Optimizing rabbit semen cryopreservation using different cryoprotectants in a Tris-based extender.

Area of Science:

• Reproductive Biology
• Cryopreservation Science
• Animal Science

Background:

• Cryopreservation of spermatozoa is crucial for artificial insemination and genetic preservation in rabbits.
• Optimizing cryoprotective extenders is essential to maintain sperm viability and function post-thaw.
• Dimethyl sulfoxide (DMSO) and glycerol (GL) are common cryoprotectants, but their efficacy can be enhanced with additives.

Purpose of the Study:

• To evaluate the effects of different cryoprotectant combinations (DMSO, glycerol, trehalose, sucrose) in a Tris-based extender on post-thawed rabbit semen quality.
• To assess the impact of these treatments on sperm antioxidant status, acrosome integrity, apoptosis, and semen microbiota.
• To determine the in vivo fertility of cryopreserved rabbit spermatozoa.

Main Methods:

• Rabbit semen was cryopreserved using Tris-based extenders with five different treatments: 4% DMSO (DM4), 4% DMSO + 50 mM trehalose (DMTR), 4% DMSO + 50 mM sucrose (DMSU), 4% glycerol (GL4), or 2% DMSO + 2% glycerol (DMGL2).
• Post-thaw sperm quality was assessed for motility, viability, membrane integrity, acrosome status, and apoptosis.
• Antioxidant capacity (TAC), malondialdehyde (MDA), reactive oxygen species (ROS), and nitric oxide levels were measured.
• Semen microbiota and in vivo fertility trials were conducted.

Main Results:

• The 4% DMSO (DM4) group showed superior progressive motility, viability, and membrane integrity.
• Sperm kinematic parameters were significantly better in all DMSO-treated groups compared to glycerol groups.
• Treatments with trehalose (DMTR) or sucrose (DMSU) significantly improved live sperm with intact acrosomes and reduced detached acrosomes.
• The DMTR group exhibited the highest total antioxidant capacity (TAC) and lowest oxidative stress markers (MDA, ROS).
• The DMSU treatment resulted in the highest conception rate in vivo.
• Bacterial counts were lowest in the DMSU group.

Conclusions:

• Supplementing Tris-based extenders with 4% DMSO combined with either 50 mM trehalose or 50 mM sucrose significantly enhances cryo-tolerance of rabbit spermatozoa.
• These optimized formulations improve post-thaw sperm kinematics, preserve acrosome integrity, reduce apoptosis, and mitigate oxidative stress.
• The synergistic action of DMSO and disaccharides offers a promising strategy for improving rabbit sperm cryopreservation outcomes.