To assess sperm quality, it is important to evaluate energy metabolism. The test substance 2.4-dinitrophenol (2.4-DNP) is an agent for destroying oxidative phosphorylation. 2.4-DNP shuts off the production of adenosine triphosphate (ATP) from oxidation and then, the respiration rate increases. If the respiratory chain is damaged, there is little or no response to adding 2.4-DNP. The aim of this study was to analyze the respiratory activity and oxidative phosphorylation in semen before and after freezing and compare the obtained data with the fertilizing ability of sperm. There was a reduction in sperm respiration rates in all species after thawing. The respiration of spermatozoa of boars, bulls, stallions, reindeers and chicken showed responses to 2.4-dinitrophenol. The only difference is in the strength of the response to the test substance. After freezing and thawing, respiratory stimulation by 2.4-DNP decreased. The results of our study show that respiration rate is not correlated with pregnancy rates and egg fertility. However, there was a high correlation between the stimulation of respiration by 2.4-dinitrophenol and pregnancy rates. The test for an increase in respiration rate after adding 2.4-dinitrophenol could be a suitable test of the fertilizing ability of sperm.

A b s t r a c tMetabolic processes in milk-producing cows are substantially modified that is the main reason of different abnormalities in the reproductive function. At the same time the pattern of metabolic changes and its influence on the reproductive capacity in high-producing dairy goats (Capra hircus) are yet unknown. The aim of the present work was to study the activity of metabolic enzymes in the blood of Saanen goats depending on the period of the reproductive cycle and the competence for pregnancy maintenance. We analyzed for the first time alterations in the serum activity of enzymes, regulating the intensity of protein-carbohydrate and energy metabolism including integration of metabolic processes, at different stages of goat gestation as well as prior to the mating period in animals with the negative outcome of the subsequent pregnancy. The enzyme status of animals was assessed during the pre-mating period, the first half of pregnancy (1.5-2.5 months), and the second half of pregnancy (3.5-4.0 months). Goats were divided into two groupswith the completed reproductive cycle (the birth of viable offspring, n = 15) and with the interrupted reproductive cycle (abortions in the second half of pregnancy, n = 6). Samples of blood serum were tested to determine concentrations of total protein and activities of several enzymes: aspartate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2 ), gamma glutamyltransferase (GGT, EC 2.3.2.2), creatine phosphokinase (CPK, EC 2.7.3.2), alkaline phosphatase (ALP, EC 3.1.3.1), lactate dehydrogenase (LDH, EC 1.1.1.27), and alpha hydroxybutyrate dehydrogenase (isoenzymes of LDH: LDH-1 and LDH-2). In goats with the completed reproductive cycle, the blood activity of AST in the second half of pregnancy was found to be 1.2 times lower (p < 0.05) than during the pre-mating period or the first half of pregnancy. The activity of ALT in the blood serum of the animals increased 1.3 times (p < 0.01) by 1.5-2.5 months of pregnancy as compared with the pre-mating period and then decreased 1.9 times (p < 0.001) by 3.5-4.0 months of pregnancy. Furthermore, there was a decline in the blood activities of GGT (1.3 times, p < 0.05), CPK (1.9 times, p < 0.01), and isoenzymes LDH-1 and LDH-2 (1.3 times, p < 0.05) and a rise in the activity of ALP (1.4 times, p < 0.05) between the first and the second half of pregnancy. During the pre-mating period, a lower activity of ALT and GGT was revealed in goats with the interrupted reproductive cycle as compared with animals retained pregnancy (14.4±2.9 vs. 20.1±0.7 U/l, p < 0.05 and 43.8±2.4 vs. 54.7±4.2 U/l, p < 0.05, respectively). The results of this research suggest a reduction in the intensity of some metabolic processes in the goats by the fourth month of pregnancy to maintain increased fetus demands. Analysis of the findings also indicates that the activity of enzymes, regulating the coupling of protein and carbohydrate metabolism, in the blood of goats during the pre-mating period may be related to their subsequent ...

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One of the causes of cell death is a violation of electron transfer through the respiratory chain of mitochondria. The aim of study was to assess the integrity of the respiratory chain sperm after freezing in roosters. The effect of 2.4 dinitrophenol (2.4 DNP) on NADH fluorescence in fresh and cryopreserved semen of roosters was investigated. If the transfer of electrons and protons along the respiratory chain functions normally 2.4 DNP enhances the nonphosphorylating oxidation of NADH and its fluorescence decreases. The sperm of 30 cocks was frozen. The NADH was measured on a LUMAM fluorescence microscope. The luminescence excitation wavelength of NADH is 365 nm. Registration of NADH was carried out at a wavelength 465 nm. The amount of NADH was expressed in conventional units and the change in fluorescence after addition of 2.4 DNP was calculated as a percentage of the initial value taken as 100 %. The fluorescence of NADH after adding 2.4 DNP to frozen semen was 9% less than in fresh semen. A relationship between the magnitude of the change in NADH fluorescence after the addition of DNP and egg fertilization was established (r =0.65; P<0.05). Respiratory stimulation by 2.4 DNP is an useful and informative indicator in assessing sperm quality. Support or Funding Information The study was supported by the Russian Science Foundation (project No. 18‐16‐00071)

Один из подходов к решению практических проблем криоконсервации клеток заключается в повышении устойчивости сперматозоидов к повреждающему действию низких температур. Мы изучили влияние производных бензимидазола этил-1-бензимидазол-2-ил-сульфанила, 2-этилсульфанилбензимидазол-1-ила и 2-бензимидазол-1-ил-1-уксусной кислоты в концентрациях 10  3 , 10  5 , 10  11 , 10  13 , 10  15 М на сохранность сперматозоидов при замораживании и оттаивании, на их устойчивость к холодовому шоку и изменению осмотического давления. Установлено, что изученные вещества повышали живучесть бычьих сперматозоидов при хранении в лактозо-цитратной среде. При этом наибольшую активность и сохранность отмечали при введении в среду для замораживания спермы 2-бензимидазол-1-ил-1-уксусной кислоты в сверхнизкой концентрации 10  13-10  15 М. Время переживания сперматозоидов до сохранения подвижности у 10 % клеток повышалось по сравнению с контролем на 73 %. Аналогичные результаты получили при замораживании-оттаивании спермы жеребцов в присутствии 2-бензимидазол-1-ил-1-уксусной кислоты в концентрации 10  13 М: доля подвижных неповрежденных клеток после замораживания-оттаивания оказалась на 15,2±3,49 млн/мл (Р < 0,01) больше, чем в среде без добавки. Выживаемость сперматозоидов быков после замораживания-оттаивания была на 8,1 % (Р < 0,01) выше, чем в контроле, сохранность акросом-на 1,9±0,63 % (Р < 0,05) выше. При изменении осмолярности среды для разбавления спермы и после холодового шока 2-бензимидазол-1-ил-1-уксусная кислота увеличивала живучесть сперматозоидов быка при повышенной температуре (40 С). Можно предположить, что наблюдаемый эффект связан с защитным действием 2-бензимидазол-1-ил-1-уксусной кислоты на плазматическую мембрану и мембранные структуры митохондрий сперматозоидов. Действительно, динитрофенол практически одинаково усиливал клеточное дыхание в опыте и контроле, тогда как сукцинат, который проникает через поврежденные мембраны, оказывал меньшее стимулирующее влияние в присутствии 2-бензимидазол-1-ил-1-уксусной кислоты. В результате проведенных исследований предложена гипотеза о механизме действия сверхмалых концентраций производных бензимидазола на криорезистентность половых клеток при замораживании. Молекулы исследуемого вещества взаимодействуют с рецептором на внешней мембране сперматозоида, что взывает перестройку мембранных структур клетки. Одновременно происходит изменение вязкости воды, ассоциированной с белками мембраны, из-за формирования водородных связей с кислотными остатками в молекуле производного бензимидазола. В результате повышается устойчивость мембранных структур сперматозоидов к повреждениям вследствие колебаний осмотического давления. Такое состояние воды, возможно, сказывается на характере кристаллообразования при замерзании в сторону уменьшения размеров кристаллов, что снижает их повреждающее действие.