217 Consequences of variation in methionine concentration on development of the bovine embryo in vitro

Methionine, an essential amino acid required for protein synthesis, also participates in biochemical processes involved in cell signaling, DNA methylation, lipid metabolism, mitochondrial function and redox balance. Nutritional products to increase methionine availability in cattle have been developed but the consequences for the early embryo are not clear. Concentrations of methionine in uterine fluid have been estimated at 31 µM (Mol. Reprod. Dev. 74, 445–454). Here it was tested whether modifying methionine concentration to cause a deficiency (0 µM) or to mimic estimated uterine concentrations in cows fed rumen-protected methionine (57 µM) would alter characteristics of the bovine embryo produced in vitro. Bovine oocytes obtained from slaughterhouse ovaries were matured for 22 ± 1 h and fertilized with a pool of three bulls for 16 ± 2 h. Putative zygotes were randomly divided into three groups cultured in medium with 0, 31, or 57 µM methionine. The number of replicates (~100 oocytes/treatment/replicate) was 17 for blastocyst development data, 4 for hatching and 3 for cell number and lipid labelling data. There was no effect (P > 0.05) of methionine concentration on cleavage rate or percent of putative zygotes that became blastocysts at Day 7.5 after fertilization. The percent of putative zygotes becoming blastocysts was 23.8 ± 1.2%, 35.8 ± 1.3% and 33.1 ± 1.3% for 0, 31 and 57 µM, respectively. Percent of cleaved embryos becoming a blastocyst was affected by methionine concentration (P < 0.0001), with values of 28.2 ± 2.4%, 42.6 ± 2.8% and 38.0 ± 2.7% for 0, 31 and 57 μM. The percent of blastocysts that hatched at Day 8.5 or 9.5 was reduced (P < 0.01) for 0 µM as compared with other concentrations. At Day 8.5, for example, percent of blastocysts that hatched was 24.0 ± 3.9%, 34.5 ± 9.8% and 33.4 ± 10.0% for 0, 31 and 57 µM. Cell number in blastocysts at Day 7.5 was determined by labelling with Hoechst 33342 and antibodies to SOX2 and CDX2 to identify total cell nuclei, trophectoderm cells and inner cell mass cells, respectively. The absence of methionine (P < 0.0001) reduced total cell number (93.1 ± 4.0) in comparison to 31 (147.8 ± 3.9) and 57 µM (158.7 ± 5.5). Number of CDX2⁺ cells was affected by methionine concentration (P < 0.001), with a concentration-dependent increase from 52.4 ± 2.7 for 0 μM to 99.7 ± 2.6 for 31 μM, and 110.4 ± 3.7 for 57 μM. Number of SOX2⁺ cells was affected by methionine concentration (P = 0.054). Numbers were lower for 0 µM (40.8 ± 2.35) than for 31 µM (48.0 ± 2.3) and 57 µM (48.4 ± 3.2). Methionine concentration affected (P = 0.011) neutral lipid content of Day 7.5 blastocysts, as determined by labelling with Nile Red. There was a concentration-dependent reduction in labelling (93.2 ± 7.0 (0), 71.4 ± 5.6 (31), and 61.4 ± 6.0 units (57)). In conclusion, concentrations of methionine affect development of the bovine embryo, with highest concentrations being associated with increased number of trophectoderm cells and reduced lipid content.