162INSULIN IMPROVES FREQUENCY OF CLEAVAGE AND IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN DEFINED MEDIUM

Abstract

Pre-elongation stage bovine embryos contain insulin receptor, ligands and receptors of IGF-I and IGF-II, and possibly EGF ligand. Cumulus cells and blastocysts also express EGF receptor; however, insulin is not produced (Watson AJ et al. 1992 Mol. Reprod. Dev. 31, 87–95; Tetens F. et al. 2000 Anat. Embryol. 201, 349–55; Yaseen MA et al. 2001 Reproduction 122, 601-10). Reported effects of external growth factors during bovine IVC are conflicting. The present study evaluated effects of EGF and insulin on the development of in vitro-produced embryos in a chemically defined IVC medium. IVM medium was TCM199 plus fetal bovine serum, LH and estradiol. IVF was done in Tyrodes solution containing BSA, lactate, pyruvate, heparin, penicillamine-hypotaurine-epinephrine and nonessential amino acids. At 18h post-insemination, ova were vortexed, washed and placed in IVC treatments. Modified KSOM (Yang BK et al. 1995 J. Reprod. Dev. 41, 213–18) with 1X MEM nonessential and 1X BME amino acids was the base IVC medium. Incubations were done at 39°C in a humidified atmosphere of 5% CO2 in air (IVM, IVF) or 5% CO2, 10% O2 and 85% N2 (IVC). Effects of three EGF (0, 50, 100ngmL−1; Experiment 1) or five insulin (0, 5, 10, 15, 20μgmL−1; Experiment 2) doses were tested, in five replicates, by supplementation during the entire IVC period (up to Day 9; Day 0=day of insemination). Embryos were placed in fresh medium on Day 4 post-insemination. Data were analyzed using one-way ANOVA and Tukey’s test. In Experiment 1 (n=646 oocytes), EGF treatment did not affect percentages of Day 2 cleavage (83±3, 82±3, and 81±2%, respectively; mean±SEM.) and Day 9 blastocysts (34±4, 33±4 and 33±5%, respectively) or cell number per blastocyst after Hoechst staining (95±5, 88±4 and 89±5, respectively). In Experiment 2 (n=687 oocytes), insulin improved the frequency of cleavage and percentage of expanding, hatching and total blastocysts. It also increased blastocyst cell number (see Table 1). Lack of effect of EGF on bovine IVC is in contrast to its widely reported beneficial effects in mice. In many bovine IVC studies, the beneficial effects of external insulin were seen at or after the morula stage. The present study demonstrates that insulin can influence embryonic development as early as the initial cleavage stage and before embryonic genome activation. Reseach was funded in part by J. Bennett Johnston Science Foundation. Table 1 Effects of insulin (μgmL−1) on development to >2-cell (Day 2), expanded, hatched and total blastocysts (Day 9: %±SEM) and on blastocyst cell number (Day 9: mean±SEM)