RU2370033C1 - Synthetic medium for boar sperm storage in cooled state - Google Patents

Abstract

FIELD: veterinary.

SUBSTANCE: invention concerns veterinary and can be applied in artificial insemination of swine. Synthetic medium for boar sperm storage in cooled state includes glucose, disodium dehydrate salt of ethylenediamine-NNNN-tetraacetic acid, tri-substituted sodium citrate pentahydrate, ammonium sulphate, sodium carbonate and distilled water and additionally features ethyl alcohol as antioxidiser at the following component ratio (wt %): glucose 3.80-3.90; disodium dehydrate salt of ethylenediamine-NNNN-tetraacetic acid 0.26-0.28; tri-substituted sodium citrate pentahydrate 0.36-0.38; ammonium sulphate 0.18-0.20; sodium carbonate 0.05-0.06; ethyl alcohol 0.10-0.20; the rest is distilled water.

EFFECT: enhanced survivability and conceiving capacity of boar spermatozoids stored cooled to 16°C.

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Description

The invention relates to artificial insemination of farm animals, in particular to synthetic media for dilution and storage of sperm of boars in a refrigerated state.

It is known that during storage of diluted sperm of boars in a state cooled to 16-18 ° C, the processes of lipid peroxidation are activated, which negatively affects the motility and fertilizing ability of sperm stored at this temperature.

In this regard, it is necessary to develop more advanced synthetic media for boar semen, allowing to increase the shelf life of diluted boar semen in a chilled state to 16-18 ° C and increase their fertilizing ability.

A known medium for storing boar semen containing tetra-substituted EDTA, medical anhydrous glucose, trisubstituted sodium citrate, ammonium sulfate and distilled water [1].

Also known is a synthetic medium for diluting boar semen containing glucose, chelaton-3, trisodium citrate pentahydrate, ammonium sulfate, sodium bicarbonate, spermosan-3, lithium lactic acid and bidistilled water [2].

The disadvantage of these media is that they do not support the high mobility of sperm stored in a refrigerated state during storage for more than 12-24 hours at 16 ° C, and also do not impede the development of lipid peroxidation, which reduces the fertilizing ability of spermatozoa.

Known synthetic medium for dilution and freezing of boar semen, containing glucose, 5.5-aqueous sodium citrate, ethylenediaminetetraacetate disodium two-water, tris (oxymethyl) aminomethane, ammonium sulfate, sorbitol, glycerin and antioxidant - an aqueous extract of sunflower oil [3].

The disadvantage of this environment is that it is designed to dilute and freeze the sperm of boars and cannot be used to store sperm of boars in a refrigerated state. Since in the process of sperm freezing, other physicochemical effects on the cell occur than when it is stored in a refrigerated state, therefore, the medium components are selected accordingly to prevent cryo-damage of the cell during deep freezing (-196 ° С). So, for example, the protective effect of glycerin is important in deep freezing of semen of farm animals. The mechanism of the protective action of glycerin is mainly as follows: it penetrates into the cell and plays the role of an anti-salt buffer; allows you to cause overcooling of the system by binding part of the free water, which contributes to the course of vitrification of ionic processes; strengthens cell membranes, protecting them from the damaging effects of water when redistributing it on both sides of the membrane. In addition, glycerin, penetrating into the cell, replaces part of the free water and displaces the electrolytes, reducing their intracellular concentration to a degree that is not harmful during freezing. When glycerol is added to the diluent, it lowers the freezing point of the solution.

Along with a positive effect, glycerin can also have a negative effect on the biological usefulness of sperm. There is evidence that glycerin contributes to the destruction of acrosome in sperm. When using glycerin, due to its high dissolving ability, cytoplasmic membranes are damaged due to the dissolution of their constituent components, in particular lipids, which leads to the loss of many vital cell compounds. Glycerin suppresses the respiratory activity of sperm. Therefore, despite the positive role of glycerol and other components necessary for freezing boar sperm, their presence in the environment for storing boar sperm in a state cooled to 16 ° C is undesirable.

The closest analogue (prototype) of our invention is (HCC) a medium for dilution of sperm of boars [4], which contains the following components: medical anhydrous glucose - 40 g; ethylene diamine-NNNN-tetraacetic acid disodium salt (Trilon-B) - 2.6 g; sodium citric acid trisubstituted pentahydrate (sodium citrate) - 3.8 g; ammonium sulfate - 1.8 g; sodium bicarbonate - 0.5 g; distilled water - 1000 ml; spermosan-3 - 250 thousand units The ratio of ingredients (wt.%):

Medical anhydrous glucose 3.80 Disodium Salt ethylenediamine-NNNN-tetraacetic acid 0.26 Sodium citric acid trisubstituted quaternary 0.36 Ammonium sulfate 0.18 Sodium bicarbonate 0.05 Distilled water rest Spermosan-3 250 thousand units

The disadvantage of this medium is that it does not contain antioxidants and does not allow to maintain the high fertilizing ability of the sperm of boars, maintained at 16-18 ° C.

It is known that during storage of spermatozoa in a cooled state, superoxide radicals can accumulate in them, which are the precursors of all reactive oxygen species in vivo. Their formation leads to damage to DNA, proteins, lipids, etc. Stationarity of lipid oxidation processes is an important condition for the stability and functioning of cell membranes, since it has been established that with the development of lipid peroxidation, both the fatty acid composition of the membranes and their physicochemical properties (microviscosity, fluidity, membrane potential, polarity of the inner regions of the membrane, etc.) change. ) The consequence of this is a change in the transport properties of the membrane and the activity of membrane-bound enzymes.

In order to prevent lipid peroxidation and the accumulation of toxic lipid peroxidation products, some researchers suggest using various antioxidants in the composition of synthetic media to dilute animal sperm.

The aim of the invention is to increase the survivability and fertilizing ability of boar sperm when diluted and stored in a refrigerated state to 16 ° C.

This goal is achieved by the fact that the medium, including: glucose; ethylene diamine-NNNN-tetraacetic acid disodium salt (trilon-B); sodium citric acid trisubstituted pentahydrate (sodium citrate); ammonium sulfate; sodium bicarbonate and distilled water, additionally contains ethyl alcohol as an antioxidant in the following ratio of ingredients (wt.%):

Glucose 3.80-3.90 Disodium Salt ethylenediamine-NNNN-tetraacetic acid 0.26-0.28 Sodium citric acid trisubstituted quaternary 0.36-0.38 Ammonium sulfate 0.18-0.20 Sodium bicarbonate 0.05-0.06 Ethanol 0.10-0.20 Distilled water rest

Ethyl alcohol is a colorless liquid, in all ratios mixes well with water, the chemical formula is C ₂ H ₅ OH, and the molecular weight is 46.069. When included in the composition of the medium in non-toxic concentrations does not significantly change the osmotic pressure and pH of the medium.

Ethyl alcohol has antioxidant properties [5]. The introduction of ethanol in the optimal amount into the composition of the medium for storing boar semen in a cooled state reduces the likelihood of activation of the lipid peroxidation process in boar semen stored. Ethyl alcohol has not been used in animal sperm media before.

Permissible boundary values of the concentrations of the components in the claimed medium are shown in table 1.

Table 1 Components of the environment and quality indicators of preserved semen of boars The composition of the media (wt.%) 1st 2nd 3rd Glucose 3.80 3.85 3.90 Ethylene diamine-NNNN-tetraacetic acid disodium salt 0.26 0.27 0.28 Sodium citric acid trisubstituted pentahydrate 0.36 0.37 0.38 Ammonium sulfate 0.18 0.19 0.20 Sodium bicarbonate 0,050 0,055 0,060 Ethanol 0.10 0.15 0.20 Distilled water up to 100 up to 100 up to 100 The absolute indicator of survivability of sperm at 16 ° C, conventional units 996 1025 985

If the content of the components goes beyond the limits of the extreme lower or upper values of the intended environment, this gives worse results and does not lead to the achievement of the goal (table 2).

table 2 Components of media and quality indicators of preserved sperm of boars Qualitative indicators of sperm with the following components Glucose 3.75 3.95 Ethylene diamine-NNNN-tetraacetic acid disodium salt 0.25 0.30 Sodium citric acid trisubstituted pentahydrate 0.35 0.39 Ammonium sulfate 0.17 0.21 Sodium bicarbonate 0,040 0,070 Ethanol 0.05 0.25 Distilled water up to 100 up to 100 The absolute indicator of survivability of sperm at 16 ° C, conventional units 860 840

An example of cooking medium.

The optimal (second) composition of the proposed medium in wt.% Is added to a sterile chemical flask with a prescription: medical anhydrous glucose - 3.85; sodium citric acid trisubstituted pentahydrate - 0.37; ethylene diamine-NNNN-tetraacetic acid disodium salt - 0.27; ammonium sulfate - 0.19; sodium bicarbonate - 0,055; ethyl alcohol - 0.15; distilled water - the rest and sanitizing preparation (according to the instructions for its use).

Freshly prepared synthetic media before diluting semen should be examined for their appearance, color, and periodically for pH and osmotic pressure.

The proposed synthetic medium according to physico-chemical properties must meet the following requirements: appearance - a transparent liquid without sediment and mechanical impurities; without smell; pH - at a temperature of 20 ° C - 6.7 ± 0.10; osmotic pressure - 7.0-8.5 atm.

The prepared medium is used to dilute sperm within 3-4 ^x hours from the date of preparation. When using the medium, 1-6 volumes of the medium are added to 1 volume of sperm depending on the concentration of sperm. The diluted boar semen is stored in a thermostat at 16 ° C.

The advantages of the claimed medium is an increase in the survivability of sperm and their fertilizing ability when storing sperm in a state cooled to 16 ° C.

The invention is illustrated by the following examples.

Example 1. The study of the effects of ethyl alcohol in the composition of the medium on the biological usefulness of boar sperm.

Sperm for the study was obtained from boars of large white breed in a manual way. After taking each ejaculate, its volume and a number of qualitative indicators were determined (sperm activity as a percentage, concentration in billions / ml, total number of sperm in the ejaculate in billions).

To study the effect of ethyl alcohol in the medium on the preservation of the biological usefulness of boar sperm, outside the body during dilution of sperm, the following concentrations were used: 0.025; 0.05; 0.075; 0.10; 0.15; 0.20 and 0.25 wt.%. In the control group, native boar semen was diluted with HCC medium.

Diluted semen was packaged in numbered vials and stored together with a control sample at 16 ° C, periodically determining sperm motility every 24 hours until they died. Then, the absolute sperm survival rate (Sa) was calculated.

The studies showed that when using ethanol in the experiment in the composition of the proposed medium at a concentration of 0.075-0.20 wt.%, An increase in the absolute sperm survivability index from 1022 to 1079 conventional units was observed. while in the control, where HCC medium (prototype) was used, the absolute sperm survivability index was equal to 762 conventional units. As a result, the absolute survivability index in the proposed environment is 34-41% higher compared to the control (prototype).

The results are presented in table 3.

Table 3 The concentration of ethyl alcohol, wt.% Number of experiments The mobility of freshly obtained sperm,% The absolute indicator of survivability of sperm at 16 ° C, conventional units 0,025 9 85 847 ± 38.9 0,050 9 85 894 ± 42.3 0,075 9 85 1022 ± 36.5 ^x 0.10 9 85 1034 ± 42.6 ^x 0.15 9 85 1068 ± 43.8 ^x 0.20 9 85 1079 ± 39.3 ^x 0.25 9 85 852 ± 43.6 Control (prototype) 9 85 762 ± 33.6 ^x - P <0.01

Example No. 2.

Testing the proposed environment in a production environment.

The proposed environment for dilution and storage of boar semen in a state cooled to 16 ° C in the following ratio of components (in wt.%): Glucose - 3.85; sodium citric acid trisubstituted pentahydrate - 0.37; ethylene diamine-NNNN-tetraacetic acid disodium salt - 0.27; ammonium sulfate - 0.19; sodium bicarbonate - 0,055; ethyl alcohol - 0.15; distilled water - the rest and sanifying preparation (according to the instructions for its use), was tested under production conditions on the fertilizing ability of sperm, stored for 24 ^hours. Insemination of sows was carried out on a commission basis at the pig complex of CJSC "Mordovian Bacon" in the Republic of Mordovia. The proposed medium inseminated 146 heads of sows, medium HCC (prototype) 128 heads, 124 and 98 sows, respectively, were farmed. The results are presented in table 4.

Table 4 Synthetic environment Inseminated pigs, goal. Farmed Average multiple pregnancy, goal. Goal. % Offered (best option) 146 124 85,4 10.9 Control of HCC medium (prototype) 128 98 79.6 10.6

Tests have shown that the proposed medium for diluting and storing boar semen in a chilled state up to 16 ° C increased the fertility of sows by 5.8% and their multiple fertility by 0.3 pigs compared to the control (prototype).

Thus, the introduction of ethyl alcohol in the composition of the proposed medium for diluting and storing boar semen in a chilled state up to 16 ° С allows to significantly improve the biological usefulness of spermatozoa (absolute survivability by 34-41%), as well as increase sow fertility by 5.8% and their multiple pregnancy by 0.3 pigs compared with the control (prototype) of the GCC medium.

Information sources

1. A.S. USSR No. 869768. Bull. No. 37, 1981.

2. A.S. USSR No. 1708327 A1. Bull. No. 4, 1992.

3. Instructions on the organization and technology of work of stations and enterprises for artificial insemination of farm animals. M .: Kolos, 1981, p. 69.

Claims (1)

A synthetic environment for storing boar sperm in a cooled state, including glucose, disodium ethylene diamine-NNNN-tetraacetic acid disodium salt, trisodium citrate pentahydrate (sodium citrate), ammonium sulfate, sodium bicarbonate and distilled water, which additionally contains distilled water ethyl alcohol in the following ratio of ingredients, wt.%:
Glucose 3.80 - 3.90 Disodium Salt ethylenediamine-NNNN- tetraacetic acid 0.26-0.28 Sodium Citrate trisubstituted quaternary 0.36-0.38 Ammonium sulfate 0.18-0.20 Sodium bicarbonate 0.05-0.06 Ethanol 0.10-0.20 Distilled water Rest