Evaluation of sperm motility across varying thawing temperatures in Jersy, Holstein Friesian, and Murrah Bulls

Background: The heating rate during the thawing of frozen semen significantly impacts the functional activation of mitochondria, which affects sperm motility assessment. Methods: This study examined the effect of thawing temperature on the motility of spermatozoa in cryopreserved semen. A total of 240 semen straws (0.25 ml each) from 24 bulls (8 Jersey, 8 Holstein Friesian (HF), and 8 Murrah breed), aged 2 to 6 years, were used. Semen was collected, analyzed, processed, frozen, and stored in liquid nitrogen using a standard protocol with a tris-citrate-egg yolk extender. Samples were thawed for 30 seconds in a water bath at temperatures T1: 32°C, T2: 34°C, T3: 36°C, T4: 38°C, and T5: 40°C. Sperm motility, progressive motility, fast motility, slow motility, and immotile percentage of the frozen semen were evaluated. A computer-assisted semen analyzer (CASA) was used for analysis. The data was entered into MS-Excel and analyzed using analysis of variance (ANOVA), with the significance between treatments assessed using Duncan’s multiple range test (DMRT) post hoc test. Results: Jersey semen showed the best motility percentage at T3 (p<0.05) (T1: 53.025±6.73, T2: 60.01±3.81, T3: 74.33±1.40, T4: 65.59±3.17, T5: 59.92±3.58). HF semen also showed the best motility percentage at T3 (p<0.05) (T1: 60.67±6.31, T2: 55.93±6.31, T3: 76.6±2.28, T4: 72.93±2.10, T5: 57.69±2.28). Murrah semen showed the best motility percentage at T4 (p<0.05) (T1: 56.63±3.93, T2: 58.58±4.34, T3: 77.09±1.59, T4: 82.72±4.03, T5: 72.87±4.00). Progressive motility (%) was highest at T3 for Jersey (52.41±2.97), T3 for HF (56.29±4.65), and T4 for Murrah (63.94±6.37) (p<0.05). Fast motility (%) was highest at T3 for Jersey (12.71±2.04), T3 for HF (18.43±1.63), and T4 for Murrah (12.589±2.74) (p<0.05). Slow motility (%) was lowest at T3 for Jersey (27.8±3.43), T3 for HF (34.23±2.72), and T4 for Murrah (31.32±4.72) (p<0.05). Immotility (%) was lowest at T3 for Jersey (25.73±1.37), T3 for HF (23.4±2.28), and T4 for Murrah (17.27±4.03) (p<0.05). Conclusion: Thawing at T3 (36°C) enhances motility, progressive motility, and fast motility while reducing slow motility and immotile percentage in Jersey semen. Similarly, thawing at T4 (38°C) improves motility, progressive motility, and fast motility while reducing slow motility and immotile percentage in Murrah semen.