Survival of Cryopreserved Stallion Spermatozoa and the Level of Oxidative Modification of Sperm Plasma Proteins

Abstract

Sperm freezing is one of the ways to preserve the sperm of rare, high-productive, physically strong stallions. Freezing negatively affects the reproductive characteristics of sperm, because of an increase in oxidative stress (OS) in particular. Macromolecules, especially proteins, are damaged in the result of OS. Aldehyde and ketone derivatives are formed, which are stable and available for diagnosis. The aim of the study was to determine the level of spontaneous oxidative protein modification (OPM) of sperm plasma in stallions with high and low sperm survival rates during hypothermic storage of cryopreserved sperm. Two experimental groups were formed: the 1st group consisted of 10 stallions, the survival rate of thawed after freezing spermatozoa of which ranged from 6 to 18h (average 11.0±2.8), the 2nd group included 12 stallions with sperm survival from 72 to 96h (average value - 84.1±2.4). Sperm survival time was defined as the ability of spermatozoa to maintain progressive motility above 5% during hypothermic (+4°C) storage of sperm. To assess the level of OPM in spermoplasma, sperctrophotometric analysis of 2,4-dinitrophenylhydrazones (DNPH) formed during the interaction of carbonyl derivatives of proteins with 2,4-dinitrophenylhydrazine (spectrophotometer “SF-2000”, Russia) was used. The R.L. Levine’ technique was modified by E.E. Dubinina. Measurements of DNPH derivatives were carried out at 14 wavelengths to record neutral aldehyde dinitrophenylhydrazones (ADNPHn) in the range 260-280nm, the main character (ADNPHb) - 258-264 and 428-520nm, to determine ketone-dinitrophenylhydrazones of a neutral nature (CDNPHn) 363-370nm, basic character (CDNPHb) - 430-434 and 524-535nm. The area under the absorption spectrum of DNPH derivatives of carbonyl derivatives of proteins (S) was analyzed. The total level of OPM in sperm plasma in group 1 stallions was statistically significantly higher than in group 2 stallions (677.7 Eod/g protein and 476.2 Eod/g protein, respectively, p<0.05). The result indicates a higher severity of OS in the sperm plasma of stallions with a low sperm survival rate, more significant damage to amino acid residues of proteins. OPM of sperm plasma leads to changes in their secondary, tertiary structure that negatively affects the functional state of proteins, the morphological and reproductive characteristics of sperm deteriorate. Damaged proteins can undergo aggregation and fragmentation processes. The accumulation of OPM products, protein aggregates resistant to proteolysis, disrupts cell metabolism, leading to apoptosis. The total level of OPM of sperm plasma in stallions with low survival rates of thawed after freezing spermatozoa is higher than in animals with high survival rates.