Effect of methionine supplementation on methylation and lipid accumulation of the preimplantation embryo in dairy cows

Abstract Text: The lipid profile of oocytes and early embryo can be influenced by the environment of the cow. Our objective was to determine effect of supplementing rumen-protected methionine on DNA methylation and lipid accumulation in preimplantation embryos of dairy cows. Lactating Holsteins entering their 2^nd or greater lactation were randomly assigned to two treatments from 30 ± 2 DIM to 72 ± 2 DIM; Control (CON; n=5, fed a basal diet with a 3.4:1 Lys:Met) and Methionine (MET; n=5, fed the basal diet plus Smartamine M to a 2.9:1 Lys:Met). On day 60, dominant follicles greater than 5 mm were aspirated using an ultrasound-guided transvaginal approach. A CIDR^® device was inserted in all cows after follicular aspiration (d 60) and superovulation began at d 61.5 using FSH treatment equivalent to 400 mg of NIHFSH-P1 (Folltropin^®) in 8 decreasing doses at 12 h intervals over a 4 d period. During the superovulatory period, all cows received two PGF2α injections at d 63 and d 64 (concomitant with the 5th and 7th FSH injections), and CIDR was withdrawn at d 65. Twenty-four hours after CIDR withdrawal, ovulation was induced with GnRH. Cows were artificially inseminated at 12 h and 24 h after GnRH using a high-fertility sire. Embryos were flushed 6.5 d after artificial insemination. Embryos with stage of development 4 or greater were used for analysis. Methylation was assessed by immunofluorescent labeling with anti-5-methylcytosine while lipid accumulation was assessed by staining with Nile Red. ImageJ software was used for image analysis to determine intensity of labeling. For methylation, fluorescence intensity of nuclear 5-methylcytosine labeling was expressed as a ratio of labeling for DNA using propidium iodide. For lipids, fluorescence intensity of Nile Red staining was compared against a negative control embryo (subtraction of background). Nuclear staining (propidium iodide or Hoescht 33342) was used to count the total number of cells/embryo. A total of 37 embryos were harvested from cows (MET= 16; CON=21). Statistical analysis was performed using the MIXED procedure of SAS. Cows receiving MET (1661) had greater (P=0.021) lipid accumulation when compared with cows receiving CON (1033). There were no treatment effects (P> 0.511) on number of cells or stage of development. In conclusion, cows supplemented with methionine produced embryos with higher lipid concentration when compared to CON which could potentially serve as an important source of energy for the early embryo. 

Keywords: Methionine, embryo, methylation, lipid