P-174: Vitrification of bovine oocytes: Parthenogenetic development as a bioassay for oocyte survival and quality

Abstract

The aim of this study was to compare two different vitrification and thawing protocols for oocyte cryopreservation and the overall efficiency for oocyte survival and chemical activation using parthenogenetic development as a bioassay for oocyte quality. Prospective, comparative, in-vitro animal study. Commercially obtained bovine oocytes (after approximately 20hr of maturation) were placed in 4mg/mL hyaluronidase for granulosa cell removal. Selected oocytes at metaphase II were vitrified and thawed using two commercially available products and methodology; the Irvine protocol (Irvine Scientific, Santa Ana, CA) or the Reprogen protocol (Reprogen, Ltd., Limassol, Cyprus). Both protocols employ similar solutions for vitrification and thawing. However, the Irvine protocol requires more procedural steps than the Reprogen protocol. Furthermore, the Irvine protocol uses special thin cryotips for cryostorage of two oocytes each, whereas the Reprogen protocol uses conventional cryostraws allowing the storage of up to eight oocytes. Frozen-thawed intact oocytes were chemically activated with 5μM ionomycin and 1.9mM 6-dimethylaminopurine and cultured in-vitro. Fresh oocytes at metaphase II were activated and cultured under the same conditions and served as controls. Results are summarized in Table 1. Briefly, using the Irvine or Reprogen vitrification-thawing protocol, 92.5% or 93.1% of bovine oocytes survived and 78.3% or 75.0% could be activated chemically. Parthenogenetic embryos developed at a rate of 44.8% or 40.7%. No statistically significant difference (Chi-square test) for oocyte survival (p=0.7) and parthenogenetic development (p=0.8) was observed between these two protocols. Fresh bovine oocytes (controls) were successfully activated (82.1%) of which 56.2% advanced parthenogenetically.Tabled 1 Chemical oocyte activation and subsequent parthenogenesis can be used as a bioassay to evaluate the viability of frozen-thawed oocytes in comparison to fresh oocytes. Both vitrification-thawing protocols seem to be equally efficient in maintaining oocyte survival and parthenogenetic development. However, the Reprogen protocol is a more simplified method requiring fewer steps and equipment. Furthermore, the Reprogen protocol allows for the storage of more oocytes in a single cryostraw and can therefore be considered as an economically better option for oocyte vitrification.