Synthetic environment for dilution and freezing of semen of rams


(57) Abstract:

The invention relates to livestock and can be used in artificial insemination of sheep. The environment contains, wt%: lactose 6,0, bone glue 1,8, xylitol 0,18, Tris-(oxymethyl)-aminomethan 0,07, Trilon B 0,1, glycerin 4,3, yolk of chicken eggs 14,2, distilled water up to 100. The environment helps to enhance the biological integrity of spermatozoa after cryopreservation and thawing. table 2.

The invention relates to agriculture, in particular the artificial insemination of farm animals, such as sheep.

It is known that the stability of the sperm to freeze the primary role of the strength and character of the intermolecular bonds of proteins and lipids, hydrogen bonds membranes.

When freezing due to the phase transition of water and lipids is a violation of these relations, the oxidation of N-groups. This leads to destabilization of membrane proteins, loss and inactivation of enzymes, lipid peroxidation and metabolism disorders. This also contributes to pre-freeze the dilution of native sperm, leading to a significant decrease in the concentration of chemicals PL is Mr. gisagara (sucrose, lactose), polysaccharides (dextrin), the yolk of chicken eggs, Tris-(oxymethyl)-aminomethan, Trilon-B, gum Arabic, glycerol [1, 2, 3].

However, these environments do not have sufficient critisicim action due to the absence in their composition of specialized ingredients that can effectively structurewith water around the sperm. This leads to the formation of ice crystals that destroy the integrity of the membranes, to reduce the activity of sperm.

Closest to the technical nature of the proposed environment, GGUK-Tris buffer medium for dilution and freezing of semen of rams: distilled water - 100 cm3, lactose - 8,4 g, dextrin corn - 5 g, xylitol - 0.26 g, Tris-(oxymethyl)-aminomethan -0,105 g Trilon-B - is 0.135 g, glycerol - 6.0 cm3the yolk of chicken eggs - 20 cm3[2].

However, this environment does not possess sufficient holodonositel action, because dextrin, which is a polysaccharide and core the extracellular cryoprotectant, is not effective structures of extracellular water and especially one that is part of the membranes of the sperm, weak impact in reducing the electrical point of crystallization of salts in solutions.

The aim of the invention awali more effective natural extracellular cryoprotector of animal origin, which is better dextrin (polysaccharide) will neutralize the harmful effects on sperm deep freeze.

The objective is achieved by increasing the duration of the biological usefulness of RAM spermatozoa after cryopreservation due to the structuring of water around the sperm, dehydration of cells, reducing electrical freezing point of water and crystallization of salts.

The invention consists in the use of bone glue (GOST 2067-80), collagen, protein complex formation, belonging to the group of scleroprotein.

For bone glue (collagen) is characterized by a high content of proteids: glycine (26%), Proline (14%), hydroxyproline (12%) and 2% polysaccharides [4].

Bone glue, having a high molecular weight of 300,000, when the medium composition does not significantly alter the osmotic pressure even when significant (20-25%) concentrations. In aqueous solutions of bone glue absorbs water, it forms several types of gels [4].

These properties are well explain his role in the regulation of water metabolism of various compounds.

Example cooking environment.

In pure chemical flask contribute prescription Lavandou water (+80)-(+90oC) and completely dissolve all the components. Then the solution is cooled to 40oTo bring back the yolk of chicken eggs, stir and bring antibiotics.

Freshly prepared environment before dilution of the semen should be evaluated for appearance, color, periodically the pH and osmotic pressure.

The proposed synthetic environment physico-chemical properties must meet the following requirements: appearance - homogeneous emulsion, no sediment and solids; color yellow-grey, odourless; pH at a temperature of 20oWith - 6,80,15; the osmotic pressure of the medium without glycerol at a temperature of 0oWith - 10,00,5 ATM.

The prepared environment is used for diluting the sperm within 4 hours after preparation. Stored at a temperature of 0 -(+ 4)oWith, before use, is heated to 28-30oC.

When using the environment to one volume of sperm add 2-3 medium volume, depending on the concentration of sperm.

The proposed environment has a higher protective properties in comparison with the prototype and increases the biological usefulness of semen during cryopreservation and thawing. Sperm motility, Piri 0oC is 374+12,3 conventional units, against respectively 4.2 and 315 conventional units in the control (prototype).

Protective effect of bone glue in the composition of the designed synthetic environment is manifested in the fact that, being a natural surface-active substance, it is adsorbed on the surface of sperm and as a biological polymer acts as a cryoprotectant - promotes dehydration of sperm, acquisitions and vitrification of water directly around them, protects sperm from contact with ice crystals, significantly reduces the eutectic point of the salts.

Thus, its protective effect is more directional than other cryoprotectants (gum Arabic, dextrin and other). The proposed environment will improve biological integrity of spermatozoa after cryopreservation and thawing (activity - by 6.8%, the absolute survival - 15.4%), and increase the fertility of ewes by 10.5% compared with control (medium with dextrin).

The optimal concentration of bone glue as part of GGUK-Tris buffer medium was installed in the laboratory experiments conducted in VNIILM, scientific-production experiments it Belgorod region.

Example 1. Sperm for research were obtained using an artificial vagina from adult sheep. Each ejaculate was determined by its volume, motility and concentration of spermatozoa in accordance with the methodological recommendations (1).

To study closewith properties of bone glue as part of GGUK-Tris buffer medium without dextrin diluted semen sheep were Packed up in numbered tubes and together with a control sample subjected equilibrate (cooling) for 2 h at a temperature of(+2)-(+4)oC. Next, the sperm was frozen on the Teflon plate in the vapor of liquid nitrogen, in the holes of 0.2 ml.

For biological control laboratory samples of frozen semen was thawed in a 3% solution of sodium citrate at 42oWith, then we determine the motility of the sperm through every hour to complete their destruction. The absolute survival of spermatozoa was determined during incubation temperature of +38oC. research closewith properties of bone glue (collagen) in the composition of the medium on the spermatozoa of the RAM shown in the table. 1.

Studies have shown (PL. 1) that the best results after thawing createservicehost - 4.54 points, the absolute measure of survivability 354. that is, This is higher than was the control group, respectively by 8% and 26%.

Received in favor of the experience, the difference is statistically highly significant with values of td1= 8,l and td2=3,9. Thus, studies have shown that createsite properties offer protector - bone glue is higher than that of dextrin (control), mobility 8%, and in absolute survival by 26%.

Example 2. Synthetic GGUK-Tris buffer medium with the proposed cryoprotectant - bone glue was tested in the research and production experience to study the fertilizing ability of cryopreserved semen of rams.

The results of these studies are presented in table 2.

Conducted research and production experiments on artificial insemination of sheep (PL. 2) indicate a higher fertilizing capacity of sperm, frozen in GGUK-Tris-buffered medium with bone glue, where the sperm motility was equal to 4.5 points, survival - 374. unit, and fertilizing ability is 55.3%. This is higher than in the control, respectively, by 7.0%, 18.7% and 10,5%.

Thus, the application with the AI semen of sheep increased biological value of sperm after thawing (mobility 7%; absolute survival - +18,7%), and improving the fertilizing ability of sperm in artificial insemination of sheep - 10.5%.

Sources taken into account

1. Methodical recommendations on technology taking a deep freeze and use of semen of sheep producers in the artificial insemination of sheep. M., 1986.

2. Guidance on the use of GGUK-Tris buffer medium for dilution, freezing, storage and use of semen of sheep producers. VNIILM, Forest glades, 1992.

3. Patent 2028796, Bulletin 33, 1995. Synthetic environment for freezing semen of rams.

4. Great medical encyclopedia. 1985.

Synthetic environment for dilution and cryopreservation of semen of rams, including lactose, xylitol, Tris-(oxymethyl)-aminomethan, Trilon-B, glycerin, yolk of chicken eggs, distilled water, wherein the medium further comprises bone glue in the following ratio of ingredients, wt. %: lactose 6,0, bone glue 1,8, xylitol 0,18, Tris-(oxymethyl)-aminomethan 0,07, Trilon-B 0,1, glycerin 4,3, yolk of chicken eggs 14,2, distilled water to 100.


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