Metformin Improves Quality of Post-Thaw Canine Semen

SIMPLE SUMMARY: Cryopreservation of semen is getting easier, however, fertilizing results after insemination with frozen-thawed semen is still not constant in canine species depending on the breed and could be still improved. In this study, we decided to modulate the mitochondrial activity through the addition of metformin in semen extender to increase germ cells’ quality. Metformin presented the absence of toxicity and an improvement in sperm motility after thawing, as well as an increase in the expression of several molecular markers associated with quality. In addition, the oxidative stress and DNA damage were reduced in semen frozen in the presence of metformin. Overall, these data suggest that metformin added in canine semen extender has beneficial effects on canine semen quality and could be associated with different components such as vitamins, to enhance the antioxidants status. ABSTRACT: Sperm cryopreservation is an assisted reproductive technique routinely used in canine species for genetic conservation. However, during cryopreservation, the DNA damages are still elevated, limiting the fertilization rate. The present study was conducted to evaluate whether supplementation of canine semen extender with a molecule limiting the metabolic activities can improve the quality of frozen-thawed canine spermatozoa. We used metformin, known to limit the mitochondrial respiratory and limit the oxidative stress. Before and during the freezing procedure, metformin (50 µM and 500 µM) has been added to the extender. After thawing, sperm exposed to metformin conserved the same viability without alteration in the membrane integrity or acrosome reaction. Interestingly, 50 µM metformin improved the sperm motility in comparison to the control, subsequently increasing mitochondrial activity and NAD(+) content. In addition, the oxidative stress level was reduced in sperm treated with metformin improving the sperm quality as measured by a different molecular marker. In conclusion, we have shown that metformin is able to improve the quality of frozen-thawed dog semen when it is used during the cryopreservative procedure.