doi: 10.15389/agrobiology.2023.4.660eng

UDC: 636.1.082.4:577.1

The equipment of the Center for Collective Use the Collection of Genetic Resources of the All-Russian Research Institute of Horse Breeding was used
Supported financially by the Russian Science Foundation, grant No. 20-16-00101-П


M.M. Atroshchenko1 , M.G. Engalycheva1, 2, A.M. Shitikova1, 2

1All-Russian Research Institute for Horse Breeding, Divovo, Rybnoe District, Ryazan Province, 391105 Russia, e-mail (✉ corresponding author);
2Pavlov Ryazan State Medical University, 9, ul. Vysokovol'tnaya, Ryazan, 390026 Russia, e-mail:,

Atroshchenko M.M.
Shitikova A.M.
Engalycheva M.G.

Final revision received September 8, 2022
Accepted November 1, 2022

Cryopreservation of stallion semen is a modern widespread method in horse breeding for preserving the genetic material of animals. Freezing and thawing reduces the reproductive characteristics of spermatozoa. Oxidative stress that causes damage to macromolecules is a factor contributing to damage to germ cells. With age, the oxidative stress and the amount of damaged proteins increase. In this work, for the first time, we quantified products of oxidative modification of proteins (OMP) in semen plasma in stallions of different ages. There is a significant increase in the content of protein carbonylation products in older animals compared to younger ones, mainly due to neutral aldehyde derivatives. This study is the first to assess the reserve-adaptive potential (RAP) of the seminal plasma of stallions. It was found that the ability to withstand oxidative stress in young stallions is significantly higher than in older stallions. The aim of this study was to assess the level of spontaneous OMP, induced OMP and the RAP values for stallion spermatozoa as influenced by the animal age. The study was carried out in 2020 on 40 purebred Arabian and Soviet draft stallions (Equus ferus caballus L.) (AO Tersk breeding stud No. 169, Stavropol Territory; Perevozsky and Pochinkovsky stud farms, Nizhny Novgorod Province). Three ejaculates of each stallion were collected with a 48-hour interval. The stallions of group I (n = 20) were from 14 to 21 years of age (mean age 15.8±1.9 years), of group II (n = 20) from 3 to 5 years of age (4.3±0.6 years). In each ejaculate, the volume and concentration of spermatozoa in 1 ml of semen was determined. Then the ejaculate was divided into two parts, one was diluted with lactose-chelate-citrate-yolk (LCCY) medium in a ratio of 1:3 and the progressive motility (PM) and survival of spermatozoa were determined at 4 °C. To assess the survival of spermatozoa during hypothermic storage of sperm, its PM was determined with a 24-hour interval up to a decrease in PM to 5 %. Sperm was frozen in liquid nitrogen vapor in 18 ml aluminum tubes according to the standard of the All-Russian Research Institute for Horse Breeding and stored in liquid nitrogen at -196 °C. The cryopreserved sperm was thawed in a water bath at 40 °C for 90 s, followed by the determination of the spermatozoa PM and survival at 4 °C. Another part of the ejaculate was centrifuged at 3500 rpm for 20 min. After microscopy of the supernatant, aliquots of seminal plasma free of spermatozoa were frozen in 2.0 ml Eppendorf tubes at -18 °C. To quantify the OMP, we used the spectrophotometric analysis of 2,4-dinitrophenylhydrazones formed by the interaction of protein carbonyl derivatives (aldehydes and ketones) with 2,4-dinitrophenylhydrazine. The total amount of carbonyl derivatives was recorded in a native sample of biological material (spontaneous OMP) and after in vitro induction of protein oxidation of biological material with a reaction mixture containing solutions of iron(II) sulfate and hydrogen peroxide (metal-catalyzed induced OMP). From metal-catalyzed and spontaneous OMP, RAP was evaluated to characterize the OS resistance. Spectrophotometric measurements were carried out at 14 wavelengths, at 260-280 nm for neutral aldehyde-dinitrophenylhydrazones, at 258-264 and 428-520 nm for basic aldehyde-dinitrophenylhydrazones, at 363-370 nm for neutral ketone-dinitrophenylhydrazones, and at 430-434 and 524-535 nm for basic ketone-dinitrophenylhydrazones. Statistically significant differences in sperm quality between animals of two age groups were found only in the survival rate of spermatozoa during hypothermic storage of diluted (p < 0.05) and cryopreserved (p < 0.01) sperm. The total amount of spontaneous OMP products in the seminal plasma of older stallions was statistically significantly higher than in young stallions (531.7 and 384.3 ODU/g protein, respectively, p < 0.05). In addition, in group I, there was a shift in the absorption spectrum towards neutral aldehyde derivatives the content of which in animals of group II was significantly lower (367.6 and 255.8 ODU/g protein, p < 0.05). The evaluation of metal-catalyzed (induced) OMP also revealed a higher total amount of carbonyl derivatives in older stallions, but its increase under the influence of an oxidizing mixture was much higher vs. the initial spontaneous OMP in young stallions. The RAP value for the seminal plasma of young stallions significantly exceeds that of mature stallions (p < 0.05), which can positively affect the reproductive characteristics of native and cryopreserved sperm.

Keywords: Equus ferus caballus, stallions, sperm, seminal plasma, cryopreservation, oxidative stress, protein oxidative modification.



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