The effects of repeated freezing and thawing on bovine sperm morphometry and function.

Refreezing the remaining genetic resources after in vitro fertilization (IVF) can conserve genetic materials. However, the precise damage inflicted by repeated freezing and thawing on bovine sperm and its underlying mechanism remain largely unexplored. Thus, this study investigates the impact of repeated freeze-thaw cycles on sperm. Our findings indicate that such cycles significantly reduce sperm viability and motility. Furthermore, the integrity of the sperm plasma membrane and acrosome is compromised during this process, exacerbating the advanced apoptosis triggered by oxidative stress. Additionally, transmission electron microscopy exposed severe damage to the plasma membranes of both the sperm head and tail. Notably, the "9 + 2" structure of the tail was disrupted, along with a significant decrease in the level of the axonemal protein DNAH10, leading to reduced sperm motility. IVF outcomes revealed that repeated freeze-thaw cycles considerably impair sperm fertilization capability, ultimately reducing the blastocyst rate. In summary, our research demonstrates that repeated freeze-thaw cycles lead to a decline in sperm viability and motility, attributed to oxidative stress-induced apoptosis and DNAH10-related dynamic deficiency. As a result, the utility of semen is compromised after repeated freezing.

ß-hydroxybutyrate impairs bovine oocyte maturation via pyruvate dehydrogenase (PDH) associated energy metabolism abnormality.

Background: Ketosis is one of the most frequent and costly metabolic disorders in high-producing dairy cows, and negatively associated with the health and reproductive performance of bovine. Ketosis is mainly caused by the accumulation of ketone body ß-hydroxybutyric acid and its diagnosis is based on ß-hydroxybutyrate (ßHB) concentration in blood. Methods: In this study, we investigated the effects of ßHB on bovine oocyte maturation in the concentration of subclinical (1.2 mM) ßHB and clinical (3.6 mM). Results: The results showed ßHB disrupted bovine oocyte maturation and development capacity. Further analysis showed that ßHB induced oxidative stress and mitochondrial dysfunction, as indicated by the increased level of reactive oxygen species (ROS), disrupted mitochondrial structure and distribution, and depolarized membrane potential. Furthermore, oxidative stress triggered early apoptosis, as shown by the enhanced levels of Caspase-3 and Annexin-V. Moreover, 3.6 mM ßHB induced the disruption of the pyruvate dehydrogenase (PDH) activity, showing with the decrease of the global acetylation modification and the increase of the abnormal spindle rate. Conclusion: Our study showed that ßHB in subclinical/clinical concentration had toxic effects on mitochondrial function and PDH activity, which might affect energy metabolism and epigenetic modification of bovine oocytes and embryos.