54 Choline alters the pattern of DNA methylation and lipid content of pre-implantation bovine embryos

Abstract

Choline is a methyl donor for DNA methylation and a precursor for phosphatidylcholine, which is the major phospholipid of cell membranes. Early embryonic development involves processes of DNA demethylation and remethylation as well as synthesis of new cell membranes. Addition of choline chloride (ChCl) to culture medium of embryos produced invitro increased birthweight of Brahman calves after embryo transfer. The objective was to determine whether the addition of ChCl to culture medium alters the pattern of DNA methylation and lipid content of pre-implantation embryos produced invitro. Embryos were incubated after fertilisation in BBH7 culture medium containing 0.0, 0.004, 1.3, or 1.8mM ChCl (adjusted with NaCl to maintain isotonicity). Concentrations were chosen to approximate free choline (0.004mM) and total choline (1.30mM) in plasma of cows at week 1 postpartum and concentration of total choline in plasma of cows fed rumen-protected choline (1.8mM). Cleavage and blastocyst rate (n=8 replicates) were evaluated at Days 3 and 7.5 post-insemination, respectively. Embryos ≥8 cells (range 8 to 24 cells; stages near embryonic genome activation) and expanded blastocysts were harvested at Day 3.75 (n=232) and 7.5 (n=204) to estimate global DNA methylation by immunostaining for 5-methyl-cytosine. Methylation of DNA was estimated by calculating the ratio of fluorescence for 5-methyl-cytosine to that of propidium iodide (DNA). Another group of expanded blastocysts was harvested (n=99) to estimate lipid content using Nile Red. Embryo development was analysed by GLIMMIX procedure and fluorescence data by GLM procedure of SAS (SAS Institute Inc.). The proportion of zygotes that cleaved after fertilisation (77.5±2.3, 78.1±2.3, 74.5±2.4, and 80.1±2.2% for 0.0, 0.004, 1.3, and 1.8mM ChCl; P=0.2736) and cleaved embryos that became blastocysts (37.8±4.4, 41.5±4.6, 42.8±4.6, and 39.6±4.4%; P=0.5764) was similar between treatments. The DNA methylation at both days was affected by treatment (P<0.001). At Day 3.75, 1.3mM choline reduced methylation and there were no effects of other concentrations (1.13a±0.03, 1.04a±0.03, 0.92b±0.03, and 1.13a±0.04; means with different superscripts differ at P<0.05). For blastocysts, in contrast, DNA methylation was increased for embryos treated with 1.3 and 1.8mM choline (0.98a±0.04, 1.04ab±0.03, 1.25c±0.03, and 1.11b±0.03). Lipid content in blastocysts was also affected by treatment (P=0.0139). In particular, lipid content was higher for embryos treated with 1.3 and 1.8mM choline (409.1a±54.3, 542.3ab±62.3, 651.3b±54.3, and 583.9b±55.0). In conclusion, addition of ChCl to culture medium altered DNA methylation in bovine pre-implantation embryos produced invitro in a manner dependent on developmental stage and choline concentration. Likewise, ChCl increased lipid content in the resultant blastocysts. Support was provided by the Red Larson Endowment.