MODIFICATIONS MADE TO CULTURE-MEDIUM BY BOVINE OVIDUCT EPITHELIAL-CELLS - CHANGES TO CARBOHYDRATES STIMULATE BOVINE EMBRYO DEVELOPMENT

Co-culture remains a common method to support the development of bovin e embryos, derived from IVM/IVF procedures. However, the mechanism by which somatic cells confer their benefit to the developing embryo rema ins undetermined. This study therefore analysed the changes made to th e culture medium TCM-199, used in bovine embryo co-culture systems, by somatic cells and determined tile effects of specific changes in medi um composition on bovine embryo development in culture. Bovine oviduct epithelial (BOE), Buffalo rat liver (BRL) and fibroblast (3T3) cells were compared. The concentrations of glucose, L-lactate, pyruvate, ami no acids, NH4+, H+ and the gas tensions of O-2 and CO2 were measured i n TCM-199 supplemented with 10% fetal calf serum (FCS) prior to and di rectly following 48 h incubation periods with each cell type. All thre e somatic cell types modified the carbohydrate composition of the medi a in a similar manner with the greatest changes made by the BOE cells. Notable alterations were an increase in the levels of L-lactate and p yruvate and a reduction in glucose concentration, which in the case of the BOE cells, fell from 5.55 mM to 2.67 mM. In order to determine th e relevance of such changes in carbohydrate concentrations on bovine e mbryo development, modifications were made to carbohydrate levels in s ynthetic oviduct fluid (SOF) medium and their effect on blastocyst dev elopment in vitro assessed. In SOF medium supplemented with amino acid s and BSA (SOFaa), significantly more zygotes developed to the blastoc yst stage (64%; P < 0.01) than in SOFaa medium with the concentrations of glucose, D/L-lactate and pyruvate equivalent to those in TCM-199 ( 11%). Interestingly, when the levels of carbohydrates in SOFaa mimicke d those present in TCM-199 following a 48 h incubation with BOE cells, 57% of zygotes reached the blastocyst stage. This improvement was asc ribed to the reduction in glucose and increases in D/L-lactate and pyr uvate concentrations in the culture system. Results from this study de monstrate that BOE cells create an environment favourable to embryonic development. The analysis of media samples by enzymatic methods meant that only the biologically active L-isomer of lactate was quantified. However, in SOFaa, both the L-isomer and inactive D-isomer are presen t in equimolar amounts. As such, culture media in which D/L-lactate sy rup is used actually contain only 50% biologically active lactate mean ing that all D/L-lactate concentrations are reported at twice the effe ctive concentration. Therefore the effect of D/L-lactate concentration on blastocyst development was subsequently determined in this study. Blastocyst development was poor (24-36%) until the total D/L-lactate w as present in the culture system at concentrations equal to or greater than 0.82 mM. However, blastocyst cell numbers remained law (60.1 +/- 6.9 - 78.5 +/- 6.6) unfit a total D/L-lactate concentration of 3.3 mM . This data reinforces that embryo morphological appearance is not sen sitive enough to be used as the sole criterion for assessing embryo de velopment. (C) 1997 Wiley-Liss, Inc.