114 VITRIFICATION OF BOVINE EMBRYOS IN MEDIUM WITH POLYVINYL ALCOHOL REPLACING BSA

Abstract

Embryos vitrified in medium supplemented with 4.25 μg/mL sodium hyaluronate (SH) and 0.1% polyvinyl alcohol (PVA) survived vitrification better than embryos vitrified in medium supplemented with 0.25% FAF-BSA (Walker and Seidel 2005 Reprod. Fert. Dev. 17, 153). The purpose of the present study was to determine if the small amount of SH was beneficial to in vitro survival and to examine the effects of different concentrations of PVA in vitrification solutions. Day 7 blastocysts (n = 360) were produced in vitro with semen from three bulls, two replicates each. Cryoprotectant solutions were prepared in a 2 × 3 factorial combination with two SH concentrations (0 or 4.25 μg/mL) and three PVA concentrations (0.05, 0.1%, or 0.2%). For vitrification, embryos were placed into chemically defined HEPES-buffered medium (HCDM-2) at room temperature (22–24°C) and then transferred to V1 (5 m ethylene glycol in HCDM-2) for 3 min. Next, embryos were placed in a 6 μL drop of V2 (7 m ethylene glycol, 0.5 m galactose, and 18% w/v Ficoll 70 in HCDM-2) for 45 s. During these 45 s, dilution medium (0.5 m galactose in HCDM-2) was aspirated into 0.25-mL straws, followed by the 6 μL drop of V2 plus embryos and a final short column of dilution medium. When 45 s had elapsed, the heat-sealed end of straw was dipped into liquid nitrogen to cover the embryo, and then the remainder of the straw was immersed slowly. Straws were thawed in air for 10 s and then in 37°C water for 20 s. Next, straws were shaken like a clinical thermometer four times to mix columns, and held in 37°C water for 10 min before embryos were expelled, rinsed and cultured in CDM-2 + 5% FCS. At 48 h, embryo survival (as determined by expansion of blastocysts), embryo quality (1 = excellent, 2 = fair, 3 = poor), inner cell mass (ICM) quality (1 = large and compact, 2 = clearly visible, 3 = not discernable) and blastocyst stage (5 = early, 6 = full, 7 = expanded, 8 = hatching, 9 = hatched) were evaluated and replicate averages were analyzed by ANOVA. Neither bull nor SH concentration nor PVA concentration significantly affected any response (P > 0.10). Averaged over PVA concentrations, vitrification of embryos in 0 μg/mL or 4.25 μg/mL SH resulted in similar survival rates (67% vs. 62%, respectively). When averaged over SH concentrations, 0.2% PVA had a numerically higher survival rate of blastocysts as compared to 0.1% or 0.05% (71% vs. 63% and 60%, respectively). The main effects of 0 μg/mL SH and 0.2% PVA also resulted in numerically higher, but nonsignificant improvements in quality score, ICM score and blastocyst stage as compared to the other doses of SH and PVA. Vitrification of Day 7 in vitro-produced bovine blastocysts in medium containing 0.2% PVA in the absence of SH resulted in a subclass mean of 80% embryo survival. Results of this experiment show no benefit of 4.25 μg/mL SH and that 0.2% PVA may be slightly better than 0.05% or 0.1% in terms of embryo survival. Therefore, our results indicate that 0.2% PVA can be used alone as an effective alternative to animal products in this vitrification procedure for in vitro-derived bovine blastocysts.