CN111406738A - A kind of chicken semen dilution for artificial fallopian tube insemination and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of poultry breeding, and discloses a chicken sperm diluent for artificial oviduct insemination and a preparation method thereof, wherein the chicken sperm diluent comprises potassium citrate, fructose, sodium glutamate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride hexahydrate, TES, sodium acetate and deionized water, the survival rate of chicken sperm preserved for 2 hours in the sperm diluent is still higher and is more than 50 percent, cock sperm can be preserved at normal temperature by using the sperm diluent, the time of artificial insemination of chickens can be prolonged, and the utilization rate of the cock sperm can be favorably prolonged⁷～2.1×10⁷In between, insemination and hatchling may be achieved.

Description

A kind of chicken semen dilution for artificial fallopian tube insemination and preparation method thereof

technical field

The invention belongs to the technical field of poultry reproduction, and more particularly, relates to a chicken semen diluent for artificial fallopian tube insemination and a preparation method thereof.

Background technique

Artificial insemination technology is the artificial introduction of sperm into the cervix or uterine cavity of female animals for the purpose of achieving in vivo fertilization by means other than natural mating. With the improvement of modern people's living standards, the supply and demand of poultry products has increased year by year. Many countries with developed poultry industries have been exploring new technologies for artificial insemination on a large scale to improve production efficiency and reduce insemination costs. With the continuous development of human society and the continuous development and utilization of the natural environment, the habitat of birds is shrinking, resulting in more and more birds being listed as endangered species (Moorman et al., 2001). Cage protection has become the main means of reproduction for rare birds, but the hatching rate of caged and protected wild birds is very low. Artificial insemination of poultry is widely used in the breeding of non-poultry birds, especially endangered birds (Sen, 2004).

Semen diluent is essential for the preservation, transportation and insemination of chicken sperm, which can significantly reduce the breeding cost of roosters and the utilization rate of sperm, and improve economic benefits. At present, the domestic chicken semen dilution is only Beijing poultry dilution (BJJX). However, for the differences in the quality of the chickens studied, the breeding environment, and the insemination sites of the chicken semen, the developed semen dilution may not necessarily achieve the expected effect.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned deficiencies and shortcomings of the prior art, the present invention provides a chicken semen diluent for artificial fallopian tube insemination.

Another object of the present invention is to provide the above-mentioned preparation method of the chicken semen diluent for artificial fallopian tube insemination.

The object of the present invention is achieved through the following technical solutions:

A chicken semen diluent for artificial fallopian tube insemination, the chicken semen diluent comprising potassium citrate, fructose, sodium glutamate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride hexahydrate, TES, sodium acetate and Sterilized deionized water.

Preferably, each 1L of sterile deionized water contains the following components: potassium citrate 5.5-6.5 g, fructose 0.04-0.06 g, sodium glutamate 7.8-9.6 g, potassium dihydrogen phosphate 0.6-0.8 g, dihydrogen phosphate 0.6-0.8 g Potassium 8.7-11 g, magnesium chloride hexahydrate 0.3-0.4 g, TES 2.7-3.3 g, and sodium acetate 2.3-2.9 g.

More preferably, each 1L of sterile deionized water contains the following composition: potassium citrate 6g, fructose 0.05g, sodium glutamate 8.7g, potassium dihydrogen phosphate 0.65g, dipotassium hydrogen phosphate 9.7g, magnesium chloride hexahydrate 0.34g g, TES 3g, sodium acetate 2.6g.

The described preparation method of the chicken semen diluent for artificial fallopian tube insemination, the method is to mix potassium citrate, fructose, sodium glutamate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride hexahydrate, TES, sodium acetate Mix, and then add deionized water and stir until it is completely dissolved, and then make up to 1L to prepare semen diluent.

The application of the chicken semen diluent for artificial fallopian tube insemination.

Preferably, during the artificial fallopian tube insemination, the number of sperm per injection is (1.8-2.1)×10 ⁷ .

Compared with the prior art, the present invention has the following beneficial effects:

1. The survival rate of the chicken spermatozoa preserved in the semen diluent for 2 hours in the present invention is still relatively high, greater than 50%. The semen diluent can be used to store rooster sperm at room temperature, which is beneficial to prolong the artificial insemination time of chickens and the utilization rate of rooster sperm.

2. The present invention uses the sperm diluent to carry out laparoscopic-mediated artificial tubal insemination, and the number of injected sperm is guaranteed to be 1.8×10 ⁷ to 2.1×10 ⁷ in each artificial tubal insemination, which can achieve insemination and hatching.

Detailed ways

The content of the present invention is further described below in conjunction with specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

1. Under sterile conditions, take by weighing potassium citrate, fructose, sodium glutamate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride hexahydrate, TES and sodium acetate respectively according to the formula described in Table 1, add 100ml of sterilized distilled water was placed in a beaker, stirred thoroughly until completely dissolved, then transferred to a 1L volumetric flask, and the volume was adjusted to 1L, and then the solution was filtered with a 0.45μm disposable filter to obtain a semen dilution, which was carried out at 4°C. save.

2. The chicken semen dilution includes potassium citrate (K ₃ C ₆ H ₅ O ₇ ), fructose (C ₆ H ₁₂ O ₆ ), sodium glutamate (C ₅ H ₈ NNaO ₄ ), potassium dihydrogen phosphate (KH ₂ PO ₄ ), dipotassium hydrogen phosphate (K ₂ HPO ₄ ), magnesium chloride hexahydrate (MgCl ₂ .6H ₂ O), TES (C ₆ H ₁₅ O ₃ Si) and sodium acetate (CH ₃ COONa).

Table 1 Composition of rooster semen diluent

3. Preservation method: Table 2 shows the effect of semen dilution on chicken sperm survival rate (%) at 37°C. The chicken sperm was preserved with the semen diluent at 37°C. It can be seen from Table 2 that the sperm survival rate of chicken sperm stored in the semen diluent for 2 hours was still high, more than 50%. Therefore, the semen diluent can be used to store rooster sperm at room temperature, which is beneficial to prolong the artificial insemination time of chickens and the utilization rate of rooster sperm. When using, the semen diluent should be preheated to 37°C in advance, and then mixed with rooster semen. The amount of semen diluent can be increased or decreased according to the required semen concentration.

Table 2 The effect of semen dilution on chicken sperm survival rate (%) at 37℃

4. How to use: observe the sperm motility count by microscopic examination of semen, and then add semen diluent to dilute it to about 2×10 ⁷ cells/mL. The number of injected sperm should be between 1.8×10 ⁷ and 2.1×10 ⁷ during each artificial tubal insemination. The hens to be fertilized were anesthetized by the subwing vein, and then restrained. Using laparoscopy, the chicken sperm diluted with the semen diluent was injected into the oviducts of the hens to be fertilized, and then the egg production rate and hatching of the hens were observed. The rates are shown in Table 3. Table 3 shows the fertilization rate of artificial tubal insemination. It can be seen that using the sperm diluent for laparoscopic-mediated artificial fallopian tube insemination, insemination and hatching can be achieved.

The molar concentration ratio of sodium and potassium in the semen diluent of the present invention is 1:2, and the content of potassium is much higher than the molar concentration ratio of Na ⁺ and K ⁺ in chicken semen fluid of 15:1, that is, every 10ml of vas deferens contains Na ⁺ 39.3mg, K ⁺ 4.3mg. K ⁺ is essential for maintaining sperm volume, semen pH, and obtaining and maintaining sperm motility. A small amount of K ⁺ can maintain the normal function and necessary vitality of sperm; in addition, a moderate amount of K ⁺ can prolong the life of sperm, and high potassium can stimulate the fertilization ability of sperm. Potassium citrate in semen diluent provides a weakly acidic environment for sperm, and continuously provides bioavailable potassium ions in the form of cations. Potassium citrate can prolong sperm lifespan and improve sperm survival index. Inorganic salts such as sodium chloride and potassium chloride, potassium citrate keep the sperm deformity rate at a low level. Therefore, using the sperm diluent for laparoscopically mediated artificial fallopian tubes, the number of injected sperm is guaranteed to be between 1.8×10 ⁷ and 2.1×10 ⁷ in each artificial fallopian tube insemination, and insemination and hatching can be achieved.

Table 3 Fertilization rate of artificial tubal insemination

Example 2

1. Under sterile conditions, weigh potassium citrate 5.5g, fructose 0.04g, sodium glutamate 8.5g, potassium dihydrogen phosphate 0.6g, dipotassium hydrogen phosphate 9.5g, magnesium chloride hexahydrate 0.3g, TES 2.5g and 2.5g of sodium acetate, add 100ml of sterilized distilled water to a beaker, stir well until completely dissolved, transfer to a 1L volumetric flask, make up to 1L, and filter the solution with a 0.45μm disposable filter to prepare The semen diluent was obtained and stored at 4°C.

2. The semen diluent should be preheated to 37°C in advance, and then mixed with rooster semen. The amount of semen diluent can be increased or decreased according to the required semen concentration.

Example 3

1. Under sterile conditions, weigh potassium citrate 6.5g, fructose 0.06g, sodium glutamate 9.0g, potassium dihydrogen phosphate 0.7g, dipotassium hydrogen phosphate 10g, six 0.4g of magnesium chloride in water, 3.5g of TES and 2.8g of sodium acetate, add 100ml of sterilized distilled water into a beaker, stir well until completely dissolved, transfer to a 1L volumetric flask, make up to 1L, and then use 0.45μm disposable The solution was filtered through a filter to prepare a semen dilution, which was stored at 4°C.

2. The semen diluent should be preheated to 37°C in advance, and then mixed with rooster semen. The amount of semen diluent can be increased or decreased according to the required semen concentration.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations and The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (6)

1. a chicken semen dilution for artificial tubal insemination, is characterized in that, described chicken semen dilution comprises potassium citrate, fructose, sodium glutamate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride hexahydrate , TES, sodium acetate, and sterile deionized water. 2. The chicken semen dilution liquid for artificial fallopian tube insemination according to claim 1, characterized in that, every 1 L of sterilized deionized water contains the following components: potassium citrate 5.5-6.5g, fructose 0.04-0.06g, 7.8-9.6 g of sodium glutamate, 0.6-0.8 g of potassium dihydrogen phosphate, 8.7-11 g of dipotassium hydrogen phosphate, 0.3-0.4 g of magnesium chloride hexahydrate, 2.7-3.3 g of TES, and 2.3-2.9 g of sodium acetate. 3. the chicken semen dilution for artificial fallopian tube insemination according to claim 2, is characterized in that, every 1L of sterilized deionized water contains following composition: potassium citrate 6g, fructose 0.05g, sodium glutamate 8.7g , potassium dihydrogen phosphate 0.65g, dipotassium hydrogen phosphate 9.7g, magnesium chloride hexahydrate 0.34g, TES 3g, sodium acetate 2.6g. 4. the preparation method of the chicken semen diluent for artificial fallopian tube insemination according to any one of claims 1-3, is characterized in that, the method is to combine potassium citrate, fructose, sodium glutamate, dihydrogen phosphate Potassium, dipotassium hydrogen phosphate, magnesium chloride hexahydrate, TES, and sodium acetate are mixed, and then deionized water is added and stirred until it is completely dissolved, and the volume is adjusted to 1L to prepare a semen diluent. 5. the application of the chicken semen diluent for artificial fallopian tube insemination according to any one of claims 1-3. 6 . The application of the chicken semen diluent for artificial tubal insemination according to claim 5 , wherein, during the artificial tubal insemination, the number of sperm injected per time is (1.8-2.1)×10 ⁷ sperm. 7 .