Preparation method of frozen diluent of sheep semen and application of preparation method

Abstract

The invention discloses a preparation method of a frozen diluent of sheep semen and an application of the preparation method. The preparation method of the frozen diluent of the sheep semen comprisesthe following steps: firstly, accurately weighing tris(hydroxymethyl) aminoethane, dihydrate trisodium citrate, fructose, an inositol compound, ethanediol or glycerinum, and oxytocin, adding the materials into ultrapure water, dissolving, adding a fresh yolk centrifuged liquid supernatant, penicillin and streptomycin, mixing evenly, fixing the volume after adjusting the pH value to 6.8-7.2, thus obtaining the frozen diluent of the sheep semen. The inositol compound is used to replace an antifreeze protein, the defect of the application of the antifreeze protein is avoided, meanwhile, the oxytocin is added when the semen is conveyed, and the sperm vitality and the acrosomal integrity are improved, so that the fertility rate is increased.

Description

technical field

[0001] The invention belongs to the technical field of animal reproduction, and in particular relates to a preparation method and application of a frozen diluent of sheep semen. Background technique

[0002] The preservation technology of livestock frozen semen has been studied for nearly a hundred years, but the viability and fertilization rate of sheep frozen semen after thawing are low, and the non-return rate after artificial insemination is significantly lower than that of fresh semen. The main reason is that during the freezing process of semen, ice crystals formed inside and outside the cells cause damage to sheep sperm directly affecting the viability of sperm after freezing. How to reduce the generation of ice crystals and improve the viability of semen is a hot spot in the field of cryopreservation of semen. Some studies have shown that antifreeze protein can improve the viability of mammalian frozen semen. However, the extraction technology and pat...

Examples

Embodiment 1

[0017] Tangshan Guye District Chensheng Animal Husbandry Co., Ltd. collected the semen of Australian white sheep by the false vagina method. After the semen quality appraisal, the motility rate was greater than 0.80, the sperm density was 3.5-4.0×108 / ml, and the semen volume was 1.5-2.0ml.

[0018] The preservation method of the frozen diluent of sheep semen of the present invention comprises the following steps:

[0019] (1) Aseptically absorb fresh egg yolk liquid, dilute it with sterilized physiological saline 1:1, centrifuge at 5000g at 4°C for 10 minutes, absorb 15ml of supernatant, and let stand at 20-30°C for 10 minutes to obtain solution A.

[0020] (2) Weigh 2.75g of trishydroxymethylaminomethane, 1.5g of trisodium citrate dihydrate, 1.5g of fructose, 5.0g of inositol compounds, 15ml of glycerin, and 25IU of oxytocin and add them to an appropriate amount of ultrapure water for dissolution, and then Add 10ml of fresh centrifuged egg yolk supernatant (solution A), 100,0...

Embodiment 2

[0024] Tangshan Guye District Chensheng Animal Husbandry Co., Ltd. collected Dorper sheep semen by the false vagina method. After semen quality appraisal, the motility rate was greater than 0.80, the sperm density was 3.5-4.0×109 / ml, and the semen volume was 1.5-2.0ml.

[0025] The preservation method of the frozen diluent of sheep semen of the present invention comprises the following steps:

[0026] (1) Aseptically absorb fresh egg yolk liquid, dilute it with sterilized physiological saline 1:1, centrifuge at 5000g at 4°C for 10 minutes, absorb 15ml of supernatant, and let stand at 20-30°C for 10 minutes to obtain solution A.

[0027] (2) Weigh 2.75g of trishydroxymethylaminomethane, 1.5g of trisodium citrate dihydrate, 1.5g of fructose, 5.0g of inositol compounds, 15ml of glycerin, and 25IU of oxytocin and add them to an appropriate amount of ultrapure water for dissolution, and then Add 10ml of fresh centrifuged egg yolk supernatant (solution A), 100,000 IU of penicillin, ...

Embodiment 3

[0032] A method for preparing sheep semen frozen diluent of the present invention comprises the following steps: first, accurately weigh trishydroxymethylaminomethane, trisodium citrate dihydrate, fructose, inositol compounds, ethylene glycol or glycerin, Add the element into an appropriate amount of ultrapure water to dissolve, then add fresh egg yolk centrifuge supernatant, penicillin, and streptomycin, mix well, adjust the pH value to 7.1, and then constant volume to obtain a frozen dilution of sheep semen.

[0033] The mass ratio of the tris-hydroxymethyl aminomethane: trisodium citrate dihydrate: fructose: inositol compound is 2.8: 2.5: 2.0: 4.0; the mass ratio of the tris-hydroxymethyl amino methane to ethylene glycol or glycerin The volume ratio is 0.5g:4L.

[0034] The preparation method of fresh centrifuged egg yolk supernatant: Aseptically absorb fresh egg yolk liquid, dilute it with sterilized physiological saline 1:1, centrifuge at 5000g at 4°C for 10 minutes, abso...

technical field

[0001] The invention belongs to the technical field of animal reproduction, and in particular relates to a preparation method and application of a frozen diluent of sheep semen. Background technique

[0002] The preservation technology of livestock frozen semen has been studied for nearly a hundred years, but the viability and fertilization rate of sheep frozen semen after thawing are low, and the non-return rate after artificial insemination is significantly lower than that of fresh semen. The main reason is that during the freezing process of semen, ice crystals formed inside and outside the cells cause damage to sheep sperm directly affecting the viability of sperm after freezing. How to reduce the generation of ice crystals and improve the viability of semen is a hot spot in the field of cryopreservation of semen. Some studies have shown that antifreeze protein can improve the viability of mammalian frozen semen. However, the extraction technology and pat...

Examples

Embodiment 1

[0017] Tangshan Guye District Chensheng Animal Husbandry Co., Ltd. collected the semen of Australian white sheep by the false vagina method. After the semen quality appraisal, the motility rate was greater than 0.80, the sperm density was 3.5-4.0×108 / ml, and the semen volume was 1.5-2.0ml.

[0018] The preservation method of the frozen diluent of sheep semen of the present invention comprises the following steps:

[0019] (1) Aseptically absorb fresh egg yolk liquid, dilute it with sterilized physiological saline 1:1, centrifuge at 5000g at 4°C for 10 minutes, absorb 15ml of supernatant, and let stand at 20-30°C for 10 minutes to obtain solution A.

[0020] (2) Weigh 2.75g of trishydroxymethylaminomethane, 1.5g of trisodium citrate dihydrate, 1.5g of fructose, 5.0g of inositol compounds, 15ml of glycerin, and 25IU of oxytocin and add them to an appropriate amount of ultrapure water for dissolution, and then Add 10ml of fresh centrifuged egg yolk supernatant (solution A), 100,0...

Embodiment 2

[0024] Tangshan Guye District Chensheng Animal Husbandry Co., Ltd. collected Dorper sheep semen by the false vagina method. After semen quality appraisal, the motility rate was greater than 0.80, the sperm density was 3.5-4.0×109 / ml, and the semen volume was 1.5-2.0ml.

[0025] The preservation method of the frozen diluent of sheep semen of the present invention comprises the following steps:

[0026] (1) Aseptically absorb fresh egg yolk liquid, dilute it with sterilized physiological saline 1:1, centrifuge at 5000g at 4°C for 10 minutes, absorb 15ml of supernatant, and let stand at 20-30°C for 10 minutes to obtain solution A.

[0027] (2) Weigh 2.75g of trishydroxymethylaminomethane, 1.5g of trisodium citrate dihydrate, 1.5g of fructose, 5.0g of inositol compounds, 15ml of glycerin, and 25IU of oxytocin and add them to an appropriate amount of ultrapure water for dissolution, and then Add 10ml of fresh centrifuged egg yolk supernatant (solution A), 100,000 IU of penicillin, ...

Embodiment 3

[0032] A method for preparing sheep semen frozen diluent of the present invention comprises the following steps: first, accurately weigh trishydroxymethylaminomethane, trisodium citrate dihydrate, fructose, inositol compounds, ethylene glycol or glycerin, Add the element into an appropriate amount of ultrapure water to dissolve, then add fresh egg yolk centrifuge supernatant, penicillin, and streptomycin, mix well, adjust the pH value to 7.1, and then constant volume to obtain a frozen dilution of sheep semen.

[0033] The mass ratio of the tris-hydroxymethyl aminomethane: trisodium citrate dihydrate: fructose: inositol compound is 2.8: 2.5: 2.0: 4.0; the mass ratio of the tris-hydroxymethyl amino methane to ethylene glycol or glycerin The volume ratio is 0.5g:4L.

[0034] The preparation method of fresh centrifuged egg yolk supernatant: Aseptically absorb fresh egg yolk liquid, dilute it with sterilized physiological saline 1:1, centrifuge at 5000g at 4°C for 10 minutes, abso...