed oocytes matured when exposed to cryoprotectants in INRA-Freeze™ medium, whereas 24% of the mares re-warmed oocytes reached metaphase II after in vitro maturation.
• However, when oocytes were exposed to cryoprotectants in TCM199-Hepes-BSA medium, 33% of the jennies re-warmed oocytes matured.

Conclusion

• In conclusion, the researchers developed a method for the vitrification of immature oocytes from jennies that allows in vitro maturation of the vitrified-warmed asine oocytes. This provides a potential solution to the preservation of female jenny genetics in the face of the endangerment faced by their breed.
• However, their competence for fertilization and development has yet to be determined.