The aim of this study was to evaluate the effect of a phytomelatonin-rich diet, including by-products from the food industry, on ram sperm quality and seminal plasma composition. Melatonin content in several by-products before and after in vitro ruminal and abomasal digestion was determined by HPLC-ESI-MS/MS. Finally, 20% of a mix of grape pulp with pomegranate and tomato pomaces was included in the rams’ diet, constituting the phytomelatonin-rich diet. Feeding the rams with this diet resulted in an increase in seminal plasma melatonin levels compared with the control group (commercial diet) in the third month of the study. In addition, percentages higher than those in the control group of morphologically normal viable spermatozoa with a low content of reactive oxygen species were observed from the second month onwards. However, the antioxidant effect does not seem to be exerted through the modulation of the antioxidant enzymes since the analysis of the activities of catalase, glutathione reductase and glutathione peroxidase in seminal plasma revealed no significant differences between the two experimental groups. In conclusion, this study reveals, for the first time, that a phytomelatonin-rich diet can improve seminal characteristics in rams.
Context Apart from the canonical cAMP-PKA pathway, ram sperm capacitation can be achieved by the MAPK ERK1/2 signalling cascade, activated by epidermal growth factor (EGF). Aims This study aims to investigate the effect of melatonin and nitric oxide (NO·) on capacitation and apoptotic-like changes in EGF-capacitated ram spermatozoa. Methods In vitro capacitation was induced by EGF in the absence or presence of melatonin (100 pM or 1 μM). Also, a NO· precursor, L-arginine, or a NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), were added to capacitation media to study the interaction of NO· and melatonin during EGF-capacitation. Sperm functionality parameters (motility, viability, capacitation state), apoptotic markers (caspase activation and DNA damage), NO· levels, and phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (assessed by Western blot), were evaluated in swim-up and capacitated samples with EGF. Key results NO· levels and the apoptotic-related markers were raised after EGF incubation. Melatonin had a bimodal role on sperm EGF-capacitation, preventing it at high concentration and promoting acrosome reaction at low concentration, but neither of the two concentrations prevented the increase in apoptotic-like markers or NO· levels. However, melatonin at 1 μM prevented the activation of JNK. Conclusions NO· metabolism does not seem to modulate the apoptosis-like events in ram spermatozoa. Melatonin at 1 μM prevents ram sperm capacitation induced by EGF independently from nitric oxide metabolism, and it could be exerted by limiting the JNK mitogen-activated protein kinase (MAPK) activation. Implications This study improves our understanding of the biochemical mechanisms involved in sperm capacitation, and ultimately, fertility.
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