Calabria, Alfonso (0009) “Beneficial effects of antioxidants in canine semen quality”. [Tesi di dottorato]

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Abstract

Artificial insemination is a routinely performed method in canine breeding programme and semen evaluation results crucial for the successful achievement of a pregnancy. Insemination can be performed by using fresh, chilled or frozen semen. However, semen storage should be performed with the presence of antioxidants, which helps in preventing damaging processes to spermatozoa. Recent studies have been conducted to investigate the feasibility of different antioxidants in several species, to improve semen viability during storage. The aim of this study was to evaluate the effect of two antioxidants, Maca and Crocin, in the supplementation of semen extender on quality-related canine semen parameters during cooling and freezing. For the first experiment ejaculates from nine dogs were cooled in the absence (control group) or the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage for chilled semen and immediately after thawing, after 1h and 2 h at 37°C. For the chilled semen, the addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 24 h of refrigerated storage with the higher concentration tested was observed. The second experiment aimed to evaluate the effect of Maca on frozen-thawed sperm quality in canine semen. Ejaculates from ten dogs were frozen in the absence (control group) or the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca and were evaluated immediately after thawing and after 1h and 2 h at 37 C° for sperm viability, motility, DNA fragmentation and lipid peroxidation. Canine sperm cells frozen with an extender supplemented with Maca exhibited higher total motility, especially the subpopulation of sperm with medium velocity 1 hour after thawing than control semen. Canine frozen semen with the supplementation of Maca is responsible for a surge in hyperactivation and WOB of sperm cells after one hour at 37°C. Movements of hyperactivation are considered part of the capacitation process and it is an event crucial for acrosome reaction and fertilization. What emerges from this study is the protective role of Maca against lipid membrane peroxidation of canine spermatozoa, which is a primary marker of oxidative stress. In conclusion, supplementation of the frozen extender with 10 μl/mL of aqueous extract of Maca improves the cold shock resistance of spermatozoa, protecting sperm against lipid peroxidation during the frozen-thawed process, and activates canine sperm motility and hyperactivation after thawing, improving the fertility. The third experiment was aimed to evaluate the effect of Crocin supplementation extender at three different concentrations (C0,5, C1 e C2) on quality-related canine semen parameters after cooling. Ejaculates from ten dogs were evaluated for sperm viability, sperm motility, membrane integrity and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage at 4 C°. The most interesting findings of the present study regard the improvement of semen quality obtained with 0.5 mM crocin. Indeed, the addition of 0.5 mM crocin in the extender significantly increased the proportion of spermatozoa with intact membranes at both 4 and 7 days compared to the control group and despite similar values of total motility and progressive motility most of the sperm kinetic parameters improved in C0.5 group compared to the control after 4 days of storage. In conclusion, we demonstrated that the enrichment of the extender with the crocin improves to a certain improved canine semen quality, particularly after 4 days of storage at 4 °C.

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