CN113115766A

CN113115766A - Semen preservation solution for improving semen preservation quality and preparation method and application thereof

Info

Publication number: CN113115766A
Application number: CN201911417865.7A
Authority: CN
Inventors: 曾申明; 高鹏; 邵永光; 于杰
Original assignee: China Agricultural University; Dong E E Jiao Co Ltd
Current assignee: China Agricultural University; Dong E E Jiao Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-16

Abstract

The invention relates to the technical field of animal breeding, in particular to a semen preserving fluid for improving semen preserving quality and a preparation method and application thereof. The invention provides a semen preserving fluid capable of improving animal semen preserving quality, which comprises the following components in parts by volume: 60-80 parts of semen dilution base liquid and 20-30 parts of yolk liquid, and also can be added with 1-10 parts of glycerol or also can be added with 1-10 parts of dimethylformamide and 1-10 parts of vitamin E; the semen preserving fluid can obviously prolong the normal temperature, low temperature and freezing preservation time of semen and the sperm quality of preserved semen, and the conception rate is obviously improved. The semen preservation solution preparation and the semen preservation method have the advantages of simple operation, low application cost, obvious effect and no harmful influence on animals, can be used for the fine breed propagation of equus animals such as horses, donkeys and the like, obviously improves the economic benefit of related industries, and has wide market application prospect.

Description

Semen preservation solution for improving semen preservation quality and preparation method and application thereof

Technical Field

The invention relates to the technical field of animal breeding, in particular to a semen preserving fluid for improving semen preserving quality and a preparation method and application thereof.

Background

The semen cryopreservation and artificial insemination technology are combined, so that the method plays a great role in the production of modern animal husbandry, can greatly reduce the feeding quantity of male livestock, reduce the production cost, improve the utilization rate of excellent male livestock and bring great economic benefits to the animal husbandry. At present, the breeding of horses and donkeys mainly adopts the copulation or immediately gives the oestrus to the female animals without diluting or diluting the collected semen (fresh semen) by a low time. The semen low temperature and freezing preservation technology can fully utilize the genetic resources of excellent breeding stock, quickly expand the improved seed proportion of the herd and is not limited by time and space.

In the equus animals, the utilization efficiency of semen can be improved by 5-20 times by artificial insemination. However, the variety of horses and donkeys is more, the breeding area is generally widely distributed, the breeding area is mostly in mountainous areas or areas with inconvenient traffic, the breeding mode is more in scattered households, the distribution of female animals is not centralized, and the popularization area of the artificial insemination technology is very small. The application of the artificial insemination technology of the horses and the donkeys is limited because the in-vitro preservation time of the fresh essences of the horses and the donkeys is very short and the vitality is nearly lost after a few hours. With the development of the horse and donkey industry, the application of artificial insemination is imperative, and high-quality semen with long storage time and convenient transportation is urgently needed to be used as technical support in production.

Semen preservation is divided into three methods of normal temperature, low temperature and freezing preservation. The normal-temperature preservation technology is developed earliest and is relatively mature, but at normal temperature (25 ℃), on one hand, the sperm quality is influenced by the rapid growth of microorganisms, and on the other hand, the sperm motility is reduced rapidly when the sperm is preserved at normal temperature, and the preservation time is 1-3 days. The low-temperature preservation temperature is 4 ℃, the preservation time is about one week, the preservation time is longer than the preservation time at normal temperature, and the preservation method is simpler and more convenient than the cryopreservation and has low cost. The freeze preservation refers to that the semen is preserved in liquid nitrogen or at the temperature of minus 80 ℃ after being specially treated, the preservation time can reach several years or even several decades, long-distance transportation can be realized through a liquid nitrogen container, and the genetic value of the improved male livestock can be fully exerted.

The semen preservation solution with excellent performance and suitability is the key for guaranteeing the quality of preserved semen, the research on the semen preservation solution of the equus animals is still less at present, wherein the research on the low-temperature and freezing preservation of the donkey semen is very limited, and the main reason is that: (1) donkey sperms are particularly sensitive to low temperature, have large difference among individuals and are extremely easy to damage in the freezing and thawing process; (2) the density of the sperms is small, and the electrolyte components in the sperms can promote the premature senility of the sperms. Therefore, the production operation degree of the frozen semen of the donkey is complex and difficult, and the application of the frozen semen is severely restricted. In recent years, the development of low-dose insemination technology for equine animals puts higher requirements on semen preservation effect. Therefore, the improvement of the semen quality after freezing, thawing and long-term in vitro liquid storage is an urgent problem to be solved in animal husbandry production.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a semen preserving fluid capable of improving the quality of animal sperms and application thereof.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a semen preserving solution for improving semen quality, wherein the semen preserving solution comprises, by volume: 60-80 parts of semen dilution base solution and 20-30 parts of yolk solution, wherein:

the semen dilution base solution comprises a solution A and a solution B (consisting of the solution A and the solution B); the liquid A comprises mixed sugar, citrate, amino acid, a buffer reagent and antibiotic; the solution B is skimmed milk.

The invention provides a semen preserving fluid capable of improving animal semen preserving quality. The preservation solution provided by the invention can obviously prolong the preservation time of the liquid state and improve the sperm motility after preservation at normal temperature and low temperature or freezing and thawing.

The semen preserving fluid of the invention relates to the semen dilution base fluid:

each 100ml of the A liquid comprises the following components:

4-9g of mixed sugar, 0.1-0.6g of citrate, 10-150mmol of amino acid, 0.5-3g of buffer reagent and 10-80 ten thousand IU of antibiotic.

In the semen dilution base solution, the mixed amino acid is selected from one or more of L-proline, alanine, glycine, glutamine, cysteine and glutamic acid. Through a great deal of research by the inventor, the effect of selecting the L-proline is best.

In the semen dilution base solution, the mixed sugar is at least two selected from glucose, lactose, raffinose and trehalose, preferably, the mixed sugar comprises the following components in a ratio of (3-8): (0.1-0.5): (0.1-0.5) glucose, lactose, and raffinose. The three kinds of sugar are compounded in proportion, so that various energy substrates can be provided for sperms, and the protection effect is improved.

Further, the ratio of the mixed sugar to the amino acid is (5-7) g: (10-150) mmol; the inventors have surprisingly found that there is a synergistic effect between sugar and amino acid, which is stably exerted when controlled in the above ratio, especially when the sugar is a mixture of glucose, lactose and raffinose, and the amino acid is L-proline, the preservation effect is stable, significantly better than other mixed sugars and/or other amino acid combinations. More preferably, the ratio of mixed sugar to amino acid is (5-7) g: (10-100) mmol.

The citrate is selected from one or more of potassium citrate, sodium citrate, calcium citrate and the like; preferably, the citrate is a mixture of sodium citrate and potassium citrate, and the mass ratio of the sodium citrate to the potassium citrate is: potassium citrate ═ (5-6): (6-10). The advantage is that it plays an important role in maintaining a relatively stable pH of the diluent phase.

The buffer reagent is selected from one or more of HEPES, Tris, TES, MOPS and TAPSO, and MOPS or HEPES is preferred.

The antibiotics are selected from two of penicillin, streptomycin and gentamicin, and preferably the penicillin and the streptomycin are selected according to the formula (1-2): 1 is compounded in proportion.

The liquid B is skimmed milk, and the fat content of the skimmed milk is lower than 0.1% and the protein content of the skimmed milk is higher than 3%; in the skimmed milk liquid, the mass volume percentage of the skimmed milk powder is 5-20%.

The invention also provides a preferable preparation method of the solution B, which comprises the following steps: calculated by 100ml, comprises 6-10g of skimmed milk powder, and the volume is fixed to 100ml by sterile pure water; preferably prepared by the following method: and after the solution B is fully dissolved, sterilizing the solution B for 10min at 115 ℃ under high pressure, and cooling the solution B to room temperature. Wherein, the fat content of the skimmed milk powder is required to be lower than 0.1 percent, and the protein content is higher than 30 percent.

In the semen dilution base solution, the volume ratio of the solution A to the solution B is (4-6): 5; preferably 1: 1.

The low-temperature and frozen preservation solution of the invention relates to the yolk solution:

in the yolk liquid, the volume percentage of yolk is not less than 15%, and the rest components are preferably the semen dilution base liquid.

Preferably, the yolk liquid contains 20-25% of yolk.

The yolk is from one or more of eggs, duck eggs and goose eggs; preferably an egg.

The egg should be produced by farmhouse or from chicken farm without epidemic diseases, and the egg should be fresh, complete and clean, and the surface of eggshell is sterilized with 75% alcohol cotton ball before taking yolk. After the alcohol is volatilized completely, a crack is knocked at the midline of the waist of the egg, the egg is divided into two halves, and the egg white is removed by using a method of alternately dumping two eggshells to leave the yolk. The yolk was rolled on clean filter paper to remove excess egg white. Yolk was extracted through the vitelline membrane with a sterilized syringe.

The yolk liquid is preferably prepared by the following method: taking 15-25mL (more preferably 20mL) of extracted yolk, adding semen to dilute the base solution to 100mL, and stirring thoroughly; centrifuging at 4 ℃ for 1h at 3000g (more preferably 2800 Xg) of 2000-; the precipitate was removed and the supernatant was retained.

As a preferred technical scheme of the invention, the invention provides a semen low-temperature and frozen preservation solution, wherein each 100mL of the solution A comprises (consists of the following components):

4-8g of glucose, 0.2-0.5g of lactose, 0.3-0.5g of raffinose, 0.06-0.1g of sodium citrate, 0.05-0.082g of potassium citrate, 0.8-1.3g of HEPES or MOPS reagent and 10-70mmol of L-proline, sterilizing and fixing the volume by pure water, and then adding 10-20 ten thousand IU of penicillin and 10-20 ten thousand IU of streptomycin;

preferably, the semen preservation solution further comprises 1-10 parts of glycerol; or the semen preserving fluid also comprises 1-10 parts of dimethylformamide and 1-10 parts of vitamin E.

The invention finds that glycerol is simply replaced by the dimethylformamide, the effect of improving the preservation quality of the frozen semen is reduced, and the preservation quality of the frozen semen can be obviously improved by adopting the dimethylformamide and the vitamin E for matching use compared with the method of independently adding the dimethylformamide.

As a preferable scheme of the invention, the semen preservation solution comprises 60-80% of semen dilution base solution, 20-30% of yolk solution and 1-10% of glycerol.

As another preferable mode of the present invention, the semen preservation solution comprises 60-80% of semen dilution base solution, 20-30% of yolk solution, 1-10% of dimethylformamide and 1-10% of vitamin E.

The semen preserving fluid provided by the invention can be used for preserving semen at normal temperature, low temperature or freezing; wherein, the semen preserving fluid without glycerol, dimethylformamide and vitamin E can be used for preserving semen at normal temperature or low temperature; the semen preservation solution containing glycerol or dimethylformamide and vitamin E can be used for cryopreservation of semen. When the semen preservation solution is used for freezing preservation of semen, the semen quality is improved more remarkably.

In a second aspect, the invention provides an application of the semen preservation solution in semen preservation, wherein the application comprises the following steps: the semen preservation solution without glycerol, dimethylformamide and vitamin E is adopted for preserving semen at normal temperature or low temperature, or the semen preservation solution containing glycerol or dimethylformamide and vitamin E is adopted for preserving semen in a freezing way.

It will be appreciated by those skilled in the art that an equal expansion or contraction of any of the above formulations is within the scope of the present invention.

In a third aspect, the invention also provides a preparation method of the preservation solution for improving semen quality, which comprises the following steps:

1) preparing solution A, diluting mixed sugar, citrate, buffer reagent and mixed amino acid with sterilized ultrapure water to constant volume, adding antibiotic, and filtering with 0.22 μm filter after all substances are fully dissolved;

2) sterilizing the solution B at 115 deg.C under high pressure for 10-15min, and cooling;

3) mixing the solution A and the solution B to obtain a semen dilution base solution;

4) preparing yolk liquid;

extracting yolk, adding the semen dilution base solution, and centrifuging for 1h at 4 ℃; removing the precipitate and retaining the supernatant;

preferably, the centrifugal rotation speed is (2000-3000). times.g, (more preferably 2800. times.g);

5) mixing the semen dilution base solution and the egg yolk solution, or mixing the semen dilution base solution, the egg yolk solution and glycerol, or mixing the semen dilution base solution, the egg yolk solution, dimethylformamide and vitamin E.

In a fourth aspect, the present invention provides a method of using the above-described semen preserving fluid.

Specifically, the semen preservation solution without glycerol, dimethylformamide and vitamin E is used for preserving the semen at normal temperature or low temperature, or the semen preservation solution containing glycerol or dimethylformamide and vitamin E is used for preserving the semen in a freezing way; the method comprises the following steps: diluting semen;

the semen is preferably collected to remove jelly, the survival rate, the volume and the density of the semen are detected, and the sperm rate of the straight-line advancing movement of the fresh semen is required to be more than or equal to 70 percent.

And the dilution is to mix the semen and the semen dilution base solution according to the ratio of 2: diluting at the ratio of (1-3), balancing, centrifuging (further balancing at 25 deg.C for 10min, centrifuging at 600g for 10min, discarding supernatant), and diluting with the semen stock solution to (2-10) × 10⁸one/mL (more preferably 2X 10)⁸one/mL). Subpackaging into 0.5mL frozen semen tubules by using a full-automatic filling machine.

When semen is cryopreserved using the semen preserving fluid containing glycerol or containing dimethylformamide and vitamin E, programmed freezing (preferably using a minitube programming freezer) is performed after the dilution, and the freezing procedure is as shown in table 1:

TABLE 1 freezing procedure for cryopreservation of semen

Programmed freezing to-140 deg.C, adding the obtained frozen semen into liquid nitrogen, standing for at least 5min, and transferring to liquid nitrogen tank for storage.

Preferably, the step of freezing is performed after pre-equilibration of the frozen tubule at 4 ℃ for 2-4 hours.

The preparation method provided by the invention is simple and convenient to operate, and cannot influence the activity of sperms.

Preferably, the semen diluent provided by the invention is suitable for semen preservation of equine animals, and therefore, the semen is preferably from equine animals.

The invention has the beneficial effects that:

the invention provides a semen preserving fluid capable of improving the quality of animal semen. The semen preservation solution provided by the invention can obviously improve the semen quality (such as sperm quality including sperm motility) of normal-temperature preservation, low-temperature preservation and frozen preservation, obviously prolong the preservation time of the semen, improve the conception rate of the preserved semen, and has better effect on improving the quality of the frozen preserved and unfrozen semen. The semen preservation method is simple and convenient to operate, low in application cost, remarkable in effect and free of any harmful influence on animals. Can be widely applied to semen preservation of equus animals such as horses, donkeys and the like and related embryo transplantation industry and embryo engineering, and has wide market prospect.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

The embodiment provides semen cryopreservation liquid and preparation thereof.

1. The components: glucose, lactose, raffinose, sodium citrate, potassium citrate, MOPS reagent and L-proline, wherein the medicines are analytically pure; ultrapure water, skimmed milk powder, penicillin, streptomycin, sterilized glycerol and fresh yolk.

2. The preparation method comprises the following steps: the semen cryopreservation solution is prepared by taking 200mL as an example.

Preparing solution A: 6.0000g of glucose, 0.3000g of lactose, 0.3000g of raffinose, 0.0600g of sodium citrate, 0.0820g of potassium citrate, 1.0463g of MOPS reagent and 10mmol of L-proline are respectively weighed by an analytical balance according to 100mL, sterilized ultrapure water is used for constant volume to 100mL, 10 ten thousand IU of penicillin and 10 ten thousand IU of streptomycin are added, and the mixture is filtered by a 0.22 mu m filter.

Preparing a sterilized skim milk B solution: 8.0000g of skimmed milk powder is weighed by an analytical balance according to 100mL, sterilized ultrapure water is added to a constant volume of 100mL, the solid is completely dissolved and then is sterilized by autoclaving at 115 ℃ for 10min, and the solution is cooled to room temperature.

Preparing a semen dilution base solution: and (3) weighing 100mL of the solution A and 100mL of the sterilized skimmed milk solution B by using a measuring cylinder in terms of 200mL, and uniformly mixing the solution A and the sterilized skimmed milk solution B to obtain 200mL of semen dilution base solution.

Preparing 20% yolk liquid: calculated as 200 mL. Eggs from chicken farms without epidemic diseases are selected, fresh, complete and clean, and 75% alcohol cotton balls are used for disinfecting the surfaces of eggshells before the yolk is taken. After the alcohol is volatilized, a crack is knocked at the midline of the waist of the egg, the egg is divided into two halves, and the egg white is removed by using a method of alternately dumping two eggshells to leave the yolk. Egg yolk was slowly rolled on sterile filter paper to remove excess egg white. Taking 40mL of extracted yolk by a sterile syringe through a yolk membrane, and adding 160mL of semen dilution base solution; the precipitate was removed after centrifugation at 2800 Xg for 1h at 4 ℃ leaving a supernatant.

Preparing a frozen preservation solution: measuring 70mL of semen dilution base solution, 25mL of 20% egg yolk solution and 5mL of glycerol by 100mL, and uniformly mixing to obtain 100mL of cryopreservation solution.

The other volumes of liquid are configured according to the corresponding proportion.

Example 2

The embodiment provides semen cryopreservation liquid and preparation thereof.

1. The components: glucose, lactose, raffinose, sodium citrate, potassium citrate, HEPES reagent and L-proline, wherein the medicines are analytically pure; ultrapure water, skimmed milk powder, penicillin, streptomycin, sterilized glycerol and fresh yolk.

Preparing solution A: 6.0000g of glucose, 0.3000g of lactose, 0.3000g of raffinose, 0.0600g of sodium citrate, 0.0820g of potassium citrate, 0.9520g of HEPES reagent and 20mmol of L-proline were weighed out in 100mL by an analytical balance, and the volume was adjusted to 100mL by using sterilized ultrapure water, 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin were added, and the mixture was filtered by a 0.22 μm filter.

Preparing 20% yolk liquid: calculated as 200 mL. The method comprises the following steps of selecting farmhouse self-laying eggs or eggs from chicken farms without epidemic diseases, wherein the used eggs are fresh, complete and clean, and disinfecting the surfaces of eggshells by using 75% alcohol cotton balls before taking yolk. After the alcohol is completely volatilized, knocking a crack at the midline of the waist of the egg, dividing the egg into two halves, and removing egg white by using a method of alternately pouring two eggshells to leave yolk. The yolk is placed on sterile filter paper and excess egg white is removed by rolling. Taking 40mL of extracted yolk by a sterilized injector through a yolk membrane, and adding 160mL of semen dilution base solution; centrifuging at 2800 Xg for 1h at 4 ℃; the precipitate was removed and the supernatant was retained.

Preparation of a cryopreservation liquid: and weighing 70mL of semen dilution base solution, 25mL of 20% egg yolk solution and 5mL of glycerol by using a measuring cylinder according to 100mL, and uniformly mixing to obtain 100mL of frozen preservation solution.

Example 3

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing solution A: 6.0000g of glucose, 0.3000g of lactose, 0.3000g of raffinose, 0.0600g of sodium citrate, 0.0820g of potassium citrate, 0.9520g of HEPES reagent and 30mmol of L-proline were weighed out in 100mL by an analytical balance, and the volume was adjusted to 100mL by using sterilized ultrapure water, and 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin were added, and the mixture was filtered by a 0.22 μm filter.

Example 4

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing solution A: 6.0000g of glucose, 0.3000g of lactose, 0.3000g of raffinose, 0.0600g of sodium citrate, 0.0820g of potassium citrate, 0.9520g of HEPES reagent and 40mmol of L-proline were weighed out in 100mL by an analytical balance, and the volume was adjusted to 100mL by using sterilized ultrapure water, 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin were added, and the mixture was filtered by a 0.22 μm filter.

Example 5

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing solution A: 6.0000g of glucose, 0.3000g of lactose, 0.3000g of raffinose, 0.0600g of sodium citrate, 0.0820g of potassium citrate, 0.9520g of HEPES reagent and 50mmol of L-proline were weighed out in 100mL by an analytical balance, and the volume was adjusted to 100mL by using sterilized ultrapure water, and 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin were added and filtered by using a 0.22 μm filter.

Example 6

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing solution A: 6.0000g of glucose, 0.3000g of lactose, 0.3000g of raffinose, 0.0600g of sodium citrate, 0.0820g of potassium citrate, 0.9520g of HEPES reagent and 60mmol of L-proline are weighed by an analytical balance based on 100mL, the volume is adjusted to 100mL by using sterilized ultrapure water, 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin are added, and the mixture is filtered by using a 0.22 mu m filter.

Example 7

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing solution A: 6.0000g of glucose, 0.3000g of lactose, 0.3000g of raffinose, 0.0600g of sodium citrate, 0.0820g of potassium citrate, 0.9520g of HEPES reagent and 70mmol of L-proline are weighed by an analytical balance based on 100mL, the volume is adjusted to 100mL by using sterilized ultrapure water, 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin are added, and the mixture is filtered by using a 0.22 mu m filter.

Example 8

The embodiment provides semen cryopreservation liquid and preparation thereof.

1. The components: glucose, lactose, raffinose, sodium citrate, potassium citrate, HEPES reagent and L-proline, wherein the medicines are analytically pure; ultrapure water, skim milk powder, penicillin, streptomycin, dimethylformamide, vitamin E and fresh yolk.

Preparing solution A: in 100mL, using an analytical balance to weigh: 6g of glucose, 0.6g of lactose, 0.6g of raffinose, 0.16g of sodium citrate, 0.164g of potassium citrate, 2.984g of HEPES and 30mmol of proline; sterilized ultrapure water was added to 100ml, and the mixture was filtered through a 0.22 μm filter.

Preparing a sterilized skim milk B solution: weighing 16g of skimmed milk powder by using an analytical balance according to 100mL, fixing the volume to 100mL by using sterilized ultrapure water, carrying out high-pressure sterilization at 115 ℃ for 10min in an autoclave after the solid is completely dissolved, cooling to room temperature, and adding 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin.

Preparation of a cryopreservation liquid: according to 100mL, 69mL of semen dilution base solution, 25mL of 20% egg yolk solution, 5mL of dimethylformamide and vitamin E1mL are weighed by using a measuring cylinder, and are uniformly mixed to obtain 100mL of cryopreservation solution.

Example 9

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing solution A: in 100mL, using an analytical balance to weigh: 6g of glucose, 0.6g of lactose, 0.6g of raffinose, 0.16g of sodium citrate, 0.164g of potassium citrate, 2.984g of HEPES and 30mmol of proline; sterilized ultrapure water was added to 100ml, and the mixture was filtered through a 0.22 μm filter. Preparing a sterilized skim milk B solution: weighing 16g of skimmed milk powder by using an analytical balance according to 100mL, fixing the volume to 100mL by using sterilized ultrapure water, carrying out high-pressure sterilization at 115 ℃ for 10min in an autoclave after the solid is completely dissolved, cooling to room temperature, and adding 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin.

Preparation of a cryopreservation liquid: based on 100mL, 68mL of semen dilution base solution, 25mL of 20% egg yolk solution, 5mL of dimethylformamide and 2mL of vitamin E are weighed by using a measuring cylinder, and are uniformly mixed to obtain 100mL of cryopreservation solution.

Example 10

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing solution A: in 100mL, using an analytical balance to weigh: 6g of glucose, 0.6g of lactose, 0.6g of raffinose, 0.16g of sodium citrate, 0.164g of potassium citrate, 2.984g of HEPES and 30mmol of proline; sterilized ultrapure water was added to 100ml, and the mixture was filtered through a 0.22 μm filter. Preparing a sterilized skim milk B solution: weighing 16g of skimmed milk powder by using an analytical balance according to 100mL, fixing the volume to 100mL by using sterilized ultrapure water, carrying out high-pressure sterilization at 115 ℃ for 10min in an autoclave after the solid is completely dissolved, and adding 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin.

Preparation of a cryopreservation liquid: weighing 66mL of semen dilution base solution, 25mL of 20% egg yolk solution, 5mL of dimethylformamide and 4mL of vitamin E by using a measuring cylinder, and uniformly mixing to obtain 100mL of cryopreservation solution.

Example 11

The embodiment provides semen cryopreservation liquid and preparation thereof.

Preparing a sterilized skim milk B solution: weighing 16g of skimmed milk powder by using an analytical balance according to 100mL, fixing the volume to 100mL by using sterilized ultrapure water, carrying out high-pressure sterilization at 115 ℃ for 10min in an autoclave after the solid is completely dissolved, and adding 20 ten thousand IU of penicillin and 20 ten thousand IU of streptomycin.

Preparation of a cryopreservation liquid: and weighing 64mL of semen dilution base solution, 25mL of 20% egg yolk solution, 5mL of dimethylformamide and 6mL of vitamin E by using a measuring cylinder according to 100mL, and uniformly mixing to obtain 100mL of cryopreservation solution.

Example 12

The embodiment provides a method for preserving semen, which comprises the following specific steps:

1. semen collection

The male donkey of 3-7 years old is required to be healthy, free from diseases, excellent in body condition and moderate in fat condition. And (3) performing the public donkey training including feeding and movement 2 months before formal semen collection, wherein 6 eggs and 2 carrots are fed after concentrate is removed every day in the feeding aspect, and the movement aspect is performed for at least 1 hour in the donkey walking machine every day. Semen collection is carried out 2-3 times before semen is used, and the semen can be used after reaching the normal and stable standard. Artificial semen collection is carried out by adding 1500ml tap water at 42-45 deg.C and 1000 ml into artificial vagina, and lubricant is applied to 1/3 part by using disposable liner. Blowing air to swell the inner tube and keep a certain pressure, wherein the pressure is adjusted according to the length and the thickness of the penis of the donkey. After the penis of the donkey is erect, the penis of the donkey is washed clean by using sterile water at 37 ℃ and wiped dry. The semen collection person stands on the right rear side of the donkey, the teacher is adjusted to pull the breeding male donkey to climb across the fake donkey, the male donkey climbs across the female donkey and then pours the penis into the semen collection cylinder, the semen collection cylinder is kept still, the male donkey discharges air after ejaculation, the semen collection device is slowly removed, and the semen collection cup is immediately taken into the laboratory through the transmission window.

2. Semen treatment

After the semen is taken into a laboratory, the colloidal substances are removed by filtering through gauze, the semen is poured into a measuring cylinder along the side wall of the measuring cylinder, the volume of the semen is measured, 120 mu L of original semen is transferred and added into a 3mL physiological saline cuvette, and the semen density is measured by using a sperm densimeter.

The warming stand, slides, coverslips were preheated to 37 ℃ in advance. And (3) taking 7 mu L of the liquid from the liquid level by using a pipette, dripping the liquid on a glass slide, covering a cover glass to prevent bubbles from being generated, and detecting the sperm movement parameters by using a sperm full-automatic analysis system. When the total moving sperm rate (TM), the straight-line advancing sperm rate (PM), the curvilinear moving speed (VCL, mum/s), the average path speed (VAP, mum/s), the straight-line moving speed (VSL, mum/s) and the forward moving sperm rate (PM) are measured to be more than or equal to 70 percent, the freezing can be carried out. And (3) putting the semen qualified for the inspection into a 50mL centrifuge tube, adding the semen with the same volume to dilute the base solution, balancing for 10min at 25 ℃ in a dark place, centrifuging for 10min at 600 Xg, and discarding the supernatant.

3. Semen tubule manufacturing

And (5) after the semen is diluted, tubing. And filling the subpackaged semen by a Minitube full-automatic filling machine, wherein each frozen semen thin tube contains 0.5mL of semen diluent.

4. Semen freezing

After the fine tube semen is prepared, the mixture can be frozen after being balanced for 2 hours at 4 ℃ in the dark. Freezing is carried out according to a set cooling route by using a program freezer, and the freezing program is shown in table 1. Stopping the procedure when the freezing temperature is reduced to-140 deg.C, opening the cover of the freezing chamber, putting the fine tube frozen essence into liquid nitrogen, standing in the liquid nitrogen for at least 5min, and transferring into a liquid nitrogen tank for preservation.

Comparative example 1

This comparative example provides a semen cryopreservation solution and its preparation, differing from example 1 only in that L-proline is not added.

Comparative example 2

This comparative example provides a semen cryopreservation solution and its preparation, differing from example 8 only in that no vitamin E was added.

Test example 1

The experimental example is used for analyzing the influence of different semen preserving fluids in examples 1-7 and comparative example 1 on the semen preserving quality, and the specific analysis method is as follows:

1. semen collection

2. Semen treatment

The warming stand, slides, coverslips were preheated to 37 ℃ in advance. Using a pipette gunAnd 7 mu L of the liquid is taken down from the liquid level and is dripped on a glass slide, a cover glass is covered to prevent bubbles from being generated, and a sperm full-automatic analysis system is used for detecting sperm motion parameters. When the total moving sperm rate (TM), the straight-line advancing sperm rate (PM), the curvilinear moving speed (VCL, mum/s), the average path speed (VAP, mum/s), the straight-line moving speed (VSL, mum/s) and the forward moving sperm rate (PM) are measured to be more than or equal to 70 percent, the freezing can be carried out. And (3) putting the semen qualified for the inspection into a 50mL centrifuge tube, adding an equal volume of semen dilution base solution (different groups are set, P0 groups are added into the semen dilution base solution in the comparative example 1, and P1-P7 groups are sequentially added into the semen dilution base solutions in the examples 1-7 respectively), balancing at 25 ℃ in a dark place for 10min, centrifuging at 600g for 10min, and discarding the supernatant. Diluting to about 1X 10 with a cryopreservation solution⁸Per mL (the cryopreservation solution of comparative example 1 was added to the group P0; and the cryopreservation solutions of examples 1 to 7 were added to the groups P1 to P7, respectively).

3. Semen tubule manufacturing

And (5) after the semen is diluted, tubing. And (3) filling the subpackaged semen by adopting a Minitube full-automatic filling machine, wherein each frozen semen thin tube is 0.5mL of the semen diluent obtained in the step (2).

4. Semen freezing

5. Thawing of frozen semen in thin tube

After the frozen semen of the donkey tubule is preserved for about 7 days, a water bath unfreezing method is adopted, the tubule frozen semen is clamped out of liquid nitrogen, the frozen semen stays in air for 5-8s, the frozen semen is put into a water bath kettle with warm water at 38 ℃, after the frozen semen is stirred for 30s, water drops outside the tubule are taken out and wiped dry, the tubule is cut open, the semen is poured into a 1.5ml centrifuge tube, and the incubation is carried out for 10min at 37 ℃.

6. Sperm motility parameter detection

The warming stand, slides, coverslips were preheated to 37 ℃ in advance. After the semen diluent is uniformly mixed by using a pipette, 7 mu L of the semen diluent is taken from the liquid level and is dripped on a glass slide, a cover glass is covered to prevent bubbles from generating, and a sperm full-automatic analysis system is used for detecting sperm motion parameters. Images were acquired using a 20 Xphase contrast microscope in conjunction with a computer, 5 fields of view were read for each sample, and sperm motility parameters were assessed using Computer Assisted Sperm Analysis (CASA). Total motile sperm rate (TM), linear progressive motile sperm rate (PM), curvilinear motion velocity (VCL, μm/s), mean pathway velocity (VAP, μm/s), and linear motion velocity (VSL, μm/s) were evaluated, respectively.

7. Data analysis

One-way analysis of variance (ANOVA) and Ducan tests were performed using the span 18.0(SPSS inc., Chicago, IL, USA) statistical software, and the results of the analysis were expressed as mean and standard deviation.

8. Results of the experiment

After the donkey semen is frozen, preserved and thawed, the results of TM, PM, VCL, VSL and VAP are shown in Table 2 after CASA detection and analysis.

Compared with a control group (P0), the experimental group (P1-P7) added with L-proline has obviously increased TM and VCL. PM of P2, P3, P4 and P5 groups is obviously higher than that of a control group, but has no obvious difference with P6 and P7 groups, and P6 and P7 groups have no obvious difference with the control group. The VSL of the P2 group is obviously higher than that of the control group, but has no obvious difference with other experimental groups, and the VSL of the P3, P4, P5, P6 and P7 groups has no obvious difference with the control group. The P2 and P3 groups had significantly improved VAP compared with the control group, had no significant difference compared with other experimental groups, and the P1, P4, P5, P6 and P7 groups had no significant difference compared with the control group. The sperm motion parameters of TM, PM, VCL, VSL and VAP in the P2 group are all higher than those of the control group, and the sperm motion parameters of TM, PM, VCL and VAP in the P3 group are all higher than those of the control group.

TABLE 2 post-thaw motility analysis of sperm using different cryopreservation fluids to preserve semen (1)

Note: the lower case letters in the upper vertical row indicate significant differences between groups (P <0.05), and abcdefg indicates descending order of the mean values of the groups. P0 is control group (no proline added), and P1, P2, P3, P4, P5, P6 and P7 are groups with concentrations of L-proline of 10mmol, 20mmol, 30mmol, 40mmol, 50mmol, 60mmol and 70mmol respectively in 100mL of A solution. Total motile sperm rate (TM), linear progressive motile sperm rate (PM), curvilinear motion velocity (VCL, μm/s), mean pathway velocity (VAP, μm/s), linear motion velocity (VSL, μm/s).

Test example 2

This test example was conducted to analyze the effect of the different semen preserving solutions of examples 8 to 11(P9-P12) and comparative example 2(P8) on the quality of semen preservation, and the specific analysis method was the same as the test example except that the semen preserving solutions were different. After the donkey semen is frozen, preserved and thawed, the results of TM, PM, VCL, VSL and VAP are shown in Table 3 after CASA detection and analysis. The result shows that the vitamin E with different concentrations has a certain protection effect on frozen semen protection of donkey, the thawing activity is improved to some extent when the concentration of 1% is added, but the thawing activity is not obvious, when the concentration is continuously added, the activity of the thawed semen is improved by the concentration of 2%, the thawing activity begins to be reduced when the concentration is continuously improved to 4% and 6%, but the activity is still improved compared with that of a control group.

TABLE 3 post-thaw motility analysis of sperm using different cryopreservation fluids to preserve semen (2)

Note: the lower case letters in the vertical columns indicate significant differences between the groups (P < 0.05).

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The utility model provides an improve semen preservation quality's semen preservation liquid which characterized in that, calculates according to the part by volume, includes: 60-80 parts of semen dilution base solution and 20-30 parts of yolk solution;

wherein:

the semen dilution base solution comprises a solution A and a solution B;

each 100ml of the A liquid comprises the following components:

4-9g of mixed sugar, 0.1-0.6g of citrate, 10-150mmol of amino acid, 0.5-3g of buffer reagent and 10-80 ten thousand IU of antibiotic;

the liquid B is skimmed milk.

2. The semen preserving fluid as claimed in claim 1, wherein the amino acid is selected from one or more of L-proline, alanine, glycine, glutamine, cysteine, glutamic acid;

preferably, the amino acid is L-proline.

3. The semen preservation solution according to claim 1 or 2, wherein the mixed sugar is selected from at least two of glucose, lactose, raffinose and trehalose;

preferably, the mixed sugar comprises the following components in a mass ratio of (3-8): (0.1-0.5): (0.1-0.5) glucose, lactose and raffinose;

more preferably, the ratio of said mixed sugar to said amino acid is (5-7) g: (10-150) mmol.

4. The semen preserving fluid as claimed in any one of claims 1 to 3, wherein in the solution A:

the citrate is selected from one or more of potassium citrate, sodium citrate and calcium citrate; preferably, the citrate is a mixture of sodium citrate and potassium citrate, and the mass ratio of the sodium citrate to the potassium citrate is: potassium citrate ═ (5-6): (6-10);

and/or the buffer reagent is selected from one or more of HEPES, Tris, TES, MOPS and TAPSO;

and/or the antibiotic is selected from one or more of penicillin, streptomycin and gentamicin, preferably the penicillin and the streptomycin are selected according to the formula (1-2): 1 is compounded in proportion.

5. The semen preserving fluid as claimed in any one of claims 1 to 4, wherein the fat content in the skim milk is less than 0.1% and the protein content is greater than 3.0%; in the skimmed milk liquid, the mass volume percentage of the skimmed milk powder is 5-20%; preferably, the volume ratio of the liquid A to the liquid B is (4-6): 5;

and/or the presence of a gas in the gas,

in the yolk liquid, the volume percentage of yolk is not lower than 15%; preferably, the remaining components in the egg yolk liquid are the semen dilution base liquid.

6. The semen preservation solution according to any one of claims 1 to 5, further comprising 1 to 10 parts of glycerol; or the semen preserving fluid also comprises 1-10 parts of dimethylformamide and 1-10 parts of vitamin E.

7. The use of the semen preservation solution according to any one of claims 1 to 6 for preservation of semen, wherein the semen preservation solution according to any one of claims 1 to 5 is used for preservation of semen at normal or low temperature, or the semen preservation solution according to claim 6 is used for cryopreservation of semen.

8. The method for preparing a semen preserving fluid as claimed in any one of claims 1 to 6, which comprises the steps of:

1) preparing solution A, fixing the volume of the mixed sugar, citrate, buffer reagent and amino acid with sterilized ultrapure water, adding antibiotics, and filtering by using a 0.22-micron filter for later use;

4) preparing yolk liquid;

5) mixing the semen dilution base solution and the egg yolk solution or mixing the semen dilution base solution, the egg yolk solution and glycerol or mixing the semen dilution base solution, the egg yolk solution, dimethylformamide and vitamin E.

9. A method for preserving semen, characterized in that the normal temperature or low temperature preservation of semen is carried out by using the semen preserving fluid as claimed in any one of claims 1 to 5; or, the semen is frozen and preserved by using the semen preserving fluid of claim 6; the method comprises diluting semen with the semen preserving fluid;

preferably, the semen is from an equine animal, including a male horse, a male donkey, a wild horse, a wild donkey, and a zebra;

more preferably, the semen is diluted with the semen dilution base according to a ratio of 2: (1-3), balancing, centrifuging, and diluting with the cryopreservation solution until semen density is (2-10) × 10⁸one/mL.

10. The method according to claim 9, wherein when the semen preservation solution of claim 6 is used for semen cryopreservation, programmed freezing is performed after the dilution, and the freezing procedure is as follows:

programmed freezing to-140 deg.C, adding the obtained frozen semen into liquid nitrogen, standing for at least 5min, and storing.