Diluent applicable to freezing of sperm of down producing goat as well as preparation method and application of diluent

Abstract

The invention provides a diluent suitable for freezing semen of down producing goats, and belongs to the technical field of biology. The diluent suitable for freezing the sperm of the down producing goat, provided by the invention, can reduce damage of low temperature to the sperm during cryopreservation, remarkably improve the activity of sperms, improve forward motion parameters of the sperms, remarkably improve indexes such as sperm plasma membranes and acrosomes, and improve the activity of the sperms during recovery and the conception rate of ewes.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a diluent suitable for freezing cashmere goat semen, and a preparation method and application thereof. Background Art

[0002] Cashmere goats are an important livestock species, known for their production of valuable cashmere. In addition to cashmere, cashmere goats also provide other products such as meat and milk, making an important contribution to the agricultural economy. As the global demand for cashmere and other goat products increases, the conservation of cashmere goat genetic resources and the optimization of breeding technology have become particularly important.

[0003] Artificial insemination technology is an important means to achieve large-scale cashmere goat breeding and management. It can achieve the goal of simultaneous breeding, simultaneous delivery, and simultaneous market release in production. This technology first requires the collection of ram semen, the inspection of semen quality, and then dilution with diluent. Finally, the semen is injected into the reproductive tract of estrus ewes to replace natural mating. Artificial insemination technology can improve the efficiency of semen use, reduce the number of rams raised, reduce the spread of diseases, and increase the conception rate of ewes.

[0004] Sperm cryopreservation technology is a widely used reproductive technology that solves the problem of long-term storage of animal semen and is often used in assisted reproduction. Cryopreserved sperm samples can be used for scientific research, as well as for the continuous improvement and development of assisted reproductive technology, which helps to improve the technical level in the field of livestock reproduction. The sperm of Inner Mongolia Cashmere Goats is extremely sensitive to freezing damage, and will still suffer certain damage during the freezing and thawing process. The extent of these damages depends on many factors, including freezing and thawing conditions, storage time, and the quality of sperm samples. During the freezing process, they experience cold shock, oxidative stress, and changes in the structure and distribution of plasma membrane phospholipids, which affect their composition and quantity, thereby damaging enzyme activity, resulting in significant sperm vitality, decreased viability, and freezing damage to structural integrity. This has limited the promotion and application of goat frozen semen and its commercialization level is low.

[0005] Chinese patent CN111771871B discloses the use of epimedium polysaccharide in preparing goat semen cryopreservation diluent, the raw materials of which include Tris, citric acid, fructose, epimedium polysaccharide, antibiotics and egg yolk. However, semen preserved with the diluent still has high cryogenic damage.

[0006] Therefore, it is necessary to provide a diluent that can cryopreserve cashmere goat semen at low temperatures and effectively reduce the damage of low temperature to semen during cryopreservation, which is a problem to be solved at present. Summary of the invention

[0007] In view of this, the present invention provides a diluent suitable for freezing cashmere goat semen, which can effectively reduce the damage of low temperature to semen during freezing, and improve the activity of sperm during recovery and the conception rate of ewes.

[0008] The diluent suitable for freezing cashmere goat semen of the present invention comprises the following components: Tris, citric acid, dextrose, egg yolk, soy lecithin, skim milk, antibiotics, water and glycerol.

[0009] Preferably, the concentration of each component in the diluent suitable for freezing cashmere goat semen is: Tris 1.6-2.0g / 50ml, citric acid 0.8-1.2g / 50ml, glucose 0.45-0.55g / 50ml, egg yolk 7-7.5ml / 50ml, soy lecithin 0.7-0.8g / 50ml, skim milk 0.07-0.12g / 50ml, antibiotics 550-570μL / 50ml, glycerol 2.5-3ml / 50ml, and the balance is water.

[0010] Preferably, the antibiotic is any one of penicillin, streptomycin and gentamicin.

[0011] The method for preparing the diluent suitable for freezing cashmere goat semen comprises the following steps: (1) Weigh the raw materials according to the proportion; (2) Tris, citric acid, glucose, egg yolk, soy lecithin, skim milk, antibiotics and glycerol are mixed evenly, and then water is added to obtain the diluent for freezing cashmere goat semen.

[0012] The present invention also provides an application of the diluent suitable for freezing cashmere goat semen, and the specific method is: placing the cashmere goat semen in the diluent and freezing and storing it at a temperature of -180 to -200°C.

[0013] Preferably, the volume ratio of the cashmere goat semen to the diluent is 1-2:9-10.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a diluent suitable for freezing cashmere goat semen, a preparation method and an application thereof. The diluent can reduce the damage of low temperature to semen during freezing, significantly improve the activity of sperm and forward movement parameters, and significantly improve indicators such as sperm plasma membrane and acrosome, thereby improving the activity of sperm during recovery and the conception rate of ewes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The comparison results of sperm motility TM of cashmere goat semen after freezing and thawing by the diluents in Example 1 and Comparative Example 1; Figure 2 The comparison results of the diluents in Example 1 and Comparative Example 1 on the forward movement of sperm after freezing and thawing of cashmere goat semen; Figure 3 The results of the effects of the diluents prepared in Example 1 and Comparative Example 1 on the plasma membrane integrity rate of cashmere goat semen after freezing and thawing; Figure 4 The results of the effects of the diluents prepared in Example 1 and Comparative Example 1 on the acrosome integrity rate of cashmere goat semen after freezing and thawing; Figure 5 The diluent prepared in Example 1 and Comparative Example 1 has an effect on the conception rate of ewes after freezing and thawing of cashmere goat semen. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with the embodiments. Example

[0017] A diluent suitable for freezing cashmere goat semen, comprising the following components in the following concentrations: Tris 1.8g / 50ml, citric acid 1g / 50ml, glucose 0.5 / 50ml, egg yolk 7.5ml / 50ml, soy lecithin 0.8 / 50ml, skim milk 0.08g / 50ml, antibiotic (gentamicin) 550μL / 50ml, glycerol 2.5ml / 50ml, and the balance is water.

[0018] The preparation method of the diluent suitable for freezing cashmere goat semen comprises the following steps: (1) Weigh the raw materials according to the proportion; (2) Tris, citric acid, glucose, egg yolk, soy lecithin, skim milk, antibiotics and glycerol are mixed evenly, and then water is added to obtain the diluent for freezing cashmere goat semen.

[0019] A diluent suitable for freezing cashmere goat semen, comprising the following components in the following concentrations: Tris 1.9g / 50ml, citric acid 0.9g / 50ml, glucose 0.7 / 50ml, egg yolk 7.5ml / 50ml, antibiotic (gentamicin) 550μL / 50ml, glycerol 2.5ml / 50ml, and the rest is water.

[0020] The preparation method of the diluent is the same as that of Example 1.

[0021] The diluents prepared in Example 1 and Comparative Example 1 were used to perform a freezing-thawing experiment on cashmere goat semen, and the specific method was as follows: Take 45 mL of the diluent prepared in Example 1 or Comparative Example 1, add 5 mL of cashmere goat semen respectively, mix well to obtain a mixed solution, and then freeze it in liquid nitrogen at -196°C for 30 days.

[0022] The frozen mixture was then thawed at 37°C and sperm motility was analyzed using a sperm analyzer. The results are shown in Table 1 and Figure 1~2 .

[0023] Table 1

[0024] Figure 1 The comparison results of sperm motility TM of cashmere goat semen after freezing and thawing by the diluents in Example 1 and Comparative Example 1; Figure 2 The PM comparison results of the sperm forward motility of cashmere goat semen after freezing and thawing by the diluents in Example 1 and Comparative Example 1 are shown.

[0025] As shown in Table 1 and Figure 1~2 As shown, the diluent of Example 1 showed a higher sperm motility (77.3) after freezing and thawing of cashmere goat semen, which was significantly different from that of Comparative Example 1 (59.2). The diluent of Example 1 showed a higher sperm forward movement ratio (58.9) after freezing and thawing of cashmere goat semen, which was significantly different from that of Comparative Example 1 (47.3).

[0026] The plasma membrane integrity rate of cashmere goat semen after freezing and thawing was tested by the diluents prepared in Example 1 and Comparative Example 1. The results are as follows: Figure 3 ,in Figure 3 (A) is the plasma membrane integrity rate of Example 1, Figure 3 (B) is the plasma membrane integrity rate of Comparative Example 1. Figure 3 M1 is a sperm with an intact plasma membrane, and M2 is a sperm with a damaged plasma membrane.

[0027] from Figure 3 It can be seen that the diluent of Example 1 showed a higher plasma membrane integrity rate (74.78%) for cashmere goat semen after freezing and thawing, which was significantly different from the control example.

[0028] The diluents prepared in Example 1 and Comparative Example 1 were used to test the acrosome integrity rate of cashmere goat semen after freezing and thawing. The results are as follows: Figure 4 ,in Figure 4 (A) is the acrosome integrity rate of Example 1, Figure 4 (B) is the acrosome integrity rate of Comparative Example 1. Figure 4 M1 is a sperm with intact acrosome, and M2 is a sperm with damaged acrosome.

[0029] from Figure 4It can be seen that the diluent of Example 1 showed a higher acrosome integrity rate (85.68%) for cashmere goat semen after freezing and thawing, which was significantly different from the control example.

[0030] After freezing and thawing the cashmere goat semen using the diluent prepared in Example 1 and Comparative Example 1, fertilization was performed using the same method to detect the conception rate of the ewes. The results are as follows: Figure 5 .

[0031] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A diluent suitable for freezing cashmere goat semen, characterized in that: Includes the following components: Tris, citric acid, dextrose, egg yolk, soy lecithin, skim milk, antibiotics, water and glycerol.

2. The diluent suitable for freezing cashmere goat semen according to claim 1, characterized in that: The concentration of each component in the diluent of the freezing of cashmere goat semen is: Tris 1.6-2.0g / 50ml, citric acid 0.8-1.2g / 50ml, glucose 0.45-0.55g / 50ml, egg yolk 7-7.5ml / 50ml, soy lecithin 0.7-0.8g / 50ml, skim milk 0.07-0.12g / 50ml, antibiotics 550-570μL / 50ml, glycerol 2.5-3ml / 50ml, and the balance is water.

3. The diluent suitable for freezing cashmere goat semen according to claim 1, characterized in that: The antibiotic is any one of penicillin, streptomycin and gentamicin.

4. The method for preparing a diluent suitable for freezing cashmere goat semen according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Weigh the raw materials according to the proportion; (2) Tris, citric acid, glucose, egg yolk, soybean lecithin, skim milk, antibiotics and glycerol are mixed evenly, and then water is added to obtain the diluent for freezing cashmere goat semen.

5. The use of the diluent suitable for freezing cashmere goat semen according to any one of claims 1 to 3, characterized in that: The specific application method is: placing cashmere goat semen in the diluent and freezing and preserving it at a temperature of -180 to -200°C.

6. The use of the diluent suitable for freezing cashmere goat semen according to claim 5, characterized in that: The volume ratio of the cashmere goat semen to the diluent is 1-2:9-10.

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