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

Abstract

This experiment investigates the potential effects of different cryoprotectant molecules, such as glycerol (GL) and dimethyl sulfoxide (DMSO), added to a Tris-based extender focusing on assessing sperm quality, antioxidant status, acrosome integrity, apoptosis, semen microbiota of post-thawed rabbit semen, and in vivo fertility trial in rabbit does. The Tris-based extender was supplemented with various cryoprotectants to form the following five experimental treatments: 4% DMSO (DM4), 4% DMSO+50 mM trehalose (DMTR), 4% DMSO+50 mM sucrose (DMSU), 4% glycerol (GL4), or a mixture of 2% DMSO + 2% glycerol (DMGL2). The results indicate that the DM4 group had better results for progressive motility, viability, and membrane integrity (p < 0.001). Sperm kinematic parameters were the greatest in all DMSO groups compared to the GL groups (p < 0.01). Fortified Tris with DMTR or DMSU significantly improved live sperm with intact acrosomes and significantly reduced live sperm with detached acrosomes (p < 0.01). DMTR group had the greatest total antioxidant capacity (TAC) and lowest malondialdehyde (MDA) and reactive oxygen species (ROS) levels compared to other groups (p < 0.01). Moreover, nitric oxide decreased in DMTR or DMSU groups compared to other groups (p < 0.05). Viable sperm was the greatest in DMSU group, while apoptotic (%) was the lowest in DMSU and DMTR groups (p < 0.01). Total bacterial count was higher in the DM4 group, while lowest in the DMSU group (p < 0.01). While the DMSU treatment resulted in the highest conception rate, no significant differences were observed in litter size across groups (p > 0.05). The cryo-tolerance of rabbit spermatozoa was significantly improved by modifying the freezing extender to include 4% DMSO enriched with either 50 mM Trehalose or 50 mM Sucrose. This optimized formulation enhanced post-thaw parameters, specifically leading to higher sperm kinematics (e.g., motility and velocity), preserved acrosome integrity, and a reduction in apoptosis-like changes. The mechanism for this protective effect is attributed to the synergistic action of DMSO and the disaccharides in mitigating oxidative stress by enhancing the activity of intrinsic antioxidant defenses within the spermatozoa.

Keywords: cryoprotectant; disaccharides; oxidative stress; rabbit; sperm cryopreservation.

Figure 1

Effects of various cryoprotectants agents in a Tris dilution on total antioxidant capacity (TAC) (A), oxidative-related biomarkers such as malondialdehyde (MDA) (B), hydrogen peroxide (H₂O₂) (C), and nitric oxide (NO) (D) in extender of post-thawed rabbit semen. Distinct symbols (^a–b) indicate significant variations between treatments (p < 0.05, Mean ± SE, n = 5). The Tris-based extender was fortified with 4% DMSO (DM4), 4% DMSO + trehalose 50 mM (DMTR), 4% DMSO + surcose 50 mM (DMSU), glycerol 4% (GL4), or 2% DMSO + 2% glycerol (DMGL2).

Figure 2

Effect of different cryoprotectants in a Tris-based extender on apoptosis-like changes (Annexin V/PI assay) involving viable (A), apoptotic (B), necrotic (C), and ROS of post-thawed rabbit semen (D). Distinct symbols (^a–e) indicate significant variations between treatments (p < 0.05, Mean ± SE, n = 3). The Tris-based extender was fortified with 4% DMSO (DM4), 4% DMSO + trehalose 50 mM (DMTR), 4% DMSO + surcose 50 mM (DMSU), glycerol 4% (GL4), or 2% DMSO + 2% glycerol (DMGL2).

Figure 3

Effects of different cryoprotectants in a Tris-based extender on total bacterial count (A) and coliform bacteria count (B) of post-thawed rabbit semen. Distinct symbols (a-b) indicate significant variations between treatments (p < 0.05, Mean ± SE, n = 7). The Tris-based extender was fortified with 4% DMSO (DM4), 4% DMSO + trehalose 50 mM (DMTR), 4% DMSO + sucrose 50 mM (DMSU), glycerol 4% (GL4), or 2% DMSO + 2% glycerol (DMGL2).

Figure 4

The conception rate (A) and litter size (B) of doe rabbits inseminated with semen extended different cryoprotectants in a Tris-based extender. Distinct symbols (^a–d) indicate significant variations between treatments (p < 0.05). The Tris-based extender was fortified with 4% DMSO (DM4), 4% DMSO + Trehalose 50 mM (DMTR), 4% DMSO + Sucrose 50 mM (DMSU), Glycerol 4% (GL4), or 2% DMSO + 2% Glycerol (DMGL2).