Application of a synthetic phenol-type antioxidant for cryopreservation of Russian sturgeon (Acipenser gueldenstaedtii) sperm

. Antioxidative and cryoprotective effects of a novel synthetic antioxidant – 1,1'-(piperazine-1,4-diyl)bis(2-(2-hydroxyphenylthio)ethanone ( AO ) is presented in comparison with commonly known antioxidant butylated hydroxytoluene ( BHT ). Superoxide anion radical scavenging activity of new AO and the increase in superoxide dismutation activity of the Russian sturgeon’s ( Acipenser gueldenstaedtii ) sperm were established in the presence of this compound. It was shown that the new phenol agent reduces level of carbonyl oxidation by-products, which can react with thiobarbituric acid (TBARS), in Russian sturgeon’s native and frozen/thawed sperm, which indicates the manifestation of antioxidant properties by this compound including under the action of damaging factors during cryopreservation. A beneficial effect of this phenol derivative on the activity indicators (the percentage of motile sperm cells and total period of sperm movement) of the Russian sturgeon’s frozen/thawed sperm was shown, which indicates the cryoprotective effect of a novel synthetic antioxidant. The efficiency of the AO exceeds the effect of BHT under cryopreservation conditions of Russian sturgeon sperm in the presence of the modified Stein’s medium, but f urther studies are needed on the effect of a new phenol agent on the fertilizing capacity of Russian sturgeon’s sperm.


Introduction
Various negative environmental pressures have caused declines in fish populations and diversity of species with currently threatened stocks, including sturgeon.Cryopreservation, long-term storage of sperm, is an important technique for conservation of endangered species, keeping genome pool of the male brood stock.However, it is well known that cryopreservation induces damage to spermatozoa, which may result in loss of motility, viability, and fertilizing capacity.It was found that cryopreservation increases reactive oxygen species (ROS) production to toxic levels in cells resulting in lipid peroxidation (LPO) [1], protein oxidation [2,3] and inactivation of enzymes associated with sperm motility [4], DNA fragmentation [5], mitochondrial dysfunction [6].Oxidative stress is a state of imbalance between ROS generation and depletion, involving enhanced ROS production as one of the damage causes [7].Fish sperm is sensitive to damage by ROS due to the high content of polyunsaturated fatty acids within the lipid structure of cell membranes [8].This requires efficient antioxidant systems to defend against LPO damage of membranes and associated sperm dysfunction.However, endogenous antioxidant capacity in sperm cells is limited [9].For this reason, the antioxidant additives effect in the cryomedium during semen cryopreservation of several fish species including sturgeon has been extensively studied [10][11][12][13].
It was previously found that supplementation with butylhydroxytoluene (BHT) analogue, such as (3,5-di-tert-butyl-4-hydroxyphenyl)methylenediphosphonic acid in cryopreservation media was efficient in the case of low-temperature cryopreservation of sturgeon sperm [14,15].In recent years a large number of pharmacologically active substances have been synthesized on the basis of phenols and various heterocyclic compounds.The antioxidant capacities of novel nitrogen-containing heterocyclic compounds) have been discovered [16].It was shown that the nitrogen-containing heterocyclic derivatives [17], as well as the sulfur-containing phenols [18] were effective as antioxidant additives in cryopreservation media in the case of low-temperature cryopreservation of Russian sturgeon (Acipenser gueldenstaedtii) sperm.
The aim of this work is to compare the antioxidative and a cryoprotective activity of a new synthetic antioxidant -1,1'-(piperazine-1,4-diyl)bis(2-(2-hydroxyphenylthio)ethanone (AO) and one of the most widely used synthetic antioxidants BHT during cryopreservation of Russian sturgeon sperm in the presence of the modified Stein's medium.The antioxidant and cryoprotective activity of these compounds was assessed experimentally in vitro.The superoxide anion radical (O2 -• ) scavenging activity of phenol derivatives, their effect on the ability of the Russian sturgeon's sperm to deactivate O2 -• , on LPO in sturgeon's sperm were investigated.Cryoprotective effect of studied compounds was compared using its influence on the LPO level of Russian sturgeon's sperm cells, on its activity in the cryopreservation conditions.

Reagents and solutions
New phenol derivative AO was synthesized by a method that was published earlier [19].The 0.1% (5.46 mМ) adrenaline (epinephrine, a pharmaceutical form) was used in this study.The reagents including BHT (99%) were purchased from Sigma Aldrich Chemical Co.In experiments with fish sperm AO and BHT were dissolved in the modified Stein's cryomedium (130 mM NaCl, 5 mM KCl, 20 mM NaHCO3, 5.5 mM glucose, 12.5% egg yolk, 12.5% Me2SO) [20], to make a stock solution with the sperm at a concentration of 0.1 mM.

Sperm collection
Russian sturgeon sperm was received from sturgeon hatcheries of the Low Volga (Bertyulsky, Lebyazhyi, Sergiyevsky) in the spawn period (from the end of April till middle of May 2022) and used in the study.Males of Russian sturgeon (10 male fish) had average mass 9.0 ± 0.9 kg (9-11 years old).For stimulation of sturgeon gametes maturation, the Luteinizing Hormone -Releasing Hormone Ethylamide (LH-RHa, Surfagon) was injected at dose of 0.5-1 mg/kg body weight at water temperature 14-16 °С.The sperm of Russian sturgeon males was collected into glass containers by catheter.The seminal fluid collected for the experiments was cooled (8 ± 2 °С) and delivered to the laboratory in the thermal container.

Determination of the superoxide anion radical scavenging activity and SOD-protective activity
О2 -• scavenging activity of a testing compounds and the superoxide dismutase (SOD)protective activity of the Russian sturgeon's sperm were determined using superoxide generating reaction of autoxidation of adrenaline in alkaline medium, registering the formation of adrenochrome at 347 nm according to the methods of Sirota (2010Sirota ( , 2015) ) [21,22] as previously described [23,24].The reaction was started by adding 10 µl of 0.1% adrenaline hydrochloride to a 0.2 ml buffer with continuous stirring.The ethanol solution of phenol derivatives was added 5 µl to the buffer prior to adding adrenaline.The reaction mixture contained 230 µM of adrenaline hydrochloride, 0.43 mM of carbonate buffer (pH 10.65) and 25 µM of phenol compounds.The percentage inhibition of adrenochrome accumulation (I) was calculated by the formula 1: where Ai is the absorbance in the presence of the testing compound and A0 is the absorbance of the blank solution.The optical density was recorded for 4 min by microplate spectrophotometer Zenyth 200rt Anthos and the measurement was repeated in triplicate.
The SOD-protective activity of a biological product is understood as its ability to utilize O2 -• .This activity was determined by Sirota method [25].In this case adrenochrome level was determined in the presence of sperm, 50 µl sperm was added to the buffer prior to adding a testing compound.The blank solution contained a biopreparation (source of SOD).Inhibition of the adrenochrome accumulation in the presence of the compounds under study (the initial concentration of these compounds was 25 M) was used to characterize their effect on the SOD-protective activity of the sturgeon's sperm.

Determination of Russian sturgeon's sperm lipid peroxidation level
Russian sturgeon's sperm LPO intensity has been assessed by carbonyl oxidation byproducts accumulation, which reacted with thiobarbituric acid (TBARS) using the published method [26].Determination method of TBARS level in sturgeon sperm before cryopreservation with cryomedium and after cryopreservation with cryomedium also was published earlier [27].To 156 ml of 1.2% solution of KCl at 0-4 °C 8 ml of sturgeon's sperm and 0.1 mM of tested compound (AО, trolox) was added.The resulting mixture was incubated for 48 h at 5 °C, the 2 ml probes of a mixture were taken after 1, 3, 24 and 48 h into the plastic tubes (4 ml) for centrifugation.0.1 ml of 2.6 mM solution of ascorbic acid and 0.1 ml of 40 lM Mohr's salt, 1 ml of 40% solution trichloroacetic acid were added to each probe.The tubes were placed for 10 min in a water bath at 37 °C, then they were centrifugated for 10 min at 3000g.On the next step 2 ml of supernatant were transferred to the clean tubes, 1 ml of 0.8% solution of thiobarbituric acid was added, the tubes were placed into a boiling water bath for 10 min and then they were cooled to the room temperature (25 °C).After cooling 1.0 ml portions of chloroform were added to the tubes to obtain receive the transparent solutions and these probes were centrifuged at 3000g for 15 min.Supernatant liquid was collected and extinction of the probe was measured using SF-103 spectrophotometer at 532 nm, the test probe was taken as a standard.The calculation was performed by the formula 2: where X (nmol) is the quantity of TBARS in native sperm; E -the extinction factor of the probe; 3.2 ml is the total volume of sperm from the tested fish; 2 ml is the volume of supernatant used for TBARS determination; 3 ml is the total volume of probes; 0.156 is the extinction factor of the 1 nmol TBARS at 532 nm.TBARS content in the sturgeon's sperm lipids was expressed as nano-moles per 10 9 cells.

Motility and duration time of sperm after activation
The percentage of motile Russian sturgeon sperm cells was estimated at room temperature (21°C) using binocular microscope Micmed-5 with video-eyepiece НВ-200 (LOMO, Russia) after river water addition as an activating solution to the post-thaw sperm at a 1:250 ratio, and the fresh sperm was activated at a 1:1000 ratio.For cryopreservation sturgeon sperm with 4 and 5 points activity was used that is determined according to the Persov scale [28], spermatozoa concentration in the sperm (Goryaev camera) was 2.01⋅10 9 cells/ml.Duration time (total period of sperm movement, sec) was defined as the time from the activation to the stop of movement of last spermatozoa in microscope field of view using stopwatch.The same operator measured motility and duration time of sperm after activation in triplicates.

General procedure for sperm freezing and thawing
Sperm cryopreservation was carried out according to the methods of Tsvetkova et al.
(2012) [29].The semen diluted with a cryoprotective medium was poured into prenumbered polyethylene containers with a volume of 1.5 and 0.6 ml and placed in a freezer of cryobank-reproductor of Southern Scientific Center of Russian Academy of Sciences (SSC RAS) with an average temperature of -20 °C for 1 hour.The ratio of sperm and cryomedium was 1:1.The freezing temperature was measured with an electronic thermometer.After that, freezing was carried out in three stages: • from 5°C to -15°C at a speed of 2-5 °C/min (freezing time 2-5 minutes); • from -15 °C to -70 °C at a speed of 20-25 °C/min (freezing time is about 3 minutes); • deep freezing in Thailor Warton (USA) in liquid nitrogen for 3 day.
During the storage, liquid nitrogen level was optimal.Thawing (defrosting) of sperm was carried out in a water bath for 30-40 seconds at a temperature of 38-40 °C.

Statistical analysis
The statistical analysis was performed using Statistica software for Windows, Version 9.0 (StatSoft, Inc.), the data were presented as mean ± SD.Motility, TBARS concentration in experiment were analyzed using a Student's t-test.Statistical significance was set at p<0.05.

The superoxide anion radical scavenging activity of studied phenol derivatives
According to the obtained results (Table 1), the same slight inhibition of the adrenochrome formation in the presence of BHT and the novel phenol-type derivative AO was observed, which indicates the O -• scavenging activity of these compounds (10 %) in the used model system.It has been shown that the addition of a new phenol derivative AO to the incubation medium with Russian sturgeon's sperm leads to inhibition of adrenaline oxidation in comparison the control experiment without the biological preparation and compounds (48.57± 0.75%).This fact demonstrates the ability of the compound to increase the SODprotective activity of the sturgeon's sperm -ability of biological product to utilize O2 -• .BHT has practically no effect on the ability of fish sperm to utilize O2 -• (p>0.01).

The effect of compounds on the Russian sturgeon's sperm TBARS level accumulation
The effect of the AO and BHT on the TBARS accumulation level in the Russian sturgeon's sperm was determined at different stages of long-term in vitro oxidation (1, 3, 24 and 48 h) (Table 2).In the control experiment, the gradual increase in the TBARS level in Russian sturgeon sperm was observed, after 48 h it increased for 31% in comparison with 1 h (p<0.0005).The addition of AO to sperm led to the reduction of TBARS accumulation level compared to the control experiment at all oxidation stages, the largest reduction (69%) was observed after 3 hours of sperm lipid peroxidation.At the subsequent LPO stages, the decrease of the TBARS level in the presence of the new phenol derivative remained rather high (66 and 55%).The addition of BHT increased TBARS level at the initial LPO stage.At the subsequent LPO stages a decrease of the TBARS level in the presence of BHT was observed.However, this change was statistically significant only after 24 h incubation (p<0.0001).Thus, unlike BHT, the new phenol-type compound demonstrated a prolonged protective effect, preventing the LPO toxic products accumulation in the Russian sturgeon's spermatozoa.

AO and BHT cryoprotective effect
According to the obtained result (Table 3), the addition of the novel phenol-type derivative to the basic cryomedium (the modified Stein's medium) at room temperature is statistically significantly (p < 0.0001) reduced the TBARS level in Russian sturgeon sperm.In the case of BHT, the decrease was not statistically significant (p>0.05).After sperm activation in the control experiment sperm samples showed 90% motility of spermatozoa within 900 seconds.In the AO presence the number of sperm cells with translational motility at room temperature increased by 5%, the total period of sperm movement increased by 21 seconds compared to the control experiments.The addition of BHT decreased the percentage of sperm cells with translational motility (by 5%) compared to the control experiments, but while there was an increase in the time of motility by 64 seconds.It was determined that the TBARS level in Russian sturgeon sperm increased after cryopreservation during 3 days of freezing in liquid nitrogen both in the control experiment (1.7 times), and in the presence of studied phenol derivatives (p<0.005), the highest rate was observed in the case of the new phenol derivative (3 times).Although when it was added to the basic cryomedium the retention of 10% spermatozoa with translational motility for 10 seconds was found.After cryopreservation the slight decrease of TBARS level compared to the control experiment was observed in the case of BHT additive (p>0.05), while no motile of post-thaw sperm cells was observed as well as in the control experiments.

Discussion
The initial ROS in living organism is O2 -• -a respiration byproduct and a crucial component of the immune defense system.Superoxide can be generated both enzymatically and non-enzymatically as a result of autoxidation reactions, for example, during quinoid oxidation of adrenaline in the body [25].Physiologic amounts of O2 -• play an important role in the biological processes regulation, including in fish sperm [30].Although the oxidizing power of superoxide itself is not so strong compared to other oxygen metabolites such as hydroxyl radicals, O2 -• can generate more dangerous species, which cause the LPO [31].Recently increased O2 -• production was observed in frozen fish sperm as compared to fresh sperm [6].Therefore, the antioxidant introduced into the basic cryoprotective medium must have O2 -• scavenging activity.Study of such activity represents important assays to screen antioxidant activity of potential additives in the cryomedium during cryopreservation.The slight scavenging activity for new phenol derivative AO towards O2 -• (10 % inhibition) (Table 1) was established in the work.It is known that the formation of O2 -• in the model system of adrenaline oxidation in an alkaline medium used in this work is accompanied by the formation of several reactive forms: hydrogen peroxide, carbon dioxide radical anion, and bicarbonate radical anion [22], then the revealed inhibitory activity of the compounds in this reaction indicates gross inhibitory activity.It has been shown that the O2 -• scavenging activity of new phenol is comparable to BHT action.
According to literature data BHT is a primary antioxidant acting mainly as a chain breaker and scavenging radical species by hydrogen donation [32].It is interesting to note, in this connection in vivo BHT prevents the O2 -• formation as the by-products of mitochondrial electron transport.it reduces superoxide levels not by directly removing it, but by reducing the probability of radical generation [33].In experiments with coho salmon (Oncorhynchus kisutch) sperm [13] when adding BHT to the base cryo-medium a decrease in the О2 -• level was found during short-term storage.Under certain conditions BHT can behave as a prooxidant, effectively reducing molecular oxygen with O2 -• formation [34].
It is known that oxidative damage to spermatozoa depends not only on the ROS production, but also on the spermatozoa antioxidative protection.Therefore, in this work, we studied the effect of AO and BHT on the ability of biopreparation based on Russian sturgeon's sperm to deactivate O2 -• .Normally, the action of O2 -• is balanced by the antioxidant enzyme SOD, which can convert superoxide into molecular oxygen and the non-radical active compound, hydrogen peroxide, which in turn is utilized by antioxidant enzymes such as catalase, glutathione peroxidase [35].The addition of fish sperm in the incubation medium where the reaction of adrenaline autooxidation proceeds, leads to inhibition of adrenaline autooxidation product formation, which indicates the O2 -• utilization by cytosol SOD (Cu/Zn SOD).According to the obtained results, the addition of AO increases the COD-protective activity of Russian sturgeon's sperm by 27 %, while BHT has no effect on the ability of the biopreparation to utilize O2 -• .
It is well known that a radical chain reaction initiated by electron from superoxide could be involved in the enhancement in the LPO [6], which is particularly important for aquatic species because they contain larger amounts of highly unsaturated fatty acids than other species.It can be seen from the data obtained in this study unlike BHT, new phenol agent demonstrated a high efficiency of the inhibition of accumulation of toxic products in Russian sturgeon's sperm during long-term in vitro oxidation, evidencing for its prolonged action.The high antioxidant activity of the novel heterocyclic compound AO containing phenol fragments and nitrogen-and sulfur-heteroatoms can be explained by the intramolecular synergism of the antioxidant actions owing to the presence of two radical scavenging phenol fragments and antiperoxide sulfide groups, for which can be realized homolytic cleavage of the С-S bond and formation of stable thiyl radicals inhibiting the development of radical chain reactions [36].
The study of the effect of phenols on the LPO of defrosted sperm showed that in the presence of a new phenol AO, the greatest increase in the level of TBARS was observed compared with the control and with the addition of BHT.However, 10% of the spermatozoa maintained forward movement for 10 seconds, which is sufficient for the use of artificial insemination in aquaculture, while in the samples in the control experiment and in the BHT presence, no sperm motility was observed after thawing.Shaliutina et al. (2013) [37] also found no motile of post-thaw Russian sturgeon sperm cells after nine days of storage under aerobic conditions at 4 °C.Our results on a decrease in the motility of fish spermatozoa in the presence of BHT contradict the data we obtained earlier for the Russian sturgeon and beluga (Huso huso Linnaeus, 1758) sperm [14,17], as well as the data of Merino et. al. (2020) [38] for coho salmon semen in short-term storage, Ogretmen and Inanan (2014) [39] for common carp (Cyprinus carpio) spermatozoa.In the latter case, it was found that BHT at concentrations of more than 1 mM caused sperm immobility during the preparatory stages of the sperm freezing.In our study, sperm motility after thawing was not observed at a significantly lower BHT concentration (0.1 mM).

Conclusion
This work demonstrates a significant antioxidant and cryoprotective effect of a novel sulfur-containing nitrogen heterocyclic phenol-type agent.It was shown that this activity of the new compound exceeds the activity of a well-known widely used antioxidant.A prolonged protective effect on lipid peroxidation of the Russian sturgeon sperm was demonstrated in in vitro experiments, which indicate the ability of the new phenol-type compound to inhibit oxidative processes under conditions of oxidative stress development.Our results showed that unlike BHT the new phenol derivative being a component of cryoprotective medium increased motility of Russian sturgeon sperm.This compound improves the cryoresistance of sturgeon sperm cells and can be considered as a modifier of cryoprotective effect of basic media.However, further studies are needed on the effect of new phenol on the fertilizing capacity of Russian sturgeon sperm.

Table 1 .
The superoxide anion radical scavenging activity of studied phenol derivatives and their effect on the SOD-protective activity of the Russian sturgeon's sperm

Table 2 .
Effect of the studied phenol derivatives on the TBARS accumulation in the Russian sturgeon's sperm during long-term in vitro oxidation

Table 3 .
Studied phenol derivatives effect on the TBARS level in the Russian sturgeon sperm, on the percentage of sperm cells with translational motility and duration time before and after cryopreservation Control value is the modified Stein's medium without additives.The average values for a series of experiments are given: † differences from the control experimental group (p<0.0001);* differences from the experimental groups before cryopreservation (p<0.0005);** differences from the experimental group before cryopreservation (p<0.005).The values are expressed as mean ± SD