A high temperature and humid environment negatively affect the productivity of commercial animal agriculture [1]. Production losses as a result of high ambient temperature are an increasing concern not only in tropical areas, but also in the northern and southern hemispheres where heat stress is a problem during the summer season, particularly during heat waves [2]. Heat stress negatively affects multiple fundamental productive traits such as growth, carcass traits, milk yields, and reproduction [3]. Notably, reduced fertility is one of the primary economic losses caused by heat stress.

Among the major effects of heat stress on reproduction is the adverse impact on gamete maturation and embryo development. Heat stress alters developmental competence, mitochondrial distribution, and genetic expression of mitochondrial related genes involved in the respiratory chain of metaphase II (MII) oocytes [4]. Similarly, heat stressed oocytes show altered expression of transcripts important in mitochondrial function [5]. At the structural level, heat stress affects chromosomes, meiotic spindle, and cytoplasmic microtubules and microfilaments, where the percentage of oocytes affected increases with the time of exposure to elevated temperatures [6]. Genes involved in oocyte maturation and early embryonic development (MOS, GDF9, and POU5F1) have also been found to present altered expression in MII oocytes, as a result of heat stress, when comparing oocytes collected during the hot versus the cold season [7]. Furthermore, in vivo and in vitro models of heat stress have shown that heat stressed oocytes present altered expression of developmentally important genes (CX43, CDH1, DNMT1, C-MOS, GDF9, GAPDH, POU5F1, and HSPA14) [8]. Additionally, decreased protein synthesis is observed in heat-stressed oocytes [9].

Effects of heat stress on embryo development that have been observed include abnormal morphology [10], reduced developmental competence [11], migration of organelles to the center of blastomeres, increased proportion of mitochondria with swollen morphology, as well as an increased distance between membranes comprising the nuclear envelope [12]. Additionally, in vivo recovered embryos subjected to in vitro heat stress resulted in migration of organelles to the center and swollen mitochondria [12], rearrangement of actin filaments and microtubules [13], and increased reactive oxygen species [14].

Gamete and embryo development are complex biological processes achieved by multiple genes interacting in a sequential and highly specific manner. The highly regulated gene expression during gamete and embryo development requires regulation in cis by transcription factors and regulation in trans by epigenetic mechanisms [15]. Epigenetic modifications play a fundamental role in the regulation of gamete and embryo development. It has been evidenced that environmental stressors (nutrient availability, temperature, environmental contaminants, social environment) can affect epigenetic modifications of developing oocytes and embryos [16]. Furthermore, it has been observed that heat stress applied during oocyte maturation or early embryo development can result in altered DNA methylation levels of bovine and mice oocytes and embryos [[17], [18], [19]]. Importantly, there are no studies evaluating the effect of in vivo and in vitro heat stress on the epigenetic profile of bovine oocytes and early embryos within the same study. In this regard, the objective of this study was to evaluate the effect of in vivo and in vitro heat stress on developmental rates, DNA methylation, and DNA hydroxymethylation of bovine oocytes and early embryos. In Louisiana, USA; heat stress significantly affects cattle reproduction during summer, especially Bos taurus derived cattle. Importantly, the main bovine commercial industry in Louisiana is beef cattle. In this study we wanted to evaluate the effect of heat stress on cows at their basic metabolic status and not include confounding factors like animals with higher metabolic status as lactating or pregnant cows. For this reason, we used non-lactating, non-pregnant Bos taurus beef cows. We hypothesized that in vivo and in vitro heat stress will not affect the global DNA methylation and DNA hydroxymethylation of bovine oocytes and early embryos. We hypothesized this based on findings of a previous study reported by our group [20], where under similar experimental conditions to this study, we did not observe an effect of seasonal heat stress (in vivo heat stress) on the DNA methylation and DNA hydroxymethylation of bovine oocytes.