WO2017139909A1 - Freezing diluent for livestock semen - Google Patents

Abstract

The present invention relates to a freezing diluent for livestock semen. Every 100 ml of the freezing diluent comprises the following components: 2.71 g of trihydroxymethyl aminomethane, 1.4 g of citric acid, 1.0 g of monosaccharide, 5-20 ml of fresh egg yolk, 0-10 ml of a permeable protecting agent, 0.1 million IU of penicillin, 0.1 million IU of streptomycin, 0.05-5 g of polyvinyl alcohol, and the remaining being ultrapure water. According to the present invention, for the first time, a common high-molecular polymer polyvinyl alcohol is used as an ice crystal inhibitor instead of a natural antifreeze protein, and is applied to the cryopreservation research of livestock semen. The survival rate of defrosted sperm is around 75%, the activity thereof is 50% or higher, the acrosome completeness is 65% or higher, the plasma membrane completeness is around 50%, and the non-return rate after artificial insemination is higher than 70%.

Description

Livestock semen cryopreservation solution Technical field

The invention relates to the technical field of animal reproductive physiology and reproduction, in particular to a frozen dilution solution of livestock semen.

Background technique

Although the semen cryopreservation study has been carried out for more than 50 years, the semen activity after thawing is only about 30%-50%. The rate of non-return and fertility after artificial insemination is significantly lower than that of fresh semen. During the freezing process of livestock semen, the mechanical damage caused by the ice crystal formed inside and outside the cell directly causes the sperm motility after freezing. How to reduce the occurrence of ice crystals The damage of sperm in livestock has been the research field of livestock breeding technology and low temperature biology research. At the hot spot, the damage to sperm can be reduced by changing the composition of the semen diluent. At present, osmotic organic solvents and sugars have been used in the cryopreservation of mammalian semen, but organic solvents and sugars cannot completely solve the problem. Internal and external ice crystals cause mechanical damage to sperm.

In 1969, DeVries and Wohlschlag discovered a new type of ice crystal inhibitor, antifreeze protein. Antifreeze proteins reduce the mechanical damage of ice crystals to tissue cells by modifying ice crystal traits and inhibiting recrystallization. Some studies have shown that antifreeze proteins can improve the survival rate of human and some mammalian semen after cryopreservation. However, the application of antifreeze proteins also has certain limitations. First, as a heterologous protein, antifreeze proteins may cause certain immune responses in animals. Secondly, the main source of antifreeze proteins is extracted from the blood of polar marine fish, and these technologies and patents are mainly in Europe and America. In the hands of the state, the price is high; finally, the antifreeze protein inhibits the formation of ice crystals by changing the shape of the ice crystals, which leads to the formation of some sharp needle-shaped ice crystals, thereby aggravating the mechanical damage of the sperm by freezing. There is still some controversy about whether antifreeze protein can improve the quality of frozen sperm.

The development of more widely used, safe and reliable ice crystal inhibitors is the key to improving the promotion of semen freezing technology in the livestock industry. It can rapidly expand the genes of excellent male animals, improve varieties and improve breeding efficiency.

Polyvinyl alcohol is a high molecular organic compound, and pharmaceutical grade polyvinyl alcohol is non-toxic and has no side effects. It is widely used as an emulsifier and film former in the fields of medicine and cosmetics. Chinese patent "a kind of pig semen diluent" patent number: 201310478237.6 announced the use of polyvinyl alcohol in the dilute preservation of pig semen at room temperature, the Chinese patent "pork semen cryoprotectant" patent number: 201210196733.8 announced the polyvinyl alcohol It is applied to the low temperature dilution of 0-4 °C of pig semen. When semen is stored at room temperature or 0-4 °C, it mainly reduces the energy loss by inhibiting sperm activity, so that the sperm can maintain the insemination ability under static state, and it can be used within 1-7 days, which expands the use of excellent male semen. Scope, the use of polyvinyl alcohol in these technologies, mainly as a film-forming agent to protect sperm cell membrane activity, while reducing the sedimentation rate of sperm, play a role in protecting sperm cells when diluted at room temperature and stored at 0-4 ° C. However, no studies have reported the use of polyvinyl alcohol as an ice crystal inhibitor for the cryopreservation of sperm.

Summary of the invention

In order to solve the above problems, the present invention finds for the first time that the non-permeable high molecular polymer polyvinyl alcohol has the same effect as the antifreeze protein for inhibiting the re-formation of ice crystals, and can be used instead of the antifreeze protein for cryopreservation of livestock semen, thereby avoiding Defects related to the application of antifreeze proteins have not been reported in the literature.

The frozen liquid dilution of livestock semen of the present invention comprises the following components per 100 ml:

The balance is ultrapure water.

Preferably, the polyvinyl alcohol has a molecular weight of 9 KD.

Preferably, the monosaccharide is glucose, fructose, mannitol, sorbitol, galactose or xylitol.

Preferably, the osmoprotectant is glycerin.

The method for preparing a frozen liquid dilution of livestock semen according to the present invention comprises the steps of weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of monosaccharide, 0-10 ml of osmotic protective agent, and 100,000 penicillin. IU, streptomycin 100,000 IU, polyvinyl alcohol 0.05-5 g, dissolved in ultrapure water, stir evenly, adjust the pH to 6.8-7.2, add 5-20ml of fresh egg yolk inactivated at 56 °C for 30 min before use. After mixing well, the volume was adjusted to 100 ml, and the mixture was centrifuged at 15,000 rpm for 1 hour at 4 ° C, and the supernatant was filtered through a 0.45 μm disposable filter for use.

The frozen liquid dilution of the livestock semen of the present invention can be subjected to one-step cryopreservation of the livestock semen by mixing the livestock semen and the frozen semen of the livestock semen of the present invention in a ratio of 1:4-1:10, and then sub-packaging. In a 0.25 ml plastic thin tube, slowly cool down to 5 ° C, then quickly move into the liquid nitrogen phase for 5 min, and finally put the thin tube into liquid nitrogen for cryopreservation.

The livestock semen freezing dilution liquid of the invention can also perform the two-step cryopreservation of the livestock semen by mixing the livestock semen and the frozen diluent without the osmotic protective agent in a ratio of 1:4, and then sub-packaging. In a 2 ml cryotube, slowly cool down to 5 ° C, then mix the above suspension and the frozen semen dilution of the livestock semen of the present invention in a ratio of 1:1, further equilibrate at 1-5 h for 5 h, using pipetting The device was pipetted into a drop of 0.2 ml of dry ice and finally frozen pellets were placed in liquid nitrogen for cryopreservation.

The two-step cryopreservation method of the present invention comprises the osmotic protective agent-free frozen diluent, which is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, and 1.0 g of monosaccharide, respectively. Penicillin 100,000 IU, streptomycin 100,000 IU, polyvinyl alcohol 0.05-5 g, soluble in ultrapure water, stir well, add 5-20ml of fresh egg yolk inactivated at 56 ° C for 30 min before use, mix well and dilute to volume 100 ml, centrifuged at 15,000 rpm for 1 hour at 4 ° C, and the supernatant was filtered through a 0.45 μm disposable filter for use.

For the cryopreservation of goat semen, in order to remove the adverse effects of phosphatase A and egg yolk interaction on sperm survival in seminal plasma, the semen and washing solution were mixed at a ratio of 1:10, centrifuged at 600 g for 15 min, and the supernatant was taken. Repeat the washing of the sperm pellet once, and the rest of the steps are the same as the sheep.

In the cryopreservation method of the present invention, the cleaning liquid for freezing the goat semen is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of monosaccharide, 100,000 IU of penicillin, and a chain. 100,000 IU of the peptide and 0.05-5 g of polyvinyl alcohol were made up to 100 ml with ultrapure water and filtered through a 0.22 μm disposable filter.

The quality of the semen used in the cryopreservation of the present invention is: the vigor is greater than 75%, the deformity rate is less than 10%, and the sperm density is higher than 1 x 10 ⁹ /ml.

In the technical solution of the present invention, the trishydroxymethylaminomethane is a buffer substance, citric acid is a solute, and may also participate in glycolysis, the monosaccharide is a sperm metabolism substrate, and the egg yolk is a low temperature shock plasma membrane. Guarantee The protective agent, the glycerin is a osmotic protective agent, the penicillin and streptomycin are antibiotics, and the polyvinyl alcohol is an ice crystal inhibitor. Our research shows that although the molecular structure is different from the antifreeze protein, polyvinyl alcohol has a significant inhibitory effect on the re-formation of ice crystals after thawing, thereby reducing the secondary damage of ice crystals to sperm cells. In addition, polyvinyl alcohol inhibits ice crystal formation. The ability is higher than other high molecular polymers, such as dextran and polyvinylpyrrolidone.

The beneficial effects of the present invention are as follows:

1. The first application of polyvinyl alcohol as an ice crystal inhibitor to the cryopreservation of livestock semen: The present invention is aimed at the main problems in the current research on the cryopreservation of livestock semen. For the first time, the common high molecular polymer, polyvinyl alcohol, is used as ice crystal suppression. The agent replaces the natural antifreeze protein for the cryopreservation study of livestock semen. Polyvinyl alcohol can effectively inhibit the formation of ice crystals during the thawing process of frozen semen, reduce the mechanical damage of ice crystals to sperm, improve the quality of frozen semen in livestock, and improve the rate of artificial insemination. A large number of experiments have shown that the sperm semen stored frozen by the frozen semen of livestock semen of the present invention has a sperm activity of about 75% after thawing, an activity of more than 50%, acrosome integrity of more than 65%, and plasma membrane integrity. At about 50%, the rate of non-return after artificial insemination is over 70%.

2. Safe and reliable, low cost: The polyvinyl alcohol used in the present invention is a common chemical raw material with wide source, low cost, guaranteed purity, stable chemical properties, and ice crystal suppression using polyvinyl alcohol as a semen frozen diluent. The agent has the advantages of no immune reaction, low toxicity, guaranteed purity, and low price.

detailed description

The technical solutions of the present invention are further illustrated by the following specific embodiments, but the technical solutions of the present invention are not limited to the embodiments.

Example 1:

The semi-fine wool sheep semen of Yunnan was collected by electric stimulation, and then the semen quality was tested. The semen index for cryopreservation must meet: viability greater than 75%, sperm density higher than 1 × 10 ⁹ /ml, deformity rate less than 10%, semen volume 1-2ml.

The semen cryopreservation solution is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 5 ml of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and polyvinyl alcohol 0.1. g, soluble in ultrapure water, stir well, add 10ml of fresh egg yolk inactivated at 56 °C for 30min before use. After mixing well, the volume was adjusted to 100 ml, and the mixture was centrifuged at 15,000 rpm for 1 hour at 4 ° C, and the supernatant was filtered through a 0.45 μm disposable filter for use.

Mix the sheep semen and frozen dilutions that meet the freezing requirements in a ratio of 1:5, then disperse them in 0.25ml plastic tubules, slowly cool down to 5 °C, further equilibrate at this temperature for 2 hours, and then transfer the liquid nitrogen. Fumigation for 5 min in the phase, and finally into liquid nitrogen for cryopreservation.

The method of cryopreservation of livestock semen, the survival rate of sperm after thawing is about 75%, the vitality is above 50%, the acrosome integrity is about 65%, the plasma membrane integrity is about 50%, and the rate of return after artificial insemination is not More than 70%.

Example 2:

The Nubian sheep semen was collected using a fake vagina, and then the semen quality test was performed. The semen index for cryopreservation must meet: viability greater than 75%, sperm density greater than 1 x 10 ⁹ /ml, malformation rate less than 10%, semen volume 0.75-2 ml.

The semen cleaning solution is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of fructose, 100,000 IU of penicillin, 100,000 IU of streptomycin, 0.2 g of polyvinyl alcohol, and dissolved in Ultra-pure water, mix well and dilute to 100 ml, and filter with 0.22 μm disposable filter for use.

The semen frozen dilution solution is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of fructose, 7 ml of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and 0.2 of polyvinyl alcohol. g, soluble in ultrapure water, stir well, add 15ml of fresh egg yolk inactivated at 56 ° C for 30 min before use, mix well and dilute to 100 ml, centrifuge at 15000 rpm for 1 hour at 4 ° C, take supernatant The solution was filtered through a 0.45 μm disposable filter for use.

The goat semen and washing solution meeting the freezing requirements were uniformly mixed at a ratio of 1:4, and centrifuged at 2500 rpm for 10 minutes at room temperature. After removing the supernatant, the sperm was washed once under the same centrifugation conditions. Add the cleaning solution and resuspend the sperm to the original volume. The washed sperm suspension and the frozen diluent are uniformly mixed in a ratio of 1:8, and then sub-packed in a 0.25 ml plastic thin tube, and then slowly cooled to 5 ° C, further equilibrated at this temperature for 2 hours, and then rapidly transferred into the liquid. Fumigation in the nitrogen phase for 5 min, and finally into liquid nitrogen for cryopreservation.

The method of cryopreserved livestock semen, the sperm survival rate after thawing is about 75%, the vitality is above 50%, the acrosome integrity is about 60%, the plasma membrane integrity is about 50%, and the rate of return after artificial insemination is not Reach More than 70%.

Example 3:

Semen terracotta ram semen was collected using a false vaginal method, and semen quality testing was performed immediately. The semen index for cryopreservation must meet: viability greater than 75%, sperm density above 1 × 10 ⁹ /ml, and semen volume 1-2 ml.

Preparing a frozen diluent without a osmotic protective agent, which is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 100,000 IU of penicillin, and 100,000 IU of streptomycin, respectively. Polyvinyl alcohol 1.0g, dissolved in ultrapure water, stirred evenly, add 20ml of fresh egg yolk inactivated at 56 ° C for 30min before use, mix well and dilute to 100ml, centrifuge at 15000 rev / min for 1 hour at 4 ° C The supernatant was filtered through a 0.45 μm disposable filter for use.

The semen cryopreservation solution is configured by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of fructose, 10 ml of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and polyvinyl alcohol 1.0. g, soluble in ultrapure water, stir well, add 20ml of fresh egg yolk inactivated at 56 °C for 30min before use, mix well and dilute to 100ml, centrifuge at 15000 rpm for 1 hour at 4 °C, take supernatant The solution was filtered through a 0.45 μm disposable filter for use.

The semen which meets the freezing requirement and the frozen diluent which does not contain the osmotic protective agent are uniformly mixed in a ratio of 1:4, and then sub-packaged in a 2 ml cryotube, and then slowly lowered to 5 ° C, and then the above suspension and the present invention The frozen liquid dilution of the livestock semen was uniformly mixed in a ratio of 1:1, further equilibrated at 5 ° C for 1 h, and a 0.2 ml drop was pipetted into dry ice to be pre-frozen, and finally the frozen pellet was placed in liquid nitrogen for cryopreservation.

The method of cryopreserved livestock semen, the sperm survival rate after thawing is about 75%, the vitality is about 60%, the acrosome integrity is about 70%, the plasma membrane integrity is about 55%, and the artificial insemination does not return. About 75%.

In the above examples, the molecular weight of the polyvinyl alcohol used is 9KD. Since the polyvinyl alcohol is a high molecular polymer, if the molecular weight is too high, it may form an "ice mass" during the freezing process, causing more serious damage to the sperm cells. If the molecular weight is too low, it may not be effective in inhibiting the formation of ice crystals.

The above embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention.

The above-described livestock semen cryopreservation formula and freezing procedure provided by the present invention are described in detail. The principles and embodiments of the present invention are described herein by way of specific embodiments. It is only used to help understand the method of the present invention and its core ideas. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims (5)

1. A frozen liquid dilution of livestock semen characterized by comprising the following components per 100 ml:

   
2. The frozen liquid dilution of livestock semen according to claim 1, wherein the polyvinyl alcohol has a molecular weight of 9 kD.
3. The frozen liquid dilution of livestock semen according to claim 1, wherein the monosaccharide is one or a combination of glucose, fructose, mannitol, sorbitol, galactose or xylitol.
4. The frozen liquid dilution of livestock semen according to claim 1, wherein the osmoprotectant is glycerin.
5. The frozen liquid dilution of livestock semen according to claim 1, wherein the fresh frozen egg yolk is subjected to inactivation treatment at 56 ° C for 30 minutes.