RU2323702C2 - Synthetic medium for ram sperm dilution and cryopreservation - Google Patents

Abstract

FIELD: agriculture; cattle breeding.

SUBSTANCE: synthetic medium contains (in mass %): lactose - 6.00, dextrin - 3.60, xylite - 0.18,tris (methylol)-aminomethane - 0.08, disodium dihydrate salt EDTA - 0.10, glycerine - 4.3, egg yolk - 14.3, sodium succinate 6-hydrate - 0.03, distilled water - the rest.

EFFECT: medium allows increasing pregnancy and multiple pregnancy of ewes at artificial insemination.

3 tbl, 2 ex

Description

The invention relates to agriculture, in particular to livestock. It can be used for artificial insemination of sheep with cryopreserved sperm of sheep to increase coagulation and multiple fertility.

For artificial insemination of ewes, freshly harvested, chilled (up to + 2-0 ° C) and cryopreserved (up to -196 ° C) sperm of sheep is used. In the last two cases, during technological processing, it is diluted 3-4 times with protective synthetic media.

In the native sperm of sheep, there are enough natural antioxidants that protect living cells from lipid peroxidation (LPO). These are phospholipids, tocopherols, ubiquinone, cystine, coenzyme Q, and others, as well as specific protection enzymes, for example, the glutathione peroxidase system.

Considering the susceptibility of ram sperm during storage to the LPO process, which negatively affects the preservation of the biological usefulness of sperm, foreign and domestic scientists suggest enriching the environment for dilution with various antioxidants.

However, in the course of further technological processing (dilution, balancing, freezing, defrosting), the concentration of sperm plasma chemicals significantly decreases.

Known media for dilution and cryopreservation of ram sperm, including disaccharum (sucrose, lactose), polysaccharides (dextrin), egg yolk, or egg powder, buffer capacity stabilizers (tris (hydroxymethyl) aminomethane) trilon-B, extracellular cryoprotectants (gum arabic, glycerin, unitiol, glutathione, antioxidants (2,6-tert-butyl toluene; butyloxytoluene, taurine, gliotaurin) (1).

However, these media do not have sufficient cryoprotective effect due to the lack of specialized ingredients in their composition that can efficiently structure water around sperm cells and effectively stabilize the structure of sperm membranes.

There is a violation of the ordering of the structure of sperm membranes, which can initiate LP. As a result of this, destructive changes in the membrane structures of spermatozoa develop. In turn, this leads to a violation of ion permeability and a decrease in the fluidity of membranes, inactivation of enzymes, and the accumulation of metabolic products, thereby negatively affecting the preservation of the biological usefulness of spermatozoa.

Obviously, these processes are the main reasons for the low effectiveness of artificial insemination of sheep with cryopreserved sperm.

The closest in technical essence to the proposed environment is GZHUK-Tris-buffer medium for dilution and freezing of sperm of sheep: lactose - 8.4 g; corn dextrin - 5 g; xylitol - 0.26 g; tris- (hydroxymethyl) aminomethane - 0.105 g; disodium EDTA (Trilon-B) disodium salt - 0.135 g; glycerin - 6.0 cm; egg yolk - 20 cm; IHFGAN-3 - 8 mg; distilled water - 100 ml (2).

The antioxidant IHFGAN-3, which has a positive effect on the survivability and fertilizing ability of ram cryopreserved sperm, is a fat-soluble phenolic antioxidant, chemical formula: N-bromide, N-dimethyl-N-decyl-4oxy-3,5-ditret-butyl-benzyl amine synthesized at the Institute of Chemical Physics RAS. IHFGAN-3 is injected into a GLC-Tris-buffer medium at a dose of 0.08 mg / ml to prevent the activation of lipid peroxidation in the membrane structures of sperm cells during sperm cryopreservation.

However, this drug cannot contribute to the full realization of the reproductive ability of sperm after defrosting. Being an antioxidant, IHFGAN-3 in the composition of the medium manifests itself only as a lipid peroxidation inhibitor. It does not contain specialized ingredients that, under hypoxic conditions, would contribute to the energy supply of live sperm.

The aim of the invention is to improve the synthetic environment to increase the safety, biological usefulness of ram sperm outside the body after sperm dilution, cryopreservation and defrosting, which ensures an increase in the percentage of coagulation and multiple pregnancy of ewes with artificial insemination.

The goal is achieved by the inclusion in the medium of a biologically active preparation (BAS) of prolonged action, which has a double (combined) positive effect on live spermatozoa during cryopreservation and sperm defrosting. In the composition of the medium, the drug used manifests itself firstly as an antioxidant, an inhibitor of lipid peroxidation, and secondly, being oxidized under hypoxia conditions, it becomes the main substrate for the energy supply of sperm, which helps preserve the structure of damaged cells during cryopreservation.

The invention consists in the use of sodium succinic 6-aqueous.

Sodium succinic 6-aqueous — a free-flowing white powder, it is readily soluble in water and has the following chemical formula: NaOOCCH ₂ CH ₂ COONa6Н ₂ О with a molecular weight of 162. When incorporated into a medium in non-toxic concentrations, the osmotic pressure and pH of the medium do not significantly change. In the composition of the medium, sodium 6-aqueous succinic acid dissociates into sodium ions and succinic acid - the chemical formula: НOOССН ₂ СН ₂ СОО (synonym - ethane-1,2-dicarboxylic acid).

The listed metabolic effects of succinic acid indicate the adequacy of the choice of this substrate for maintaining the energy supply of sperm in our designed environment and maintaining their structure.

Previously, this drug was not used in synthetic media.

The advantage of the present invention in comparison with the prototype is that the proposed environment has higher cryoprotective properties compared to the prototype and control, increases the biological usefulness of sperm during cryopreservation and defrostation. In the experimental group, sperm motility during thawing after cryopreservation (-196 ° C) is 4.5 ± 0.20 points, and the absolute survival rate at 0 ° C is 368 ± 13.0 conventional units, against 4.25 ± 0.18 score and 340 ± 12.4 conventional units of the prototype, respectively.

Sperm motility in the control group (medium without sodium succinic 6-aqueous) was 4.1 ± 0.15 points, the absolute survivability index was 305 ± 10.5 conventional units.

A synthetic medium with sodium succinic 6-aqueous more efficiently preserves the biological usefulness and fertilizing ability of cryopreserved sperm of sheep and stimulates the reproductive function of ewes - fertilization and multiple pregnancy.

The proposed environment provides higher cryoprotective properties in comparison with the prototype due to the fact that the new protector includes not only sodium ions, which increase sperm motility, but also succinic acid, which, being a natural antioxidant, inhibits lipid peroxidation and oxidizes under hypoxic conditions , becomes a source of energy sperm.

These properties well explain its role in preventing the growth of metabolic disorders initiating lipid peroxidation and irreversible damage to sperm membranes during cryopreservation.

Thus, the protective effect of sodium 6-aqueous succinic acid is more directed than that of the antioxidant IHFGAN-3 due to the fact that it has a wider spectrum of physiological effects on sperm - sodium ions increase sperm motility, and succinic acid inhibits lipid peroxidation under conditions hypoxia is becoming a source of energy for sperm.

As a result, the proposed environment allows to more effectively increase the biological usefulness of sperm after cryopreservation and defrostation, as it has better cryoprotective properties.

The method is as follows. For cryopreservation of sperm, GLC-Tris buffer medium was used, in which, instead of IHFGAN-3, the preparation of sodium succinic 6-aqueous at a concentration of 0.40 mg / ml was added.

Medium Preparation Example

In a sterile chemical flask, make a prescription (in wt.%): Lactose - 6.00; dextrin 3.60; xylitol - 0.18; tris- (hydroxymethyl) amino-methane - 0.08; disodium EDTA (Trilon-B) disodium salt - 0.10; glycerin - 4.30; egg yolk - 14.30; sodium succinic, 6-aqueous - 0.03; distilled water the rest. All of the above ingredients in total amount to 100 wt.%.

Freshly prepared synthetic medium before diluting sperm should be examined in appearance, color, periodically - in terms of pH and osmotic pressure.

The proposed physical and chemical properties environment must meet the following requirements: appearance - a homogeneous emulsion, without sediment and mechanical impurities: Color - odorless yellow-gray; pH at a temperature of 20 ° C - 6.8 ± 0.15; osmotic pressure of the medium without glycerol at a temperature of 0 ° C - 10.0 ± 0.5 atm.

The prepared medium is used to dilute sperm within 4 hours from the time of preparation. It is stored at a temperature of 0 ° -4 ° C; it is heated to +28 - + 30 ° C before use.

When using the medium, 2-3 volumes of the medium are added to one volume of sperm, depending on the concentration of sperm in the ejaculate.

The invention is illustrated by the following examples.

Example No. 1. Sperm for research was obtained from Salsk sheep using an artificial vagina. After taking each ejaculate, its volume and a number of qualitative indicators were determined (sperm activity in points, concentration in billion / ml, total number of sperm in the ejaculate in billion). In the experiments, ejaculates were used that met the requirements of the “Instructions for the use of GJUK-Tris-buffer medium for dilution, freezing, storage and use of ram sperm” (3), sperm was also diluted 3-4 times according to this instruction. To study the effect of 6-aqueous succinic sodium in the medium on the preservation of the biological usefulness of ram sperm outside the body after cryopreservation, the following drug concentrations were used: 0.20, 0.40, 0.80 and 1.20 mg per 1 ml. In the control group, native sheep sperm was diluted with GZHUK-Tris buffer medium without preparation.

Diluted semen was packaged in numbered vials and, together with a control sample, was subjected to balancing (cooling) for 2 hours at a temperature of + 2-4 ° C. Next, the sperm was frozen on a fluoroplastic plate in pairs of liquid nitrogen, in 0.2 ml wells, according to the methodology of VNIIplem, according to the manual. For biological control, experimental samples of frozen sperm were defrosted in a 3% solution of sodium citrate at 42 ° C, then sperm motility was determined every hour until they died. The absolute indicator of survivability (Sa) of spermatozoa was determined at an incubation temperature of + 2 + 4 ° С. The experiments were carried out in six replicates.

The optimal concentration of sodium 6-aqueous succinic acid in diluted semen was determined after its defrosting by the indicator of sperm motility and survivability in experimental and control samples (Table 1).

Table 1. The effect of sodium succinic 6-aqueous in GZHUK-Tris-buffer medium on the biological parameters of ram sperm after cryopreservation Drug Concentration (mg / ml) Indicators Mobility Survivability Conc. water. ions Score % Sa (conventional units) % pH % Experience: sodium succinic 6-aqueous 0.25 4.2 ± 0.14 105 315 ± 15.9 112.5 6.80 99.3 0.40 4.5 ± 0.10 ^x 113 340 ± 18.7 ^x 121.5 6.78 99.0 0.80 3.8 ± 0.12 95 256 ± 20.1 91.4 6.65 97.0 1.20 3.6 ± 0.10 90 230 ± 20.5 82.1 6.50 95.0 Control (without drug) 4.0 ± 0.25 one hundred 280 ± 20.0 one hundred 6.85 one hundred ^x - statistically significant value (p <0,01)

The data in table 1 indicate that the inclusion of sodium succinic 6-aqueous in the composition of the medium significantly affects the biological indicators (motility and vitality) of ram sperm. The optimal concentration of sodium succinic acid in the medium should be considered 0.40 mg / ml. In this case, sperm motility exceeds the control group by 12.5%, and the absolute survivability by 21.5%. (p <0.01).

The cryoprotective properties of our recommended medium with sodium succinic 6-aqueous and a prototype medium with the preparation of IHFGAN-3 compared with the control (GLC-Tris-buffer medium without the preparation of IHFGAN-3 and sodium succinic 6-aqueous) are illustrated in Table 2.

Table 2. The effect of sodium succinic 6-water and IHFGAN-3 in the composition of GZHUK-Tris-buffer medium on increasing the biological usefulness of cryopreserved ram sperm The concentration of drugs (mg / ml) Indicators Mobility Survivability Conc. water. ions point % Sa (conventional units) % pH % Sodium succinic 6-aqueous 0.40 4.5 ± 0.20 ^x 109.7 368 ± 13.0 ^x 120 6.75 ± 0.1 99.3 IHFGAN-3 (prototype) 0.08 4.25 ± 0.18 103.6 340 ± 12.4 ^x 111 6.78 ± 0.1 99.7 Control (without drugs) 4.10 ± 0.15 one hundred 305 ± 10.5 one hundred 6.80 ± 0.1 one hundred ^x is a statistically significant value (p <0.05)

The above data (Table 2) show that the synthetic medium with sodium succinic 6-aqueous increases the biological usefulness of cryopreserved ram sperm by a statistically significant amount compared to the prototype (antioxidant IHFGAN-3) and control (medium without sodium succinate and IHFGANA-3) .

So, if in the control group sperm motility was equal to 4.1 points, and with the use of IHFGAN-3 it increased to 4.25 points, then in a medium with sodium succinic 6-aqueous sperm motility increased to 4.50 points, which is higher than the control and prototype by 9.7% and 3.6%, respectively.

More significant differences were established in the study of sperm vitality. When using the IHFGAN-3 preparation, the absolute survivability index increased by 11% compared with the control, and in an environment with sodium succinic 6-aqueous, this indicator increased by 20%. The obtained difference in the survivability indices of the experimental and control groups (20% and 11%) is statistically significant in both cases at p <0.05.

Thus, the studies showed that 6-aqueous sodium succinic acid in the GLC-Tris-buffer medium has a complex effect on ram spermatozoa (as an LPO inhibitor and as a substrate for energy supply to cells). Better than the prototype IHFGAN-3, it affects the preservation of the biological usefulness of ram sperm outside the body after cryopreservation and defrostation, increasing mobility by 9.7%, and the absolute survivability by 20%.

Example No. 2. The effect of sodium succinic 6-aqueous on the percentage of ejaculation and multiple pregnancy of ewes was studied on a commission basis in a scientific and production experiment at a concentration of 0.40 mg / ml. This concentration of the drug in the composition of GLC-Tris-buffer medium was chosen due to the fact that it increases by a statistically significant value the qualitative indicators of defrosted ram sperm.

The results of these studies are presented in table 3.

The data obtained (Table 3) show that the fertility of ewes in the experimental group, where the optimal dose of sodium succinic 6-aqueous (0.40 mg / ml) was used, was higher than in the control group by 11.2%, and multiple pregnancy - 12 goals - 142 lamb per 100 ewes, against 130 goals in the control group (p <0.05).

Table 3.
The effect of sodium succinic 6-water and IHFGAN-3 in GLC-Tris buffer medium on the results of artificial insemination of ewes with cryopreserved ram sperm. Dose of the drug, mg / ml. Mobility score Vitality (conventional units) Inseminated sheep (goals) Lounged Goal. % Number of lambs per 100 queens, goal. Sodium succinic 6-aqueous, (0.40) 4.5 ± 0.10 ^x 370 ± 15.3 ^x 192 109 59.8 ± 3.2 ^x 142 IHFGAN-3 (0.08) 4.2 ± 0.12 355 ± 18.4 190 95 53.5 ± 3.6 135 Control (without drug) 4.0 ± 0.15 310 ± 20.1 183 90 48.6 ± 3.2 130 ^x is a statistically significant value (p <0.05)

Thus, the introduction of sodium succinic 6-aqueous in a concentration of 0.40 mg / ml into a GLC-Tris buffer medium can significantly improve the biological usefulness of sperm after defrosting (mobility by 9.7%; absolute survival rate by 20%), as well as increase the fertility of ewes by 11.2%, their multifertility - by 12 goals (for every 100 queens) compared with the control (medium without antioxidants). The efficiency of using the medium with sodium succinic 6-aqueous (at a concentration of 0.40 mg / ml) was higher by 6.3% and 7 goals for every 100 uterus, compared with the prototype (medium with IHFGAN-3 at a concentration of 0.08 mg / ml).

Claims (1)

A synthetic medium for dilution and cryopreservation of ram sperm, including lactose, dextrin, xylitol, tris- (hydroxymethyl) aminomethane, disodium ethylene diamine tetraacetic acid disodium salt, glycerin, chicken egg yolk and distilled water, which additionally contains succinic acid 6 water in the following optimal ratio of ingredients, wt.%: Lactose 6.00 Dextrin 3.60 Xylitol 0.18 Tris- (hydroxymethyl) -aminomethane 0.08 Disodium EDTA Salt 0.10 Glycerol 4.3 Egg yolk 14.3 Sodium succinic 6-aqueous 0,03 Distilled water rest.