Grape extracts of the Greek species Vitis vinifera possess potent antioxidant properties in vitro. The freeze/thaw process and the preparation of semen during assisted reproductive techniques can adversely affect the functional integrity of spermatozoa. The objective was to assess the effect of three different concentrations (1 μg ml−1, 2 μg ml−1 and 5 μg ml−1) of a polyphenol-rich grape pomace extract on motility, viability, acrosomal and lipid peroxidation status of thawed bovine spermatozoa after 2 and 4 hrs of incubation. The results indicate that the percentage of “Rapid” spermatozoa remained significantly increased (p <0.05) in the presence of 5 μg ml−1 of the extract, compared to the control after 2 hrs of incubation. Additionally, the incubation of spermatozoa with 2 μg ml−1 and 5 μg ml−1 of the extract for 2 hrs resulted in a significantly better maintenance of viable spermatozoa with intact acrosome (p <0.05). The other parameters did not show statistically significant changes. Moreover, the presence of 2 μg ml−1 and 5 μg ml−1 of the extract kept the levels of Malondialdehyde (MDA) production in significantly lower level, compared to the other groups, after 2 hrs and 4 hrs of incubation (p <0.05). Particularly, a dose-dependent effect was noticed after 2 hrs of incubation. Our results suggest that the grape pomace extract exerts a powerful antioxidant role, by suppressing lipid peroxidation, and provides protection in terms of motility and acrosomal integrity, which are correlated with in vivo fertility. The optimal extract concentration is 5 μg ml−1.

Cryopreserved semen is widely used in assisted reproductive techniques. Post-thawing spermatozoa endure oxidative stress due to the high levels of reactive oxygen and nitrogen species, which are produced during the freezing/thawing process, and the depletion of antioxidants. To counteract this depletion, supplementation of sperm preparation medium with antioxidants has been widely applied. Melatonin is a hormone with diverse biological roles and a potent antioxidant, with an ameliorative effect on spermatozoa. In the present study, we assessed the effect of melatonin on thawed bovine spermatozoa during their handling. Cryopreserved bovine spermatozoa were thawed and incubated for 60 min in the presence or absence of 100 μΜ melatonin. Also, the effect of melatonin was assessed on spermatozoa further challenged by the addition of 100 μΜ hydrogen peroxide. Spermatozoa were evaluated in terms of kinematic parameters (CASA), viability (trypan blue staining) and antioxidant capacity (glutathione and NBT assay, determination of iNOS levels by Western blot analysis). In the presence of melatonin, spermatozoa presented better kinematic parameters, as the percentage of motile and rapid spermatozoa was higher in the melatonin group. They also presented higher viability and antioxidant status, as determined by the increased cellular glutathione levels and the decreased iNOS protein levels.

Endocrine-disrupting chemicals (EDCs) have raised increasing concern due to their potential effects on reproductive health. This review focuses on the impact of EDCs, particularly bisphenol A (BPA) and its analogues, and per- and polyfluoroalkyl substances (PFAS), on domestic ruminants (cattle and sheep) by integrating findings from both in vitro and in vivo studies. The analysis highlights how exposure to EDCs affects steroidogenesis, oxidative stress responses, apoptosis, epigenetic regulation, and overall fertility markers, such as oocyte maturation, sperm motility, and embryo developmental competence. While most data originate from in vitro bovine studies, in vivo research in sheep offers valuable insights. Importantly, given the potential for EDCs to bioaccumulate in animal tissues, these findings hold significant implications for animal health, particularly regarding reproductive physiology and fertility rates.

Reactive Oxygen Species (ROS) play an important role in sperm physiology. They are required in processes such as capacitation and fertilization. However, the exposure of spermatozoa to ROS generated from internal or external sources may create a potentially detrimental redox imbalance. Antioxidant supplementation in semen is now a rather common approach to protect spermatozoa from oxidative stress (OS) during their handling and/or cryopreservation. Supplementation with pterostilbene, a potent antioxidant, protects spermatozoa from OS and ameliorates their post-thawing characteristics and viability. In the present study, we used freezing/thawing as a model of natural ROS overproduction and investigated the molecular mechanisms modulated by pterostilbene. Specifically, bovine frozen/thawed spermatozoa were incubated with 10 or 25 μM pterostilbene for 60 min. Results have shown that in a dose-independent manner, pterostilbene decreased lipid peroxidation and increased intracellular GSH levels. Moreover, pterostilbene ameliorated energy production, as ATP and AMP/ATP levels were restored, and increased autophagy levels through AMP-activated protein kinase (AMPK) activation, which finally resulted in the inhibition of apoptotic cell death in bovine spermatozoa when exposed to OS. This study sheds light on spermatozoa redox state, the crosstalk between apoptotic and autophagic pathways, and its role in determining the beneficial or detrimental effect of ROS in spermatozoa.