The relationship between grade and metabolism for in vivo and ICSI derived embryos

Abstract

The objective of this study was to describe the metabolism of glucose, pyruvate and lactate by in vitro- (ICSI derived) and in vivo-derived equine embryos and the relationship with embryo grade and sex. During the course of this study, a simplified embryo grading system was developed for both in vivo- and in vitro-derived embryos that proved to be significantly associated with both embryo metabolism and survival rates following transfer into a recipient mare. A total of 15 in vivo derived embryos and 32 ICSI-derived embryos were cultured in protein free synthetic oviduct fluid (SOF) for 24h between day 8 and day 9 of development. Aliquots of 10µL of media before culture (t=0) and spent media after culture (t=24h) were snap frozen for subsequent analysis by GC-Mass Spectrometry for glucose, lactate and pyruvate. The embryos were photographed and classified as grade 1, 2 or 3 based on the percentage of extruded debris between the trophoblast and the zona pellucida. In this study the sex of the embryos (in vivo n = 5, ICSI n = 25) was determined by PCR. At day 8 of development, the embryo metabolism of glucose, pyruvate and lactate in relation to embryo origin (in vivo vs in vitro), grade and sex wasevaluated following 24h of culture in protein free media. Overall, there was a positive correlation between embryo volume and lactate peak area (r2 = 88.2%, p ≤ 0.001) and a negative correlation with both glucose peak area (r2 = 29.8%, p =0.023) and pyruvate peak area (r2 = 46.2%, p =0.003) in the spent media. These results illustrated that as embryo volume increased, the amount of glucose and pyruvate in the media decreased and the production of lactate increased. After accounting for embryo volume, general linear model analysis revealed an effect of embryo grade (p =0.031), but no significant effect of embryo sex or origin on lactate consumption over 24h. Grade 2 embryos, however, consumed more lactate than Grade 1 embryos (p = 0.029). In conclusion, the metabolic relationships between glucose, lactate and pyruvate were described for both in vivo- and in vitro-derived equine embryos in this study. Understanding the optimal metabolic conditions for in vitro-derived embryos in comparison with in vivo-derived embryos will enable the development of models to investigate early embryonic death and to optimize pregnancy rates in the mare.