A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

SIMPLE SUMMARY: For more productive and sustainable livestock activity, various reproductive biotechnologies are being incorporated into breeding programs to accelerate genetic improvement. Among these strategies, embryo cryopreservation is a key technique for the conservation and dissemination of genetic resources while also optimizing animal production and biosafety. Though vitrification techniques are rapidly gaining acceptance due to their speed, simplicity, and feasibility, their practical applications in veterinary reproduction are limited because there is no standard protocol that facilitates warming in field conditions. Moreover, working time increases when a large number of embryos has to be cryopreserved. In-straw warming/dilution methods allow for the vitrification of embryos and their direct transfer to the uterus of recipients. In order to increase vitrification efficiency by reducing the working time and simplifying warming in field conditions, in vitro-derived cattle embryos at the expanded blastocyst stage were vitrified by using two different protocols (short equilibration vitrification and long equilibration vitrification) and in straw diluted/warmed. The short equilibration protocol improved vitrification outcomes in terms of embryo survival and hatching ability, and it improved embryo quality in terms of higher total cell number and lower apoptosis rate. A gene expression analysis of surviving embryos also indicated that the short equilibration treatment could lead to the production of more high-quality blastocysts. ABSTRACT: This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.