The influence of caffeine supplementation and concerted utilization of enzymatic and mechanical semen liquefaction on freezability of dromedary camel spermatozoa

Assisted reproductive technologies have been reported to improve reproductive efficiency and genetic potential in camelids. Two experiments were carried out to determine efficiency of centrifugation in the presence of a mucolytic agent for liquefaction of dromedary semen. In the first experiment, three groups, namely: I. Tris lactose (TL, control), II. Tris lactose supplemented with amylase (TL_A) and III. Tris lactose supplemented with amylase followed by seminal plasma removal via centrifugation (TL_A_(Cent.)) and re-suspension into enzyme free TL media. After equilibration, control group recorded 62.85 ± 4.28% motility and 1.3 ± 0.13 viscosity score, while TL_A group values were 72.88 ± 3.30% and 0.83 ± 0.07%, respectively. TL_A_(Cent.) group showed significant viscosity reduction (0.33 ± 0.05) and motility decline 47.85 ± 3.04% with increment in abnormalities and detached acrosome. The second experiment investigated the effect of caffeine addition to tolerate enzymatic and mechanical stress. Using 4 mM caffeine in amylase-treated semen (TL_AC) improved post-thaw motility 50.0 ± 1.29% and recovery rate 77.8 ± 3.83% compared to the control (40.17 ± 2.79% and 62.55 ± 8.39%), respectively. Caffeine supplemented centrifuged samples (TL_AC_(Cent.)) showed superiority (P < .05) in post-thaw motility and recovery rate (38.33 ± 6.41%, 62.76 ± 8.10%) compared to centrifuged samples TL_A_(Cent.) without caffeine addition (25.00 ± 2.88% and 40.47 ± 3.48%), respectively. Sperm kinetics showed that TL_A exhibited high (p < .05) values for mostly all sperm kinetics. Caffeine treatments showed superiority in velocity curved line (VCL, µm/s) 94.24 ± 8.44 for TL_AC and 104.25 ± 8.72 for the TL_AC_(Cent.) group compared to 86.8 ± 5.54 for TL_A, and 85.73 ± 5.99 for the TL_A_(Cent.) groups. In conclusion, preforming a combined enzymatic-mechanical protocol in the presence of an antioxidant may be crucial for refinement of camel semen cryopreservation.