Abstract:Odontobutis yaluensis is a small benthic fish endemic to northeastern China. In its natural habitat, the fish breeds within a temperature range of 8 ℃ to 20 ℃, with optimal embryo hatching occurring at 16–20 ℃. The development and utilization of O. yaluensis fishery resources have faced significant challenges due to the vulnerability of their fertilized eggs to external factors, leading to low hatching rates. In previous artificial breeding experiments, we observed that excessively high water temperatures caused substantial mortality before the embryos reached the gastrula stage, contributing to low hatching rates. Elevated temperatures can impair tissue differentiation and organogenesis in a fish’s early embryonic developmental stages, affecting physiological activity and development. When organisms are subjected to environmental stress, excessive production of reactive oxygen species can surpass the capacity of the antioxidant defense system, resulting in oxidative stress. This induces lipid peroxidation and DNA damage. In vertebrates, oxidative stress has been implicated in embryonic damage and is potentially associated with developmental arrest. To further investigate the mechanisms underlying high-temperature stress-induced mass mortality of early embryos in O. yaluensis, we examined the causes of high embryonic mortality by observing abnormal development in early embryos under high-temperature stress and analyzing changes in antioxidant enzyme activities and growth-related gene expression. The experiment was conducted using three water temperatures (15 ℃, 20 ℃, and 25 ℃). Embryos at the 2-cell stage were selected for a 24-hour temperature treatment. After the experiment, the embryonic development was monitored microscopically for abnormalities, survival rates were recorded, and antioxidant-related enzyme activities were measured. Additionally, the mRNA levels of key growth-related genes, including the eukaryotic translation initiation factor 4E binding protein 1 (4e-bp1), MYC proto-oncogene (c-myc), ribosomal protein S6 kinase B1 (s6k1), and insulin-like growth factor 1 receptor (igf-1r), were quantified. We found that O. yaluensis embryos were more prone to abnormal development at 25 ℃ than at the other temperatures. The observed abnormalities included uneven blastomere size, cytoplasmic extrusion into the perivitelline space, blastomere detachment from the yolk, and cytoplasm filling the perivitelline space as the eggs gradually turned opaque. Disruption of membrane permeability led to water absorption and egg swelling, followed by cytoplasmic extrusion. These abnormally developed embryos failed to progress to the blastocyst stage. The survival rate of O. yaluensis embryos was significantly higher at 15 and 20 ℃ than at 25 ℃ (P<0.05). Embryos at 15 ℃ and 20 ℃ had higher total antioxidant capacity and lower malondialdehyde content and superoxide dismutase activity than those at 25 ℃ (P<0.05). The catalase activity was in the order of 15 ℃>25 ℃>20 ℃ (P<0.05). Glutathione peroxidase activity did not differ significantly among the three groups (P>0.05). Temperatures of 15–20 ℃ favored early embryonic development in O. yaluensis. However, as temperature increased, oxidative stress occurred, activating the embryonic antioxidant system and mitigating the damage caused by oxidative stress. The expression level of 4e-bp1 was significantly lower in embryos at 15 and 20 ℃ than at 25 ℃, whereas c-myc expression was highest at 15 ℃, followed by the 20 ℃ group, both significantly higher than at 25 ℃ (P<0.05). The expression levels of s6k1 and igf-1r were significantly higher in embryos at 20 ℃ than in the other two groups (P<0.05). The genes s6k1 and igf-1r are associated with accelerated development and improved protein synthesis efficiency. Identified as a rapid-response gene during early embryogenesis, c-myc has low expression levels that may lead to developmental arrest. Additionally, the increased expression of 4e-bp1 has been shown to inhibit the initiation of protein translation in embryos. In summary, at 15–20 ℃ water temperatures, O. yaluensis embryos exhibited higher survival rates and enhanced antioxidant capacity to regulate oxidative stress levels than at 25 ℃. Oxidative stress induced by high temperatures has been identified as a key factor causing oxidative damage to embryonic cells and leading to abnormal development and significant mortality in O. yaluensis embryos. Additionally, the expression of genes related to embryonic development and protein synthesis is downregulated to prevent the progression of abnormal development in damaged embryos, thereby slowing the developmental process and suppressing further abnormalities.
