RU2317703C1 - Method for cryogenic preservation of sturgeon fish semen - Google Patents

Abstract

FIELD: fish industry, in particular, artificial rearing of sturgeon fish.

SUBSTANCE: method involves diluting semen with freezing protective solution before freezing procedure, said solution containing saccharose, potassium chloride, and methanol; freezing in liquid nitrogen vapor; introducing into protective solution glycerin as additional freezing protective agent; freezing diluted semen in three stages; using protective solution components in the following ratio, wt%: saccharose 0.05-0.5; potassium chloride 0.01-0.15; methanol 6-12; glycerin 0.05-5; distilled water the balance.

EFFECT: retention of semen impregnation capacity after cryogenic preservation process owing to utilization of more effective composition of freezing protective solution and moderate freezing mode.

2 cl, 3 tbl, 3 ex

Description

The invention relates to the fishing industry and can find application in the artificial breeding of fish, mainly sturgeon, and in the creation of low-temperature genetic banks in order to preserve the biodiversity of fish.

Known methods for cryopreservation of sperm of Siberian sturgeon and sterlet (Tsvetkova LI, Cosson J., Linhart O., Billard R. Motility and fertilizing capacity of fresh and frozen-thawed spermatozoa in sturgeons Acipenser Baeri and A. ruthenus // J. Appl. Ichthyol. - 1996. - V.12. - P.107-112), as well as Siberian sturgeon, Sakhalin sturgeon and stellate sturgeon (Drokin SI, Kopeika EF Cryopreservation and phospholipids content of spermatozoa of some sturgeon species // 3 Int. Symp. Sturgeon, Piacenza, Italy, July 8 - 11, 1997. - Book. Abstr., Piacenza, 1997. - P.143-144), in which protective solutions based on Tris-HCl buffer (pH 8.0) with by adding sucrose, chicken yolk and dimethyl sulfoxide as cryop rotors. However, the cryoprotectants used dimethyl sulfoxide and yolk are not effective enough: semen cryopreserved by these methods fertilized only 20-60% of sturgeon caviar.

The closest method of the same purpose to the claimed invention is a method for cryopreservation of sperm of Siberian, Russian and Atlantic sturgeons, which involves diluting sperm with a protective solution prepared in Tris-HCl buffer (pH 8.0) containing 23.4 mm sucrose, 0.25 mm potassium chloride and 10% methanol, and freezing diluted semen in plastic straws in pairs of liquid nitrogen (3-4 cm above its surface) for 3 minutes, followed by immersion in liquid nitrogen (Urbanyi B., Horvath A., Kovacs B . Successful hybridization of Acipenser specie s using cryopreserved sperm // Aquaculture International. - 2004. - V.12. - P.47-56). The application of this method made it possible to obtain from fertilizing eggs with canned sperm from 17.4 to 50.0% hatching of larvae.

This method does not allow to maintain the high fertilizing ability of cryopreserved sperm, which is due to both the composition of the protective solution and the method of freezing. The content of sucrose and potassium chloride in the used protective solution in the indicated concentrations is not effective enough. Excess sucrose content reduces its protective effect, and a lack of potassium chloride does not provide the necessary reduction in motor activity of cells before freezing. The preparation of a protective solution in Tris-HCl buffer does not have a positive effect on the safety and fertilizing ability of thawed semen. In this case, the use of a buffer system is not necessary, since the addition of the remaining components at the indicated concentrations does not change the pH of the protective solution, and Tris (oxymethyl) aminomethane (Tris) itself does not have cryoprotective properties. Rapid freezing of sperm at a constant temperature is not advisable, since the rapid crystallization of intracellular fluid and the formation of large ice crystals with a sharp decrease in temperature, causing damage to cell structures, leads either to loss of fertilizing ability, or to the death of a significant amount of sperm.

The present invention is aimed at increasing the safety of the fertilizing ability of sturgeon semen during cryopreservation due to a more effective composition of the protective solution and a more gentle freezing regimen.

The specified technical result is achieved by the fact that in the known method of cryopreservation of sturgeon semen, which involves diluting the semen before freezing with a protective solution containing sucrose, potassium chloride and methanol, and subsequent freezing in liquid nitrogen vapors, the peculiarity is that they are introduced into the protective solution into as an additional cryoprotectant glycerin, the dilution of sperm is frozen in three stages, while the components of the protective solution are taken in the following ratio, wt.%: sucrose 0.05-0.5; potassium chloride 0.01-0.15; methanol 6-12; glycerin 0.05-5; distilled water - the rest.

It is advisable to freeze from 5 to minus 15 ° C at a speed of 2-5 ° C / min, from minus 15 to minus 70 ° C at a speed of 10-20 ° C / min, and then gently immerse in liquid nitrogen.

The introduction of an additional cryoprotectant glycerol into the protective solution, which has pronounced antifreeze properties and is able to bind intracellular water, reduces the likelihood of cell damage due to the formation of large ice crystals, thereby increasing the protective effect of the solution. In addition, glycerol stabilization of proteins and enzymes prevents their cold denaturation.

The use of two cryoprotectants penetrating into the cell through the membrane in a protective solution can reduce the risk of temperature shock for spermatozoa and their damage due to the synergistic effect of lowering the crystallization temperature of intracellular fluid. The combined effect of the used cryoprotectants, due to the different sizes and structure of the molecules, also contributes to the prolongation of the protective action of the solution due to their different diffusion rates through the membrane and interaction with different cell structures.

The use of sucrose in a protective solution in lower concentrations in comparison with the known method reduces the osmotic stress on the membranes, prevents osmotic shock and dehydration of cells due to exosmosis. The use of potassium chloride in significantly higher concentrations can reduce the motor activity of cells before freezing, which reduces the likelihood of damage.

The proposed composition of the protective solution prevents damage to cell structures due to premature intracellular crystallization, which, combined with the proposed freezing mode, ensures the preservation of the fertilizing ability of 60-80% of the cells (depending on the quality of native sperm).

Freezing sperm in three stages with a slow decrease in temperature avoids damage to cell structures and thereby contributes to the preservation of sperm and their fertilizing ability.

The recommended limits for the content of the components of the protective solution and the freezing modes were established during numerous experiments.

Thus, the set of distinctive features of the described method ensures the achievement of the specified technical result.

The analysis of the prior art made it possible to establish that the source is not detected, characterized by signs identical to all the essential features of the claimed invention, therefore, the present invention meets the condition of "novelty."

An additional search for known solutions showed that the claimed invention does not follow explicitly from the prior art for a specialist, since the combined action of a protective solution of the specified composition and the proposed sperm freezing mode exhibits new properties from the point of view of exposure to biological objects. Therefore, the claimed invention meets the condition of "inventive step".

Information confirming the possibility of carrying out the invention with obtaining the above technical result.

The method is as follows.

A protective solution for cryopreservation of sperm of sturgeon fish is prepared immediately before use, dissolving in distilled water in appropriate concentrations, wt.%: Sucrose 0.05-0.5; potassium chloride 0.01-0.15; methanol 6-12; glycerin 0.05-5; distilled water - the rest.

Sperm is cooled to 10-15 ° C in the refrigerator for 30 minutes. To the cooled sperm with continuous stirring, a protective solution cooled to the same temperature is added in a volume ratio of 1: 1. A suspension of sperm in a protective solution is left for equilibration for 1 hour at a temperature of 0-5 ° C. The diluted semen is poured into 1.5 ml vinyl chloride tubes, which are sealed and mounted on the freezer disk. Sperm freezing is carried out by slowly lowering sperm tubes to the surface of liquid nitrogen installed on a freezer disk equipped with a temperature sensor and a recorder that records sperm temperature in the tube.

Sturgeon semen is frozen according to the following program:

I stage: from +5 to minus 15 ° C at a speed of 2-5 ° C / min,

Stage II: from minus 15 to minus 70 ° C at a speed of 10-20 ° C / min,

Stage III: smooth immersion in liquid nitrogen.

Sperm thawing is carried out by removing the tubes from liquid nitrogen and placing them in a water bath or defrosting device МТА-70 with a temperature of 38-40 ° С, where they are shaken for 20-40 s until the liquid phase appears in them.

In thawed samples, the number of motile spermatozoa is determined. Immediately after this, thawed sperm of satisfactory quality is used for insemination of eggs. Technological water is used to activate thawed sperm. Inseminated eggs are de-gummed and incubated according to sturgeon reproduction technology.

The invention is illustrated by examples:

Example 1

At the Konakovsky plant for commercial sturgeon breeding and in the cryobiology laboratory of the All-Russian Research Institute of Freshwater Fisheries, cryopreservation of sperm of Siberian sturgeons (Lena population) of different quality was carried out using different compositions of protective solutions. Sperm samples cooled to 15 ° С were diluted in the ratio 1: 1 with protective solutions cooled to the same temperature. The resulting suspension was calibrated for 1 hour at 0 ° C, then poured into 1.5 ml vinyl chloride tubes and frozen according to a three-stage program: from 5 to -15 ° C at a rate of 2 ° C / min, from -15 to -70 ° C at a rate of 20 ° C / min, then gently immersed in liquid nitrogen. The tubes were thawed in an MTA-70 device at a temperature of 38 ° C. The effectiveness of cryopreservation was evaluated by fertilizing eggs with thawed sperm. The experiments were carried out in laboratory conditions on Petri dishes. The dependence of the results of the fertilization of Siberian sturgeon eggs with thawed sperm on the composition of the protective solution is presented in table. one.

In all experiments where the qualitative and quantitative composition of the protective solutions corresponded to the component content limits recommended in the invention, the level of fertilizing eggs with cryopreserved sperm was high, and in some cases even exceeded the level of fertilization with fresh sperm in control, which indicates the high efficiency of the applied cryopreservation method, which allows maintain viability and high fertilizing ability of sperm.

Table 1. Dependence of the results of the fertilization of Siberian sturgeon eggs with thawed sperm on the composition of the protective solution Experience number The content of the components of the protective solution,% Fertilization of eggs with thawed sperm Sucrose Kcl Methanol Glycerol Tris-HCl pH8 Distilled water % fertilization % of control one 0.4 0.05 12 0.1 up to 100 - 11.1 12.9 0.4 0.05 12 0.1 - up to 100 62.1 72,4 0.5 0.1 12 0.1 - up to 100 61,4 71.6 0.6 0.1 12 0.1 - up to 100 50,5 58.7 2 0.05 0.05 12 0.1 - up to 100 62,4 93.1 3 0.1 0.02 8 0.2 - up to 100 64.7 73.5 0.1 0.04 8 0.2 - up to 100 70.5 80.1 0.1 0.15 8 0.5 - up to 100 74.7 84.9 0.1 0.20 8 0.5 - up to 100 49.8 56.7 0.1 0.01 6 0.1 - up to 100 63.5 72,2 0.1 0.01 8 - up to 100 52.1 59.3 one 0.1 10 5 - up to 100 64.9 73.8 one 0.1 10 6 - up to 100 51,2 58.2 four 0.1 0.06 8 0.1 - up to 100 81.0 96.4 5 0.2 0.06 8 0.1 - up to 100 73.3 102,4 0.2 0.05 7 0.1 - up to 100 66.7 93.2 0.2 0.04 7 0.1 - up to 100 72.0 100.6 0.2 0.1 7 0.1 - up to 100 72,2 100.8 6 0.1 0.06 8 0.5 - up to 100 65.8 91.0 7 0.1 0.08 8 0.05 - up to 100 81.4 102.6 0.1 0.08 8 0.1 - up to 100 82.8 104.4 0.1 0.08 8 0.5 - up to 100 85.6 107.9 0.1 0.08 8 one - up to 100 79.2 99.9 0.1 0.08 8 2 - up to 100 73.1 92.2

Example 2

At the Konakovsky commodity sturgeon breeding plant, in a cage warm-water economy at the Krasnodar TPP, at the Adygea sturgeon factory, at the live-fishing base of the Kras-NIIRKh Chituk, as well as in the cryobank VNIIPRH, sperm cryopreservation was carried out with the aim of creating a collection of cryobanks of different types of sturgeon. Sperm samples cooled to 10 ° C were diluted in a 1: 1 ratio with a protective solution cooled to the same temperature, the content of the components of which corresponded to the limits recommended in the claimed invention. The resulting suspensions were equilibrated for 1 hour at 0 ° C, then poured into 1.5 ml vinyl chloride tubes and frozen according to the three-stage program described in Example 1, except that cooling from 5 to minus 15 ° C was carried out at a rate of 5 ° C / min, from minus 15 to minus 70 ° C at a speed of 15 ° C / min. The tubes were thawed in an MTA-70 device at a temperature of 40 ° C. The fertilization ability of the sperm samples was checked under laboratory conditions on Petri dishes. The verification results, depending on the compositions of the used protective solutions, are presented in table. 2.

Table 2. The quality of samples of cryopreserved sturgeon semen stored in a cryobank Kind of fish The content of the components of the protective solution,% Fertilization of eggs with thawed sperm Saha rose Kcl Methanol Glycerol Distilled water % fertilization % of control Russian sturgeon 0.5 0.05 10 0.05 up to 100 62.7 - 78.0 91.8 Siberian sturgeon (Lena population) 81.0 96.4 Sterlet 82.9 - Beluga 60.0 - Siberian sturgeon (Baikal population) 0.2 0.1 8 0.1 up to 100 64,2 - 60.1 - Siberian sturgeon (Lena population) 63.7 88.1 69.9 106.0 64.0 92.3 Sterlet 64.6 - Beluga 60.8 130.0 Stellate sturgeon 75.0 - Siberian sturgeon (Lena population) 0.1 0.08 8 0.5 up to 100 68.7 179.4 92.3 121.1 77.9 95.6 63.5 87.1 66,4 77.8 72.3 97.4 73.3 98.9 Siberian sturgeon (Ob population) 72.6 89.0 Russian sturgeon 62.3 120.5

The data indicate that the fertilizing ability of the cryopreserved by the claimed method of sperm of different types of sturgeon fish remains at the level of native sperm in the control, and sometimes exceeds it.

Example 3

Under the production conditions of the Konakovsky Commodity Sturgeon Plant, insemination of industrial lots of Siberian sturgeon caviar was inseminated with a semen mixture of several males, cryopreserved by the method described in example 1, except that the protective solution contained, wt.%: Sucrose - 0.1, KCl - 0 , 08, methanol - 8, glycerin - 0.7, distilled water - the rest. Insemination and incubation of fertilized eggs was carried out in accordance with the sturgeon reproduction technology adopted in fish farming practice. The results of the fertilization of batches of caviar of 100 and 300 g of different numbers of thawed sperm of Siberian sturgeons are presented in table. 3.

Table 3. The results of the fertilization of Siberian sturgeon caviar with cryopreserved sperm under industrial conditions Experience number Caviar volume, ml Semen volume, ml Activator volume, ml Activator sperm dilution Fertilization of eggs with thawed sperm % fertilization % of control one one hundred 3 300 1: 100 50,0 73,4 2 6 300 1:50 69.1 101,4 3 300 9 900 1: 100 58.0 76.7 four 13.5 1350 58.7 77.6 5 eighteen 1800 75,2 99,4

The results indicate the possibility of the successful use of cryopreserved by the claimed method of sperm for insemination of eggs on an industrial scale with different ratios of volumes of sperm, caviar and activator.

The above examples illustrate the high efficiency of cryopreservation of semen of sturgeon fish of the claimed method, ensuring the survival and high fertilizing ability of sperm.

Thus, the above information indicates that when using the claimed invention the following combination of conditions:

- the method of cryopreservation of sperm of fish according to the claimed invention is intended for use in industry, in particular for cryopreservation of sperm of sturgeon fish using a protective solution, the components of which at the indicated concentrations in conjunction with the proposed freezing regimen ensure the survival and high fertilizing ability of thawed spermatozoa;

- for the claimed method in the form described in the independent clause of the claims, the possibility of its implementation using the means and methods described in the application is confirmed.

Therefore, the claimed invention meets the condition of "industrial applicability".

Prototype

Urbanyi B., Horvath A., Kovacs B. Successful hybridization of Acipenser species using cryopreserved sperm // Aquaculture International. - 2004. - V.12. - P. 47-56.

The successful hybridization of sterlet (A. ruthenus) × Siberian sturgeon (A. baeri), sterlet × Russian sturgeon (A. gueldenstaedti) and sterlet × Atlantic sturgeon (A. sturio) using cryopreserved sperm of male sturgeon and sterlet sterlet × sterlet was carried out for the first time. quality control. Sperm of all three species was diluted with a protective solution containing 23.4 mm sucrose, 0.25 mm KCl, 30 mm Tris (pH 8.0) and 10% methanol. Samples were frozen in plastic straws in pairs of liquid nitrogen at a height of 3 cm above the level of liquid nitrogen for 3 minutes. After thawing, approximately 3,000 sterlet eggs were fertilized with thawed sperm from six straws. The hatching of larvae after fertilization with sterlet sperm was 30.6%, while for hybrids A. ruthenus × A. baeri, A. ruthenus × A. gueldenstaedti and A. ruthenus × A. sturio, these indices were 50%, 17, 4% and 34%. To confirm interspecific hybridization, morphometric markers were used, as well as the evaluation of randomly amplified polymorphic DNA (RAPD).

Aquaculture International 12: 47-56, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Successful hybridization oi Acipenser species using cryopreserved sperm

BELA URBANYI ¹ *, AKOS HORVATH′d BALAZS KOVACS ² Department of Fish Culture, Faculty of Agriculture and Environmental Sciences, Stent 1st van University, PdterK. u. 1., GodMS, H-2103, Hungary, ^ -Agricultural Biotechnology Center, Szent-GySrsyi Albert ut 4, Gdaollo. H-2100, Hungary; * Author for correspondence (e-mail: urbanvib (a), nt.kts.sau.hu: phone: + 36-28-522-000 / 1657; fax: + 36-28-410-804).

Received 10 December 2002; accepted 20 June 2003

Abstract Successful hybridization of sterlet (Acipenser ruthenus) x Siberian sturgeon (Acipenser baeri), sterlet x Russian sturgeon (Acipenser ^ ueldenstaedti) and sterlet x European sturgeon (Acipenser sturid) was carried out for the first time using cryopreserved semen of sturgeon males x sterlet crosses as controls. Sperm of all three species was diluted wim a cryodituentent composed of 23.4 mM sucrose, 0.25 mM KCl, 30 mM Tris (pH 8.0) and 10% methanol. The samples were frozen in plastic straws in the vapor of liquid nitrogen at the height of 3 cm above the level of nitrogen for 3 min. Following thawing approximately 3000 sterlet eggs were fertilized with six straws of frozen-thawed sperm. The hatching rate with sterlet sperm was 30.6% while the hatching rate of A. ruthenus x A. baeri, A. ruthenus x A. suelden-staedti and A ruthenus, x A. sturio hybrids was 50, 17.4 and 34%, respectively. Morphometric markers as well as random amplified polymorphic DNA (RAPD) assay was used to verify interspecific hybridization.

Key words: Cryopreservarion, Hybrids, RAPD, Sperm, Sturgeon

Claims (2)

1. The method of cryopreservation of sturgeon sperm, including diluting the sperm before freezing with a protective solution containing sucrose, potassium chloride and methanol, and subsequent freezing in liquid nitrogen vapors, characterized in that glycerol is introduced into the protective solution as an additional cryoprotectant, the diluted semen is frozen in three stages, while the components of the protective solution are taken in the following ratio, wt.%: sucrose  0.05-0.5 potassium chloride  0.01-0.15 methanol  6-12 glycerol  0.05-5 distilled water  rest 2. The method according to claim 1, characterized in that the sperm are frozen from 5 to -15 ° C at a rate of 2-5 ° C / min, from -15 to -70 ° C at a rate of 10-20 ° C / min, and then gently immersed in liquid nitrogen.