In vitro maturation of equine oocytes followed by two vitrification protocols and subjected to either intracytoplasmic sperm injection (ICSI) or parthenogenic activation.

The research article is about a study examining the viability and developmental competence of horse oocytes after undergoing in vitro maturation and vitrification via the Rapid-I method. The goal of the study is to develop an effective protocol for preserving equine oocytes.

Objective and Methodology

• The purpose of this study was to establish the viability and developmental capability of equine oocytes after undergoing in vitro maturation (IVM) and vitrification (the process of turning a liquid into a glass-like solid to avoid the formation of ice crystals) using a method called Rapid-I. The aim was to establish an effective equine oocyte vitrification process to assist in preserving genetic material in horses.
• The process began with the retrieval of equine oocytes by scraping ovarian follicles from slaughtered mares. In total, 1052 ovaries were used, from which 3135 oocytes were obtained. Of these, 2853 oocytes were retrieved and 2557 underwent in vitro maturation for approximately 36 hours.
• 1202 oocytes (47%) had a first polar body after in vitro culture, suggesting successful maturation.

Experimentation and Results

• The researchers conducted three distinct experiments to assess different aspects of the oocytes’ survival and development. In the first experiment, the researchers sought to evaluate the toxicity of the vitrification media by exposing the oocytes to vitrification media without cryopreservation. The best results were obtained when IVM oocytes were exposed to EquiproVitKit media, with a survival rate of 69.5%.
• In the second experiment, oocytes were collected directly from the ovaries or after undergoing IVM. These were then vitrified using either the EquiPro VitKit or an in-house medium. Viability was evaluated by staining with fluorescein diacetate and ethidium bromide. Oocytes that were matured in vitro and then vitrified using EquiproVitKit media showed a survival rate of 63%.
• In the third experiment, IVM oocytes that had been vitrified were activated either chemically, using ionomycin in TCM-199, or artificially inseminated using ICSI. The researchers observed that the cleavage rate (an indicator of successful fertilization) was 10.2% for oocytes activated in TCM-199 medium, compared to 22.5% of oocytes activated in a different medium, G1/G2. Meanwhile, ICSI of vitrified IVM oocytes resulted in 20% embryo development to the 16-cell stage, compared to 33.3% in a control group.

Conclusion

• In conclusion, this complex series of experiments and analyses led the researchers to determine that the vitrification of oocytes after IVM using the Rapid-I method is a good method for preserving equine genetic(material). The combination of in vitro maturation and vitrification showed promising results, creating potential implications for future advancements in equine reproduction and genetical preservation.