Abstract
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.
Highlights
At least one 100 kDa band was observed in every bull analyzed using western blotting with an anti-angiotensin-converting enzyme (ACE) monoclonal antibody, and band intensity decreased by 70% (p < 0.05) after cryopreservation
Nauc and Manjunath [5] reported a 70% - 80% decrease in the concentration of BSP-A1-A2, BSP 0A3, and BSP-30 kDa proteins in bovine seminal plasma after cryopreservation when compared to fresh bovine semen, and these proteins were later positively correlated with the freezing capacity of bovine semen [3]
To confirm if the same amount of protein was added to all samples, western blotting was performed with an anti-β-tubulin antibody
Summary
Artificial insemination with frozen semen achieves satisfactory pregnancy rates, but the process of cryopreservation can still lead to approximately 50% loss in sperm viability [1]. During this process, the plasma membrane undergoes structural alterations, mainly during the cooling stage (from 19 ̊C to 8 ̊C), where it passes from a liquid to a gel state [2], and these changes are the main cause of intracellular ice crystal formation, excessive influx of calcium ions, and cell dehydration, resulting in decreased sperm fertilization capacity [3]. Westfalewicz et al [3] studied protein changes in sperm membrane due to cryopreservation, and reported altered expression of 16 proteins, which mostly decreased after the freezing/thawing process
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