73 Fatty Acid Supplementation in Culture Medium with Reduced Nutrient Concentrations Improves Bovine Blastocyst Development Compared with Standard Culture Medium

Lipids are a potent source of cellular energy and are metabolized within mitochondria via fatty acid β-oxidation, a process that also requires carnitine. Embryos metabolize lipids during pre-implantation development, but relatively little is known about the effect of fatty acid supplementation for early bovine embryogenesis in culture. The objective of this study was to evaluate the effect of lipid supplementation (via albumin) and l- carnitine(C; 5 mM) during embryo culturein a novel medium with reduced concentrations of nutrients, compared with our standard culture medium (control). Following in vitro maturationand IVF, zygotes were cultured using a serum-free sequential media system (0-72 h step 1; 72-168 h step 2). Concentrations of salts, bicarbonate, and protein [2.5 mg mL−1 fatty acid-free (FAF) or fraction V (FrV) BSA] were the same in all treatments to maintain consistent osmolarity and pH. Nutrients (glucose/fructose, citrate, lactate, pyruvate, amino acids, vitamins, and EDTA) were diluted to 6.25% of control. In addition to the control medium (100%+FAF; n = 587), experimental treatments included 6.25%+FAF+C (essentially lipid free; n = 573) and 6.25%+FrV+C (lipid rich; n = 585). Following in vitro culture (7 reps), hatching blastocystswere stained to determine inner cell mass(ICM; SOX2+) and trophectoderm (TE; CDX2+) cell numbers. Lipid content of single expanded blastocystswas determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS). Data (mean ± SEM) were analysed by ANOVA. Embryo cleavage did not differ between treatments. Blastocystdevelopment (per cleaved embryo) was higher (P < 0.05) after culture in lipid rich (38.3 ± 1.5%) compared with control (29.6 ± 2.2%) and lipid free (28.1 ± 3.6%). Blastocysthatching was reduced (P < 0.05) in lipid free (1.4 ± 0.7%) but not in lipid rich (5.2 ± 1.7) compared with control (9.8 ± 2.1). However, blastocystsdeveloped in lipid rich and lipid free had reduced cell numbers compared with control: TE, 98.7 ± 5.9 and 98.8 ± 9.1 v. 160.3 ± 9.0; ICM, 19.2 ± 2.9 and 25.2 ± 6.1 v. 43.3 ± 4.0; and total cell number, 117.9 ± 7.3 and 124.0 ± 8.7 v. 203.6 ± 10.2, respectively. Analysis by GC-MS identified 40 annotated lipids (i.e. triacylglycerols and phosphatidyl cholines) that were significantly reduced in blastocystscultured in lipid rich compared with control. In summary, blastocystdevelopment was significantly improved after supplementation of fatty acids and l- carnitineto a medium with reduced nutrient concentrations. The mechanism underlying this phenomenon may be related to increased lipid metabolism in the low nutrient environment. Although more embryos developed in this novel medium, these blastocystshad reduced cell numbers even though blastocystexpansion and hatching were not affected. This reduced nutrient medium may provide an experimental model in which to independently studypathways controlling cell proliferation and blastocystdevelopment. Future studies will investigate whether embryo cell number can be rescued while maintaining improved blastocystdevelopment.