84 PORCINE EMBRYOS UTILIZE SMALL AMOUNTS OF PYRUVATE, LACTATE, AND GLUCOSE IN VITRO

Abstract

Porcine embryo culture systems are suboptimal to the in vivo environment, and significant effort has been made to improve development to the blastocyst stage in vitro. Since metabolism of the early embryo has many similarities to the Warburg effect, our goal was to determine the role of glucose on development, gene expression, and metabolism of other energy substrates in the blastocyst stage embryo. Pig embryos were in vitro produced and cultured in MU1 containing pyruvate, lactate, amino acids, and either 0, 7.5, 15, or 250 µM glucose, N = 1164, 4 replications. There was no difference in blastocyst percentage between the 0 µM and 7.5 µM glucose (34% ± 6.5 v. 29% ± 8.2), but there was a decrease in development in response to 15 and 250 µM compared with 0 µM glucose (25% ± 8.5, 23% ± 8.7 v. 34% ± 6.5; P ≤ 0.01). Glucose transporters (SLC2A1 and SLC2A2) and hexokinases (HK1 and HK2) were analysed by qPCR to detect differences in gene expression, 3 replicates containing 10 blastocyst pools. The abundance of both HK1 and HK2 was decreased in blastocysts cultured with 7.5 µM glucose compared with 0 µM (P ≤ 0.04). Glucose transporters were not affected by glucose supplementation (P ≥ 0.5). Metabolic data were collected to determine if embryos were adjusting their energy substrate use in response to glucose. Two assays were completed to determine lactate and pyruvate consumption or release into the media by embryos, in comparison with media without embryos. In vitro-produced embryos were cultured in MU1 with 0 or 7.5 µM glucose N = 360, 4 replications. Both treatments consumed lactate, but there were no differences between treatments (6.8 ± 9.4 pmol/blastocyst/h v. 12.5 ± 1.6 pmol/blastocyst/h; P = 0.6). Blastocysts cultured in 7.5 µM glucose consumed pyruvate, whereas blastocysts without glucose produced pyruvate (–0.34 ± 0.3 pmol/blastocyst/h v. 0.73 ± 0.2 pmol/blastocyst/h; P < 0.01). It has been suggested that fructose is a more efficient replacement for glucose in pig embryo culture. Therefore, we produced pig embryos in vitro and cultured these embryos in MU1, MU1 + 2 mM glucose, or MU1 + 2 mM fructose to the blastocyst stage, 4 replications, N = 389. Again, there was a decrease in embryos that developed to the blastocyst stage in 2 mM glucose compared with MU1 control blastocysts (26% ± 5.8 v. 11% ± 2.5; P = 0.001), but there was only a trend for a decrease in development in response to 2 mM fructose (17 ± 2.3%; P = 0.06). There was no difference in total cell number between MU1, 2 mM glucose, and 2 mM fructose (30.6 ± 2.2, 30.5 ± 3.7, and 32.6 ± 3.0, respectively; P ≥ 0.9) 3 replications, N = 32. Because there is very little consumption of lactate and very low levels of pyruvate are being consumed when glucose is present, it does not appear that any of these energy substrates are major players for the developing pig embryo. Future experiments should be conducted to determine other means of energy production and metabolism in these embryos.

The research was funded by Food for the 21st Century.