252 VITRIFICATION OF BOVINE BLASTOCYSTS PRODUCED AFTER OOCYTE MATURATION IN MEDIA CONTAINING FATTY ACIDS

Abstract

High concentrations of non-esterified fatty acids (NEFA) during negative energy balance (NEB) in high yielding dairy cows have been proven to be partially responsible for reduced fertility. This hypothesis has been tested by the addition of NEFAs to in vitro maturation medium at concentrations present in follicular fluid during NEB. We aimed to evaluate whether high concentrations of palmitic acid (C16:0) (PA), stearic acid (C18:0) (SA), or oleic acid (C18:1) (OA) during oocyte maturation could have a carry-over effect on embryo quality and could subsequently affect embryo cryotolerance. Cumulus–oocyte complexes (n = 4600) were matured in serum-free TCM199 plus epidermal growth factor (EGF, 20 ng mL^–1; negative control), supplemented with ethanol alone (positive control) or with 0.133 mmol L^–1 PA, 0.067 mmol L^–1 SA, or 0.200 mmol L^–1 OA (NEFAs dissolved in ethanol). The three NEFAs were tested separately in 4 replicates for PA and 5 replicates for OA or SA. Each fatty acid tested per replicate including a negative and a positive control group. After the embryos were cultured for 7 days in SOF medium, the number of blastocysts was recorded and classified as expanded, hatching, or hatched. Then, blastocysts were cryopreserved by open pulled straw vitrification using the two-step approach described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Vitrified warmed embryos were cultured in groups of <25 per 50-μL droplet of modified SOF medium with 5% fetal calf serum (FCS) under mineral oil for 48 h and examined for re-expansion and hatching. The percentages of survival in the different treatment groups were analyzed using logistic regression analyses, including the effect of replicates. Survival or not was included as the dependent variable and group was the independent variable. For every fatty acid a separate model was used. For all analyses, differences were considered to be statistically significant at the P < 0.05 level. Addition of OA to in vitro maturation media had no significant effects on cryotolerance of embryos. However, addition of PA or SA to in vitro maturation media (Table 1) significantly (P < 0.05) decreased the survival of bovine blastocysts from 79% in the positive control to 57% in PA and from 61% to 53% in SA. The results of the present study indicate that maturation of oocytes in the presence of NEB-associated concentrations of PA and SA can have carry-over effects on embryo quality, leading to reduced cryotolerance. We suggest that elevated NEFA concentrations in the follicular fluid may be one of the factors through which NEB exerts its negative effects on fertility in high yielding dairy cows.

Table 1. Survival percentage (mean ± SD) of vitrified expanded bovine blastocysts matured in palmitic acid (C16:0) or stearic acid (C18:0)

The authors thank J. Mestach and G. Spaepen for their excellent technical support. This research was supported by the Ministry of the Flemish Community, Belgium, in cooperation with the Ministry of Higher Education, Egypt.