Survival of vitrified in vitro–produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure

Abstract

Vitrification is an alternative to slow-rate freezing for cryopreserving bovine embryos. However, this technology requires simplification if it is to be used under field conditions. The main objective of this work was to develop a new system for the direct transfer of vitrified embryos to be used under farm conditions. For this, three objectives were set: (1) to compare the effect of vitrification, using the cryologic vitrification method (CVM), and slow-rate freezing on bovine embryo development and quality; (2) to develop a one-step warming procedure for bovine in vitro–produced (IVP) vitrified (by CVM) embryos; and (3) to assess the effects on embryo survival of a new method for the direct transfer of vitrified IVP bovine blastocysts. In vitro–produced blastocysts were initially either vitrified by CVM or subjected to slow freezing to compare embryo survival and quality (experiment 1). No differences were detected between these cryopreservation techniques in terms of the survival and quality variables at 24 hours or in terms of the proteins expressed. However, at 48 hours the vitrified embryos showed higher hatching rates, greater total cell numbers, and lower apoptotic indices (P < 0.05). In experiment 2, CVM-vitrified IVP blastocysts were warmed by the conventional two-step or one-step warming procedure by incubating them at 41 °C in 0.25 M sucrose for 10 minutes, 0.15 M sucrose for 10 minutes, or 0.25 M sucrose for 5 minutes. In addition, embryo transfer (ET) was performed using vitrified embryos warmed by the one-step procedure in 0.25 M sucrose solution for 5 minutes. As a control group, IVP fresh embryos were transferred to recipient females. No differences were observed in embryo survival or total cell number between any of the warming procedures. Moreover, no significant differences for pregnancy at 60 days were found between the ET groups. In experiment 3, expanded IVP blastocysts were then either vitrified using a conventional or a modified fiber plug designed to allow direct ET after in-straw cryoprotectant (CP) dilution. They were warmed using the one-step process (0.25 M sucrose, 5 minutes) in a 0.25 mL French straw. Embryo recovery associated with the modified fibreplug system was less reliable than with the conventional system. However, no differences were seen between the systems in terms of in vitro embryo survival among those finally recovered. Finally, IVP blastocysts were vitrified using conventional fibreplugs to maintain a high embryo recovery rate, and then warmed using the one-step warming in-straw CP dilution procedure, but using an adapter with a wider opening coupled to the French straw and a heated metal chamber to protect and keep the straw at 41 °C (experiment 4). No differences were seen in embryo survival rates between the two groups. The CVM combined with this new one-step warming in-straw CP dilution procedure could be used for direct ET under field conditions.