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 \u3b2-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\u2009mM) during embryo culture in a novel medium with reduced concentrations of nutrients, compared with our standard culture medium (control). Following in vitro maturation and IVF, zygotes were cultured using a serum-free sequential media system (0-72\u2009h step 1; 72-168\u2009h step 2). Concentrations of salts, bicarbonate, and protein [2.5\u2009mg\u2009mL 121 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\u2009=\u2009587), experimental treatments included 6.25%+FAF+C (essentially lipid free; n\u2009=\u2009573) and 6.25%+FrV+C (lipid rich; n\u2009=\u2009585). Following in vitro culture (7 reps), hatching blastocysts were stained to determine inner cell mass (ICM; SOX2+) and trophectoderm (TE; CDX2+) cell numbers. Lipid content of single expanded blastocysts was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS). Data (mean\u2009\ub1\u2009SEM) were analysed by ANOVA. Embryo cleavage did not differ between treatments. Blastocyst development (per cleaved embryo) was higher (P\u2009<\u20090.05) after culture in lipid rich (38.3\u2009\ub1\u20091.5%) compared with control (29.6\u2009\ub1\u20092.2%) and lipid free (28.1\u2009\ub1\u20093.6%). Blastocyst hatching was reduced (P\u2009<\u20090.05) in lipid free (1.4\u2009\ub1\u20090.7%) but not in lipid rich (5.2\u2009\ub1\u20091.7) compared with control (9.8\u2009\ub1\u20092.1). However, blastocysts developed in lipid rich and lipid free had reduced cell numbers compared with control: TE, 98.7\u2009\ub1\u20095.9 and 98.8\u2009\ub1\u20099.1\u2009v. 160.3\u2009\ub1\u20099.0; ICM, 19.2\u2009\ub1\u20092.9 and 25.2\u2009\ub1\u20096.1\u2009v. 43.3\u2009\ub1\u20094.0; and total cell number, 117.9\u2009\ub1\u20097.3 and 124.0\u2009\ub1\u20098.7\u2009v. 203.6\u2009\ub1\u200910.2, respectively. Analysis by GC-MS identified 40 annotated lipids (i.e. triacylglycerols and phosphatidyl cholines) that were significantly reduced in blastocysts cultured in lipid rich compared with control. In summary, blastocyst development was significantly improved after supplementation of fatty acids and L-carnitine to 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 blastocysts had reduced cell numbers even though blastocyst expansion and hatching were not affected. This reduced nutrient medium may provide an experimental model in which to independently study pathways controlling cell proliferation and blastocyst development. Future studies will investigate whether embryo cell number can be rescued while maintaining improved blastocyst development