Mammalian sperm is highly susceptible to the reactive oxygen species (ROS) stress caused by biochemical and physical modifications during the cryopreservation process. 5′AMP-activated protein kinase (AMPK) is involved in regulating both cell metabolism and cellular redox status. The aim of the present study was to investigate whether the resveratrol protects boar sperm against ROS stress via activation of AMPK during cryopreservation. Boar sperm was diluted with the freezing medium supplemented with resveratrol at different concentrations (0, 25, 50, 75, 100, and 125 μM). It was observed that the addition of 50 μM resveratrol significantly improved the postthaw sperm progressive motility, membrane integrity, acrosome integrity, mitochondrial activity, glutathione (GSH) level, activities of enzymatic antioxidants (glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase), and the phosphorylation of AMPK. Meanwhile, the lipid peroxidation, ROS levels, and apoptosis of postthaw sperm were reduced in the presence of 50 μM resveratrol. Furthermore, when fresh boar sperm was incubated with the medium in the presence of 50 μM resveratrol and 30 μM Compound C (an AMPK inhibitor), the effects of the resveratrol were partly counteracted by the Compound C. These observations suggest that the resveratrol protects boar sperm via promoting AMPK phosphorylation. In conclusion, the addition of resveratrol to the freezing extenders protects boar sperm against ROS damage via promoting AMPK phosphorylation for decreasing the ROS production and improving the antioxidative defense system of postthaw sperm. These findings provide novel insights into understanding the mechanisms of resveratrol on how to protect boar sperm quality contrary to the ROS production during cryopreservation.

Sperm motility patterns are continuously changed after ejaculation to fertilization in the female tract. Hyperactivated motility is induced with high glucose medium in vitro or the oviduct fluids in vivo, whereas sperm maintain linear motility in the seminal plasma or the uterine fluids containing low glucose. Therefore, it is estimated that sperm motility patterns are dependent on the energy sources, and the mitochondrial oxidative phosphorylation is activated to produce ATP in low glucose condition. To elucidate these hypotheses, boar sperm was incubated in different energy conditions with the transcription and translation inhibitors in vitro. Sperm motility parameters, mitochondrial activity, ATP level, gene expression and protein synthesis were analyzed. Sperm progressive motility and straight-line velocity were significantly increased with decreasing glucose level in the incubation medium. Moreover, the mitochondrial protein turnover meaning transcription and translation from mitochondrial genome in sperm is activated during incubation. Incubation of sperm with mitochondrial translation inhibitor (D-chloramphenicol) suppressed mitochondrial protein synthesis, mitochondrial activity and ATP level in sperm and consequently reduced the linear motility speed, but not the motility. Thus, it is revealed that the mitochondrial central dogma is active in sperm, and the high-speed linear motility is induced in low glucose condition via activating the mitochondrial activity for ATP generation.

The process of cryopreservation results in high concentration of reactive oxygen species which is detrimental to spermatozoa. The aim of this study was to investigate whether addition of vitamin E analogue to freezing extender can facilitate the cryosurvival of spermatozoa in rabbits, and how vitamin E protects spermatozoa against damages during the process of preservation. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of Trolox (a vitamin E analogue). The level of radical oxygen species (ROS) in spermatozoa that was exposed to Trolox was significantly lower than that of the control during each step of the process of preservation. The percentage of frozen-thawed spermatozoa with lipid peroxidation in the Trolox treatments was significantly lower than that of the control. The motility, intact acrosome, membrane integrity and mitochondrial potentials of the frozen-thawed spermatozoa in the treatment of 200 μM Trolox were significantly higher than those of the control. These observations suggest that addition of vitamin E to a freezing extender leads to higher integrity of acrosome, plasma membrane and mitochondrial membrane potential as well as higher motility. Vitamin E protects spermatozoa through its capacity to quench ROS accumulation and lipid peroxidation during the process of preservation. Addition of Trolox is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry.