Comparison of vitrification protocols in immature equine oocytes
Oocyte cryopreservation would facilitate the conservation of female genetic material and allow more flexibility in the application of assisted reproductive techniques in domestic animals and endangered species. The overall success rate of this technique in the horse is low compared with other species. Therefore, further research is required to elucidate the species-specific mechanisms responsible for poor survivability following vitrification. This study aimed to evaluate the effect on maturation rate, cleavage and blastocyst development of vitrified immature equine oocytes, using a three-step vitrification protocol with ethylene glycol (EG) and dimethyl sulfoxide (DMSO); and comparing the effect of media without freezing. The vitrification protocol was designed based on the results of preliminary experiments. Oocytes were recovered from immature follicles of live mares. Oocytes were held overnight at room temperature (14-24 hrs) in a holding medium. Oocytes were then denuded and placed in a base solution (BS) composed of 20% fetal bovine serum (FBS) + M199/Hanks’ salts. Oocytes were randomly allotted to control, vitrification, and cryoprotectant agents (CPAs)-exposed groups. Control oocytes were cultured directly for in-vitro maturation (IVM). Three oocytes were exposed to a three-step vitrification protocol composed of a pre-vitrification solution (PVS) 1 (5% EG/ 5% DMSO); PVS 2 (10% EG/ 10% DMSO) during 40s each; and finally vitrification solution (VS) (17.5% EG/ 17.5% DMSO/ 3 M sucrose), during 10s. All media were diluted in M199/Hanks’ salts + 20% FBS. Oocytes were then transferred to a 75-μm sterile stainless steel mesh. The oocytes were warmed at 42 °C in the BS for 5 minutes. Oocytes from the vitrified group were plunged into liquid nitrogen, while oocytes from CPA-exposed groups were only exposed to cryoprotectants. Oocytes were then subjected to IVM, fertilization and embryo culture. Fisher's Exact Test analyzed differences in maturation, cleavage and blastocyst rates between groups. The maturation rate of vitrified and CPA-exposed groups did not differ significantly from control oocytes. However, no blastocysts were obtained from CPA-exposed and vitrified groups. Vitrification and control groups showed that immature equine oocytes could maintain viability and meiotic competence; moreover, cryoprotectant exposure did not show any blastocyst formation as compared to control. Further investigation is necessary to understand the overall physiology of equine oocytes in order to optimize the developmental capacity of embryos.
Vitrification Ovocytes immatures Cryopreservation Vitrification Equine Immature oocytes Cryoprotectants