Many factors affect development of mammalian preimplantation embryos in vitro. It is well known that in vitro development of bovine embryos is highly affected by culture condition including energy source, growth factors, pH or gas environment. Many efforts have been made towards the suitable environments which can successfully support embryo development in vitro. For a rapid growth and differentiation, embryo requires energy by utilizing ATP, NADPH with oxygen molecules. These energy substrates are produced from the electron transport chain in the mitochondria. In addition to energy production, reactive oxygen species (ROS) are also generated as by-product of such energy production system. ROS production is sensitively controlled by the balance of oxidizing and reducing status and affected by several antioxidant enzymes such as superoxide dismutase (SOD), Catalase, glutathione peroxidase (GPx) or low molecular weight thiols such as glutathione (GSH). Imbalance of oxidation and reduction causes production of excess ROS, which causes the developmental arrest, physical DNA damage, apoptosis induction or lipid peroxidation. Environmental oxygen condition during embryo culture also highly affects embryo development as well as intracellular redox balance. Several studies have revealed that regulation of intra- and extra- cellular reducing environment by reducing excess ROS by using antioxidants, reducing oxygen concentration are effective for improving embryo development. Also, recent studies have demonstrated the difference in gene expression affected by oxidative stress. This review briefly summarizes the effects of ROS and the role of redox balance on preimplantation embryos for improving the efficiency of in vitro production of mammalian embryos.

In most mammalian species including cattle, heat stress has deleterious effects on nutritional, physiological and reproductive functions. Exposure of animals to a hot environment causes an increase in body temperature in mammals, including domestic animals. High ambient temperature also causes a decrease in the length and intensity of estrus by disturbing ovarian function as well as decreasing pregnancy rate after artificial insemination. Therefore, it is important to understand the effects of heat stress on reproductive function in order to improve the production of domestic animals. Heat stress decreases appetite, weight gain, and milk yield in dairy cattle. It also adversely affects the reproductive performance of both sexes. In males, it reduces spermatogenic activity, while in females it adversely impacts oogenesis, oocyte maturation, fertilization development and implantation rate. Detection and evaluation of the deteriorating effects of heat stress on reproductive organs and cells can help to design measures to prevent them and improve reproductive functions. In this review, we discuss the impacts of heat stress on reproductive functions.

The effects of β-mercaptoethanol (β-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine

Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus–oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM. Abstract Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus–oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM.

For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.