Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolitic 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 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 acrosome reaction (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 of lysophosphatidylcholine (LPC), as positive control and to 0.01 μg/mL 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 % formaldheyde 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 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 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 incubation (90, 75 and 75 %, respectively; P<0.05) and at 4 h 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.

The effect of plasmin on acrosome reaction of buffalo (Bubalus bubalis) spermatozoa in vitro / I., Venditto; E., Mariotti; L., Boccia; M., Rubessa; M., De Blasi; A., Sattar; Gasparrini, Bianca. - In: REPRODUCTION FERTILITY AND DEVELOPMENT. - ISSN 1031-3613. - STAMPA. - 22 (I):(2010), pp. 317-317.

The effect of plasmin on acrosome reaction of buffalo (Bubalus bubalis) spermatozoa in vitro

GASPARRINI, BIANCA
2010

Abstract

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolitic 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 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 acrosome reaction (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 of lysophosphatidylcholine (LPC), as positive control and to 0.01 μg/mL 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 % formaldheyde 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 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 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 incubation (90, 75 and 75 %, respectively; P<0.05) and at 4 h 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.
2010
The effect of plasmin on acrosome reaction of buffalo (Bubalus bubalis) spermatozoa in vitro / I., Venditto; E., Mariotti; L., Boccia; M., Rubessa; M., De Blasi; A., Sattar; Gasparrini, Bianca. - In: REPRODUCTION FERTILITY AND DEVELOPMENT. - ISSN 1031-3613. - STAMPA. - 22 (I):(2010), pp. 317-317.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/357234
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