Counteracting oxidative stress improves quality of chilled stallion semen

Stallion semen experiences oxidative stress during cooling and transport, and is consequently damaged by reactive oxygen species. Two different approaches have been investigated to enhance the intrinsic antioxidant defense mechanisms against oxidative stress in liquid-preserved semen. First, a dietary antioxidant supplementation to improve antioxidant status in tissue, seminal plasma and spermatozoa was tested. Second, the combined addition of three enzymatic antioxidants to semen extender in order to increase the antioxidant status of seminal plasma was evaluated. In the first experiment, the effect of dietary supplementation with Lepidium meyenii (Maca) on fresh and chilled stallion semen characteristics were evaluated. Maca is a traditional Andean crop used as a nutraceutical for its fertility-enhancing properties which is linked with its antioxidant activity. The diet of five stallions was supplemented daily with 20 mg of Maca powder for a total of 60 days. A control group of another five stallions received the same diet without Maca. Semen was collected once before the administration of Maca (D0), twice during the administration at 30 and 60 days (D30-D60), and finally twice at 30 and 60 days after the end of the administration (D90-D120). Ejaculates were processed for cooled shipping at 5°C and evaluated in the laboratory for total and progressive motility, acrosome integrity and lipid peroxidation after collection and after 24 hours, 48 hours, and 72 hours storage, respectively. Dietary supplementation with Maca significantly improved sperm concentration and total sperm (p<0.05). The beneficial effects of Maca supplementation on motility and acrosome integrity in the raw semen appeared from the end of treatment with Maca (D60) until the end of the study (D120). Furthermore, total motility, progressive motility, and acrosome integrity decline slower during storage in the Maca-treated group than in the control group. The lipid peroxidation did not change during cooling storage in each group and did not show a significant difference between two groups. The results from this study indicated that the dietary supplementation with Maca was able to increase sperm production and in stabilizing semen quality during storage of chilled semen. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GPX) constitute the principal enzymatic components of the endogenous antioxidant system of equine spermatozoa and seminal plasma. The objective of the second experiment was to evaluate the effect of adding a combination of SOD, CAT and GPX to a semen extender on the quality of stallion semen stored at 5°Cfor 72 hours. Ejaculates from seven stallions were split in two aliquots and diluted with semen extender without (control) or with the addition of 15 IU/mL SOD, CAT, and GPX. Semen analysis was performed within 3 hours after semen collection (T0) and every 24 hours during storage of chilled semen (T24, T48, T72). At each time point, total and progressive motility, kinetic parameters of sperm movement, DNA fragmentation and the levels of activated caspase-3 were evaluated. In the first 24 h of storage, almost no difference between control samples and treated samples was evident. After 48 and 72 hours, beneficial effects of a combination of antioxidants became evident. The antioxidant supplementation significantly inhibited the activation of activated caspase 3 and concomitantly maintained total motility and the percentage of rapid moving sperm cells at a higher level (p<0.05). A storage-dependent increase in DNA damage was alleviated only to a minor extend after prolonged storage time, i.e. 72 hours. The results suggest that the tested combination of SOD, CAT and GPX added to a cooling extender improves viability, motility and kinetic features and reduce DNA fragmentation in semen stored for more than 24 hours. In conclusion, a dietary supplementation of stallions with an antioxidant or measures to supplement the semen extender with a combination antioxidants are both valid tools to counteract oxidative stress and maintain the quality of chilled equine semen at a high level for a prolonged time.