157 EFFECTS OF FETAL CALF SERUM OR PHENAZINE ETHOSULFATE AND FRUCTOSE OR GLUCOSE ON EMBRYONIC DEVELOPMENT AND LIPID ACCUMULATION OF BOVINE EMBRYOS

Abstract

Slaughterhouse oocytes (n = 6222) were maturated in a chemically defined medium (CDM) similar to SOF plus 0.5% fatty acid-free BSA (FAF-BSA) and hormones (M-CDM) for 23 h at 38.5°C in 5% CO₂ in air. Oocytes and frozen-thawed sperm, centrifuged through a Percoll gradient, were co-cultured for 18 h in F-CDM (CDM + heparin). Zygotes were cultured at 38.5°C in 5% CO₂/5% O₂/90% N₂ in CDM-1 (CDM + nonessential amino acids, 10 μM EDTA, 0.5% FAF-BSA, and 0.5 mM fructose or glucose in Expt 1 and glucose in Expt 2). In both experiments, after 48 h, 8-cell embryos were cultured 135 h in CDM-1 (CDM-1 + essential amino acids, no EDTA, and 2 mM fructose or glucose). A factorial design with two hexoses and three additives in CDM-2 (control; 10% fetal calf serum (FCS); and 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH) was used for both experiments, each replicated eight times. For Expt 1, Day 7.5 blastocysts were fixed and stained with Sudan Black B to quantify cytoplasmic lipid droplets. A digital photo at 600× of the equatorial part of the embryo was evaluated by classifying lipophilic droplet diameters as small (S, <2 μm), medium (M, 2 to 6 μm), or large (L, >6 μm), reported as number of lipid droplets (LD) per 1000 μm². Data were analyzed by ANOVA. For Expt 1, 8-cell embryo production per oocyte matured was not affected by fructose or glucose (P > 0.1) (70 vs. 68%, respectively); however, blastocyst rates per oocyte matured (B/O) and per 8-cell embryo (B/E) were higher (P < 0.01) for fructose than glucose (Table 1). There were no differences between control, PES, and FCS (P > 0.1) for B/O, or B/E. For Expt 2, B/O and B/E were higher (P < 0.01) for fructose than for glucose. No differences were found for additives (P > 0.1) control, FCS, or PES for B/O or B/E. There was an interaction (P < 0.05) between additives and hexoses for blastocyst production, because the benefit of fructose compared to glucose was greater for controls than for FCS or PES (means not presented). Accumulations of each size of LD were less for PES (P < 0.05) than for control and FCS. Control and PES were lower than FCS (P < 0.05) for S, M, and L droplets. There was no effect of fructose or glucose (P > 0.1) on numbers of S, M, or L droplets (Table 1). In conclusion, fructose produced more blastocysts than glucose after 8-cell development, but there was no hexose effect either before this stage or in lipid accumulation. PES reduced and FCS increased lipid accumulation relative to controls.

Table 1. Main effects of additives and hexoses on development of bovine embryos (±SE)