Effect of Melatonin Addition During Cryopreservation of Chinchilla Rabbit Spermatozoa in Mexico

Abstract

In recent years, efforts have been made to make rabbit production more efficient, and reproductive strategies, such as artificial insemination, have been developed to make it more productive. However, unlike in other species, there are currently no protocols to ensure that sperm quality is maintained during thawing. As the main problem faced by spermatozoa during cryopreservation is free radical formation, the addition of antioxidants, such as melatonin (MLT), to extenders for sperm cryopreservation has been tested. This study aimed to evaluate the effects of different MLT concentrations on the cryopreservation of Chinchilla rabbit sperm in Mexico. Ejaculates were collected from six males with proven fertility, and a pool was formed from those that had at least 70% progressive motility (PM) (18 pools in total). The pooled semen samples were microscopically assessed for wave motion (WM), PM, viability (VB), morphology, membrane functionality (MF), and acrosomal integrity. A two-step freezing protocol was applied using INRA-82 as the extender supplemented with Orvus Paste (0.5%), dimethyl sulfoxide (DMSO) (4%), and dimethylformamide (DMF) (4%) as cryoprotectants (final concentration). Aliquots were taken to form six MLT concentrations (0.0, 0.1, 0.75, 1.5, 2.25 and 3.0 mM) at room temperature (23°C). The sperm samples were packaged in 0.25 mL straws, cooled to 5°C, and then exposed to nitrogen vapours before being immersed in liquid nitrogen for storage. For thawing, the straws were immersed in a water bath for 30 s at 37°C. Thawed sperm samples were microscopically assessed for VB using the NucleoCounter (NC) system, following the manufacturer’s instructions. Analysis of variance (ANOVA) was conducted to compare the effects of different MLT concentrations on sperm parameters. No significant differences (p  > 0.05) were observed among the concentrations for any of the evaluated variables. In conclusion, MLT addition during Chinchilla rabbit sperm cryopreservation had no effect on sperm motility, VB, acrosomal integrity, MF, membrane integrity, or morphology.