Cryopreservation of rabbit semen: impacts of permeable and non-permeable mixture of cryoprotectant, male group individuality, freezing rate, semen package size and antioxidant bovine serum albumin on rabbit semen freezability

In the present study, three experiments were designed to identify the most appropriate technique for freezing rabbit semen. Experiment 1 aimed to determine the optimal levels of dimethyl sulfoxide (DMSO) contents in freezing medium and their effects on individual bucks. Semen ejaculates for each buck (n=15 bucks) were mixed and split into three portions for extension with a freezing medium containing varying concentrations of DMSO (0.75, 1.0, and 1.4 M). Diluted semen samples were packaged in 0.25 mL straws and suspended above liquid nitrogen (LN) for 10 min, then dipped in LN. A few days after freezing, post-thaw semen evaluation was assessed, and according to the results, six bucks and an extender containing 0.75 M of DMSO were used for experiments 2 and 3. In experiment 2, the pooled semen from 6 bucks was divided into two portions for packaging in two straw sizes (0.25 and 0.50 mL). Each straw size was divided into five groups and suspended at different heights above LN (2, 4, 6, 8, and 10 cm) for 10 minutes before being preserved in LN. In experiment 3, the pooled semen was divided into four portions for dilution with freezing medium containing different concentrations of bovine serum albumin (BSA; 0, 2.5, 5.0, and 7.5 mg/mL). Semen samples were packaged in a 0.50 mL straw and suspended 10 min, 4 cm above LN for freezing. Pre-freezing and post-thawing, semen samples were evaluated for semen quality. Results showed that the extender containing 0.75 M DMSO had higher significant values for post-thaw sperm motility, longevity, acrosome integrity and sperm plasma membrane permeability. Bucks’ individuality had significant effects on post-thaw motility, acrosome and sperm plasma membrane integrity. A significant interaction was recorded between DMSO concentrations and bucks’ individuality on sperm longevity. Semen package sizes had no significant effects on the evaluated parameters. Semen was frozen at 2 and 4 cm above LN had significantly better post-thaw quality. BSA at concentrations 5 and 7.5 mg/mL improved recovery rates of acrosome integrity and sperm membrane permeability. DMSO 0.75 M and freezing 4 cm above LN seem to be more adequate for rabbit semen cryopreservation. The appropriate level of DMSO differs between bucks, as the post-thaw sperm longevity is affected. BSA enhanced acrosome and sperm membrane integrity. Results obtained will need further investigation to be confirmed in the field.