28 Evaluation in vitro of two protocols of vitrification from alpaca (Vicugna pacos) embryos

Abstract

The reproductive efficiency of South American camelids as the alpaca is low, with a few number of animals having a good genetic characteristic. The transfer of cryopreserved embryos has great potential to disseminate valuable genetic, but the suitable protocol for such cryopreservation still needs to be developed. In this study, two protocols of vitrification of alpaca embryos were tested. Day 6.5 post-mating, embryos (n = 66) were recovered from 14 female alpacas through a non-surgical technique and classified according to the characteristics of old world camelids reported by Skidmore et al. 2004 (Reprod. Fertil. Dev. 16, 605–609). Only quality 1 and 2 embryos were used for the study. They were placed together in 50-µL drops of holding medium for 30 min and transferred to a 100-µL drop of equilibration solution 1, consisting of 7.5% (v/v) ethylene glycol (EG) + 0.25 M sucrose. After 1 min, embryos were transferred to equilibration solution 2, consisting of 15% (v/v) EG + 0.5 M sucrose. After 2 min, embryos were transferred into 2 consecutive drops of vitrification solutions A [SA: 30% (v/v) EG + 1 M sucrose] for 20 s each, then in 2 other drops of vitrification solution B [SB: 30% (v/v) EG + 3% glycerol + 1 M sucrose] for 20 s each. Thereafter, embryos were quickly loaded into open pulled straws (OPS) in a volume of 10 µL and then plunged into liquid nitrogen. For warming, the OPS were held in air for 5 s and subsequently thawed at 37°C for 50 s. Straws were emptied into 1 mL of prewarmed holding medium solution (HMS1) containing 1 M sucrose for wash and the thawed blastocysts were transferred into a second 1 mL of prewarmed HMS1. After 5 min incubation at 37°C, the blastocysts were transferred into 1 mL of warmed Holding medium solution 2 (HMS2) containing 0.5 M sucrose maintained at room temperature (∼24°C) for evaluation. Data were analysed by the Chi-squared test. Post-thaw embryo expansion results were 81.3% and 58.8% for SA and SB (P < 0.05), respectively. Post-thaw embryo quality (1 and 2) were found at 62.5% and 29.1% with SA and SB, respectively (P < 0.05). In conclusion, the vitrification of alpaca embryos with the ethylene glycol:sucrose solution results in better post-thaw outcomes than the ethylene glycol:sucrose:glycerol. Further experiments with embryo transfer are needed.

This research was funded by FONDECYT project no. 149-2017.