RU2577882C1 - Medium of cryopreservation of bull semen and method of its preparation - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: medium for bull semen preservation is composed with use of the following components (wt %): tris-(hydroxymethyl)-aminomethane 1.9, fructose 0.9, citric acid 0.7, sucrose 2, glycerol 4.5-6.5, soybean phospholipids 0.04-2.00, macromolecular surfactant 0.01-1, bidistilled water - the rest. The method of preparing the medium for bull semen cryopreservation comprises preparation of lipid-free base for the medium for bull semen cryopreservation by dilution in hot bidistilled water with a temperature of 80-90°C of tris-(hydroxymethyl)-aminomethane, citric acid, fructose, adding glycerol, and cooling to room temperature, adjusting the solution pH to 6.8. The liposome concentrate is separately prepared by adding in 10% sucrose solution of 0.2-20.0 wt % of soybean phospholipids, the surfactant is applied, it is maintained at room temperature, stirring occasionally for 4-24 hours, then the suspension is treated in the ultrasonic disintegrator for 5-30 minutes and the lipid-free base of the medium is mixed for cryopreservation of bull semen and the liposome concentrate at a ratio of 4:1, sterilised by autoclaving at a temperature of 115°C and pressure of 1.4 of the atmosphere for 1 hour, and sterilized by filtration through a filter with pores of 0.22 mcm.

EFFECT: proposed medium for cryopreservation has improved biological safety, has optical transparency and has a long-term use.

2 cl, 3 tbl, 2 dwg, 4 ex

Description

The invention relates to veterinary medicine, in particular to artificial insemination of cattle.

Known media for freezing bull's seed, including lactose, glycerin, penicillin, streptomycin, polymyxin, EDTA-calcium, EDTA-zinc, EDTA-copper, water and chicken egg yolk (patent RU 2355358, A61D 19/02, publ. 20.05 .2009);

medium for dilution and freezing of bull seed containing fructose, sodium citrate, TRIS, low-density lipoproteins from fresh yolk, glycerin, penicillin and distilled water (patent CN 101220345, C12N 5/6, published on July 16, 2008);

a medium for diluting and freezing bull seed, containing lactose, glycerin, antioxidants - sorbitol or sodium thiosulfate and yolk (patent UA 10894, A61D 19/02, publ. 29.12.1999);

medium for dilution and freezing of bull seed containing lactose, chicken egg yolk, glycerin, sodium citrate, distilled water and tissue extract obtained from the genitals of cows (patent UA 31304, A61D 19/02, 04/10/2008).

A known method of preparing the seed of animal producers for its use, including mixing lactose, citric acid, tris (oxymethyl) aminomethane, glycerol, bromide-N, water and yolk of chicken eggs, mixing with sperm and freezing the mixture (patent RU No. 2393816, A61D 19/02, publ. 07/10/2010).

A known method of treating bull seed is when chitosan succinate is introduced into the lactose-glycerol-vitelline medium and whole bull sperm is diluted with the resulting synthetic medium (patent RU No. 2223718, A61D 19/02, publ. 02.20.2004).

However, all of the above media contain a substance of animal origin (chicken egg yolk), which, with high cryoprotective efficiency, has a number of significant drawbacks: inconstancy of composition, difficulty in sterilization, rapid loss of cryoprotective properties during storage, the ability to cause immune sensitization when introduced into the genital tract of a female, and the danger of pathogenic virus transmission.

A known method of cryopreservation of animal cells and tissues, comprising mixing fructose, citric acid, tris- (hydroxymethyl) aminoethane, glycerin, purified water and phosphotidylcholine from soybean phospholipids (patent WO 2003/022046 A1, A01N 1/02, publ. 20.03.2003 );

A method of cryopreservation of animal cells and tissues is known, including mixing cells and tissues of fructose, citric acid, tris (hydroxymethyl) aminoethane, glycerin, purified water, hyaluronic acid and soybean phospholipids with animal cells, including soybean seed (patent US 2003 (0077566 A1, A01N 1/02, publ. 04.24.2003).

However, all known methods do not provide long-term storage of the environment for cryopreservation, because the developed methods do not include sterilization of the cryopreservation medium, and the cryopreservation medium also contains lecithin in the form of a coarse suspension, which makes the cryopreservation medium completely opaque, which makes visual analysis of the quality of the frozen-thawed seed extremely difficult.

Known methods of preparing a medium for diluting the seeds of producers: a method comprising mixing carbohydrate, cryogenic-protective and lipid-protein components in an aqueous medium followed by sterilization of a diluent solution, while sterilizing the diluent solution is carried out in hermetically sealed containers with an electromagnetic field of a certain frequency, specific energy consumption at a heating temperature of 70-75 ° C (patent RU 1769422, 6 A61D 19/02, publ. 06/27/1995);

a method comprising mixing carbohydrate, cryogenic-protective and lipid-protein components in an aqueous medium, the mixture of components being sealed in containers that are placed in an aqueous medium with a temperature of 18-20 ° C and affect the mixture with an electromagnetic field of the microwave range, then the data the containers are kept for 16-20 hours, and then they are heated to 58-60 ° C for 20-25 minutes (patent RU 2073499, A61D 19/02, publ. 02.20.1997).

The main disadvantages of these solutions are the need for complex and expensive equipment, and, therefore, the high cost of their implementation, as well as the inconsistency of the composition of the medium and insufficient for the complete destruction of pathogenic microorganisms that cause chlamydial and mycoplasma infections, the degree of sterilization.

The closest in technical essence and the achieved positive effect is a synthetic environment for freezing bull seed, which includes tris- (hydroxymethyl) aminomethane - 2.65-2.85%, fructose - 0.70-0.80%, lemon acid - 1.3-1.5%, glycerin - 5-7%, dry soya lecithin Centrolex F - 8-12%, distilled water - the rest (patent RU 2323701, A61D 19/02, publ. 10.05.2008).

This environment, taken as a prototype, is prepared in the following way. Tris (hydroxymethyl) aminomethane, fructose, citric acid are added to a clean chemical flask according to the recipe and poured with boiled distilled water at a temperature of 80-90 ° C. Thoroughly dissolve all components. Then the solution is cooled to 40 ° C and glycerol and dry soya lecithin are added there. Soy lecithin in the proposed variant of the cryoprotective medium hydrates spontaneously and forms large liposomes with an average diameter of more than 1000 nm.

The medium has high cryoprotective efficiency, does not contain components of animal origin, which ensures its sanitary and epidemiological safety, but has two significant drawbacks. This medium is not intended for long-term storage and is intended for use within 6 hours after preparation; the medium contains lecithin in the form of a coarse suspension, which makes the medium completely opaque. This property of the medium complicates the visual analysis of the quality of the frozen-thawed seed and makes it impossible to use a more advanced instrumental analysis of the quality of the seed, which requires an optically transparent medium.

The aim of the invention is to increase the biological safety of the synthetic environment for cryopreservation of bull seed and increase its technological properties (transparency, increase shelf life).

The technical result of the invention is achieved by the fact that the proposed environment for cryopreservation of bull seed, including tris- (hydroxymethyl) -aminomethane, fructose, citric acid, sucrose, glycerin, dry soya lecithin (soybean phospholipids), which can be used as a source of commercial vegetable lecithin of various brands (Lipoid C75, Lipoid C100, Lipoid C40, Azolectin, Lezi-Pro-90, Lecigran and bidistilled water, characterized in that it additionally contains a high molecular weight non-toxic surfactant, as Which uses methyl cellulose, or pluronic (F68, or F127, or F168), or carrageenan, or sodium alginate, or hyaluronic acid, or bovine serum albumin in the following ratios of components, wt.%:

tris (hydroxymethyl) aminomethane 1.9 fructose 0.9 lemon acid 0.7 sucrose 2 glycerol 4,5-6,5 soybean phospholipids 0.04-2.00 surface-active substance 0.01-1 bidistilled water rest

A method of preparing a medium for cryopreservation of bull seed is proposed, which includes preparing a non-lipid base for the medium for cryopreservation of bull seed by diluting tris- (hydroxymethyl) -aminomethane, citric acid, fructose, adding glycerin and cooling to room temperature in hot bidistilled water at a temperature of 80-90 ° C temperature, bringing the pH of the solution to 6.8, characterized in that the liposome concentrate is separately prepared by adding 0.2-20.0 wt.% soy phospholipids to a 10% sucrose solution, a surfactant the substance is kept at room temperature, stirring occasionally for 4-24 hours, then the suspension is treated with an ultrasonic disintegrator for 5-30 minutes and the non-lipid base medium for cryopreservation of bull seed and liposome concentrate are mixed in a ratio of 4: 1, sterilized by autoclaving at a temperature of 115 ° C and a pressure of 1.4 atmospheres for 1 hour or sterilized by filtration through a 0.22 μm pore filter.

It is well known that media containing egg yolk are mainly used to freeze bull seed. Egg yolk reduces sperm sensitivity to cold shock and freezing. The carrier of the cryoprotective properties of the egg yolk is a light lipoprotein fraction of the yolk. The main part of the lung lipoproteins of SJ is organized into small conglomerates with an average molecular weight of about 5 × 106 (Cook, Martin, 1969). The light lipoprotein fraction of the yolk consists of phospholipids (egg lecithin) and a protein component. It is believed that egg lecithin has cryoprotective properties. The purified proteins of the light lipoprotein fraction showed no cryoprotective properties. Nevertheless, purified egg phospholipids have weaker cryoprotective properties compared to the full fraction of light lipoproteins. Egg yolk is highly effective in protecting spermatozoa from cold shock during both cooling and freezing. Suffice it to say that the removal of egg yolk from a standard medium for freezing bull seed leads to a decrease in sperm survival in the freeze-thaw cycle by about 6 times, while the removal of the traditional cryoprotectant, glycerol, from the same medium is only 2 times. But, with high cryoprotective efficiency, the yolk has a number of undesirable properties: inconstancy of composition, difficulty in sterilization, rapid loss of cryoprotective properties during storage (several hours), and the ability to induce immune sensitization when a female is introduced into the genital tract. In addition, in recent years, in connection with the massive import of breeding bulls into Russia, cases of abortion caused by chlamydial and mycoplasma infections have become more frequent. Currently, there is no clear methodology for identifying the causative agents of these infections, and the chain polymerization reaction used does not always provide the possibility of species-specific determination of the pathogen. Thus, the yolk of a chicken egg in the presence of antibodies to mycoplasma in it is often the reason for rejection of the benign cryopreserved seed of manufacturing bulls.

The most promising substitute for yolk is plant-derived phospholipids (soya lecithin). It is known that the cryoprotective effectiveness of phospholipids significantly depends on the organization form of phospholipid particles in a cryoprotective solution. We propose to introduce plant phospholipids in the form of small liposomes (30-350 nm). Lecithin in the form of small liposomes forms a more stable suspension, is not prone to the formation of sediment, interacts more easily with the sperm membrane. In addition, phospholipids in the form of small liposomes make it possible to obtain an optically transparent suspension, which is important for visual and instrumental assessment of the quality of the seed diluted with cryomedium. In addition to phospholipids, a high molecular weight surfactant is introduced into the medium. Such a substance can be dry purified albumin or high molecular weight surfactants of a non-protein nature, namely methyl cellulose, sodium alginate, hyaluronic acid, carrageenan, pluronic and some others. The listed substances not only stabilize liposomes, but also change the interaction of liposomes with the sperm cell membrane, increasing the cryoprotective effect of liposomes.

The preparation of the medium for cryopreservation of bull seed was as follows.

A non-lipid base is prepared for the cryoprotective environment for bull seed. To do this, tris (hydroxymethyl) aminomethane, citric acid, fructose, and glycerol are diluted in hot bidistilled water with a temperature of 80-90 ° C. The resulting solution was cooled to room temperature, the pH of the solution was adjusted to 6.8.

Additionally, a liposome concentrate of the following composition is prepared: sucrose 9-10 g, soy phospholipids (commercial soy lecithin of the brands Lipoid C75, Lipoid C100, Lipoid C40, Azolectin, Lezi-Pro-90, Lecigran) 0.2-10 g. To obtain a suspension of phospholipids a portion of dry lecithin in a solution of sucrose and incubated at room temperature, stirring occasionally for 4-24 hours. After hydration of lecithin for a specified time, an initial coarse suspension of phospholipids is obtained containing multilamellar liposomes with an average diameter of 1000-2000 nm (the size depends on the duration of hydration, the intensity of stirring, and the temperature of the solution). The primary coarse suspension is treated on an ultrasonic disintegrator for 5-30 minutes. As a result of ultrasonic treatment, large liposomes in suspension are crushed and transform into the form of predominantly monolamellar liposomes with an average diameter of 30-350 nm. Then, a stabilizer is added to the suspension of liposomes, which can be: methyl cellulose at a concentration of 0.05-0.25%, or pluronic (F68, or F127, or F168) at a concentration of 0.05-0.25%, or carrageenan at a concentration of 0.05 -0.25%, or sodium alginate at a concentration of 0.05-0.5%), or hyaluronic acid at a concentration of 0.1-0.5%), or bovine serum albumin at a concentration of 1-5%).

The prepared non-lipid base (first or second options) is mixed with liposome concentrate in a ratio of 4: 1.

The obtained cryoprotective environment for bull seed can be used without additional processing or sterilized.

The sterilization method depends on the type of stabilizer added and on the average particle size, which depends on the particular brand of commercial lecithins and on the ultrasonic treatment regimen.

The cryoprotective medium is sterilized by filtration or autoclaving. The cryoprotective medium is sterilized by filtration through a 0.22 nm filter. The method is suitable for suspension of liposomes with an average particle size of up to about 150-170 nm. With a higher average particle size in the suspension, the filters clog quickly and further filtering becomes impossible.

Cryoprotective medium is sterilized by autoclaving at a temperature of 115 ° C and a pressure of 1.4 atmospheres for 1 hour. Higher temperature autoclaving is unacceptable, as will cause caramelization of sugars. Autoclaving is unacceptable if bovine serum albumin is used as a liposome stabilizer.

The finished medium is stored at a temperature of + 4 ° C in a dark place, because light and high temperatures can cause peroxidation in liposomes. Without sterilization, the medium remains stable and cryoprotective for at least 5 days. Sterilization can increase the shelf life of the finished cryoprotective environment for bull seed up to 2-3 months.

The invention is illustrated by the following examples.

Example 1. The determination of particle size in a suspension of soybean phospholipids of the brand Lecy-Pro-90 in a solution of sucrose.

In the experiment used food lecithin brand Lecy-Pro-90, purchased in the company "Protein plus". The primary (coarse) suspension of Lecy-Pro-90 “dry lecithin” was obtained by spontaneous hydration of dry lecithin. It is known that “dry” soya lecithins, which contain at least 95% polar lipids, are capable of spontaneously forming a suspension of multilamellar liposomes when placed in an aqueous medium. To obtain a suspension, a sample of Lecy-Pro-90 dry lecithin at the rate of 6% was placed in a sucrose solution and kept at room temperature with constant stirring for 4 hours. Then part of the suspension was placed in a refrigerator (+ 4 ° C) for 20-24 hours. During this time, almost complete hydration of lecithin occurs and a suspension containing multamellar liposomes forms. This suspension is hereinafter referred to as suspensions obtained by spontaneous hydration. The second part was subjected to sonication on an ultrasonic disintegrator UZDN-2T. The suspension was treated with ultrasound at 22 Hz, 60 W / cm ² for 5 minutes using an immersion probe with an end diameter of 2 mm. To prevent lecithin oxidation during ultrasonic treatment, the suspension was cooled to + 10 ° C in an ice bath. Then, the resulting suspension was stored in a refrigerator (+ 4 ° C) until measurements. This solution is hereinafter referred to as a suspension obtained by ultrasonic crushing.

Particle sizes were measured one day after preparation.

The sizes of liposomes or lipid micelles in the resulting suspension were determined by measuring dynamic light scattering on a BeckmanCoulter N5 Submicron Particle Size Analyzer. The sizes of lipid particles in suspension were determined 1 day after preparation. The appearance of the suspension was also visually evaluated.

The measurement results are presented in table 1.

Table 1 Characterization of LeCy-Pro-90 lectin suspensions obtained by spontaneous hydration of lecithin and ultrasonic crushing of suspension particles. The method of preparation of the suspension The average particle size, nm. Visual characteristic of the suspension Spontaneous Hydration Suspension 1788.3 ± 55.4 Opaque mixture of milk consistency, yellowish, slight nutty odor The suspension obtained by ultrasonic crushing 74.6 ± 12.9 Translucent opalescent liquid, slight nutty smell. In a thin layer (about 0.5 cm), the printed text can be distinguished by the clearance.

In addition to studying the characteristics of the suspension, visual studies of spermatozoa were made in a 5% LeCy-Pro-90 lecithin suspension obtained by spontaneous hydration and in a 1% LeCy-Pro-90 lecithin suspension obtained by ultrasonic crushing. As can be seen in figure 1, the suspension treated with ultrasound does not interfere with the visual examination of sperm (Fig. 1B), while in the suspension obtained by spontaneous hydration, sperm are difficult to distinguish (Fig. 1a).

Example 2. Determination of the optimal concentration of LeCy-Pro-90 lecithin in a suspension treated with ultrasound to provide cryoprotective effects on bovine spermatozoa during cryopreservation.

In the experiment, food grade lecithins of the LeCy-Pro-90 grades purchased from the Protein Plus company were used. Lecithin suspensions were prepared based on a 10% sucrose solution. The primary (coarse) suspension of LeCy-Pro-90 “dry lecithin” was obtained by spontaneous hydration of dry lecithin. To obtain a suspension, a sample of dry lecithin in a sucrose solution was kept at room temperature, stirring occasionally for 4 hours. Then the suspension was processed on an ultrasonic disintegrator UZDN-2T (22 Hz, 60 W / cm ² for 5 minutes). Seed of Holstein bulls was diluted 1/4 or more (depending on the concentration of sperm in the ejaculate) with lactose-glycerin medium (10% lactose, 0.2% HEPES, 5% glycerol with the addition of either 20% egg yolk (control) or 20 % liposome concentrate with different liposome concentrations so that the final concentration of lipids in the medium was from 0.03 to 3%. Freezing was carried out in open granules on plates of solid carbon dioxide (dry ice) according to the standard method (V.N. Vinogradov et al. National technology of freezing and using Bani sperm breeding bulls manufacturers // Moscow, 2008, 160 pp.). Thawing was performed in granules deicing solution (130 mM NaCl, 4 mM KCl, 1 mM CaCl _2, 0.5 mM MgCl _2, 14 mM fructose, 10 mM Hepes, 0.1% bovine serum albumin) in a water bath at 39–40 ° C.

The cryoprotective efficacy of soybean phospholipids was determined by sperm survival during cryopreservation. Viability was judged by the mobility of frozen thawed sperm 5 minutes after thawing and after 5 hours of keeping frozen thawed sperm at 38 ° C. Motility was determined on a Biola SFA-500 sperm analyzer.

The research results presented in figure 2, indicate that the optimal concentration of soya lecithin in the form of a suspension of liposomes with an average diameter of 80-100 nm lie in a fairly wide range, from 0.1 to 1% lecithin in a cryoprotective environment. Sperm motility 10 minutes after thawing in the experimental groups with the indicated concentrations did not have significant differences from the control group with the addition of egg yolk.

Example 3. Comparison of the cryoprotective efficacy of tris-citrate-fructose-glycerol medium with egg yolk and tris-citrate-fructose-glycerol medium with the addition of plant phospholipids in the form of small liposomes.

In this example, a comparison was made of the cryoprotective effect on sperm of a bull of egg yolk and a liposomal concentrate made from soy phospholipids against a background of tris-citrate-fructose-glycerol medium. The control was bull seed, cryopreserved in Tris-citrate-fructose-glycerol medium with the addition of 20% egg yolk. In the experiment, 20% liposome concentrate of the following composition was added to tris-citrate-fructose-glycerol medium instead of yolk: sucrose 10 g, soy phospholipids (azolectin) 1.8 g, sodium alginate 0.5 g, bidistilled water 88 ml. Liposomes were crushed by ultrasound on an ultrasonic disintegrator UZDN-2T (22 Hz, 60 W / cm ² for 5 minutes).

Freezing was carried out in open granules on plates of solid carbon dioxide (dry ice) according to a standard method (V.N. Vinogradov et al. National technology for freezing and using semen from breeding bulls of producers // M., 2008, 160 s). Granules were thawed in a thawing solution (130 mm NaCl, 4 mm KCl, 1 mm CaCl ₂ , 0.5 mm MgCl ₂ , 14 mm fructose, 10 mm Hepes, 0.1% bovine serum albumin) in a water bath at a temperature of 39- 40 ° C.

The cryoprotective efficacy of soybean phospholipids was determined by sperm survival during cryopreservation. Viability was judged by the mobility of frozen thawed sperm 5 minutes after thawing and after 5 hours of keeping frozen thawed sperm at 38 ° C. Motility was determined on a Biola SFA-500 sperm analyzer. The experimental results are presented in table 2.

Table 2. Comparison of frozen thawed bull seed cryopreserved in tris-citrate-fructose-glycerol medium and in non-yellowish tris-citrate-glycerol medium with soy phospholipids in the form of liposomes with an average diameter of 80-100 nm. Cryoprotective medium Mobile sperm immediately after thawing,%. The average speed of sperm movement immediately after thawing Mobile sperm after 5 hours of cultivation at 38 ° C,%. Visually Sperm analyzer Tris-citrate-fructose-glycerol-yolk medium 39.0 ± 2.0 39.5 ± 2.8 73.1 ± 3.5 21.1 ± 5.5 Tris-citrate-fructose-glycine medium with soybean phospholipids 38.5 ± 1.1 36.5 ± 1.2 69.0 ± 2.2 19.5 ± 2.3

As can be seen from the table, the motor characteristics of a seed cryopreserved in tris-citrate-fructose-glycerol medium with egg yolk (control) and with a liposome concentrate containing sucrose, plant phospholipids in the form of liposomes with an average diameter of 80-100 nm and sodium alginate, not have significant differences. So the mobility after thawing in the control and the experimental group was 39.5 ± 2.8% and 36.5 ± 1.2% of motile sperm cells, respectively, mobility 5 hours after thawing, 21.1 ± 5.5% in the control and 19, 5 ± 2.3 in the experimental group. The data indicate the effectiveness of the proposed cryoprotective environment.

Example 4. The study of the effect of storage of cryoprotective medium before use on its cryoprotective effectiveness.

In the experiment, the seed of bulls of Holstein and black-motley breeds was used. A liposomal concentrate was prepared on the basis of a 10% sucrose solution; the soybean extract Azolectin (Sigma) was used as a source of plant phospholipids. The lecithin suspension was processed on an ultrasonic disintegrator UZDN-2T (22 Hz, 60 W / cm ² for 5 minutes). After sonication, 0.1% pluronic P68 was added to the liposome concentrate as a stabilizer. The non-liposomal base of the following composition was used (in weight percent): tris- (hydroxymethyl) aminomethane - 2.4; fructose - 0.9; citric acid - 1.3; glycerin - 6, double-distilled water. The non-liposomal base and liposome concentrate were mixed in a 4: 1 ratio. The medium was autoclaved at 115 ° C for 1 hour and sealed until use. The medium was stored in the refrigerator (+ 4 °). Cryopreservation of bull seed was carried out according to the standard method of freezing in granules. For freezing, we used the medium after 1 day (control) and 10 days of storage (V.N. Vinogradov et al. National technology for freezing and using sperm of breeding bulls of producers // M., 2008, 160 pp.). Granules were thawed in a thawing solution (130 mm NaCl, 4 mm KCl, 1 mm CaCl ₂ , 0.5 mm MgCl ₂ , 14 mm fructose, 10 mm Heps, 0.1% bovine serum albumin) in a water bath at a temperature of 39- 40 ° C.

Cryoprotective effectiveness of the medium was determined by the survival of sperm during cryopreservation. Viability was judged by the mobility of frozen thawed sperm 5 minutes after thawing and after 5 hours of keeping frozen thawed sperm at 38 ° C. Motility was determined on a Biola SFA-500 sperm analyzer.

Table 3. Effect of storage of the medium before use (storage at + 4 ° C) Indicators of frozen and thawed semen in Tris-citrate-fructozoglycerin medium with liposomes, alginate and antioxidants. Mobility before freezing,%. Sperm counts after thawing Mobility after defrosting Mobility after cultivation at 38 ° C, 5 hours,%. The number of motile sperm,%. average speed Seed cryopreservation in the medium 1 day after
cooking * 80 ± 1 39 ± 5 62 ± 7 15 ± 5 Seed cryopreservation in the medium 10 days after
cooking *. 79 ± 1 36 ± 6 66 ± 7 17 ± 6

The research results presented in table 4 indicate that the medium retains cryoprotective effectiveness for 10 days of storage. So, the motor characteristics of the seed were: after cryopreservation in a freshly prepared medium, the mobility immediately after thawing is 80 ± 1% of motile spermatozoa, motility 5 hours after thawing -15 ± 5% of motile spermatozoa; after cryopreservation in an environment remaining at +4 for 10 days, mobility immediately after thawing is 79 ± 1% of motile spermatozoa, motility 5 hours after thawing is 17 ± 6% of motile spermatozoa. Thus, ten-day storage of medium with soya lecithins in the form of liposomes small size did not affect its cryoprotective characteristics.

The proposed environment has increased biological safety, because does not contain egg yolk or other complex components of animal origin, which can be carriers of dangerous pathogens.

Unlike the prototype, a medium with plant lecithin in the form of liposomes, up to 350 nm in size, has optical transparency. The new environment for cryopreservation of bull seed does not interfere with visual and instrumental quality control of the seed.

Due to the more effective interaction of small liposomes with the sperm cell membrane, the optimal concentration of phospholipid in the cryoprotective medium decreases from 4-8% to 0.1-1%.

Due to the high stability of liposome sizes and sterilization, the period of possible use of the finished cryo-medium increases from 5 hours to 2-3 months, which allows optimizing the labor costs of technologists at breeding enterprises.

The invention is applicable in enterprises whose activity is to obtain and cryopreserve the seed of bulls.

Claims (2)

1. A medium for cryopreservation of bull seed, including tris (hydroxymethyl) aminomethane, citric acid, sucrose, fructose, glycerin, soy phospholipids, which can be produced from various types of commercial plant lecithin, for example Lipoid C75, Lipoid C100, Lipoid C40 , Azolectin, Lezi-Pro-90, Lecigran and bidistilled water, characterized in that it additionally contains a high molecular weight non-toxic surfactant, which is used methyl cellulose, or pluronic (F68, or F127, or F168), or car Raginan, or sodium alginate, or hyaluronic acid, or bovine serum albumin in the following ratios of components, wt.%:
Tris (hydroxymethyl) aminomethane 1.9 Fructose 0.9 Lemon acid 0.7 Sucrose 2 Glycerol 4,5-6,5 Soybean Phospholipids 0.04-2.00 Surface-active substance 0.01-1 Bidistilled water rest 2. A method of preparing a medium for cryopreservation of a bull’s seed, comprising preparing a non-lipid basis for a medium for cryopreservation of a bull’s seed by diluting tris- (hydroxymethyl) -aminomethane, citric acid, fructose, adding glycerin and cooling to hot in double-distilled water at a temperature of 80-90 ° C room temperature, the pH of the solution is adjusted to 6.8, characterized in that the liposome concentrate is separately prepared by adding 0.2-20.0 wt.% soy phospholipids to a 10% sucrose solution, and a surfactant is introduced oh, kept at room temperature, stirring periodically, 4-24 hours, then treated with a suspension on an ultrasonic disintegrator for 5-30 minutes and mixed non-lipid base medium for cryopreservation of bull seed and liposome concentrate in a ratio of 4: 1, sterilized by autoclaving at a temperature of 115 ° C and a pressure of 1.4 atmospheres for 1 hour or sterilized by filtration through a 0.22 μm pore filter.

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