96 VITRIFICATION OF PORCINE OOCYTES — A NEW APPROACH TO GAMETE CRYOPRESERVATION

Abstract

Cryopreservation of porcine oocytes has a special importance in reproductive biotechnologies owing to the high lipid content of the oocytes. The objective of this study was to investigate the efficiency of a new and simple method of oocyte vitrification for cryopreservation of porcine oocytes. Abattoir ovary-derived prepubertal porcine oocytes at germinal vesicle (GV) or metaphase II (MII) stage were exposed for 10-15 min to vitrification solution I that contained 4% (v/v) ethylene glycol (EG) in either TCM-199 supplemented with 20% FCS or phosphate-buffered NCSU23 medium supplemented with 0.4% BSA. Oocytes were then washed thrice in EG (35%; v/v)-based vitrification solution II containing sucrose (13.7%; w/v) and polyvinyl pyrrolidone (5%; w/v) and placed in groups of 25-50 oocytes per 1-2 µL droplet onto aluminum foil kept directly over the liquid nitrogen (LN₂). After visually observed vitreous formation, they were transferred to cryovials and directly plunged into LN₂ for storage. Thawing was performed in sucrose- or EG-based media. After warming, the viability was assessed by fluorescein diacetate (FDA) staining and in vitro maturation (IVM) of GV-stage oocytes was determined based on cumulus expansion and polar body extrusion. In addition, the developmental capacity of oocytes was assessed by in vitro fertilization (IVF). Effects of pretreatment with cytochalasin B (CB) on the post-thaw viability, nuclear maturation, and developmental capacity were also investigated. The results demonstrated that the rate of oocytes having normal morphology was more than 90% in all groups. Viability based on esterase enzyme activity and oolemma integrity, as determined by FDA staining, ranged from 80% (without CB) to 86% (with CB) for MII-stage oocytes and 79% (without CB) to 90% (with CB) for GV-stage oocytes. The IVM rate of pelleted GV-stage oocytes reached 70% based on cumulus expansion and 55% based on polar body extrusion. Sucrose-based thawing media was found to be superior to EG-based media. Although the vitrification method resulted in a high survival rate based on the morphological appearance, FDA staining, and IVM rate, developmental rates of pelleted oocytes were found to be compromised. However, we believe that there is a scope for improving the developmental rates with further modifications in the method.