RU2436299C1 - Medium for cryopreservation of goat sperm - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to animal husbandry. The medium for cryopreservation of goat sperm contains crystalline glucose, tris-(hydroxymethyl)-aminomethane, citric acid 1-hydrate, egg yolk, glycerin, distilled water, amber acid and sodium alginate, with the following ratio of components, wt %: crystalline glucose 0.6000-0.667; tris-(hydroxymethyl)-aminomethane 3.635-4.039; citric acid 1-hydrate 1.887-2.084; amber acid 0.197-0.232; sodium alginate 0.020-0.040; glycerin 5.000-5.500; egg yolk 2.500-3.000; distilled water - the rest.

EFFECT: invention improves stability of sperms to cryopreservation and stabilises their biological full-value in freezing-thawing processes.

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Description

FIELD OF THE INVENTION

The invention relates to agriculture, in particular to artificial insemination of goats, and can be used in biotechnology for the reproduction of other closely related species of agricultural and wild animals.

State of the art

Known medium for dilution and freezing of semen of bulls, including per 100 ml: 0.8-1.2 g of fructose, 1.4-1.6 g of sodium citrate, 2.3-2.6 g of TRIS'a, 7.5 -9.5 g of low density lipoproteins, 5-8 ml of glycerol, 0.085-0.12 million IE of penicillin and distilled water (see Qingwang Li; Jianhong Nu; Hongwei Zhao; Zhongliang Jiang, Patent No. CN101220345 (A) 2008.07. 16). The disadvantage of this medium is the inaccuracy of the recipe, in particular it is unclear: “TRIS” means tris- (oxymethyl) -aminomethane or tris- (hydroxymethyl) -aminomethane, the concept of “low density lipoproteins” unites a whole class, and the mechanism of their isolation from an organism, a ready-made drug for sale does not exist. Thus, it is difficult to use this recipe. In addition, the environment is intended for bulls, which means that it cannot be extrapolated to goats.

Known: a synthetic medium for diluting and freezing ram sperm containing lactose, dextrin, tris- (hydroxymethyl) aminomethane, disodium dichloride salt of ethylenediaminetetraacetic acid, glycerin, chicken egg yolk and distilled water, while it additionally contains xylitol, bromide N-dimethyl-N-decyl-4-hydroxy-3,5-di-tert-butyl-benzylamine in the following ratio of ingredients, wt.%:

lactose - 5.0-8.0,

dextrin - 3.5-7.0,

Tris- (hydroxymethyl) -aminomethane - 0.065-0.13,

disodium bisodium salt of ethylenediaminetetraacetic acid - 0.08-0.16,

glycerin - 3.5-6.0,

egg yolk - 12.0-15.0,

xylitol - 0.1-0.3,

bromide-N, N-dimethyl-N-decyl-4-hydroxy-3,5-di-tert-butyl-benzylamine - 0.002-0.008,

distilled water - the rest (see Pat. RU No. 2083183, IPC A61D 19/02, publ. 07/10/1997) and a synthetic medium for dilution and cryopreservation of ram sperm, including lactose, xylitol, tris (hydroxymethyl) -aminomethane, trilon-B , glycerin, chicken egg yolk, distilled water, while it additionally contains bone glue in the following ratio of ingredients, wt.%: lactose - 6.0, bone glue - 1.8, xylitol - 0.18, tris- (oxymethyl) aminomethane - 0.07, trilon-B - 0.1, glycerin - 4.3, chicken yolk - 14.2, distilled water to 100 (see US Pat. RU No. 2198622, IPC A61D 19/02, о UBL. 20.02.2003).

The disadvantage of these media is their specificity for sperm of sheep. Sheep sperm freezing media can be used for cryopreservation of goat sperm, but only during the sexual season, and after thawing, sperm activity is rather low (3.7-4.2 points). With year-round use of goats, in the non-sexual season, when the quality of native sperm is worse, after cryopreservation and thawing, sperm motility is lower than 4 points, or necrospermia.

Given the variety of diluents for sperm of bulls and rams, there are few media for diluting goat sperm. From the finished products, OVIX-CELL synthetic non-yellowing medium (France) and milk diluent (France) are used, including per 100 ml: 0.194 g glucose, 10.0 g milk, 7.0 g glycerol, 0.03 g penicillin, 0.05 streptomycin .

However, these diluents are technologically difficult to use, since their use is associated with multiple multi-stage dilution, preliminary washing and centrifugation of sperm. The use of this technology increases the cost of a dose of sperm 20-25 times, in addition, it can only be used stationary.

For conservation of goat sperm without washing sperm there is a synthetic medium containing per 100 ml: 10.0 g of sucrose, 2.0 g of complexonate Na ₂ Ca EDTA, 0.03-0.06 g of polygen, 7.0 g of glycerol, 20 ml chicken yolk and distilled water (Russia, Manual on the technology of organizations for artificial insemination and transplantation of embryos of farm animals, 2000).

However, when freezing goat sperm in a given environment, its activity after thawing varies and amounts to 1.5-4.0 points with low fertility, therefore, this medium is not used in practice.

The closest in technical essence and the achieved positive effect and accepted by the authors for the prototype is yolk-tris-glucose diluent for goats (Australia), consisting of glucose (0.625-1,000 wt.%), Tris- (hydroxymethyl) -aminomethane (3,786-6,057 wt.%), citric acid (2.172-3.475 wt.%), chicken yolk (2.5-4.0 wt.%), glycerin - 5-8 wt.% and distilled water up to 100 ml (G. Evans, WMC (Maxwell Salamon's Artificial Insemination of Sheep and Goats. - Sydney: Butterworths, 1987).

The disadvantage of this diluent is the low quality of sperm after freezing and thawing: sperm activity - 4-4.5 points with a fertilizing ability of 44-48%.

Disclosure of invention

The objective of the invention is to develop a medium for cryopreservation of goat sperm, with the ability to increase the resistance of sperm to cryopreservation and subsequent thawing.

The technical result that can be obtained using the present invention is to improve the resistance of sperm to cryopreservation and stabilization of their biological usefulness in the processes of freezing and thawing.

The technical result is achieved using a medium for cryopreservation of goat sperm, including tris- (hydroxymethyl) aminomethane, citric acid 1-water, crystalline glucose, glycerin, chicken yolk, distilled water, while it additionally contains succinic acid and sodium alginate in the following ratio components in wt.%:

Crystalline glucose 0.600-0.667 Tris (hydroxymethyl) aminomethane 3,635-4,039 Lemon acid 1,887-2,084 succinic acid 0.197-0.232 Sodium Alginate 0,020-0,040 Glycerol 5,000-5,500 Chicken Yolk 2,500-3,000 Distilled water rest

The essence of obtaining a medium for diluting goat sperm is as follows. Take yolk-tris-glucose diluent for goats (G. Evans, WMC Maxwell Salamon's Artificial Insemination of Sheep and Goats. - Sydney: Butterworths, 1987), consisting of glucose, tris (hydroxymethyl) aminomethane, citric acid, chicken yolk and glycerol, and succinic acid and sodium alginate are additionally introduced in the following ratio of components in wt.%:

Crystalline glucose 0.600-0.667 Tris (hydroxymethyl) aminomethane 3,635-4,039 Citric Acid 1-Aqueous 1,887-2,084 succinic acid 0.197-0.232 Sodium Alginate 0,020-0,040 Glycerol 5,000-5,500 Chicken Yolk 2,500-3,000 Distilled water rest

Succinic acid (butanedioic acid, ethane-1,2-dicarboxylic acid) is found in all organs and tissues of the body, including seminal fluid. It is the product of the fifth reaction and the substrate of the sixth reaction of the tricarboxylic acid cycle. (T.T. Berezov, B.F. Korovkin. - Biological chemistry M .: Medicine, 1990. - 528 p.) Its cytoprotective effect is due to the following properties:

- the ability to increase the capacity of buffer systems;

- antihypoxic effect due to its effect on the transport of mediator amino acids,

- antioxidant effect.

Violation of energy metabolism in sperm as a result of freezing-thawing leads to a change in transmembrane ion fluxes and the accumulation of intracellular calcium. At the same time, an attack develops with active oxygen species, associated with the leakage of electrons accumulating on the intermediate links of the respiratory chain, proteins, nucleic acids and lipids, proceeding by the mechanism of free radical oxidation. Free radicals (highly reactive forms of oxygen, hydrogen peroxide, aldehydes) formed during the incomplete reduction of oxygen, which change the functional properties of a number of enzymes, carbohydrates, proteins, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as a result, the cell loses its functions and, in addition to the direct damaging effect of crystallization and recrystallization, secondary destructive processes are stimulated. Succinic acid has a modulating effect on membrane-bound enzymes, ion channels, receptor complexes, including benzodiazepine, GABA and acetylcholine. The restoration of the respiratory function of sperm in the presence of succinic acid proceeds in two ways: by restoring the NAD-dependent portion of the Krebs cycle (Leninger A. Fundamentals of biochemistry. - M .: Mir - 1991-384 p.) Or by stimulating an alternative pathway, which is succinate oxidation oxidation or succinic oxidation acid that replaces the oxidative phosphorylation cycle sections that were interrupted due to crystallization and recrystallization processes (Vinogradov A.D. Measurement of the activity of succinate dehydrogenase / Reactions of living systems and the state of energy exchange metabolism. - Pushchino, 1979. - p. 98-125; Kondrashova MN Interaction of the processes of transamination and oxidation of carboxylic acids under different functional conditions of tissues // Biochemistry. - 1991. - v. 56. - issue 3. - S.388-405).

Sodium alginate is the sodium salt of natural alginic acid released from brown algae. Forms highly viscous solutions, stabilizes emulsions. It is highly soluble in water in the presence of glycerol and sugars, pH of a 1% solution = 6.5-7.0 (Afanasyev N.I., Parfenova L.N. Viscosity of aqueous solutions of polyelectrolytes. // Izv. Universities. Forest Journal. - 1991 No. 5. S.150-153).

Due to its unique physicochemical properties and good binding abilities, sodium alginate is widely used in preventive and clinical medicine and biotechnology (L.K. Dobrodeeva, V.P. Belozerov, N.I. Kondakov, N.V. Tsimbalenko, L.P. . Shilina, KG Dobrodeev. Food additives of algal origin for the prevention and treatment of immunodeficiency states. Arkhangelsk, 1996. 12 p .; Yu.S. Ovodov. Selected chapters of bioorganic chemistry. // Syktyvkar, Syktyvkar un-t. 1998, 222 s; Yu.S. Khotimchenko, V.V. Kovalev, O.V. Savchenko, O.A. Ziganshina. nomic properties, physiological activity and the use of alginates - polysaccharides of brown algae // Marine Biology 2001 №3 S.151-162 T.22)....

Sodium alginate in the composition of the medium has two functions.

The first is due to its ability to increase the viscosity of the solution. This is necessary because, as a rule, sperm diluted with medium has a viscosity lower than native, and during artificial insemination part of it flows out, which reduces the effectiveness of fertilization.

The second function is reconstructive.

Sodium alginate in weakly concentrated solutions exists in the form of aggregates of different sizes. A trimodal distribution of particle sizes in the concentration range of 10 ^-6 -10 ^-5 g / dl was established, which indicates the presence of three groups of particles with sizes of hydrodynamic radii (GDR) falling within the intervals: R ₁ = 2-4 nm (individual alginate molecules sodium); R ₂ = 20-60 nm and R ₃ = 130-370 nm (associates of macromolecules). Above a concentration of 10 ⁻⁵ g / dl, a two-modal distribution is observed, and particles of groups R ₂ and R ₃ are present in the solution. The number of particles of the second group increases sharply to a concentration of sodium alginate solution equal to 5 · 10 ^-4 g / DL. The number of particles of the third group increases gradually and at a concentration of sodium alginate solution equal to 2 · 10 ^-3 g / dl, equilibrium occurs in a system where only particles of the second and third groups are present, the percentage in the solution of which is 70-90% and 30-10% respectively (I.A. Oberyukhtina, K.G. Bogolitsyn, N.R. Popova, L.N. Parfenova. Application of laser correlation spectroscopy in the study of the hydrodynamic properties of dilute solutions of sodium alginate. All-Russian conference "Chemistry and Plant Technology substances ”, oral report, Kazan, June 24-27, 2002).

One of the main reasons for the decrease in fertilizing ability during cryopreservation and defrostation is damage to the outer protoplasmic membrane of the sperm head, destruction of the acrosome, as well as significant loss of amino acids of lipoproteins and damage to enzyme systems (N.A. Zheltobryukh, 1973, D.T. Danov et al. , 1984; P. Watson, 1975; H. Wiggin, J. Almguist, 1975; M. Mollova, 1980).

The idea is that sodium alginate macromolecules, having a polyelectrolyte effect, will “seal” damage to the sperm membrane, preventing the “leakage” of enzymes, in particular hyaluronidase and constituent substances, and, in addition, to prevent the formation of conglomerates, which is observed in sperm with damaged shell as a result of disruption of electrostatic repulsion forces.

The implementation of the invention

Examples of specific performance of obtaining medium for cryopreservation of goat sperm.

Examples 1-16 are summarized in tables 1-6.

The selection of optimal concentrations was carried out taking into account changes in pH and osmotic pressure of the diluent.

The media were prepared in the following ratio of components (table 1, 2):

Table 1 Recipes of experimental environments Components Yolk-Tris Glucose Diluent Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Crystalline fructose - - - - - - - - Crystalline glucose 0.667 0.667 0.667 0.667 0.667 0,600 0,600 0.734 0.734 Tris (hydroxymethyl) aminomethane 4,039 4,039 4,039 4,039 4,039 3,635 3,635 4,442 4,442 Citric acid 1-aqueous 2,316 2,200 2,084 1,853 1,158 1,922 1,887 2,280 2,235 succinic acid - 0.116 0.232 0.463 1,158 0.162 0.197 0.267 0.302 Glycerol 5,300 5,500 5,500 5,500 5,500 5,000 5,000 6,000 6,000 Chicken Yolk 2,600 2,500 2,500 2,500 2,500 3,000 3,000 2,500 2,500 Distilled water rest

table 2 Recipes of experimental environments (continued) Components Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 Example 16 Crystalline glucose 0,600 0,600 0,600 0,600 0.667 0.667 0.667 0.667 Tris (hydroxymethyl) aminomethane 3,635 3,635 3,635 3,635 4,039 4,039 4,039 4,039 Citric acid 1-aqueous 1,887 1,887 1,887 1,887 2,084 2,084 2,084 2,084 succinic acid 0.197 0.197 0.197 0.197 0.232 0.232 0.232 0.232 Sodium Alginate 0.010 0,020 0,040 0,080 0.010 0,020 0,040 0,080 Glycerol 5,000 5,000 5,000 5,000 5,500 5,500 5,500 5,500 Chicken Yolk 3,000 3,000 3,000 2,000 2,500 2,500 2,500 2,500 Distilled water rest

To determine the effectiveness and the optimal ratio of the ingredients of the experimental medium, a series of experiments was carried out.

Sperm (all comparative experiments were carried out on mixed ejaculates) are diluted with test synthetic media in a ratio of 1: 3. As a control medium take a prototype - vitelline-tris-fructose diluent. Diluted sperm is poured into glass vials and placed in a refrigerator at 0 ... + 3 ° C for 3 hours for equilibration, then frozen in liquid nitrogen (at -196 ° C) according to the generally accepted method (Instructions for the technology of work of organizations for artificial insemination and transplantation of embryos of farm animals, 2000). Thaw semen in a special thawer, in accordance with the same instruction. To determine the survival of sperm, an assessment is made of mobility in points. Sperm is evaluated for motility on a ten-point scale after additional dilution in a 1: 2 ratio with isotonic sodium citrate solution (3.1%), for which a drop of the studied sperm is examined under a microscope at a temperature of + 38 ° C.

According to the results of the first series of experiments (Table 3), the best was the medium with succinic acid (Example 2) (Table 1).

Table 3 Sperm motility upon dilution with an experimental medium with succinic acid (experiment 1) Sperm motility, points Yolk-Tris Glucose Diluent The experimental environment (n = 5) example 1 example 2 example 3 example 4 Before dilution 8.84 ± 0.22 8.84 ± 0.22 8.84 ± 0.22 8.84 ± 0.22 8.84 ± 0.22 Immediately after dilution 8.54 ± 0.21 8.61 ± 0.23 9.12 ± 0.18 9.04 ± 0.22 7.43 ± 0.23 After 3 hours of balancing 7.64 ± 0.18 7.95 ± 0.27 8.51 ± 0.25 6.75 ± 0.27 3.51 ± 0.24 Immediately after freezing-thawing 4.16 ± 0.22 4.27 ± 0.27 5.05 ± 0.25 3.16 ± 0.26 units 2 hours after thawing 3.85 ± 0.25 3.90 ± 0.29 4.13 ± 0.18 units n

Note: indicators with P <0.05 relative to vitelline-glucose diluent are highlighted

Table 4 presents the results of experiment 2 to determine the optimal succinic acid content and component ratio.

Table 4 Sperm motility upon dilution with an experimental medium with succinic acid (experiment 2) Sperm motility, points The experimental environment (n = 5) Example 5 Example 6 Example 2 Example 7 Example 8 Before dilution 8.75 ± 0.25 8.75 ± 0.25 8.75 ± 0.25 8.75 ± 0.25 8.75 ± 0.25 Immediately after dilution 8.38 ± 0.25 8.96 ± 0.26 9.09 ± 0.21 9.13 ± 0.23 8.86 ± 0.12 After 3 hours of balancing 7.78 ± 0.17 8.30 ± 0.22 8.45 ± 0.23 8.02 ± 0.18 7.03 ± 0.27 Immediately after freezing-thawing 4.32 ± 0.19 4.98 ± 0.25 5.10 ± 0.19 4.68 ± 0.18 3.74 ± 0.36 2 hours after thawing 4.00 ± 0.25 4.10 ± 0.22 4.16 ± 0.21 3.77 ± 0.16 1.85 ± 0.31

When sperm is diluted with these media (examples 2 and 6), sperm motility slightly increases, then, after 3 hours of equilibration, it decreases by 3.4-5.1%, while in the yolk-tris-glucose diluent - by 13.6% . Immediately after freezing-thawing, 56.9-58.3% of sperm remain active, while in vitelline-tris-glucose diluent - 47.1%. 2 hours after thawing while maintaining semen at 38 ° C, 46.7-47.5% of sperm remain active, while in vitelline-tris-glucose diluent, 43.6% remain.

Examples 5, 7 and 8 were less effective.

After sodium alginate was added to the medium, the best (by sperm motility) were Examples 10, 11, 14, and 15 (Table 5). The mobility of sperm immediately after freezing and thawing is higher by 34.4-39.1% than in the vitelline-tris-glucose diluent, and 12.5-17.2-% than in examples 2 and 6. Sperm motility 2 hours after thawing, it was higher by 42.3-47.1% than in the yolk-tris-glucose diluent, and by 30.6-36.3%) than in examples 2 and 6.

According to the results of the experiments, the sperm stored in the subjects (examples 10, 11, 14, 15) and in the yolk-tris-glucose diluent was subjected to acroscopic evaluation according to the method of I.I.Sokolovskaya et al. (1981).

Before immobilization, a 1% aqueous solution of sodium fluoride is used to immobilize sperm. A drop of sperm prepared for use is applied to a glass slide, covered with a coverslip. Excess moisture is removed with filtered paper. In this case, sperm in the remaining fluid layer were located freely, in one layer. The edges of the coverslip are treated with petroleum jelly so that the drug does not dry out during viewing. Viewing is carried out in an optical microscope at a magnification of × 400-500 using a dark field capacitor OI-10 or OI-13. In each preparation, 200 sperm are counted, separately recording immobile sperm and sperm with damaged acrosomes (including the complete absence of acrosome).

The nature and extent of structural damage is evaluated as follows. In intact sperm, the brightly glowing contour of the head and flagella is clearly visible. Among the remaining sperm, there are 4 categories of their inferiority:

- I category - general swelling of the acrosome, which is expressed by its dull contours;

- Category II - the beginning of the detachment of the acrosome - the absence of glow in the region of the equatorial segment;

- Category III - loss of acrosome when only the posterior nuclear cap and flagella are visible;

- IV category - complete loss of head, only flagellum visible.

As can be seen from table 6, when cryopreservation of sperm in the yolk-Tris-glucose diluent was 82% of sperm with damage to the acrosome, while 36% with exfoliation and complete loss of acrosome. After 2 hours of preservation of thawed sperm at 38 ° C, the number of damaged sperm increased to 86%, at 38% with exfoliation and complete loss of acrosome.

When cryopreservation of sperm in an experimental medium (examples 10, 11, 14, 15) of sperm with damaged acrosome was 13-15% less immediately after thawing and 14-17% less 2 hours after thawing.

Environmental test

The biological usefulness of sperm after cryopreservation in various environments was tested in a scientific and production experiment on clinically healthy goats of the Saanen breed, selected on the basis of analogues.

The purpose of the experiment is to establish the quality of a new environment for cryopreservation of goat sperm in comparison with existing ones.

Sperm is obtained in an artificial vagina, examined according to the “Instructions on the technology of organizations for artificial insemination and transplantation of farm animal embryos” (2000).

For dilution of sperm were taken: glucose-citrate-yolk diluent for sheep, recommended by the "Instructions on the technology of organizations for artificial insemination and transplantation of farm animal embryos" (2000), milk diluent for goats (France), vitelline-glucose diluent for Goats (Australia) and experimental environment. Sperm is diluted in a ratio of 1: 3.

Artificial insemination is carried out in the cervix twice during sexual hunting with an interval of 12 hours at a dose of 0.2 ml / goal.

According to the results of the goat, fertility and fertility are recorded.

From the experiment it was found that the fertility of goats during insemination with cryopreserved sperm diluted with experimental medium is higher by 28-12% than with insemination with sperm diluted with existing media. Fertility is above 6.7-15.0%, respectively.

Table 7 Results of insemination of goats with cryopreserved sperm Diluent Inseminated goats goal. Of them, the goal was pissed off. Fertility,% Received kids, goal. Fertility Glucose-citrate-vitelline 25 eleven 44.0 fourteen 127.3 Lactic 25 8 32,0 10 125.0 Vitelline Tris Glucose 25 12 48.0 16 133.3 Experimental environment 25 fifteen 60.0 21 140.0

Thus, from the experiment it follows that the claimed invention in comparison with the prototype and other known technical solutions has several advantages.

1. The use of the medium increases the protective properties of sperm during freezing (T = -196 ° C) and helps to maintain the fertilizing ability of sperm after thawing.

2. The species characteristics of animals for which the medium is intended are taken into account.

3. The environment is simple in composition and technological in preparation.

4. The environment does not require the use of expensive drugs.

Claims (1)

Goat sperm cryopreservation medium, including crystalline glucose, tris- (hydroxymethyl) aminomethane, citric acid 1-water, chicken yolk, glycerin, distilled water, characterized in that it additionally contains succinic acid and sodium alginate in the following ratio of components, wt .%:
Crystalline glucose 0.600-0.667 Tris (hydroxymethyl) aminomethane 3,635-4,039 Citric Acid 1-Aqueous 1,887-2,084 succinic acid 0.197-0.232 Sodium Alginate 0,020-0,040 Glycerol 5,000-5,500 Chicken Yolk 2,500-3,000 Distilled water Rest