322 EFFECT OF PLASMIN ON ACROSOME REACTION OF BUFFALO (BUBALUS BUBALIS) SPERMATOZOA IN VITRO

Abstract

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolytic enzymes are involved in different steps of the fertilization process; among these, a critical role may be played by the plasminogen activator-plasmin system. It has been demonstrated that plasmin, the active enzyme of this system, induces acrosome reaction (AR) in bull spermatozoa (Taitzoglou IA et al. 2003 Andrologia 35, 112-116). The aim of this study was to investigate the effect of plasmin on the ability of buffalo sperm to undergo the AR. Frozen- thawed sperm from 4 buffalo bulls were treated by swim-up and incubated with 0.01 mM heparin for 4 h. At 0, 2, and 4 h, aliquots of spermatozoa were exposed for 10 min to 60 μg mL^-1 of lysophosphatidylcholine (LPC), as positive control, and to 0.01 μg mL^-1 of plasmin. This concentration was chosen after a preliminary dose-response trial. Another sample from each treatment was incubated with IVF medium (negative control). After 10 min, sperm motility was evaluated and sperm were fixed in 37% formaldehyde and stained with trypan blue-Giemsa for subsequent microscopic examination. The total number of sperm counted, over 3 replicates, was 1269 for the negative control, 1293 for LPC, and 1238 for plasmin, equally distributed among incubation times. Differences among groups were analyzed by chi-square test. After swim-up, acrosomal loss was observed only in 4% of the sperm. The addition of 0.01 μg mL^-1 of plasmin for 10 min to buffalo spermatozoa at time 0 significantly (P < 0.01) enhanced (23%) AR compared with the control (7.8%), with the same efficiency of LPC (17.1%). After 2 h of incubation with heparin, both plasmin and LPC increased the AR compared to the control (24.4, 20.1, and 14.0%, respectively; P < 0.01). After 4 h, plasmin gave higher percentages of AR (27.2%) compared to both the control (21.0%; P < 0.05) and LPC (19.2%; P < 0.01). Another interesting result is the improved motility recorded with plasmin compared to both the control and LPC groups at 2 h of incubation (90, 75, and 75%, respectively; P < 0.05) and at 4 h of incubation (75, 60, and 60%, respectively; P < 0.05). Finally, no differences in sperm viability were observed between plasmin and the control, whereas a decreased viability was found when LPC was used at 0 h (96.2, 95.0, and 89.0%, respectively, for plasmin, control, and LPC; P < 0.05), at 2 h (85.0, 87.5, and 77.0%, respectively, for plasmin, control, and LPC; P < 0.01), and at 4 h (85.0, 93.3, and 81.1%, respectively, for plasmin, control, and LPC; P < 0.01). In conclusion, we found that addition of plasmin to capacitated sperm increases the percentage of acrosome-reacted spermatozoa and improves motility. Our results suggest that plasmin may play a role in events surrounding fertilization and suggest to evaluate in further studies whether the addition of plasmin during IVF improves the IVEP efficiency in buffalo.