The effect of vitrification of immature bovine oocytes to the subsequentin vitrodevelopment and gene expression

Abstract

Immature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P < 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P < 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.

Gene expression, oocyte nuclear maturation and developmental competence of bovine oocytes and embryos produced after in vivo and in vitro heat shock

“…For each gene, cDNA dilution curves were generated and used to calculate the individual real-time PCR efficiencies by E = 10(–1/slope) – 1. Relative gene expression analysis was performed using the Relative Expression Software Tool (REST©) with the Pair Wise Fixed Reallocation Randomization Test© method (Pfaffl et al , 2002; Faheem et al , 2014) with the calculated E values introduced. The relative expression values (C t ) are presented as mean ± SEM.…”

Cryodevices developed for minimum volume cooling vitrification of bovine oocytes

“…The membrane permeability of DMSO is lower than that of EG, probably because of the involvement of different aquaporin channels (Edashige, 2016 ). In Cryotop vitrification of bovine immature oocytes, the use of EG/PG‐based VS instead of EG/DMSO‐based VS resulted in a higher IVM outcome (90% vs. 76%), but a comparable blastocyst yield (10% vs. 14% per matured oocytes; Faheem et al, 2015 ). Recently, Somfai and Hirao ( 2021 ) reported that the replacement of DMSO with PG in protein‐free VS with a few additional modifications resulted in a statistically comparable blastocyst yield from post‐warmed bovine immature oocytes (12% vs. 8%).…”

The effect of kinetic heat shock on bovine oocyte maturation and subsequent gene expression of targeted genes

“…Data analysis was performed as described by Faheem et al (2014) and Pavani et al (2016) for embryos. For comparisons between genes, data from GV stage COC's collected during winter were used as a calibrator group for relative gene expression analysis.…”