206 Effect of L-Ascorbic Acid, Polydatin, and In-Straw Rehydration of Slow-Frozen In Vitro-Produced Bovine Embryos

Abstract

In vitro-produced (IVP) bovine embryos have poor cryotolerance due to high lipid content and reactive oxygen species levels that hinder post-thaw survival. We hypothesised that in-straw rehydration of slow-frozen embryos with sucrose and the addition of the antioxidant polydatin and l-ascorbic acid would increase post-thaw survival. The IVP embryos (n = 116) were generated in 7 replicates by aspirating oocytes from 2- to 8-mm follicles of abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 3 bulls using standard procedures, and cultured in SCF1 medium for 7 days (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Stage 7 embryos were slow-frozen using 1 of 4 protocols in a 2 × 2 factorial design: embryos were equilibrated in conventional slow-freezing media for 20 min [1.5 M ethylene glycol (EG) and 0.5 M sucrose] with 1 mm l-ascorbic acid or 1 μM polydatin, and loaded in the straw adjacent to columns of freezing medium or 0.75 M EG and 0.6 M sucrose and then seeded at −6°C, cooled at 0.5°C min−1, and plunged at –32°C. Embryos were thawed in air for 10 s followed by 30 s in 32 to 35°C water bath. Once straw columns were disrupted, embryos were allowed to equilibrate for 5 min. Subsequently, embryos were washed and placed back in culture and re-expansion was assessed at 24 and 48 h. Data (Table 1) were analysed by ANOVA with means separated by Tukey’s HSD. Results indicate that there was no main effect between the 2 antioxidants or the use of rehydration columns (P < 0.05); however, there was higher (P < 0.05) re-expansion for embryos frozen with polydatin and with rehydration column than embryos frozen with l-ascorbic acid and no rehydration column. This suggests that polydatin coupled with in-straw rehydration (0.75 M EG and 0.6 M sucrose) may improve post-thaw survival of IVP bovine embryos. Table 1.Post-thaw re-expansion rates of embryos exposed to antioxidants and in-straw rehydration (± SEM)