JP2021066719A - Lake sheep semen cryopreservation solution and use thereof - Google Patents

Abstract

To provide a lake sheep semen cryopreservation solution capable of effectively improving the survival rate, mobility and completeness in preservation, having excellent fertilization capability even after a preserved semen was melted, having a stable conception effect, and satisfying requirements of artificial fertilization to a lake sheep cryopreservation semen.SOLUTION: A lake sheep semen cryopreservation solution contains tris, glucose, trehalose, an egg yolk liquid, sodium citrate, glutathione, glycerol, a membraneous milkvetch extract, penicillin, and streptomycin as components.SELECTED DRAWING: None

Description

The present invention belongs to the technical field of lamb farming, and specifically relates to a lamb semen cryopreservation solution and its use.

Lambs are one of the important livestock in the Taihu Plain, and are the first-class protected local livestock and poultry species in China. The goat is medium-sized, has no horns on both males and females, has a long and narrow head, a high nasal bridge, most ears hang down, an elongated neck, a long and narrow body, and a lumbar region. The back is flat and straight, the abdomen is slightly sagging, the tail is flat and round, the tip of the tail is curved, and the limbs are relatively thin and long. White hair is covered throughout the body, the abdominal hair is coarse, thin and short, and the body is strong. Lamb is a rare white lamb skin variety that matures quickly, is in estrus all year round, can give birth twice a year, has a large number of babies per lactation, and lactates. High ability, fast growth and development rate, excellent slaughter after improvement, excellent properties such as high temperature resistance and humidity resistance, distributed in the Taihu region of China, and kept in a house all year round It is a breed of Chinese sheep for lamb skin that has been slaughtered and peeled off within 1 to 2 days after birth. Sheepskin of lamb has a beautiful pattern and is world famous.

Semen cryopreservation and artificial insemination techniques play a vital role in modern livestock production, and artificial insemination with frozen semen is not limited by time, region, breed or individual and significantly increases the number of male livestock. To reduce production costs, increase the utilization rate of good male livestock for seeding, and improve the economic benefits of aquaculture. The technology also has important implications for breed introduction, crossbreeding improvement, improvement of reproductive performance, disease control and protection of genetic diversity. Sheep sperm are weaker than chicken and cow sperm and are more likely to be damaged by freezing and thawing and to lose fertility. Traditional frozen sheep semen has low post-thaw motility and is rich in "aged" and dead sperm, has few effective sperm, low motility, low quality, and meets the requirements of artificial insemination. Hateful.

Chinese Patent Application Publication No. 105638643 Chinese Patent Application Publication No. 108094409

In view of the above, an object of the present invention is to be able to effectively improve the survival rate, motility and completeness in the preservation of goat semen in order to overcome the deficiencies of the above-mentioned prior art, and the preservation has been preserved. It is to provide a goat semen cryopreservation solution which has a good fertilizing ability even after the semen is thawed, has a stable conception effect, and meets the requirements for artificial insemination of the goat frozen semen.

The technical proposal adopted by the present invention in order to achieve the above object is as follows. The goat semen cryopreservation solution contains Tris, glucose, trehalose, egg yolk, sodium citrate, glutathione, glycerol, astragalus propininus extract, penicillin and streptomycin as components.

Astragalus propininus extract is an extract of the root of Astragalus propininsis, which is a leguminous plant, and contains components such as saponin, flavon, polysaccharides and amino acids, which enhances immunity, increases energy, suppresses fatigue, and prevents mutation. It also protects the liver and inhibits the action of osteoclasts. Kibana Ogi polysaccharide has the effect of lowering blood lipids, which are cholesterol and triglyceride, and increasing high-density lipoprotein. For example, heart and brain such as atherosclerosis, coronary artery disease, peripheral vascular disease and hyperlipidemia. Can prevent and treat vascular disease. Astragaloside has the effect of significantly reducing blood glucose, glycated hemoglobin, and urinary protein, and can reduce AGEs in the renal cortex and serum. Astragaloside has an antioxidant effect and is an aldose reductase. It has an inhibitory effect on mesangial cells, suppresses the proliferation of mesangial cells, and reduces renal hypertrophy. As a result of research by the inventor of the present application, a cryopreservation solution containing an astragalus propinin extract can effectively improve the cryopreservation effect of goat semen, and the concentration of the astragalus propinin extract in the cryopreservation solution is 0.1 g / L. In the case of, it was found that the effect of cryopreservation obtained was optimal.

Preferably, the cryopreservation solution of goat semen has a concentration of Tris 30 to 35 g / L, glucose 8 to 15 g / L, trehalose 30 to 50 g / L, egg yolk solution 15 to 20% (v / v), and sodium citrate. 15-20 g / L, glutathione 0.5-1 g / L, glycerol 5-8% (v / v), citrate extract 0.05-0.15 g / L, penicillin 0.8-1 g / L and streptomycin 0 .8 to 1 g / L is contained as an ingredient.

Preferably, the concentration of the Astragalus propininus extract in the lake sheep semen cryopreservation solution is 0.1 g / L.

Preferably, the yolk liquor preparation method comprises the step of repeatedly pipetting fresh sterile egg yolks and mixing them uniformly to obtain the yolk liquor.

In the present invention, Tris, glucose, trehalose, sodium citrate, glutathione and Kibana-ougi extract are weighed in a predetermined ratio, added to distilled water, thoroughly dissolved and uniformly mixed, and then a 0.2 um filter membrane. Then, add penicillin and streptomycin and mix uniformly to obtain solution A (1), and add egg yolk solution to solution A at a predetermined ratio and mix uniformly to obtain solution B. A method for preparing the goat semen cryopreservation solution, which comprises step (2) and step (3) in which glycerol is added to the solution B at a predetermined ratio and mixed uniformly to obtain the goat semen cryopreservation solution. Further provide.

The present invention further provides the use of the goat semen cryopreservation solution in the goat semen cryopreservation.

The present invention prepares fresh raw semen from goat, evaluates the motility of the semen, and if the motility is greater than 0.8, the step (1) is used for cryopreservation, and any one of claims 1 to 4. The goat semen cryopreservation solution described in the above is added to the semen prepared in step (1), mixed uniformly, then added to the cryopreservation tube, and the cryopreservation tube is left at 4 ° C. for 1 to 2 hours, and then liquid nitrogen. Further provided is a method for cryopreserving goat semen, which comprises a step (2) of leaving the product at a position 4 to 6 cm away from the liquid surface of the above, and finally putting it in liquid nitrogen for storage.

Preferably, the density of semen in the cryopreservation tube of step (2) is 6 × 10 ⁸ pieces / mL.

Compared with the prior art, the beneficial effects of the present invention are as follows. (1) The cryopreserved solution according to the present invention can effectively improve the viability, motility and completeness of the lamb semen in the preservation process, and the preserved semen has a good fertility even after thawing. It has a stable conception effect, can meet the requirements for artificial insemination of frozen lamb semen, and is expected to have future potential. When the liquid can effectively improve the cryopreservation effect of goat semen, and when the concentration of the Kibana-ougi extract in the cryopreservation liquid is 0.1 g / L, the obtained cryopreservation effect is optimal. I found it.

Hereinafter, the present invention will be described in more detail in order to facilitate understanding of the present invention. However, the invention may be implemented in a variety of other forms and is not limited to the examples described herein. Rather, the provision of these examples is to enable a more thorough and complete understanding of the disclosure of the present invention.

Example 1
In one example of the goat semen cryopreservation solution of the present invention, the goat semen cryopreservation solution has a concentration of Tris 30 g / L, glucose 10 g / L, trehalose 40 g / L, and egg yolk solution 16% (v / v). , Sodium citrate 18 g / L, glutathione 0.6 g / L, glycerol 5% (v / v), Kibana Ogi extract 0.1 g / L, penicillin 1 g / L and streptomycin 1 g / L as components.

In the method for preparing the goat semen cryopreservation solution, Tris, glucose, trehalose, sodium citrate, glutathione and Kibana-ougi extract are weighed in a predetermined ratio, added to distilled water, thoroughly dissolved and uniformly mixed. Then, filter with a 0.2 um filter membrane, then add penicillin and streptomycin and mix them uniformly to obtain solution A (1), and add egg yolk solution to solution A at a predetermined ratio to make it uniform. It includes a step (2) of mixing to obtain a solution B, and a step (3) of adding glycerol to the solution B at a predetermined ratio and mixing uniformly to obtain the goat semen cryopreservation solution.

Example 2
In one example of the goat semen cryopreservation solution of the present invention, the goat semen cryopreservation solution has a concentration of Tris 33 g / L, glucose 15 g / L, trehalose 30 g / L, and egg yolk solution 15% (v / v). , Sodium citrate 20 g / L, glutathione 1 g / L, glycerol 6% (v / v), Kibana Ogi extract 0.06 g / L, penicillin 1 g / L and streptomycin 1 g / L as components.

The method for preparing the goat semen cryopreservation solution was the same as in Example 1.

Example 3
In one example of the goat semen cryopreservation solution of the present invention, the goat semen cryopreservation solution has a concentration of Tris 35 g / L, glucose 12 g / L, trehalose 35 g / L, and egg yolk solution 20% (v / v). , Sodium citrate 16 g / L, glutathione 0.8 g / L, glycerol 8% (v / v), Kibana Ogi extract 0.13 g / L, penicillin 0.8 g / L and streptomycin 0.8 g / L as components. ..

The method for preparing the goat semen cryopreservation solution was the same as in Example 1.

Example 4
In one example of the goat semen cryopreservation solution of the present invention, the goat semen cryopreservation solution has a concentration of Tris 30 g / L, glucose 8 g / L, trehalose 50 g / L, and egg yolk solution 20% (v / v). , Sodium citrate 20 g / L, glutathione 0.5 g / L, glycerol 7% (v / v), Kibana Ogi extract 0.15 g / L, penicillin 0.8 g / L and streptomycin 0.8 g / L as components. ..

The method for preparing the goat semen cryopreservation solution was the same as in Example 1.

Example 5
In one example of the goat semen cryopreservation solution of the present invention, the goat semen cryopreservation solution has a concentration of Tris 35 g / L, glucose 10 g / L, trehalose 45 g / L, and egg yolk solution 18% (v / v). , Sodium citrate 20 g / L, glutathione 0.8 g / L, glycerol 5% (v / v), Kibana Ogi extract 0.05 g / L, penicillin 0.9 g / L and streptomycin 0.9 g / L as components. ..

The method for preparing the goat semen cryopreservation solution was the same as in Example 1.

Example 6
In one example of the goat semen cryopreservation solution of the present invention, the goat semen cryopreservation solution has a concentration of Tris 32 g / L, glucose 15 g / L, trehalose 40 g / L, and egg yolk solution 15% (v / v). , Sodium citrate 18 g / L, glutathione 0.6 g / L, glycerol 8% (v / v), Kibana Ogi extract 0.08 g / L, penicillin 1 g / L and streptomycin 1 g / L as components.

The method for preparing the goat semen cryopreservation solution was the same as in Example 1.

Example 7
Cryopreservation liquid semen adult Mizuumihitsuji sires sheep preservative effect false vaginal method was taken in, was measured movement rate of semen, satisfying the frozen, preservation method, the density of semen 6 × 10 ⁸ The lamb semen cryopreservation solution of Examples 1 to 6 was added to the collected semen and mixed uniformly, then added to the cryopreservation tube, and the cryopreservation tube was left at 4 ° C. for 2 hours. Next, it was left for 10 minutes at a place 4 to 6 cm away from the liquid surface of liquid nitrogen, and finally it was put into liquid nitrogen and stored.

Thawing and inspection Immediately after taking out the cryopreservation tube that had been cryopreserved from liquid nitrogen, it was placed in a water bath pot at 37 ° C. and shaken until the semen melted and became transparent, and the semen was taken out and the quality index was inspected.

上記結果からわかるように、本発明の実施例１〜６の上記凍結保存液は、湖羊精液に対して良好な保存効果を有し、融解後でも精子の運動率、運動性及び先体の完全率がいずれも高いレベルに維持され、冷凍した精子の品質を向上させた。 The results are shown in Table 1.
Table 1 Quality index inspection results

As can be seen from the above results, the above-mentioned cryopreservation solutions of Examples 1 to 6 of the present invention have a good preservation effect on goat semen, and sperm motility, motility and acrosome even after thawing. Both completeness rates were maintained at high levels, improving the quality of frozen sperm.

Example 8
The cryopreservation solution of the present invention contains an Astragalus propininus extract, which is an extract of dried roots of Astragalus propininum containing a plurality of components such as saponin, flavon, polysaccharides and amino acids, and is an extract of astragalus propininus. The cryopreservation effect can be effectively improved.

Experimental groups 1 and 2 were designed, except that experimental group 1 preserved goat semen with the cryopreservation solution of Example 1, and the cryopreservation solution used in experimental group 2 did not contain an extract of Kibana-ougi. The remaining components were the same as in Example 1.

False vagina method collected adult Mizuumihitsuji sires sheep semen, measure the motility of sperm, satisfying the frozen, preservation method, as the density of the semen is 6 × 10 ⁸ cells / mL , The lamb semen cryopreservation solution of Experimental Groups 1 and 2 was added to the collected semen, mixed uniformly, added to the cryopreservation tube, and the cryopreservation tube was left at 4 ° C. for 1.5 hours, and then liquid nitrogen. It was to leave it for 8 minutes at a place 4 to 6 cm away from the liquid surface of No. 1 and finally put it in liquid nitrogen for storage.

Thawing and inspection Immediately after taking out the cryopreservation tube that had been cryopreserved from liquid nitrogen, it was placed in a water bath pot at 37 ° C. and shaken until the semen melted and became transparent, and the semen was taken out and the quality index was inspected.

上記結果からわかるように、キバナオウギ抽出物を含む実験群１の前記凍結保存液の湖羊精液に対する保存効果は、キバナオウギ抽出物を含まない実験群２の前記凍結保存液の湖羊精液に対する保存効果よりも顕著に優れ、実験群１の前記凍結保存液で保存された湖羊精液は、融解後でも精子の運動率、運動性及び先体の完全率が実験群２より大幅に高く、キバナオウギ抽出物が凍結保存液の湖羊精液に対する凍結保存効果を効果的に向上できることを示した。 The results are shown in Table 2.
Table 2 Quality index inspection results

As can be seen from the above results, the preservation effect of the cryopreservation solution of the cryopreservation solution containing the Kibana-Ogi extract on the goat semen is the preservation effect of the cryopreservation solution of the experiment group 2 not containing the Kibana-Ogi extract on the goat semen. The goat semen stored in the cryopreservation solution of Experimental Group 1 was significantly superior to that of Experimental Group 2, and the sperm motility, motility, and predecessor completeness rate were significantly higher than those of Experimental Group 2 even after thawing. It was shown that the substance can effectively improve the cryopreservation effect of the cryopreservation solution on goat semen.

Example 9
The concentration of the Astragalus propininus extract in the cryopreservation solution of the present invention was 0.05 to 0.15 g / L, and in this example, the effect of the concentration of the Astragalus propininus extract on the preservation effect of the cryopreservation solution was studied.

偽膣法で大人の湖羊種雄羊精液を採取し、精液の運動率を測定し、条件を満たすと凍結保存し、保存方法は、精液の密度が６×１０⁸個／ｍＬとなるように、実験群３〜９の前記湖羊精液凍結保存液を採取した精液に加え、均一に混合した後、凍結保存管に加え、凍結保存管を４℃で１ｈ放置し、次に液体窒素の液面から４〜６ｃｍ離れる箇所に１０ｍｉｎ放置し、最後に液体窒素に投入して保存することであった。 Experimental groups 3 to 9 were designed, and the cryopreservation solution of each experimental group had the same remaining components except that the concentration of the Astragalus propininus extract was different, and specifically shown in Table 3.
Table 3 Design of concentration of Astragalus propininus extract in cryopreservation solution

False vagina method collected adult Mizuumihitsuji sires sheep semen, measure the motility of sperm, satisfying the frozen, preservation method, as the density of the semen is 6 × 10 ⁸ cells / mL , The semen semen cryopreservation solution of the experimental groups 3 to 9 was added to the collected semen, mixed uniformly, added to the cryopreservation tube, and the cryopreservation tube was left at 4 ° C. for 1 hour, and then a liquid nitrogen solution. It was left for 10 minutes at a place 4 to 6 cm away from the surface, and finally put into liquid nitrogen for storage.

Thawing and inspection Immediately after taking out the cryopreservation tube that had been cryopreserved from liquid nitrogen, it was placed in a water bath pot at 37 ° C. and shaken until the semen melted and became transparent, and the semen was taken out and the quality index was inspected.

上記結果からわかるように、前記凍結保存液におけるキバナオウギ抽出物の濃度が０．０５〜０．１５ｇ／Ｌ（実験群４〜８）である場合、凍結保存液の湖羊精液に対する凍結保存効果が好適であり、特にキバナオウギ抽出物の濃度が０．１ｇ／Ｌである場合、融解後の精液の品質指標が最適であり、キバナオウギ抽出物の濃度が０．０５ｇ／Ｌ（実験群３）より低く又は０．１５ｇ／Ｌ（実験群９）より高い場合、凍結保存液の保存効果に影響を与えて、保存品質が低下した。 The results are shown in Table 4.
Table 4 Quality index inspection results

As can be seen from the above results, when the concentration of the Kibana-ougi extract in the cryopreservation solution is 0.05 to 0.15 g / L (experimental groups 4 to 8), the cryopreservation effect of the cryopreservation solution on the goat semen is effective. It is suitable, especially when the concentration of Kibana-Ogi extract is 0.1 g / L, the quality index of the semen after thawing is optimal, and the concentration of Kibana-Ogi extract is lower than 0.05 g / L (Experimental Group 3). Or, when it was higher than 0.15 g / L (Experimental Group 9), the preservation effect of the cryopreservation solution was affected, and the preservation quality deteriorated.

Example 10
In this example, the fertilization effect of the goat semen stored in the cryopreservation solution of the present invention after thawing was studied by taking the goat semen stored in the cryopreservation solution of Example 1 as an example.

False vagina method collected adult Mizuumihitsuji sires sheep semen, measure the motility of sperm, satisfying the frozen, preservation method, as the density of the semen is 6 × 10 ⁸ cells / mL , The goat semen cryopreservation solution of Example 1 was added to the collected semen, mixed uniformly, then added to the cryopreservation tube, and the cryopreservation tube was left at 4 ° C. for 2 hours, and then from the liquid surface of liquid nitrogen. It was left for 10 minutes at a distance of 4 to 6 cm, and finally put into liquid nitrogen for storage.

Thawing Immediately after taking out the cryopreservation tube that had been cryopreserved from liquid nitrogen, it was placed in a water bath pot at 37 ° C. and shaken until the semen melted and became transparent, and the semen was taken out and artificial insemination was performed.

As a result of the examination, the thawed lambs semen reached 26 hours of fertility, and the conception rate of female lambs in estrus reached 85%.

It should be noted that the above examples are merely for explaining the technical proposal of the present invention, do not limit the scope of claims of the present invention, and have described the present invention in detail with reference to preferred examples. It can be understood that a person skilled in the art can make modifications or equivalent substitutions to the technical proposal of the present invention without departing from the spirit and scope of the technical proposal of the present invention.

Claims (8)

A goat semen cryopreservation solution, which is a goat semen cryopreservation solution containing Tris, glucose, trehalose, egg yolk solution, sodium citrate, glutathione, glycerol, Kibanaougi extract, penicillin and streptomycin as components. is there. Concentrations: Tris 30-35 g / L, glucose 8-15 g / L, trehalose 30-50 g / L, egg yolk solution 15-20% (v / v), sodium citrate 15-20 g / L, glutathione 0.5-1 g / L, glycerol 5-8% (v / v), glutathione extract 0.05-0.15 g / L, penicillin 0.8-1 g / L and streptomycin 0.8-1 g / L as components. The characteristic cryopreservation solution of goat semen according to claim 1. The lake sheep semen cryopreservation solution according to claim 2, wherein the concentration of the Astragalus propininus extract is 0.1 g / L. The lake yolk semen according to claim 1 or 2, wherein the method for preparing the egg yolk liquor includes a step of repeatedly pipetting the egg yolk of a fresh sterile egg and uniformly mixing the egg yolk liquor to obtain the egg yolk liquor. It is a cryopreservation solution. Tris, glucose, trehalose, sodium citrate, glutathione and Kibana-ougi extract were weighed in a predetermined ratio, added to distilled water, thoroughly dissolved and uniformly mixed, and then filtered through a 0.2 um filter membrane. Next, penicillin and streptomycin are added and mixed uniformly to obtain solution A (1), and egg yolk solution is added to solution A at a predetermined ratio and mixed uniformly to obtain solution B (2). Any one of claims 1 to 4, wherein glycerol is added to the solution B at a predetermined ratio and mixed uniformly to obtain the goat semen cryopreservation solution (3). It is a method for preparing the cryopreservation solution of goat semen described in the section. It is an application of the goat semen cryopreservation solution according to any one of claims 1 to 4 in the goat semen cryopreservation. It is a method of cryopreserving lake sheep semen.
Step (1) of preparing fresh raw semen of goat, evaluating the motility of the semen, and using it for cryopreservation when the motility is greater than 0.8, and (2) any one of claims 1 to 4. The goat semen cryopreservation solution described in the above is added to the semen prepared in step (1), mixed uniformly, then added to the cryopreservation tube, and the cryopreservation tube is left at 4 ° C. for 1 to 2 hours, and then liquid nitrogen. It is a method for cryopreserving goat semen, which comprises a step of leaving it at a place 4 to 6 cm away from the liquid surface of No. 1 for 8 to 10 minutes, and finally putting it in liquid nitrogen for storage. The method for cryopreserving lamb semen according to claim 7, wherein the density of semen in the cryopreservation tube of the step (2) is 6 × 10 ^{8 pieces / mL.}