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

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.

Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos

“…Improved cryosurvival has been associated with more advanced embryonic stages on a given in vitro culture day [36]. In concordance with previous studies conducted in our laboratory [36,37], D7 blastocysts derived from cow oocytes had a significantly higher re-expansion rate than blastocysts vitrified/warmed after 8 days in culture, regardless of the vitrification treatment. In bovine species, early cleavage is associated with better embryo quality and implantation [38][39][40], while the timing of blastocyst formation has been correlated to the in-vitro developmental potential of embryos, assessed by total cell number [39], expression of genes related to embryo quality [41], and pregnancy rates [42].…”

“…Vitrification/warming has been found to have a negative effect on TCN and TE cell numbers [37] and ICM cell number [53,54]. When vitrification media were supplemented with AFPs, Liang et al [30] found a significantly higher TCN in blastocysts vitrified/warmed with AFGP8 supplementation than in the non-supplemented group.…”

“…At 24 h post-warming, expanded and hatched blastocysts surviving vitrification in each group underwent immunostaining plus the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay to quantify TCN, ICM cell number, TE cell number, and AR [37]. All steps were done at 38.5 • C, unless otherwise stated.…”

Development of Optimized Vitrification Procedures Using Closed Carrier System to Improve the Survival and Developmental Competence of Vitrified Mouse Oocytes

“…In blastocyst vitrification using the OC, changing the CPA exposure time did not affect the clinical and neonatal outcomes of human blastocysts [ 48 ], while increasing the CPA exposure time improved DNA integrity in mouse blastocysts [ 49 ]. Due to the reduced cytotoxicity and osmotic stress, short-term equilibration time improved the re-expansion and hatching rates [ 50 ] and increased the total cell number with a reduced mRNA expression of apoptosis-related genes, such as Bax and Bcl2l1 , in bovine blastocysts [ 51 ]. Although a shorter CPA exposure has been proposed to reduce CPA cytotoxicity [ 31 , 32 , 52 , 53 ], other studies have recommended a longer exposure time for human oocytes using OCs [ 54 , 55 , 56 , 57 ].…”

Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in‐straw warming of in vitro‐produced bovine embryos

“…VitTrans is a device that allows field‐warming/dilution and embryo transfer directly to female recipients. Vitrification of IVP day 7 bovine blastocysts using the VitTrans vitrification/warming device and short exposure to the CPA equilibration solution results in post‐warming outcomes comparable to those of fresh non‐vitrified blastocysts (Martinez‐Rodero et al, 2021).…”