114 Effect of in vivo heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes

Cattle under the effect of heat stress have reduced fertility, with negative effects on the oocyte observed at the morphological, biochemical, transcriptional and developmental levels. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes, which plays a fundamental role in the regulation of gamete development. The objective of this study was to evaluate the effect of in vivo heat stress during the spring to summer transition on DNA methylation and DNA hydroxymethylation of bovine oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Ten Bos taurus crossbred nonlactating beef cows located at Saint Gabriel, Louisiana, USA (30°16′11.1″ N, 91°06′12.1″ W), were used for oocyte collection once monthly from April to August. Dominant follicle removal was performed 5-7 days before oocyte collection. Cumulus-oocyte complexes were collected through ovum pick-up from follicles >2 mm. Germinal vesicle (GV)-stage oocytes (50% of total obtained per cow) were subjected to a standard bovine in vitro maturation protocol to obtain metaphase II (MII) stage oocytes. The DNA methylation and DNA hydroxymethylation of GV and MII oocytes was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine. Secondary antibodies utilised were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG. Oocytes were visualised utilising a fluorescence deconvolution microscope and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. The polar body was not included for fluorescence quantification when evaluating MII stage oocytes. Results (least squares means ± standard error) were evaluated as cold months (April and May) and hot months (June, July, and August). Results were analysed by the type III test of fixed effects and Tukey media separation utilising Proc Glimmix of SAS 9.4 (P < 0.05; SAS Institute Inc., Cary, NC, USA). Maturation rates and percent of grade 1, grade 2, and grade 3 oocytes were square root arcsine transformed for statistical analysis. The number of total oocytes obtained per cow was higher in cold compared to hot months (21.88 ± 2.34 and 14.23 ± 2.17, respectively). Percent of grade-1 oocytes was higher in cold compared to hot months (38.25 ± 3.69 and 27.59 ± 3.09, respectively). There was no difference in percent of grade-2 oocytes between cold and hot months (21.80 ± 2.44 and 22.60 ± 2.20, respectively). There was a lower percent of grade-3 oocytes in cold compared to hot months (39.82 ± 4.54 and 55.87 ± 3.98, respectively). Maturation rate (in vitro maturation) was not different between cold and hot months (81.92 ± 4.04 and 91.11 ± 3.36, respectively). There was no difference between cold and hot months in DNA methylation (417,218.90 ± 71,793.86 and 313,819.88 ± 55,528.01, respectively) and DNA hydroxymethylation (444,931.10 ± 67,920.78 and 352,254.68 ± 56,425.96, respectively) of GV-stage oocytes. There was no difference between cold and hot months in DNA methylation (87,122.36 ± 14,449.47 and 89,807.26 ± 11,303.72 AU, respectively) and DNA hydroxymethylation (102,933.83 ± 15,517.70 and 137,622.45 ± 11,826.86 AU, respectively) of MII-stage oocytes.