Effect of Mycotoxins on the Spermatozoa and Embryos of Animals

Recently, several reports have been focused on the decline of productivity either in humans or animals (Luconi et al., 1999, 2004; Tesarik et al., 2000; Tsakmakidis et al., 2006). In different countries, every third to fifth couple is forced to resort to the services of theriogenology clinics to give birth for a child (Arvidson et al., 1989). With that, the theriogenology clinics face the same problems as the livestock breeding including reduced efficiency of sperm cryopreservation (Milano et al., 1991; Ronquist and Brody, 1985; Savchenkova and Vasilieva, 2016; Sousa et al., 2002). The reduced efficiency of sperm cryopreservation in humans and animals may be caused by the presence of even the permissible level of mycotoxins in food products for humans and animal (Diaz, 2006; Tkachev, 2015a).

One of the factors that may reduce the biological indicators of spermatozoa may be the effect of permissible concentrations of mycotoxins in the feed in case of prolonged chronic toxicosis passing without obvious clinical symptoms (Diaz, 2006; Tkachev, 2014a). A group of researchers in 1994 proved that prolonged animal intake of feed with acceptable concentrations of Zearalenone into provoked its accumulation in the tissues of the prostate, testes, ovaries in females, kidneys, hypothalamus and urinary excretion in the amount of up to 7 mM (Erasmuson et al., 1994). Hence, the probability of high concentration of mycotoxins getting into the sperm during ejaculation is high, which can reduce the biotechnological fitness of sperm cells to cryopreservation (Tkachev, 2013a). In 2011, Italian researchers proved the negative effect of Zearalenone and its derivatives on the acrosome reaction and the mobility rates of freshly obtained stallion sperm in vitro (Filannino et al., 2011). Earlier, the negative effect of mycotoxins in vitro had been shown on sperm cells of rams and boars (Farnworth and Trenholm, 1983). Previous studies reported that massive abortion in mares in the USA between 40 and 100 days of pregnancy, caused by the intake of allowed dosages of Zearalenone within the feed within two weeks; which amounted to 336 million U.S. dollars (Diaz, 2006). Collectively, these studies highlight the significance effect of mycotoxins on the biological indicators of spermatozoa after cryopreservation.

Likewise, the high contamination of wheat, barley, maize, etc. with mycotoxins (Tkachev, 2014b), has negative effect on the hormonal and cytogenetic status of animals and humans (Carlini et al., 1997; Francavilla et al., 2003; Lukoseviciute et al., 2007; Tkachev, 2013b; Tkachev, 2015b), along with low cryoresistance of spermatozoa (Graham, 1996). So, there is an urgent need to determine the minimum concentration of mycotoxins that affects the biological properties of germ cells in stallions.

Animal breeding worldwide including Russia, a sharp decrease in the reproductive qualities for males and females of farm animals is observed. Previous studies showed that average across the horse breeding industry, the number of actually born foals per 100 mares did not exceed 50 which means that today only three out of 12 officially registered breeds have sufficient breeding reproductive population (Tkachev, 2013b; Tkachev, 2014b). Therefore, the primary scientific and practical task of the biology of reproduction is to increase the yield of young animals. It is known that up to half of the stud males both in Russia and worldwide provide biotechnologic sperm that either dies in the process of freezing and thawing or loses its fertilizing ability after depreservation (Casey et al., 1997; Katila, 2001; Magistrini et al., 1996; Mottershead, 2000; Tkachev, 2014b).

The current study aims to determine the invitro cytotoxic effect of various dosages of Zearalenone and T-2 toxin, which would reduce cryoresistance of the spermatozoa of bulls and horses following the Kharkov technology of sperm cryopreservation.

The current study was performed in Russia (the Belgorod and Kemerovo regions) on separated ejaculates of six stud stallions; three from the Belgian breed and three from the Ukrainian riding breed, and three stud bulls of the black motley breed. The sperm of the stud bulls and stallions was obtained and cryopreserved following the Kharkov technology developed by the authors (Tkachev, 2013a).

The fundamental difference of the Kharkov technology for sperm cryopreservation is the use of an experimental device for two-step freezing with the possibility of fieldwork, and a large amount of semen dose 4 to 5 ml (instead of 0.25 – 0.5 ml used in the Western European technologies). The ejaculates were obtained twice a week. After obtaining and before cryopreservation, Zearalenone (Sigma, USA) and T-2 toxin (Sigma, USA) were introduced into the sperm diluent at a concentaration 0.5– 0.01 mM. Zearalenone (C18N22O5, molecular weight 318.36) is a nonsteroidal mycotoxin, which is produced by the fungi of the genus Fusarium; and has an estrogenic action and causes abortion, infertility, gynecomastia, and appearance of mammary gland tumors either in human or animals (Filannino et al., 2011). The T-2 toxin (C24N34O8, molecular weight 466.52) is a highly toxic trichothecene produced by the fungi of genus Fusarium; when injected into the human body, it provokes the development of alimentary toxic aleukia and polyorganic pathology; in some historical events. The diluent for sperm was the SMED formulation (100 ml of distilled water, 37 mM of NaCl, 10 mM of KCl, 0.07 mM of KH2PO4, 35.7 mM of NaHCO3, 2.4 mM of MgSO4, 10 mM of HEPES, 1.7 mM of CaCl2, 84.3 mM of fructose, 5.5 mM of glucose, and 0.3 g of bovine serum albumin, pH 7.2), and upon freezing, the penetrating cryoprotectant glycerol (Sigma, USA) in the amount up to 7 % of the final volume was added to the diluent (Backman et al., 2004). To establish the cytotoxic effect of Zearalenone and T-2 toxin, each ejaculate was divided into four equal parts where the first part was used as non treated negative control, the second one Zearalenone was added, the third one T-2 toxin was added, and the fourth one Zearalenone and T-2 toxin were added simultaneously in equal amounts. After that, the results obtained and compared on native freshly diluted and thawed sperm. In the native (freshly obtained) and thawed sperms using conventional methods (GOST 20909.4-75; GOST 24168-80; GOST 26030-83; Filannino et al., 2011; Tkachev, 2013a), the main indicators generally adopted in biology of animal reproduction were determined. The activity of the sperm cells was determined visually under Jenaval light microscope (Germany) in points (1 point was equal to 10 % of the live sperm cells with the uniform rectilinear translation) under the magnification of 10 – 20×. The survival rate of the sperm cells was determined in hours in a thermostat at 37 °C which considers the most important biological indicator used for predicting the result of insemination. A 5% decrease in the sperm cells activity in a thermostat was the final point of determining the survival rate in hours. The absolute index of survival rate is calculated, which objectively characterizes the preservation of sperm cells activity during exposure to the conditions in the thermostat; and it is expressed in arbitrary units and calculated according to the standard formula of the state standards (GOST 20909.4-75; GOST 24168-80; GOST 26030-83; Tkachev, 2013a).

The damage of the sperm cells’ membranes under the action of mycotoxins after cryopreservation was examined using SU8000 Hitachi electronic microscope (Japan) at the magnification of 3,000 – 3,500 times.

The statistical processing was performed using generally accepted methods of variation statistics, the statistical significance of differences was evaluated according to Student t-criterion (Plokhinsky, 1969). The tables show mean (M) and mean deviations (±m). The dispersion analysis was performed using a specialized application program package SPSS for Windows (IBM, USA).

To determine the minimal dosages of mycotoxins that can reduce the basic biological parameters of sperm cells in stallions after cryopreservation, the in vitro effect of Zearalenone and T-2 toxin was studied for determining the quantitative and qualitative indicators for native and thawed sperm cells on diluted ejaculates (Table 1).

Table 1: The cytotoxic effect of Zearalenone and T-2 toxin on the performance of native and thawed spermatozoa (M ± m, p= 11).

The current study aims to show the differences between the cytotoxic effect of Zearalenone and T-2 toxin on the main biological indicators of the sperm of bulls and stallions after cryopreservation. The practical need for such a study is due to a sharp decrease in the efficiency of sperm cryopreservation either in animals and in humans (Francavilla et al., 2003). Previous studies reported that sperm of 50 % of the stallions in the world cannot survive after freezing and thawing (Graham, 1996; Katila, 2001; Mottershead, 2000; Tkachev, 2013a). Based on the foregoing, we studied the cytotoxic effect of mycotoxins in the feed on increasing the chromosomal aberrations in somatic cells of stallions. It was reported that the long-term feeding of horses on the feed with even acceptable levels of mycotoxins significantly decreased the content of testosterone and increased the content of estradiol in the blood of stallions (Tkachev, 2014a). Our hypothesis suggest that the efficiency of animal sperm cryopreservation might depend on Zearalenone and T-2 toxin that were most often present in the animal feed. It has been widely known that the sperm survival rate indirectly depends on the level of testosterone in the blood (Filannino et al., 2011). The less is testosterone, the less sugar is in the sperm, and lower survival rate and the fertilizing capacity for the spermatozoa (Filannino et al., 2011). Previous studies showed that adding Zearalenone and its derivatives to the freshly obtained stallion sperm increased its activity and decreased the survival rate in vitro, possibly due to more rapid consumption of nutrients; however, in this experiment, the exposure to the toxins did not exceed two hours (Filannino et al., 2011).

Other studies showed that mycotoxins gives with the fodder even within acceptable concentrations can be accumulated in the prostate, testes, ovaries, and even in the hypothalamus, interacting with the glucuronic acid (Gromadzka et al., 2008). Mycotoxins can reduce the initial characteristics of sperm in humans and animals, which subsequently may influence the efficiency of freezing and thawing for the sperm (López-Fernández et al., 2007). Presumably, this happens due to the destruction of the structure of the chromatin of the sperm cells (López-Fernández et al., 2007). Moreover, with the accumulation in the hypothalamus, mycotoxins can cause serious disruption of the hormonal regulation in the entire host (Diaz, 2006). Meanwhile, the possibility of urinal excretion of large amounts of mycotoxins (up to 7 mM) in horses reasonably allows assuming the possibility of their getting into the sperm during ejaculation (Erasmuson et al., 1994). Our results have convincingly proven the necessity of determining the minimum concentrations of mycotoxins, which would reduce the main biological indicators of the sperm of bulls and stallions during cryopreservation, since the fertility of animals and humans may decrease in the future.

The established fundamental difference of the cytotoxic effect of mycotoxins during cryopreservation was that, T-2 toxin alone was more toxic than the simultaneous addition of Zearalenone and T-2 toxin. While, simultaneous addition of Zearalenone and T-2 toxin into the diluent was more toxic to the sperm of horses than the addition of T-2 toxin alone. We assume that this may be mainly due to the difference in biochemical metabolism of stallion sperm (respiration-dominated) and bull sperm (glycolysis-dominated) with the prevalence of glycolysis and a greater number of sperm cells in the sperm of bulls (800 – 1,200 million sperm cells per cm3 against 100 – 200 million sperm cells per cm3 of ejaculate in stallions). There is no synergism between Zearalenone and T-2 toxin occurs. The obtained data suggest that the synergistic action of Zearalenone and the T-2 toxin is observed in the case of the predominance of the respiration processes in the presence of glycerol. Indirectly, this hypothesis is confirmed by previous literature data that describe the competitive binding of Zearalenone to the receptors of hormones of the cells (López-Fernández et al., 2007), and by the information that estrogen-like effect of Zearalenone may be associated with the increased activity of aromatase which transforms androgens into estrogens (Sanderson and van den Berg, 2003).

In this study, we succeed to prove for the first time that the cytotoxic properties of mycotoxins increase dramatically during cryopreservation of bulls and stallions’ spermatozoa in the form of severe damage to the membranes which might be due to low efficiency for freezing and thawing. At the same time, the destruction of the acrosome of sperm cells by mycotoxins deprives them of the important enzymes (hyaluronidase, acrosin) that are required for fertilizing the ovum in vitro and in vivo. We assume that this may also be related to the fact that Zearalenone and T-2 toxin in the presence of glycerol increase the intensity of lipid peroxidation by increasing the amount of diene conjugates and malondialdehyde, which damage the bi-lipidic layer of the plasmalemma. This hypothesis is based on the previous studies about increasing the content of diene conjugates and malondialdehyde in the blood of horses under the action of acceptable levels of mycotoxins in the fodder (Tkachev, 2014a; 2015a).

So, there is an urgent need to look into compounds that will be able to neutralize the cytotoxic effect of mycotoxins during cryopreservation of the sperm of animals and humans.

The cytotoxic effect of Zearalenone and T-2 toxin on the sperm of bulls and stallions in vitro after cryopreservation has been studied in vitro after cryopreservation. It has been found that the minimum cytotoxic dosage of Zearalenone and T-2 toxin during the cryopreservation was 0.01 mM. Likewise, the species differences have been shown in the sensitivity of the spermatozoa of bulls and stallions for the cytotoxic effect of Zearalenone and T-2 toxin during cryopreservation, which is evidence to the fact that complex addition of Zearalenone and T-2 toxin to the diluent has lower toxicity to the plasmalemma, acrosome, and the biological parameters of the spermatozoa of bulls than adding T-2 toxin alone, which is not observed in the sperm of stallions.

The article was prepared within the framework of Agreement with the Ministry of Education and Science of Russia No. 05.607.21.0208 “Development of a technology of genome editing for the reproduction of high-value pedigree dairy cattle resistant to the virus of leucosis”, unique identifier of agreement: RFMEFI60718X0208.

All authors contributed equally to the manuscript.

The authors have declared no conflict of interest.