220 RELATIONSHIP BETWEEN FOLLICULAR FLUID METABOLOME AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE

Abstract

The follicle is a unique microenvironment within which the oocyte can develop and mature to a fertilizable gamete. The aim of this study was to examine the predictive value of the intrafollicular metabolomic profile on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from the ovaries of slaughtered animals. Following measurement of diameter (5 to 8 mm), follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba et al. 2010 Reprod. Fertil. Dev. 22, 839–851). Cleavage and blastocyst rates were assessed on Day 2 and Day 9, respectively. Follicular fluid was recovered and stored at –80°C until metabolomic analysis. Only samples that cleaved and developed to the blastocyst stage (n = 10) or that cleaved and subsequently degenerated (n = 10) were analysed. Aqueous and organic compounds were isolated and profiled separately using an Agilent 7890A gas chromatograph coupled with a 5975C mass spectrometer (Agilent, Palo Alto, CA). Both aqueous and organic data sets were divided into compounds identified and quantified using external standards and compounds semiquantified relative to the internal standard only. The percentage of oocytes that cleaved after IVF was 79.1% and the percentage that developed to the blastocyst stage was 28.3%. A total of 55 aqueous compounds were analysed, of which 19 were quantified and 36 were semiquantified. Principal component analysis of the quantified data showed separation between oocytes that formed blastocysts and oocytes that degenerated (R² = 0.561). Further analysis using partial least squares–discriminant analysis generated a robust model that could predict which oocytes would form blastocysts (R²X = 0.552; R²Y = 0.874; Q² = 0.722). Analysis of variance of the data confirmed a positive correlation of L-alanine, glycine, and L-glutamate with blastocyst formation. A total of 16 fatty acids were identified and quantified, and the total saturated fatty acids, total monounsaturated fatty acids, total polyunsaturated fatty acids (PUFA), n-3 PUFA, and n-6 PUFA contents were determined. Analysis of variance of the fatty acid data showed that follicular fluid from competent oocytes was significantly lower in palmitic acid (P = 0.023) and total saturated fatty acids (P = 0.031) than in follicular fluid from degenerate oocytes. No differences in total monounsaturated fatty acids, total PUFA, n-3 PUFA, and n-6 PUFA were found between blastocysts and oocytes that degenerated. In conclusion, our data suggest that aqueous and organic compounds in follicular fluid may be predictive of oocyte developmental outcome in an in vitro embryo production system.