Standardizing semen cryopreservation protocol in rabbit: impact of sperm concentration on in vitro quality and reproductive performances.

Cryopreserving rabbit semen is a powerful tool for breeding management via artificial insemination and biodiversity conservation through semen cryobanks. Creating cryobanks of Italian rabbit breeds is a significant step towards promoting sustainability, given their adaptability to alternative farming methods like organic farming. Exploring rabbit freezing protocols, emerge that one key aspect needs to be standardised is the concentration of spermatozoa. Accurate determination of the number of sperm within straws is essential for enhancing existing protocols and reducing result variability. Our objective was to evaluate the influence of different sperm concentrations on the in vitro quality and reproductive performances of cryopreserved rabbit semen. In the first experiment, semen pools were split into three aliquots, and prediluted with Tris–citrate–glucose (TCG) and then further extended with freezing extender (TCG with cryoprotectants i.e. 16% dimethylsulfoxide and 0.1 mol/L sucrose) to reach 15, 35, and 75×10^6 spermatozoa inside the straws. The processed semen was equilibrated at 5°C for 45 min and cryopreserved through exposure to liquid nitrogen vapour. Sperm motility parameters (CASA system) and membrane integrity (SMI, flow-cytometry) were assessed in freshly diluted, equilibrated, and thawed semen. No significant impact of sperm concentration was observed in the fresh semen. During equilibration, the concentration of 15×10^6 showed the highest motility values (p<0.05). After thawing, higher total motility (p<0.05) occurred at 35×10^6 sperm/straw (47.4±3.9%) compared to 75×10^6, with significantly higher progressive motility at 35×10^6 (20.8±2.4%). SMI remained unaffected by concentration. In the second experiment, four groups of 20 does were inseminated with fresh and frozen semen using the three sperm concentrations/straw respectively. No significant differences in fertility and prolificacy between fresh and frozen semen were found. However, frozen semen showed higher fertility at a concentration of 35×10^6 and better prolificacy at 15×10^6, although no significant differences were registered. This study demonstrates that sperm concentration per straw plays a key role in rabbit semen freezing and it primarily impacts in vitro quality. Notably, it emphasizes that frozen semen yielded fertility and prolificacy rates comparable to those of fresh semen. These findings offer valuable indications for refining and enhancing the semen freezing protocol in rabbit.