Effect of Leptin and Melatonin as Protective Additives to Tris Extender on Frozen Semen Quality of Buffalo Bulls

This study was conducted at Animal Production Research Station, El-Gemmezah, belonging to Animal Production Research Institute, Egypt, in cooperation with Department of Animal Production, Faculty of Agriculture, Mansoura university. Aim of the current study was to evaluate the possible effects of leptin and melatonin as a protective additive in Tris-extender on frozen semen quality of buffalo bulls. Five sexually mature Egyptian buffalo bulls (aged 4 to 5 years) with an average live body weight of (450-500 kg). All bulls were fed on the same diet. Semen was collected, twice a week for 4 wk (40 ejaculates for each bulls), using artificial vagina. The collected ejaculates (mass motility of more than 70%) were pooled, evaluation, divided into 7 replicates and frozen. Semen was extended in Tris- egg yolk extender as unsupplemented (T1), or supplemented with leptin (10, 20 and 50 ng/ml in T2, T3 and T4, or melatonin (10-3M, 10-6M and 10-9M in T5, T6 and T7), respectively. Semen was diluted , equilibrated at 50C for 4 h and frozen in liquid nitrogen. After storage for 4 weeks, frozen straws were thawed at 370C for 30 sec. Semen was evaluated in post-dilution, post-equilibrated and post-thawed semen. Various sperm parameters including percentage of individual motility (IM), dead sperm (DS) and sperm abnormality (SA) were determined. In the seminal plasma of thawed semen, activity of AST, ALT and LDH was measured. Total of 12 buffalo cows in heat were inseminated by semen extended with T1, T3 and T5 to determine conception rate (CR). Pregnancy rate was measured by rectal palpation on day 50 after (AI). The obtained results show that IM and SA percentages improved (P<0.05) in T3 and T5. Percentage of DS improved (P<0.05) in all treatments as compared to control. T3 showed the best (P<0.05) results concerning sperm characteristics studied in post-diluted as compared to other additives and T1. IM, DS and SA percentages showed the best (P<0.05) results in post-equilibrated semen (T3), followed T5 in comparison with other additives and control. IM, DS and SA improved (P<0.05) in post-thawed semen diluted (T3) or T5, being better with T3 more than with T5. Enzyme activity of AST, ALT and LDH reduced (P<0.05) in seminal plasma of post-thawed semen diluted with all additives as compared to control, being the lowest (P<0.05) in T3. Buffalo cows inseminated with semen (T3) showed higher (P<0.05) CR (91.6%) than T5 (75.0%) or control (T1, 66.6%).In conclusion, supplementation of  20 ng/ml leptin and 10-3 M melatonin to the freezing semen extender improved semen quality and reduce cryodamage  of the buffalo bull spermatozoa.