152 EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO
N.W.K. Karja A , S. Medvedev B , D. Fuchimoto B , A. Onishi B , M. Iwamoto C , T. Otoi B and T. Nagai BA Laboratory of Animal Reproduction and Biotechnology, Department of Veterinary Sciences, Yamaguchi University, Yamaguchi, Japan;
B Developmental Biology department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
C Prime Tech Ltd., Hangzhou, Zhejiang, China. email: taku@affrc.go.jp
Reproduction, Fertility and Development 16(2) 198-198 https://doi.org/10.1071/RDv16n1Ab152
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48 h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17 mM sodium pyruvate, and 2.73 mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55 mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118 h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48 h, 53 h, 58 h, or 63 h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72 h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24 h or 48 h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P < 0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P > 0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58 h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P < 0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63 h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P < 0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58 h of culture for the in vitro development of pig embryos to the blastocyst stage.
