Doctor of Philosophy (PhD)
Climate is one of the most important limiting factors in animal production. Cattle under the effect of heat stress have reduced fertility. Negative effects during gamete maturation and embryo development have been observed at the morphological, biochemical, transcriptional and developmental levels. Epigenetic mechanisms play a fundamental role in the regulation of gamete and embryo development. Several genes activated during gamete maturation and early embryo development are controlled by epigenetic mechanisms. There are no studies evaluating the effect of heat-stress on the epigenetic profile of bovine oocytes and embryos. Similarly, there is no information on the effect of heat stress on Anti-Mullerian Hormone (AMH) secretion and antral follicle count. In this regard, the objective of this study is to evaluate the effect of in vivo and in vitro heat stress on the DNA methylation and DNA hydroxymethylation of bovine oocytes and embryos. Additionally, to evaluate the effect of heat stress on AMH secretion and antral follicle count.
The objective of the first experiment was to assess the effect of heat stress on developmental rate, DNA methylation and DNA hydroxymethylation of bovine oocytes at the germinal vesicle and metaphase II stage. Ten Bos taurus non-lactating non-pregnant crossbred beef cows and heifers were used as oocyte donors. Oocytes were collected once monthly from April to August through Ovum Pick-Up. DNA methylation and DNA hydroxymethylation of GV and MII oocytes was assessed by fluorescence immunohistochemistry. Results of the experiment showed there was a significantly higher number of oocytes collected during spring compared to summer. Heat stress significantly reduced the number of grade 1 oocytes and significantly increased the number of grade 3 oocytes. There was no difference in maturation rates of oocytes subjected to in vitro maturation between treatments. No effect of heat stress on DNA methylation and DNA hydroxymethylation of germinal vesicle and metaphase II oocytes was detected.
The objective of experiment two was to assess the effect of in vivo and in vitro heat stress on developmental rate, DNA methylation and DNA hydroxymethylation of bovine oocytes and embryos. A group of Bos taurus non-lactating non-pregnant crossbred beef cows and heifers were used as oocyte donors. A total of 5 repetitions were performed in the experiment. Samples for this experiment were collected during the summer (August) and winter (February).Three treatments were utilized: in vivo heat stress (August samples), in vitro heat stress (February samples subjected to 41°C during the first 12 hours of IVM and then to 38.5 °C during the next 12 hours of IVM) and control (February samples in vitro matured at standard temperature of 38.5 °C). The total number of oocytes collected in each repetition in February were divided between the treatments in vitro heat stress and control. The total oocytes collected per treatment were divided into three developmental stages: oocytes at metaphase II (MII) stage, presumptive zygotes at the pronuclear stage and embryos at the 2-4 cell stage. Fluorescence immunohistochemistry methods as described for experiment 1 were used in experiment 2. Results of the experiment showed that neither in vivo nor in vitro heat stress affected maturation rate, two-pronucleus formation rate and 2-4 cell rate. Similarly, in vivo or in vitro heat stress did not result in altered DNA methylation or DNA hydroxymethylation in metaphase II oocytes, pronuclear stage embryos or 2-4 cell embryos compared to controls.
The objective of experiment three was to evaluate the effect of heat stress on the antral follicle count and AMH serum concentration levels during the spring to summer transition. Ten non-lactating non-pregnant beef Bos taurus heifers and cows (n=10) were used in the experiment. Ovarian measurements, follicle count and samples for this experiment were collected during the spring to summer transition from April to July. Blood samples for AMH serum concentration were collected once monthly. Ovarian measurements and follicle count was done three times per month during dominant follicle removal and Ovum Pick-Up for Experiment 1 and 2. For the calculations of AMH production per follicle, the follicle count of the day of blood sampling was counted. Quantification of AMH was performed through a bovine AMH enzyme-linked immunosorbent assay kit. Results of the experiment showed that there was no effect of heat stress on ovary length, height or cross-sectional area. Similarly, heat stress did not affect the antral follicle count for left and right ovary antral follicle count or total antral follicle count. Furthermore, heat stress did not affect the total AMH secretion or AMH secretion per follicle.
Diaz, Fabian Andres, "EFFECT OF HEAT STRESS ON BOVINE FOLLICULAR DEVELOPMENT AND EPIGENETIC CHANGES" (2018). LSU Doctoral Dissertations. 4766.
Available for download on Wednesday, October 22, 2025