Simple SummarySuccessful cryopreservation of semen of South American camelids has been limited, hindering the application of artificial insemination. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve sperm freezability. Thus, the objective of this study was to evaluate the effect of the addition of black maca in the freezing medium of epididymal spermatozoa of alpaca species. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. The sperm motility, viability, plasma membrane integrity, DNA integrity, total ROS production, and mitochondrial function were analyzed. The results show that maca (20 mg/mL) improved the parameters associated with sperm function. Similarly, ROS production decreased, although DNA integrity was similar among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers associated with fertility.AbstractArtificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility.