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Simple Summary: In bovine reproduction, genetically superior semen is customarily frozen to ensure its conservation and broad use. Freezing/thawing and handling compromise the quality and fertilizing capacity of spermatozoa, mainly because they induce oxidative stress, an imbalance between reactive oxygen or nitrogen species and antioxidants. The first are highly reactive molecules that alter almost all cellular components (proteins, lipids, DNA) and damage cells. Antioxidants, on the other hand, neutralize these reactive species, thus protecting cells and their components. Supplementation of media with antioxidants has been tested to reinforce the antioxidant defenses of spermatozoa and prevent oxidative stress. In this study, the antioxidant/protective role of the hormone melatonin on post-thawing spermatozoa was assessed. The inclusion of melatonin in the media, even after exposure of spermatozoa to hydrogen peroxide, enhanced the antioxidant status, preserved the semen characteristics and potentially the fertilization capacity of bovine spermatozoa. We anticipate that these results will contribute to the optimization of sperm handling protocols in ART. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: The present study was designed to investigate the effect of pterostilbene, a phenolic compound found in fruits and red wine, on bovine sperm quality characteristics. The spermatozoon was safely handled in the laboratory before subjecting it to Assisted Reproductive Techniques, such as in vitro fertilization, which generates free radicals that might negatively affect structural integrity and fertilizing capacity. An approved approach to protect spermatozoa is the supplementation of the media with exogenous antioxidants. The multiple benefits and pleiotropic actions of antioxidants make them scientifically interesting and popular. In this study, the effect of two different concentrations (10 μΜ and 25 μΜ) of pterostilbene have been tested during two incubation periods, 60 min and 240 min, after thawing. The addition of 25 μM pterostilbene in the bovine sperm preparation media had advantageous effects on motility, viability, intracellular superoxide anion concentration, and acrosomal status of spermatozoa. Thus, the role of pterostilbene seems potentially beneficial for spermatozoa and should be further investigated. Reactive Oxygen Species (ROS), primarily produced by cellular metabolism, are highly reactive molecules that modify cellular compounds. During sperm preparation in Assisted Reproductive Techniques (ARTs), intrinsic and extrinsic sources of ROS can impact spermatozoa's oxidative status. The modification of the media with compounds that enhance sperm quality characteristics is of great significance. The current study investigated the effect of pterostilbene, a phenolic compound, on bovine sperm quality. Cryopreserved spermatozoa from six bulls were thawed, supplemented with pterostilbene (0, 10 μΜ, 25 μΜ) and incubated for 60 min and 240 min. Spermatozoa were analyzed in terms of motility, viability, acrosomal status and intracellular concentration of superoxide anion in each time point. The incubation of spermatozoa with 25 μΜ pterostilbene resulted in the preservation of quality parameters through superoxide anion mitigation, while its presence in capacitating conditions resulted in higher percentage of acrosome-reacted spermatozoa. The results of the present study indicate that the addition of pterostilbene prevents oxidative insult to spermatozoa and preserves the sperm quality parameters. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) and oxidative stress (OS), the imbalance between the production of free radicals and the cellular antioxidant defenses, are discussed in relation to their role in bovine sperm physiology. Oxidative stress has been associated to male infertility and low fertility rates in Assisted Reproductive Techniques (ART). Antioxidant supplementation is an interesting approach to overcome OS-related infertility and assisted reproduction drawbacks. Several studies have been conducted to identify the potential sources of ROS in a typical ART setting and the impact of antioxidant supplementation on semen quality and pregnancy outcome. Procedures such as freezing and thawing, centrifugation and incubation are thought to produce significant amounts of ROS with a negative impact on sperm quality parameters and reproductive competence. Given the important role of ROS in sperm function, the addition of antioxidants in sperm media to prevent OS and to improve the reproductive outcome requires attention. Currently, there is limited evidence to support the ameliorative effect of antioxidant supplementation on fertilization and embryo development in farm animals. This review summarizes the different types and concentrations of antioxidants used in sperm preparation media of bovine species and their effectiveness in neutralizing excessive ROS production while preserving physiological sperm function. • Spermatozoa are exposed to free radicals during their handling in vitro. • Sperm freezing/thawing perpetrates oxidative stres on semen quality is provided. [ABSTRACT FROM AUTHOR]