No 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.

The development of neurodegenerative pathologies, among which the leading positions worldwide belong to Alzheimer's disease, is accompanied by the activation of free radical processes in the tissues of the brain and in the patient's body as a whole, which leads to the accumulation of products of oxidative modification of proteins, lipids, nucleic acids, and the formation of a syndrome of endogenous intoxication. An integral indicator reflecting the severity of this syndrome is the level of substances of low and medium molecular weight in various biological media. It is known that changes at the molecular level in Alzheimer's disease occur 5-10 years before the clinical manifestation of pathology. The development of polymodal panels for biochemical screening, diagnostics, monitoring of the course of pathology is underway. The aim of this study was to determine the severity of endogenous intoxication in patients with Alzheimer's disease and vascular dementia. The object of the study was the blood plasma of patients, as well as fractionated leukocytes - mononuclear and polymorphonuclear. The choice of biological material for the study is dictated by the need to search for a prognostic marker of the disease available for diagnosis, as well as by the accumulated theoretical material on various metabolic changes in peripheral blood cells in neurodegenerative diseases. According to the results of the study, it was found that in patients with Alzheimer's disease in mononuclear leukocytes of peripheral blood, the level of substances of low and medium molecular weight is higher than in patients with vascular dementia and patients without signs of neurodegeneration. In addition, in this fraction of leukocytes, the concentration of hypoxanthine, inosine, xanthosine and their derivatives is high, which may be a consequence of the oxidative modification of nucleic acids in the cells under study.

During the aerobic metabolism of sperm, various reactive oxygen species are produced. Sperm cells are extremely vulnerable to oxidative stress (OS), which causes loss of membrane integrity, resulting in increased cell permeability, enzyme inactivation, structural DNA damage and cellular apoptosis. The aim of this study was to assess the reserve‐adaptive potential (RAP) of sperm plasma in stallions of various ages. RAP is the difference between the level of spontaneous and induced oxidative protein modification (OMP), reflecting the degree of OS. Spontaneous OMP reflects the accumulation of OS products in vivo. To determine the level of spontaneous OMP, a sperctrophotometric analysis of 2,4‐dinitrophenylhydrazones (DNPH) formed during the interaction of carbonyl derivatives of sperm plasma proteins with 2,4‐dinitrophenylhydrazine was carried out (spectrophotometer “SF‐2000”, Russia). R.L. Levine, modified by E.E. Dubinina. Measurements of DNPH derivatives were carried out at 14 wavelengths: in the range 260‐280nm for the registration of aldehyde dinitrophenylhydrazones of a neutral nature (ADNPHn), of a basic nature (ADNPHb) – 258‐264 and 428‐520nm, for the determination of ketone‐dinitrophenylhydrazones of a neutral nature (CDNPHn) 363‐370nm, basic character (CDNFGb) – 430‐434 and 524‐535nm. After that, the total amount of carbonyl derivatives was found. Induced OMP was determined in a similar way, but after adding iron (II) sulfate and hydrogen peroxide solutions to the reaction mixture. This provoked the formation of new carbonyl groups. The more RAP, the higher the ability of the biological material to resist the OS. Two groups of stallions were formed: the first group consisted of 15 stallions aged 13 to 20 years (average age 15.2±1.8 years), the second group consisted of 15 stallions aged 3 to 6 years (average age 4.0±0.5 years). The spontaneous OMP of sperm plasma in aged stallions was 495.6 Eod/ g protein, the induced one – 573.4 Eod / g protein. In the group of young stallions, spontaneous OMP – 347.2 Eod / g protein, induced – 485.3 Eod / g protein. The OMP level of sperm plasma in older stallions is initially higher than in young stallions (p = 0.04), which indicates the accumulation of carbonyl derivatives in vivo as a result of the prevalence of OS over the antioxidant capabilities of the organism. But when additional oxidizing components are added to the reaction mixture, the OMP of aged stallions does not increase as significantly as in young ones. RAP of sperm plasma of old stallions is 13.56%, of young stallions – 28.75%. The ability to withstand OS, the reserve of antioxidant capabilities of sperm plasma in young stallions is 2.1 times higher than in older stallions.