The aim of this study was to characterize the angiotensin-converting enzyme (ACE) in Gir semen before and after cryopreservation. The ejaculate of five sexually mature bulls was used. After collection, one 1-mL aliquo...The aim of this study was to characterize the angiotensin-converting enzyme (ACE) in Gir semen before and after cryopreservation. The ejaculate of five sexually mature bulls was used. After collection, one 1-mL aliquot of fresh semen was analyzed immediately, and the rest of the semen was cryopreserved in liquid nitrogen for subsequent analysis. Freshly collected semen and thawed cryopreserved semen were centrifuged twice with Tyrode’s albumin lactate pyruvate medium (TALP) to remove plasma and extender, respectively. Samples were then subjected to western blotting, immunocytochemistry, and enzymatic activity techniques. At least one 100 kDa band was observed in every bull analyzed using western blotting with an anti-ACE monoclonal antibody, and band intensity decreased by 70% (p < 0.05) after cryopreservation. Immunocytochemistry showed periacrosomal ACE localization, and the area stained by the fluorescent antibody significantly decreased (p < 0.05) after cryopreservation. Enzyme activity was evaluated using FAPGG substrate hydrolysis, which was significantly lower (p < 0.05) in cryopreserved semen than in fresh semen. Therefore, the process of cryopreservation decreases ACE band intensity and enzyme activity in Gir bull semen, and reduces the stained area in immunocytochemistry.展开更多
The aim of this study was to characterize the testicular isoform of angio-tensin-converting enzyme (tACE) before and after semen cryopreservation, and in the acrosome reaction of sperm from Nelore bulls in vitro. Ejac...The aim of this study was to characterize the testicular isoform of angio-tensin-converting enzyme (tACE) before and after semen cryopreservation, and in the acrosome reaction of sperm from Nelore bulls in vitro. Ejaculates of 10 sexually mature Nelore bulls were used. After semen was collected, 1.0 mL of the ejaculate was used for the analysis and the rest was subjected to cryopreservation. Fresh semen before freezing, and frozen/thawed semen were centrifuged twice and the pellet was resuspended intyrode’s albumin lactate pyruvate (TALP). Thereafter, 100 μL aliquots containing 100 × 106 spermatozoa were prepared. Aliquots of samples were used for western blot analysis, subjected to capacitation, and thereafter, acrosome reaction assays were performed in vitro. With the help of an anti-ACE monoclonal antibody, a 100 kDa protein band was identified in the spermatozoa of Nelore bulls. Cryopreservation reduced the intensity of the protein bands obtained by western blot assay to less than half of that observed prior to freezing (P P < 0.05), indicating the involvement of ACE in these processes. It is concluded that tACE can be found in the spermatozoa of Nelore bulls, and cryopreservation process decreases the intensity of bands of this enzyme;and that the inactivation of tACE reduces the capacity of spermatozoa to undergo the acrosome reaction.展开更多
文摘The aim of this study was to characterize the angiotensin-converting enzyme (ACE) in Gir semen before and after cryopreservation. The ejaculate of five sexually mature bulls was used. After collection, one 1-mL aliquot of fresh semen was analyzed immediately, and the rest of the semen was cryopreserved in liquid nitrogen for subsequent analysis. Freshly collected semen and thawed cryopreserved semen were centrifuged twice with Tyrode’s albumin lactate pyruvate medium (TALP) to remove plasma and extender, respectively. Samples were then subjected to western blotting, immunocytochemistry, and enzymatic activity techniques. At least one 100 kDa band was observed in every bull analyzed using western blotting with an anti-ACE monoclonal antibody, and band intensity decreased by 70% (p < 0.05) after cryopreservation. Immunocytochemistry showed periacrosomal ACE localization, and the area stained by the fluorescent antibody significantly decreased (p < 0.05) after cryopreservation. Enzyme activity was evaluated using FAPGG substrate hydrolysis, which was significantly lower (p < 0.05) in cryopreserved semen than in fresh semen. Therefore, the process of cryopreservation decreases ACE band intensity and enzyme activity in Gir bull semen, and reduces the stained area in immunocytochemistry.
文摘The aim of this study was to characterize the testicular isoform of angio-tensin-converting enzyme (tACE) before and after semen cryopreservation, and in the acrosome reaction of sperm from Nelore bulls in vitro. Ejaculates of 10 sexually mature Nelore bulls were used. After semen was collected, 1.0 mL of the ejaculate was used for the analysis and the rest was subjected to cryopreservation. Fresh semen before freezing, and frozen/thawed semen were centrifuged twice and the pellet was resuspended intyrode’s albumin lactate pyruvate (TALP). Thereafter, 100 μL aliquots containing 100 × 106 spermatozoa were prepared. Aliquots of samples were used for western blot analysis, subjected to capacitation, and thereafter, acrosome reaction assays were performed in vitro. With the help of an anti-ACE monoclonal antibody, a 100 kDa protein band was identified in the spermatozoa of Nelore bulls. Cryopreservation reduced the intensity of the protein bands obtained by western blot assay to less than half of that observed prior to freezing (P P < 0.05), indicating the involvement of ACE in these processes. It is concluded that tACE can be found in the spermatozoa of Nelore bulls, and cryopreservation process decreases the intensity of bands of this enzyme;and that the inactivation of tACE reduces the capacity of spermatozoa to undergo the acrosome reaction.
