RU2480221C1 - Method for spermatogenesis correction in animals in chronic natural gas poisoning - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to molecular physiology and biochemistry, and may be used for normalizing spermatogenesis in animals in chronic natural gas poisoning. The method for spermatogenesis correction in animals suffered chronic natural gas poisoning, consisting in the use of selenium-containing preparations, and a correcting preparation is Selexen used in a combination with ascorbic acid administered orally to male albino rats in doses of 1.5 mg/kg and 500 mg/kg of animal's body weight, respectively once a day for 50 days, and at the same time from the third week of the administration of Selexen and ascorbic acid, the animals are exposed to sulphurous natural gas in the concentrations of 10 mg/m³ for 30 days, 240 minutes a day, and upon the completion of the experimental exposure, the morphological and kinetic parameters of spermatogenesis are to be examined.

EFFECT: implemented reduction of the toxic effects of natural gas on the reproductive function of male gonads.

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Description

The invention relates to medicine, in particular to the field of molecular physiology and biochemistry and can be used to normalize spermatogenesis in animals under conditions of chronic intoxication with natural gas.

A known method of improving the reproductive function of animals according to the patent 2352110 of the Russian Federation from 04.20.09, Bull. No. 11, which consists in the use of a vitamin-mineral complex added to animal food. However, the known method has the following disadvantages:

- the proposed biocomplex is too capacious in composition, and therefore its effectiveness is due not so much to the direction of the action as to its general stimulating effect on the whole organism;

- in addition, the vitamin component does not contain such important antioxidants that can compensate for the processes of oxidative radical formation, such as vitamin E and C;

- the mineral base is devoid of such elements important for spermatogenesis as zinc Zn and selenium Se.

Spermatogenesis correction methods are also known, which consist in the complex use of selenium and vitamin E, which are added to the daily diet of hogs (Marin-Guzman J., Mahan DC, Chung YK, Pate JL, Pope WF Effects of dietry selenium and vitamin E on boar performance and tissue responses, semen quality, and subsequent fertilization rates in mature gilts // J. Anim. Sci. - 1997. - Vol.75, No. 11. - P.2994-3003; Echeverria-Alonzo S., Santos-Ricalde R., Centurion-Castro F., Ake-Lopez R., Alfaro-Gamboa M., Rodriquez-Buenfil J. Effects of dietary selenium and vitamin E on semen quality and sperm morphology of young boars during warm and fresh season // Journal of Animal and Veterinary Advances. - 2009. - Vol.8, No. 11. - P.2311-2317).

However, the known methods have the following disadvantages:

- these correction methods have not been studied under the influence of toxic agents causing the development of oxidative stress;

- The well-known vitamin E is proposed as an antioxidant, exhibiting antioxidant properties against a number of body systems, including the reproductive system.

There is also known a prototype method for correcting spermatogenesis, which consists in using a selenium-containing compound (sodium selenite), added under conditions of chronic intoxication with hydrogen sulfide-containing gas from the Astrakhan gas field (Ushakova M.V. Functioning of the reproductive system of male rats under chronic exposure to natural toxicants: abstract of dissertation ... Biol. Sciences. - Astrakhan, 2002 .-- 22 p.).

The prototype has the following disadvantages:

- as a protector, the author proposes an inorganic form of selenium (sodium selenite), which, unlike organic, is difficult to digest;

- the selenium-containing tread itself is not supplemented by any known vitamin antioxidant (vitamin E, ascorbic acid), which can enhance the protective properties of selenium.

The present invention is aimed at reducing the toxic effects of natural gas in relation to the reproductive function of male gonads (on models with experimental animals). The proposed method allows to more noticeably reduce the toxic effects of natural gas on the reproductive apparatus by morphological and kinetic indicators of spermatogenesis. This is especially noticeable in relation to mobile forms of sperm, the relative and absolute number of which can be increased using the proposed method, compared with similar indicators after exposure to gas. Our proposed method manages to a much greater extent to achieve improved sperm motility than the method described in the prototype.

The specified technical result is achieved by the fact that selexene is used in combination with ascorbic acid, which are administered orally to male white rats, respectively, at doses of 1.5 and 500 mg / kg of animal body weight 1 time per day for 50 days, and in parallel with third week administration Celexa and ascorbic acid affect hydrogen sulfide in natural gas at a concentration of 10 mg / m ³ for 30 days at 240 minutes a day, then at the end of the experimental effects examined morphological and kinetic Indicators of spermatogenesis.

We chose selexen as a corrector, since selenium in it is in a well-digestible organic form. In addition, in organic form, selenium is low toxic (Sanotsky I.V. Irreplaceable selenium. - M.: AKM Association, 2001, - 96 p.). At present, the presence of synergism of selenium and ascorbic acid, as well as the ability of the latter to excrete excess selenium, has been proven (Bebne D. Studies in the distribution and characteristics of new mammalian selenium-containing proteins // Analyst. - 1995. - Vol.120. - P. 823-825). The animals were injected with selenium at a rate of 350 μg of selenium per 1 kg of animal body weight per day, which is adequate for sexually mature rats in terms of their metabolic rate (Ushakova M.V. Biol. Sciences. - Astrakhan, 2002 .-- 22 p.). Ascorbic acid was given to animals in a moderately elevated dose, relying on the long-standing studies of L. Pauling and his school on the role of high doses of vitamin C in the regulation of physiological processes, including reproductive ones (Pauling L. Vitamin C and health. M .: Science , 1974.- 74 p.). Vitamin C improves the “quality” of sperm, has a beneficial effect on androgenesis, and increases sperm motility (Sharaf A., Gomaa N. Androgenicity in vitamins // Qualitas plantarum et material vegetabiles. - 1970. - Vol.19, No. 4. - P.369 -374; Yousef MI, Abdallah GA, Kamel KI Effect of ascorbic acid and vitamin E supplementation on semen quality and biochemical parameters of male rabbits // Anim. Reprod. Sci. - 2003. Vol.76, No. 1-2. - P .99-111). High doses of vitamin C transform sodium selenite (inorganic selenium) into virtually unassimilable elemental selenium excreted in feces, which is not observed in the case of selenium in organic form. It also determined our choice of selexen as the source of selenium. Ascorbic acid itself, in addition, is able to regulate the level of selenium in the body and remove its excess from the body (Bebne D. Studies in the distribution and characteristics of new mammalian selenium-containing proteins // Analyst. - 1995. - Vol.120. - P .823-825).

Selexen in combination with vitamin C was administered once a day in the indicated doses, which completely covered the daily requirement of animals for these nutrients, which were evenly distributed throughout the body throughout the day. Given that the spermatogenic cycle in rats is 48 days (WHO, 1967), the animals were given a 50-day diet enriched with selexen and vitamin C. 2 weeks after the start of the administration of ascorbic acid and selexen, they began to act with hydrogen sulfide gas at a concentration of 10 mg / m ³ . The indicated gas concentration in the exposure up to several hours a day for a long period (usually more than two weeks) determines the qualification category of exposure to chronic intoxication (Asfandiyarov R.I., Buchin V.N., Lazko A.E. et al. Acute poisoning with sulfur-containing gases. - Astrakhan, 1995. - 156 p.). The exposure to hydrogen sulfide-containing gas itself was carried out “inside” the selecene and ascorbic acid injection course (from the third week within 30 days), which allowed the toxicant to be smoothly introduced into the experimental model, thus avoiding errors caused by toxic gas effects in the absence of correctors (selexene and ascorbic acid).

The creation of a method for correcting spermatogenic function was carried out on 40 sexually mature male white rats weighing 200 ± 10 g at the Astrakhan State Medical Academy of the Ministry of Health and Social Development of the Russian Federation, Department of General and Bioorganic Chemistry. Animals were divided into 4 groups. The first group was control (10 pcs.). The second group (10 pcs.) Included animals that received orally selexene in combination with ascorbic acid in doses of 1.5 and 500 mg / kg of animal body weight per day for 50 days, respectively. The third group consisted of animals exposed to hydrogen sulfide (within 30 days for 4 hours), the concentration of which in the seed chamber was maintained at 10 mg / m ³ . The fourth group included animals that received selexen in combination with ascorbic acid in doses of 1.5 and 500 mg / kg, respectively, of the animal’s body weight per day for 50 days, and in parallel with the third week of the administration of these drugs, they were exposed to hydrogen sulfide-containing gas at a concentration of 10 mg / m ³ for 30 days, 240 minutes daily.

To evaluate the protective properties of selexen in combination with ascorbic acid under the influence of a hydrogen sulfide-containing toxicant, we studied white rat ejaculate. To do this, at the end of the experiments, from the tail of the epididymis, cutting them lengthwise, we washed the seminal fluid with a dosed amount of physiological saline (empirically for rats this amount is 2-4 ml) (IV Sanotsky, VN Fomenko. Long-term effects of chemical compounds on the body. - M .: Medicine, 1979. - P.79-80) and a suspension was obtained (epididymal spermatozoa in animals of the third group were isolated on the 7th day after exposure to gas). On the watch glass, the epididymis is actively moved for 2 minutes to release part of the sperm (to avoid artifacts, glass sticks can not be used, segments of the washed rubber tube are used). A suspension of sperm is applied to a glass slide, dried in air, fixed over a flame and stained with any dye. The degenerative forms are counted in relation to the number of sperm counted in the fields of view. The total number of epididymal spermatozoa in suspension was calculated in the Goryaev chamber under the eyepiece of a light microscope at 600 × magnification. The number of spermatozoa was counted in 5 large squares of Goryaev’s chamber diagonally. In addition, in the native sperm, the percentage ratio between various morphological forms of spermatozoa (defective, motile, and dead) was determined, and a general characteristic of the morphofunctional state of the seminiferous tubules themselves was determined based on histological sections. Sections of testis 7 μm thick were made on a Microm NM-400 microtome (Germany). Sections were stained with hematoxylin-eosin. The resulting preparations were studied on a universal microscope "Nu" (Germany), connected to a color television camera "Pixera" (USA).

In the proposed method, the following results are achieved. The number and morpho-functional state of epididymal spermatozoa in white rats is normal and under experimental conditions is shown in table 1. The change in the number of epididymal spermatozoa under the influence of hydrogen sulfide-containing natural gas, selenium-containing biocomplex (selexene + vitamin C) and their combination are shown in Figure 1. U of animals exposed to gas, on the 7th day after the termination of exposure, a sharp decrease in the total number of epididymal sperm cells by more than 1.7 was noted al, in comparison with the control (P <0.001) (Table 1, Figure 1). In the sperm population, an increase in the percentage of defective forms was noted (44.4%), motile sperm cells were almost absent (0.4%). The content of dead sperm cells increased by almost 11.5 times (55.2%) as compared with the control (4.8%) (P <0.001). The lack of sperm motility is associated with a defect such as sperm tail fracture, which can be explained by an increase in the process of lipid peroxidation under conditions of the development of oxidative stress caused by a hydrogen sulfide-containing gas. At the same time, preliminary consumption of selenium-containing bioimplex by animals contributed to a decrease in the toxic effects of hydrogen sulfide-containing gas. We did not record any significant deviations from the control in the number of sperm in animals exposed to gas and previously treated with this bioprotector. In the group of animals exposed to gas and previously treated with a selenium-containing complex, there was some improvement in the ratio between different forms of spermatogenic cells in comparison with the group of animals exposed to only gas. To the normal level, the ratio between different forms of spermatogenic cells did not have time to recover on the 7th day after the withdrawal of toxic effects, which can be explained by the kinetics of the reparative processes.

The structure of the convoluted tubules of the testes of animals exposed to hydrogen sulfide-containing gas is shown in Figure 2 (stained with hematoxylin-eosin; increased 200 ×). Under conditions of exposure to a hydrogen sulfide-containing gas, interstitial tissue edema, vascular congestion of the testes, and death of germ cells were observed. The seminiferous tubules were located at a considerable distance from each other. There was a detachment of the basement membrane and a random arrangement of spermatogenic epithelial cells (and, in some cases, even desolation of the seminiferous tubules) (Fig. 2).

The structure of the convoluted tubules of the testes of animals exposed to gas and pre-treated with selexene in combination with vitamin C is shown in Figure 3 (hematoxylin-eosin stain; increased 200 ×), from which it follows that the preliminary introduction of a selenium-containing biocomplex contributes to a noticeable decrease in toxic effects, caused by the toxicant alone.

All of the above circumstances are in favor of the protective properties of selexen in combination with increased doses of ascorbic acid in relation to male reproductive function under extreme conditions of hydrogen sulfide-containing pollutants, causing the development of oxidative stress.

Thus, the proposed method for correcting spermatogenesis in conditions of chronic intoxication with natural gas reduces the toxic effects of gas in relation to the reproductive function of male experimental animals (rats), which is reflected in an improvement in the morphological and functional parameters of epididymal spermatozoa and means an increase in sperm quality in adverse environmental conditions.

Table 1 Epididymal sperm counts Control (n = 10) Biocomplex (selexen + vitamin C) (n = 10) Exposure to hydrogen sulfide-containing gas (n = 10) Exposure to gas + biocomplex (n = 10) Total amount, million 50.0 ± 6.51 54.3 ± 6.0 *** 28.4 ± 2.44 ΔΔ 41.0 ± 4.51 Defective,% 20.2 ± 2.22 *** 13.1 ± 0.62 *** 44.4 ± 3.83 ΔΔΔ 42.0 ± 3.52 Mobile% 81.0 ± 6.2 88.0 ± 6.11 *** 0.4 ± 0.12 ΔΔΔ 54.5 ± 4.22 Dead% 4.8 ± 0.82 4.7 ± 0.72 *** 55.2 ± 4.11 45.4 ± 3.31 *** P <0.001 - in comparison with the control; ΔΔ P <0.01; ΔΔΔ P <0.001 - in comparison with a group of animals exposed to gas

Claims (1)

A method for correcting spermatogenesis in animals under conditions of chronic natural gas intoxication, which involves the use of selenium-containing preparations, characterized in that selexene is used as a corrector in combination with ascorbic acid, which are administered orally to male white rats at doses of 1.5 mg / kg and 500, respectively mg / kg of animal body weight 1 time per day for 50 days, and in parallel with the third week of administration of selexene and ascorbic acid, they are exposed to hydrogen sulfide-containing natural gas at a concentration of 10 mg / m ³ in those 30 days for 240 minutes daily, then at the end of the experimental effects, the morphological and kinetic parameters of spermatogenesis are examined.

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