RU2552924C1 - Method for assessing spermatogenesis correction efficacy in animals exposed for microwave radiation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: testicular germ cells are measured quantitatively. That is ensured by 50-day oral administration of selexen and ascorbic acid into male white rats in doses 1.5 and 500 mg/kg of animal's body weight respectively once a day. 14 days later, administering the selenium-containing biocomplex is accompanied by the 30-minute daily exposure to microwave radiation at 42 GHz (λ=7.1 mm) for 30 days. Once the experimental exposures are completed, the corrective properties of the biocomplex as having an effect on the morphofunctional state of epididymal sperm cells are assessing by formula: MFSI=A+B, wherein MFSI is a morphofunctional state index, A is a portion of normal sperm cells in relation to the reference, and B is a portion of moving sperm cells in relation to the reference. If the MFSI value is 1.3 or more, the spermatogenesis correction is considered to be ineffective, while the MFSI value being more than 1.3 shows the effective spermatogenesis correction if exposed to microwave radiation.

EFFECT: invention enables assessing the spermatogenesis correction efficacy with underlying administration of the biocorrector.

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Description

The invention relates to medicine, namely to the field of molecular physiology and biochemistry and can be used to improve spermatogenesis in animals under conditions of chronic exposure to microwave radiation of extremely high frequencies.

A known method of correcting spermatogenesis, which consists in the use of selenium-containing compound (sodium selenite), added under conditions of chronic intoxication with a hydrogen sulfide-containing gas of the Astrakhan gas field (Ushakova M.V. Functioning of the reproductive system of male rats under chronic exposure to natural toxicants: abstract of dissertation ... Cand. Biol. Sciences. - Astrakhan, 2002. - 22 p.). However, the known method 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 with any known vitamin antioxidant (vitamin E, ascorbic acid) that can enhance the protective properties of selenium;

- the chemical toxicant Astrakhan hydrogen sulfide-containing natural gas was used as a stress factor.

Known methods for the correction of spermatogenesis of animals using the combined effects of selenium in an organic form and antioxidants (vitamin E, ascorbic acid) (Echeverria-Alonzo S., Santos-Ricalde R., Centurion-Castro F., Ake-Lopez R., Alfaro-Gamboa M., Rodriquez-Buenfll 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; Loginov P.V., Nikolaev AA, Vetoshkin PB Method for the correction of spermatogenesis in animals under conditions of chronic intoxication with natural gas: Pat. 2480221 Russian Federation. No. 2012107565/15; claimed. 02.28.2012; publ. 27.04 .2013, Bull. No. 12. - 8 p.). However, the known methods have the following disadvantages:

- the following factors act as extreme conditions of exposure: a) the thermal factor (temperature) associated with the seasonal rhythm, and b) the chemical factor (natural gas);

- no quantitative criterion for the effectiveness of spermatogenesis under the influence of adverse factors on the background of the use of biocorrector is not given.

A known method for assessing spermatogenesis by immature germ cells of an ejaculate (Kurilo L.F., Dubinskaya V.P., Ostroumova T.V. et al. Assessment of spermatogenesis by immature germ cells of an ejaculate // Reproduction Problems. - 1995. - T. 1, No. 3 .-- S. 33-36). However, the known method has the following disadvantages:

- assessment of spermatogenesis is given on the basis of the analysis of immature spermatogenic cells that are not involved in the fertilization process;

- not given a quantitative criterion for the effectiveness of the spermatogenous process as a whole.

There is also known a prototype method for quantitative cytological evaluation of the effectiveness of spermatogenesis according to patent No. 2231979 dated 06.15.2001 (publ. 07/10/2004). The prototype has the following disadvantages:

- a quantitative assessment of the morphological forms of germ cells of testicular tissue as a whole;

- do not take into account the kinetic characteristics of mature germ cells - sperm;

- no integrated assessment of sperm morphology and kinetics is given to interpret the effectiveness of the spermatogenic process.

The present invention allows to evaluate the effectiveness of the correction of spermatogenesis under conditions of exposure to microwave radiation of extremely high frequencies against the background of the introduced biocorrector using the integrated criterion for the morphological and functional state of epididymal spermatozoa. The proposed method manages to quantify the effectiveness of spermatogenesis in various experimental conditions relative to control parameters.

The specified technical result is achieved by the fact that male white rats are orally administered selecen and ascorbic acid, respectively, at doses of 1.5 and 500 mg / kg of animal body weight 1 time per day for 50 days, and after 14 days, against the background of the introduction of selenium-containing biocomplex, they are exposed to microwave radiation with a frequency of 42 GHz (λ = 7.1 mm) for 30 days for 30 minutes daily, then, at the end of the experimental exposure scheme, the correcting properties of the biocomplex with respect to the morphofunctional state of epididymal sperms are evaluated tozoidov by the formula:

IMFS = A + B, where

IMFS - index of morphofunctional state,

A is the proportion of normal sperm relative to control,

In - the proportion of motile sperm relative to the control,

and with IMFS values of less than or equal to 1.3, an ineffective correction of spermatogenesis is judged, and with IMFS greater than 1.3, an effective correction of spermatogenesis under conditions of exposure to microwave radiation is judged.

In small rodents, microwave radiation even of the millimeter range causes inhibition of spermatogenic function (MG Kuznetsova. The functioning of the reproductive system of male rats under the influence of electromagnetic radiation of the millimeter range. Author's abstract. Cand. Biol. Sciences. - Astrakhan, 2009. - 24 p. .; Nikolaev AA, Kuznetsova MG, Serdyukov VG Gonadotoxic effect of millimeter radiation: Monograph. - Astrakhan: Publishing house GBOU VPO AGMA, 2013. - 78 p.). In this regard, the problem arises of correcting spermatogenesis under conditions of exposure to microwave radiation. A bioelement selenium can serve as a reliable corrector (Polunin A.I., Lutsky D.L., Miroshnikov V.M. et al. Selenium and zinc in the correction of male subfertility: A manual for doctors. - Astrakhan: AGMA, 2002. - 42 p.). Organic selenium has a high degree of bioavailability and is also slightly toxic (Sanotsky I.V. Irreplaceable selenium. - M.: AKM Association, 2001. - 96 p.). The animals were injected with selenium at the 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. Functioning of the reproductive system of male rats under chronic exposure to natural toxicants. Abstract of diss. ... cand 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 other researchers on the role of high doses of vitamin C in the regulation of reproductive processes (Pauling L. Vitamin C and health. M .: Nauka, 1974. - 74 p. ; 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). Supplementation of selexen with ascorbic acid allows the removal of excess selenium from the body and increases its antioxidant status (Bebne D. Studies in the distribution and characteristics of new mammalian selenium-containing proteins // Analyst. - 1995. - Vol. 120. - P. 823- 825). Given that the spermatogenic cycle in rats is 48 days (WHO, 1967), the animals were given a 50-day diet enriched with selexen and ascorbic acid. 2 weeks after the start of administration of selexene and ascorbic acid, they began to act with microwave radiation at a frequency of 42 GHz. The indicated radiation frequency refers to the millimeter range of electromagnetic waves (microwave radiation of an extremely high frequency of EHF) emanating from a number of sources (radars, medical equipment, radio communications) that a person deals with in everyday life (GOST 24375-80). The exposure to microwave radiation itself was carried out “inside” the course of administration of selexene and ascorbic acid (from the third week within 30 days), which made it possible to smoothly introduce this factor of physical stress into the experimental model, thus avoiding errors caused by the primary effects of radiation in combination with the concomitant animal manipulations in the absence of correctors (selexen and ascorbic acid). Exposure to radiation was carried out for 30 minutes, which simulates the conditions of real life (effects of radio communications, exposure to medical devices, exposure to magnetic fields created by radar installations).

Since sperm maturation occurs in the epididymis of the testis, a suspension of epididymal spermatozoa is analyzed. To develop an integrated criterion for assessing the morphofunctional state of the testes, two approaches were taken as a basis: 1) a quantitative assessment of the morphologically normal forms of spermatozoa; 2) assessment of the kinetic properties (motility) of epididymal sperm. To create an integrated criterion, it is convenient to express quantitative indicators relative to the adopted standard. In the classical sense, such a standard can be a control (intact) group. Further, the obtained relative indicators (coefficients) are added up and a criterion is obtained that takes into account both the kinetics and morphology of the cells.

The percentage of normal sperm forms is calculated by subtracting from 100% of the total number of defective and dead sperm in percent. The relative amount of normal sperm is calculated by dividing the percentage of normal forms of sperm in the experimental group by the percentage of normal forms in the control according to the formula (1):

where A is the proportion of normal sperm relative to the control, P and P _to are the percentage of defective sperm in the experimental and control groups, respectively, D and DK are the percentage of dead sperm in the experimental and control groups, respectively.

The sperm motility coefficient will be estimated by calculating the proportion of motile sperm relative to the control according to the formula (2):

where B is the proportion of motile sperm relative to the control, M is the motility of the epididymal sperm in the experimental group,%, M _to is the motility of the epididymal sperm in the control group,%.

Then the integrated index of morphological and functional state (IMFS) of epididymal sperm will take into account the coefficients A and B according to the formula (3):

In addition, you can also calculate the average sperm quality factor (SCS) in percent, halving the IMFS score and multiplying by 100% by the formula (4):

Here, dividing the IMFS index in half means calculating the average of the sum of A + B, followed by the expression of the resulting value in percent.

The creation of a method for assessing the correction of spermatogenic function was carried out on 40 sexually mature male Wistar rats weighing 210 ± 10 g at the Department of Chemistry of SBEI HPE Astrakhan State Medical Academy of the Russian Ministry of Health. 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 millimeter-wave microwave radiation (λ = 7.1 mm, frequency f = 42 GHz) for 30 days, 30 minutes each. To create an electromagnetic field, a Yav-1-7.1 generator of monochromatic electromagnetic waves (Russia) was used. The fourth group included animals that received selexene in combination with ascorbic acid in the above daily doses for 50 days, and in parallel 14 days (from the third week) from the start of administration of these drugs - exposed to microwave radiation at a frequency of 42 GHz for 30 days 30 minutes daily.

To evaluate the protective properties of selexene in combination with ascorbic acid under the influence of microwave radiation, white rat ejaculate was studied. For this, at the end of the experiments, the seminal fluid was washed out with the dosed amount of physiological saline from the tail of the epididymis, cutting them along (empirically for rats this amount is 2-4 ml) (IV Sanotsky, VN Fomenko. Long-term effects of the influence of chemical compounds on the body. - M .: Medicine, 1979. - 232 pp.) and a suspension was obtained (epididymal spermatozoa in animals of the third group were isolated on the 7th day after the end of exposure to microwave radiation). On the watch glass for 2 minutes, the epididymis was actively moved 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 was applied to a glass slide, dried in air, fixed over a flame and stained with any dye. The degenerative forms were 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 400x magnification. The number of spermatozoa was counted in 5 large squares of the Goryaev’s chamber diagonally. In addition, the percentage of different morphological forms of spermatozoa (defective, motile and dead) was determined in native sperm. Additionally, to confirm the quantitative indicators, a general characteristic was given of the morphofunctional state of the seminiferous tubules based on histological sections. Sections of testes 7 μm thick were made on a Microm NM-400 microtome (Germany). Sections were stained with hematoxylineosine. The obtained preparations were studied using a universal Nu microscope (Germany) connected to a Pixera color television camera (USA). All the data obtained during the experiment were statistically processed using Student's criterion (Lakin G.F. Biometrics: Textbook for biol. Special universities. - M.: Higher School, 1990. - 352 p.).

In the proposed method, the following results are achieved. The number and morphological and functional indicators of epididymal spermatozoa in white rats are normal and under experimental conditions are shown in table 1. The change in the number of epididymal spermatozoa under the influence of microwave radiation of selenium-containing biocomplex (selexen + vitamin C) and their combination are shown in Figure 1. In animals exposed to microwave radiation, a decrease in the total number of epididymal spermatozoa was 1.5 times in comparison with the control (P <0.05) (table 1, Fig. 1). The number of defective sperm (30.5%) increased 1.7 times in comparison with the control (18.2%). The number of dead spermatozoa increased 3 times (30.2%) compared with the control (9.8%) (P <0.001). The lack of sperm motility is mainly associated with a defect such as tail fracture, which can be explained by an increase in the process of lipid peroxidation under the influence of microwave radiation. The consumption of selenium-containing biocomplex caused an improvement in motility and contributed to an increase in the number of epididymal sperm, compared with the control group of animals. The number of dead sperm cells decreased by more than 2 times compared with the control (P <0.001). The improvement of the kinetic properties and viability of spermatozoa when taking this biocomplex can be explained by the fact that the semenopeptide is contained in the tail of rat spermatozoa, which is of great structural importance in the assembly of the tail of spermatozoa (Ushakova M.V. Functioning of the reproductive system of male rats under chronic exposure to natural toxicants). Thesis ... Candidate of Biological Sciences. - Astrakhan, 2002. - 22 p.). At the same time, preliminary consumption of selenium-containing biocomplex by animals helped to reduce the destructive effects of microwave radiation. We did not record significantly significant deviations from the control in the number of spermatozoa in animals exposed to microwave radiation and previously treated with this bioprotector. There was a trend towards an increase in the total number of epididymal sperm. The number of defective spermatozoa in the group of animals exposed to microwave radiation while receiving selenium-containing biocomplex did not differ significantly from that of the control group. The selenium-containing biocomplex contributed to some improvement in sperm motility under conditions of exposure to microwave radiation compared with a group of animals exposed only to radiation. The number of dead spermatozoa was noticeably lower under exposure to microwave radiation while taking a biocomplex compared with a group of animals exposed only to radiation, although it did not reach control values.

To confirm the nature of the change in the morphofunctional state of epididymal spermatozoa under various experimental conditions, we consider examples of quantitative assessment of the morphofunctional state of epididymal spermatozoa using the IMFS criterion, using table 1 and formulas (1-3).

Example 1. The control group

Here, the control group itself will act as an experimental group (in the numerator), therefore, the coefficients A and B will be equal to unity, which corresponds to a 100% standard.

1) The proportion of normal sperm relative to control:

2) The proportion of motile sperm relative to control:

3) IMFS = A + B = 1 + 1 = 2

Conclusion: the control value of IMFS in the absence of experimental effects, when the indicators A and B are equal to unity (control standards), is 2.

Example 2. The effect of selenium-containing biocomplex

Animals were injected with selexen and ascorbic acid at doses of 1.5 and 500 mg / kg body weight in accordance with the scheme defined for the second experimental group.

1) The proportion of normal sperm relative to control:

2) The proportion of motile sperm relative to control:

3) IMFS = A + B = 1.1 + 1.1 = 2.2

Conclusion: the increase in IMFS compared with the control as a result of consumption of selenium-containing biocomplex reflects the fact of increasing the effectiveness of spermatogenesis.

Example 3. The influence of microwave radiation

Animals were exposed to microwave radiation at a frequency of 42 GHz in accordance with the scheme developed for the third experimental group.

1) The proportion of normal sperm relative to control:

2) The proportion of motile sperm relative to control:

3) IMFS = A + B = 0.55 + 0.74 = 1.29≈1.3

Conclusion: Microwave radiation with a frequency of 42 GHz in the indicated exposure caused a decrease in the IMPS index to 1.3, which reflects the negative effects of radiation on both the morphology and kinetics of epididymal spermatozoa.

Example 4. The effect of microwave radiation on the background of the biocorrector.

Male white rats were orally administered spleens and ascorbic acid at doses of 1.5 and 500 mg / kg body weight for 50 days. 14 days after the start of the introduction of this selenium-containing biocomplex, they began to act with microwave radiation at a frequency of 42 GHz (λ = 7.1 mm) for 30 days for 30 minutes daily. The exposure was carried out in a seed chamber made of materials not shielding electromagnetic radiation. After exposure to microwave radiation and the background introduction of selenium-containing biocomplex in animals, the morphological and kinetic parameters of epididymal spermatozoa were evaluated according to formulas (1-3) based on the obtained indicators shown in table 1.

1) The proportion of normal sperm relative to control:

2) The proportion of motile sperm relative to control:

3) IMPS = A + B = 0.85 + 0.82 = 1.67≈1.7.

Conclusion: the obtained value of IMPS exceeds that for a group of animals exposed only to microwave radiation (IMFS = 1.3), although slightly lower than the control value of IMFS, equal to 2. This circumstance testifies to the effectiveness of correction of spermatogenesis using the proposed selenium-containing biocomplex under conditions exposure to microwave radiation of extremely high frequencies.

The obtained IMFS values also make it possible to calculate the average sperm quality factors (SCS) in percent, taking into account the morphology and kinetics of epididymal spermatozoa according to formula (4).

Then we get the following average sperm quality factors for the above examples.

1) Control group:

2) The effect of selenium-containing biocomplex:

3) The effect of microwave radiation:

4) The effect of microwave radiation on the background of biocorrector:

In a generalized form, all the data obtained for the calculation of IMFS and SCKS are shown in table 2.

Thus, under conditions of exposure to microwave radiation, the IMPS index is 1.3, while against the background of the selenium-containing biocomplex consumed by animals, the IMFS index increases to 1.7. Consequently, the IMFS value is greater than 1.3 under the conditions of exposure to microwave radiation against the background of the biocorrector consumed, indicating an effective correction of spermatogenesis by this biocomplex, which is also confirmed by morphological analysis of testicular tissue sections (Fig. 2, 3). A value of IMFS less than or equal to 1.3 indicates an ineffective correction of spermatogenesis under conditions of exposure to microwave radiation of extremely high frequencies.

The structure of the convoluted tubules of the testes of animals exposed to microwave radiation is shown in Figure 2 (stained with hematoxylineosine; increased 200x). Under the influence of microwave radiation, plethora of the testes vessels and a general decrease in germ cells were observed. A random arrangement of spermatogenic epithelial cells was observed against the background in some cases of exfoliation of the basement membrane. In some cases, multiple ruptures of the basement membrane, desolation of the seminiferous tubules, or uneven height of the spermatogenic epithelium were observed, which generally suggests signs of spermatogenic epithelium necrosis (Fig. 2). The structure of the convoluted tubules of the testes of animals exposed to microwave radiation and having received a pre-selenium-containing biocomplex is shown in Figure 3 (stained with hematoxylineosine; increased 200x). The seminiferous tubules themselves have a regular rounded shape. The preliminary introduction of the biocomplex contributed to a noticeable decrease in the destructive effects caused by microwave radiation alone. The seminiferous tubules are filled with different types of spermatogenic cells. Closer to the lumen of the seminiferous tubules, sperm can be seen. The height of spermatogenic epithelium is visually noticeably higher compared to that in the group of animals exposed to only microwave radiation.

Thus, the proposed method for evaluating the correction of spermatogenesis under conditions of exposure to microwave radiation allows us to conclude that the corrector used is effective based on an integrated indicator that takes into account the morphological and kinetic characteristics of epididymal spermatozoa. The proposed method also manages to quantify the effectiveness of spermatogenesis in different experimental conditions.

Claims (1)

A method for assessing the effectiveness of correction of spermatogenesis in animals under conditions of exposure to microwave radiation, which consists in a quantitative assessment of germ cells of the testis, characterized in that male rats are orally administered selexen and ascorbic acid, respectively, at doses of 1.5 and 500 mg / kg of animal body weight 1 time per day for 50 days, and after 14 days, against the background of the introduction of a selenium-containing biocomplex, they are exposed to microwave radiation with a frequency of 42 GHz (λ = 7.1 mm) for 30 days for 30 minutes daily, then at the end of the circuit s experimental corrective actions evaluated regarding properties biocomplex morphofunctional state epididymal sperm from the formula:
IMFS = A + B, where
IMFS - index of morphofunctional state,
A is the proportion of normal sperm relative to control,
In - the proportion of motile sperm relative to the control,
and with IMFS values of less than or equal to 1.3, an ineffective correction of spermatogenesis is judged, and with IMFS greater than 1.3, an effective correction of spermatogenesis under conditions of exposure to microwave radiation is judged.

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