146 THE EFFECT OF CULTURE TEMPERATURE ON THE CLEAVAGE, DEVELOPMENT, AND GENE TRANSCRIPTION PATTERNS OF BOVINE EMBRYOS

Abstract

Bovine oocytes matured and fertilized at 39°C had significantly higher rates of fertilization than at 37°C (Lenz et al. 1983 Biol. Reprod. 29, 173–179). However, embryo culture temperature that may affect molecular composition and metabolism of embryo membranes was not investigated. The objective of this study was to determine the effect of two culture temperatures, 37 and 39°C on the cleavage, in vitro development, and gene transcription patterns in bovine embryos. A total of 794 oocytes (5 replicates) were matured in TCM-199 medium containing 10% FCS and 10 ng/mL epidermal growth factor and inseminated with 1 × 10⁶/mL spermatozoa in groups of 50 in 250 μL in Fert-Talp supplemented with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 μg/mL heparin at 39°C. After 24 h of oocyte-sperm co-incubation, presumptive zygotes were divided into two groups and cultured at 37 or 39°C under paraffin oil and 5% CO₂ in humidified air. Embryos were cultured in SOFaa medium supplemented with 4% BSA in 50-μL drops (25 zygotes per drop). Cleavage rates were recorded on Day 2 and zygote development on Days 4, 7, 8, and 9. At least 5 blastocysts from each replicate from each treatment were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 6 groups of pools of 5–7 embryos. A total of 11 developmentally important mRNA transcripts were examined. The quantification of all gene transcripts was performed by real time quantitative RT-PCR in three replicates. Embryo development was analyzed by chi-square analysis and data on mRNA expression by one-way repeated-measures ANOVA. There was no effect of culture temperature on embryo cleavage (65.2 and 72.0% for 37 or 39°C, respectively) but at Day 4 significantly (P < 0.05) more embryos developed to <8 cells at 39°C (32.5%) than at 37°C (25.9%). There was no difference in the total number of blastocysts produced in either temperature (22.4% at 37°C and 22.1% at 39°C), and significantly (P < 0.05) more zygotes that were at <8-cell stage at Day 4 progressed to the blastocyst stage at 37°C (86.4%) than at 39°C (66.6%). Transcript levels for genes related to response to stress (SOX, IFNτ) and glucose metabolism (Glut-1, G6pd) were higher (P < 0.05) in the blastocysts cultured at 39°C, and levels of Glut-5 (glucose metabolism) and Oct-4 (factor related to pluripotency) were higher (P < 0.05) in the blastocysts cultured at 37°C. No difference was found in mRNA transcription of genes related to apoptosis (BAX), response to heat (Hsp70), compaction (Ecad, Na/K, DcII), and cell connection (Cx43) at either temperature tested. It can be concluded that culture temperature may affect embryo molecular composition and the kinetics of embryo development.