Fertilization and Early Embryogenesis
(W303) EFFECTS OF GLUCOSE OR FRUCTOSE AND METABOLIC REGULATORS (DNP, PES, CERULENIN) ON BOVINE EMBRYO DEVELOPMENT AND LIPID ACCUMULATION.
Barcelo-Fimbres, Moises1, Seidel, George1, 1 Colorado State University, Fort Collins, Colorado
ABSTRACT- Slaughterhouse oocytes (n=1800) were matured and fertilized in vitro by standard procedures. Zygotes were cultured at 38.5oC in 5%CO2/5%O2/90%N2 in CDM-1 (similar to SOF) with nonessential amino acids, 10M EDTA, 0.5% fatty acid-free BSA, and 0.5mM glucose. After 48h, 8 cell embryos were cultured 120h in CDM-2 (CDM-1+essential amino acids and 2 mM hexose). A factorial design with glucose or fructose; and 5 treatments (0.3M phenazine ethosulfate (PES); 0.3M PES+30M dinitrophenol (DNP); cerulenin (fatty acid synthase inhibitor) 1g/mL (C1) and 3g/mL (C3); and control) was replicated 6 times. Day 7.5 blastocysts were fixed and stained with Sudan Black B to quantify lipid droplets in the cytoplasm. A digital photo at 600x of the equatorial part of the embryo was evaluated by classifying lipophilic droplet diameters as small (<2 m), medium (2 to 6 m), or large (>6 m), reported as number of lipid granules per 1,000 m2. Blastocyst rates per oocyte matured, and per 8 cell embryo (B/E) were higher (P<0.05) for fructose than glucose (30.4% vs 26.5% and 52.8% vs 47.6%). There were differences in B/E for PES, C1, PES+ DNP, and control vs C3 (P<.05): 35.8%, 32.8%, 32%, 32.8%, and 13.6%. Numbers of small droplets were different for PES, C1, and PES+DNP, vs C3 (32, 34, 38, and 55, respectively) (P<.05), but the control did not differ from other groups (P>.14). Medium size lipid droplets were lower (P<.05) for PES, and PES+DNP than C1, C3 and the control (21, 18, 28, 34, and 34, respectively). Large droplets were similar for PES, and PES+DNP; PES was lower (P<.05) than control, C1 and C3, but no difference was found between the control and PES+DNP. C3 was not different from the control, but higher than the other treatments (P<.05). There were no effects of glucose or fructose on small or large lipid droplet sizes (P>.16), but Fructose was lower for the medium size (23.6 vs 30.4) (P<.01). In conclusion, fructose produced more blastocyst and less medium size droplets than glucose. PES reduced lipid accumulation, and cerulenin did not.
KEY WORDS: in vitro, glucose, fructose, lipids