Cryopreservation of equine oocytes using glycerol as the cryoprotectant and cumulus investment as a parameter

Abstract

A study was conducted to determine the viability of equine oocytes cryopreserved in glycerol and to determine whether stage of cumulus investment at freezing had an effect on their survival. Ovaries were collected from mares at an abattoir and transported to the lab in physiologic saline solution. Follicles were aspirated using a syringe and needle. Recovered oocytes were assigned to one of three categories based on cumulus investment. These categories were: 1) complete cumulus layer; 2) partial cumulus layer; and 3) corona radiata only. All oocytes were stained with fluorescein diacetate (FDA) to determine viability before freezing. Forty viable oocytes from each group were frozen to be evaluated post-thaw using FDA and orcein stains. None of the 120 oocytes showed fluorescent activity, thus establishing they were nonviable. The oocytes were then placed in maturation media and evaluated visually for cumulus expansion as a second check of viability. None of the oocytes exhibited cumulus expansion after exposure to the maturation media. The oocytes were then stained with orcein to determine metaphase state. None of the oocytes were in metaphase 11 after 36 h in the maturation media, further verifying that none of the oocytes were viable post-thaw. Therefore, the effect of cumulus morphology on viability of cryopreserved equine oocytes could not be determined. One viable oocyte from each group was frozen to be evaluated postthaw by transmission electron microscopy. The most significant ultrastructural differences observed in the frozen equine oocytes as compared to fresh equine oocytes were disrupted plasma membranes of cumulus cells and oocyte, loss of microvilli on oocyte, disruption of single membrane bound organelles such as endoplasmic reticulum and Golgi, migration of intact organelles away from the periphery of the oocyte and lack of cortical granule alignment at oocyte cell membrane. The protocol evaluated in this study was not successful in maintaining viability of equine oocytes throughout the freeze-thaw process as evaluated by the methods in this experiment.