CRYOCAPACITATION AND ITS ASSOCIATION WITH OXIDATIVE FEATURES IN CRYOPRESERVED BOVINE SPERMATOZOA

Abstract

The goal of our research was to evaluate the impact of cryopreservation on the antioxidant activity, lipid peroxidation and protein carbonylation as well as its connection with the progress of cryocapacitation in bovine spermatozoa. As biological material we used semen obtained from 20 sexually mature Holstein bulls. Each ejaculate was divided into three equal aliquots as follows: the first part or control (CTRL) was incubated in physiological saline solution while the second part (CAP) was incubated in a capacitation medium at specific conditions. The third part of each sample was cryopreserved (CRYO) and stored in liquid nitrogen at -196°C for further analysis. The motility of spermatozoa was assessed with CASA (computer assisted sperm analysis), while the capacitation status was evaluated by the chlortetracycline (CTC) fluorescent staining. Total antioxidant capacity (TAC) and the level of lipid peroxidation (LPO) was measured with a combined spectro-fluoro-luminometer. The presence of protein oxidation was detected by the traditional DNPH (2,4-dinitrophenylhydrazine) method and evaluated spectrophotometrically. Based on our data, the motility was significantly decreased (P<0.0001; P<0.01) in the CRYO group against CAP and CTRL. There was a significant increase (P<0.01; P<0.05) of acrosome-reacted spermatozoa (AR-pattern) in the CRYO group when compared to CTRL and CAP, while TAC was statistically decreased (P<0.05) between the CRYO and CAP experimental group. In the case of protein oxidation and LPO, both parameters were significantly higher (P<0.0001) in the CRYO group when compared to CAP or CTRL. In summary, cryopreservation induces capacitation-like changes and promotes oxidative damage in frozen-thawed bovine spermatozoa.