Use of animal sperm liquid preservation diluent

Abstract

The application relates to application of an animal sperm liquid storage diluent. The animal sperm liquid storage diluent can be used for storing animal sperm; the animal sperm liquid storage diluent is prepared by the following method: (1) mixing glucose, fructose, tris-hydroxymethyl aminomethane, citric acid and water to obtain a mixture 1; the mass-volume ratio of the glucose, the fructose, the tris-hydroxymethyl aminomethane, the citric acid and the water is (10-20) g:(10-20) g:(20-30) g:(10-20) g:(50-100) ml; (2) mixing edible mixed plant oil and the mixture 1 of the step (1), stirring, the volume ratio of the edible mixed plant oil and the water is 50:(50-100), to obtain a mixture 2; standing, discarding the upper oil, refrigerating, diluting with sterilized distilled water. The application has the advantages of wide application range, high pregnancy rate, long animal sperm liquid storage time, good storage activity and the like.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number was 202311772753.X, the application date was December 21, 2023, and the patent title at the time of application was: A liquid preservation diluent for animal sperm and its preparation method. Technical Field

[0002] This invention belongs to the field of sperm preservation technology, and in particular relates to a method for preserving animal sperm. Background Technology

[0003] Currently, farms raising deer, foxes, raccoon dogs, dogs, and sheep lack the facilities for freezing animal semen. Furthermore, except for bovine capillary semen freezing, which is commercially and professionally available, existing techniques for other animals involve insemination immediately after collection or preservation for 2-3 days before fresh semen insemination. To avoid inbreeding, farms often want to exchange male animals for different bloodlines. However, if the distance is far, to ensure conception rates during the estrus season, male animals need to be transported to another farm for mating. This is costly, labor-intensive, and stressful for the male animals, impacting the farm's economic efficiency. Farms urgently need a diluent for preserving animal semen in liquid form with a long preservation time and high viability.

[0004] An invention patent entitled "A Method for Cryogenic Preservation of Animal Sperm" (application number CN201910990863, publication number CN111066775A) discloses a method for cryogenic preservation of animal sperm. The method uses an animal sperm diluent containing glycerol, defatted wheat protein powder, defatted pea protein powder, defatted peanut protein powder, and defatted soy protein powder. In this method, large molecular proteins, bacteria, and viruses are lysed using ultrasound. Dog sperm preserved using this method showed a sperm motility of 0.6. However, in practical applications, it has been found that while the method uses ultrasound for sterilization and microbial elimination, the lysed bacteria or microorganisms remain in the diluent. A consistently low-temperature environment must be maintained during the lysing process. Furthermore, the method has strict requirements for lysing time, but human error inevitably occurs during actual operation, affecting the final result. Insufficient lysing time leads to inadequate sterilization, while excessively long ultrasonic sterilization time can easily cause temperature rise and destroy the activity of nutrients in the diluent. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an animal sperm liquid preservation diluent, preparation method and application, which has the advantages of wide application range, high conception rate, long liquid preservation time of animal sperm and good preservation motility.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides a liquid preservation diluent for animal sperm. The components for preparing the liquid preservation diluent for animal sperm include glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid, and a variety of edible vegetable blends. The volume-to-mass ratio of the edible vegetable blends to glucose, fructose, tris(hydroxymethyl)aminomethane, and citric acid is 50 ml: (10-20) g: (10-20) g: (20-30) g: (10-20) g.

[0008] Furthermore, the components for preparing the liquid preservation diluent for animal sperm also include water, with a mass-to-volume ratio of glucose to water of (10-20) g: (50-100) ml.

[0009] Furthermore, the components for preparing the liquid preservation diluent for animal sperm also include dilution water.

[0010] For example, the volume of dilution water can be nine times the volume of the mixture, which is the product of mixing glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid, various edible vegetable blends, and water, after discarding the upper layer of oil.

[0011] The beneficial effects of adopting the above technical solution include: This invention uses a blend of various edible vegetable oils to replace plant proteins, and the prepared liquid preservation diluent for animal sperm can pass through a filter membrane; the preparation and use methods are simple. The unsaturated fatty acids contained in the blended vegetable oils can act as cryoprotectants, and their various nutrients can effectively increase antioxidant capacity and reduce oxidative stress in sperm. The diluent provided by this invention does not contain glycerol, thus avoiding the weak toxicity of glycerol to sperm. This invention has the advantages of simple preparation method, wide applicability, high conception rate, long preservation time, and good sperm motility preservation.

[0012] This invention provides a method for preparing a liquid preservation diluent for animal sperm, comprising the following steps: mixing the components.

[0013] The beneficial effects of adopting the above technical solution include: the animal sperm liquid preservation diluent prepared by the above method has the advantages of wide applicability, high conception rate, long preservation time and good preservation viability.

[0014] Furthermore, the following steps may be included:

[0015] (1) Mix glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid and water to obtain mixture 1;

[0016] (2) Mix the blended vegetable oils with the mixture from step (1) to obtain mixture 2.

[0017] The beneficial effects of adopting the above technical solution include: it is more conducive to thorough mixing.

[0018] Furthermore, in step (1), the mass-volume ratio of glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid and water is (10-20) g: (10-20) g: (20-30) g: (10-20) g: (50-100) ml.

[0019] In step (2), the volume ratio of edible blended oil and water is 50:(50-100).

[0020] The beneficial effects of adopting the above technical solution include: the above preparation method is simple to operate and economical in terms of materials; the animal sperm liquid preservation diluent prepared by the above method has the advantages of wide applicability, high conception rate, long shelf life and the ability to improve sperm motility.

[0021] Furthermore, after mixing in step (2), the step of discarding the upper layer of grease is also included.

[0022] The beneficial effects of adopting the above technical solution include: the animal sperm liquid preservation diluent prepared by this invention easily passes through filter membranes, has high clarity under a microscope, and is safer for animal mating. Compared with non-GMO soybean oil, peanut oil, and cold-pressed olive oil, the blended vegetable oils used in this invention have the best effect.

[0023] Furthermore, it also includes the step of diluting with dilution water.

[0024] The beneficial effects of adopting the above technical solution include: diluting the animal sperm preservation solution to a suitable concentration to facilitate its use.

[0025] This invention also provides the application of the above-mentioned animal sperm liquid preservation diluent in the preservation of animal sperm. For example, it can be used to preserve sperm from animals such as cattle, deer, foxes, raccoon dogs, dogs, and sheep, and has advantages such as wide applicability, high conception rate, long preservation time, and good preservation motility.

[0026] This invention provides a method for preserving sperm, comprising the following steps:

[0027] (1) Filter the above-mentioned animal sperm liquid preservation diluent, dilute the collected sperm with the filtered liquid to obtain a solution containing sperm, and put it into a container and seal it;

[0028] (2) Store after cooling.

[0029] The beneficial effects of adopting the above technical solutions include: using the above-mentioned sperm preservation methods can significantly improve sperm preservation time and motility.

[0030] Furthermore, a 0.25µm filter membrane is used for filtration.

[0031] Furthermore, the sperm is kept at a temperature of 20-35℃ from collection until storage.

[0032] Furthermore, in step (1), the collected sperm is diluted with the filtered liquid at a sperm count of 3 million / ml to 10 million / ml.

[0033] Furthermore, in step (2), the temperature is reduced at a rate of 0.1-0.5℃ / min.

[0034] Furthermore, in step (2), the temperature is lowered to 0-17℃ for storage.

[0035] The beneficial effects of adopting the above technical solution include: using a 0.25µm filter membrane solves the problem of poor filtration effect. Filtering the animal sperm liquid preservation diluent through a 0.25µm syringe filter can remove microorganisms, etc., and the filtered animal sperm liquid preservation diluent is less prone to bacterial growth during sperm preservation. Detailed Implementation

[0036] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0037] This invention provides a liquid preservation diluent for animal sperm, the components of which include glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid, a variety of edible vegetable blends and sterilized water, or one or more of these components.

[0038] Preferably, the animal sperm liquid preservation diluent includes glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid, a blend of various edible vegetable oils, and sterile water. The volume-to-mass ratio of the blended edible vegetable oils to glucose, fructose, tris(hydroxymethyl)aminomethane, and citric acid is 50 ml : (10-20) g : (10-20) g : (20-30) g : (10-20) g; the volume-to-mass ratio of glucose to sterile water is (10-20) g : (50-100) ml.

[0039] That is, the proportions of each component are as follows: per 50ml-100ml of water, glucose 10g-20g, fructose 10g-20g, tris(hydroxymethyl)aminomethane 20g-30g, citric acid 10g-20g, and 50ml of edible blended vegetable oil.

[0040] The components for preparing a liquid preservation diluent for animal sperm may also include dilution water; the volume of the dilution water may be 9 times the volume of the mixture, wherein the mixture is a product obtained by mixing glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid, a variety of edible vegetable blends, and sterile water, and then discarding the upper layer of oil.

[0041] The animal sperm liquid preservation diluent provided by this invention has advantages such as long preservation time and good sperm motility. This invention uses a blend of edible vegetable oils instead of glycerin and various plant powders, simplifying the preparation process. It can be prepared on-site, eliminating the need for expensive equipment like ultrasonic lysis equipment, making it highly practical and widely applicable. It also eliminates the need for precise control of ultrasonic lysis time, reducing operational errors. The inventors unexpectedly discovered during their research that the addition of a blend of edible vegetable oils to the animal sperm liquid preservation diluent effectively increases antioxidant capacity and reduces sperm oxidative stress. Sperm oxidative stress can cause DNA damage, primarily DNA breakage, and chromatin cross-linking in sperm through free radicals, leading to sperm structural damage, impaired function, and even sperm death. The animal sperm liquid preservation diluent provided by this invention has a significant effect on improving sperm motility and extending preservation time during refrigeration. Experimental verification shows that, compared with existing technologies, the animal sperm liquid preservation diluent provided by this invention, when refrigerated at 4℃, increases sperm motility by 5% after 5 days of storage, and extends sperm survival time from 4-9 days to 10 days.

[0042] This invention provides a method for preparing the above-mentioned liquid preservation diluent for animal sperm, comprising the following steps: mixing the components in a specified proportion. Specifically, the preparation can be carried out using the following steps:

[0043] (1) Weigh out glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid and sterile water, mix and dissolve each component in a volumetric flask to obtain mixture 1; the proportions of each component are as follows: per 50ml-100ml of water, tris(hydroxymethyl)aminomethane 20g-30g, citric acid 10g-20g, glucose 10g-20g, fructose 10g-20g;

[0044] (2) Measure out a blend of edible vegetable oils, with a volume ratio of edible vegetable oil to water of 50:(50-100).

[0045] (3) Mix the mixture 1 from step (1) with the blended edible vegetable oil from step (2) and shake well to obtain mixture 2. After standing at room temperature, the oil and water are separated, the upper layer of oil is discarded, and the mixture is refrigerated at 4°C to obtain a novel liquid preservation diluent for animal sperm.

[0046] Removing the upper layer of oil makes it easier to observe sperm motility under a microscope, avoiding the inability to observe sperm motility due to fat droplets.

[0047] Furthermore, it may also include a dilution step, in which the novel animal sperm liquid preservation diluent obtained in step (3) is diluted with water, and the dilution ratio can be 10 times, that is, the novel animal sperm liquid preservation diluent is diluted with sterile water at a volume ratio of 1:9.

[0048] In existing technologies, the formulation of the chosen sperm diluent often makes filtration difficult or even impossible. The diluent provided by this invention can be filtered using a 0.25µm filter membrane, solving the problem of poor filtration efficiency. Filtering the animal sperm liquid preservation diluent through a 0.25µm syringe filter removes microorganisms, and the filtered animal sperm liquid preservation diluent is less prone to bacterial growth during sperm preservation.

[0049] This invention also provides a method for preserving sperm using the above-mentioned liquid sperm preservation diluent, comprising the following steps:

[0050] (1) Place the collected animal semen in a thermos and keep the temperature at 20-35℃, then bring it to the laboratory;

[0051] (2) Measure the density of the animal semen in step (1) using a spectrophotometer (i.e., a sperm density meter), filter the above animal sperm liquid preservation diluent with a 0.25um filter membrane, dilute the animal sperm with the filtered diluent according to a sperm count of 3 million / ml-10 million / ml, aspirate the diluted semen into a 0.25ml capillary tube or fill the capillary tube with the diluted semen using a filling and sealing machine and seal it.

[0052] (3) Place the semen from the thin tube in step (2) into a cooling cabinet and cool it down at a rate of 0.1-0.5℃ / min until it reaches 0-17℃ for long-term storage.

[0053] Unless otherwise specified, the experimental methods used in this invention are all conventional experimental methods in the field; the materials, reagents and instruments used are all conventional materials, reagents and instruments in the field, which can be obtained through commercial channels or prepared by conventional methods.

[0054] This blended edible vegetable oil was purchased from Yihai Kerry Arawana Holdings Co., Ltd. The ingredients include: soybean oil 65%, rapeseed oil 21%, peanut oil 6%, sunflower seed oil 4%, corn oil 2%, rice bran oil 1%, sesame oil 0.6%, and flaxseed oil 0.4%, all percentages by weight. The raw materials for the corn oil, soybean oil, and rapeseed oil are non-GMO corn, non-GMO soybeans, and non-GMO rapeseed, respectively. The mass ratio of saturated fat, monounsaturated fat, and polyunsaturated fat in this blended edible vegetable oil is 14:34:52.

[0055] The following is a description through specific embodiments.

[0056] Example 1

[0057] This invention provides a method for preparing a liquid preservation diluent for animal sperm, comprising the following steps:

[0058] (1) Weigh 25g of tris(hydroxymethyl)aminomethane, 15g of citric acid, 17g of glucose and 15g of fructose using an analytical balance, measure 50ml of sterile distilled water using a graduated cylinder, mix the components to obtain mixture 1;

[0059] (2) Measure 50ml of edible blended vegetable oil;

[0060] (3) Mix the mixture from step (1) and the blended vegetable oil from step (2), stir on a magnetic stirrer for 60 minutes to obtain mixture 2, let stand at room temperature, discard the upper layer of oil, and refrigerate at 4°C to obtain a novel liquid preservation diluent for animal sperm that is 10 times concentrated.

[0061] (4) When using, dilute the new animal sperm liquid preservation diluent, which is concentrated 10 times, with sterile distilled water 10 times, that is, the volume ratio of the new animal sperm liquid preservation diluent to sterile distilled water is 1:9.

[0062] This invention provides a method for preserving sperm, comprising the following steps:

[0063] (1) Place the collected animal semen in a thermos and keep the temperature at 20-35℃, then bring it to the laboratory;

[0064] (2) Measure the density of the animal semen in step (1) using a spectrophotometer. Filter the novel animal sperm liquid preservation diluent prepared by the above method using a 0.25um filter membrane. The liquid preservation diluent can easily pass through a 0.25um needle filter. Dilute the fresh semen with the filtered diluent to make the sperm count density reach 3 million / ml-10 million / ml. Aspirate the diluted semen into a 0.25ml capillary tube or fill the capillary tube with a filling machine and seal it.

[0065] (3) Place the thin tube semen or the filled semen from step (2) into a cooling cabinet and cool it down at a rate of 0.5℃ / min until it reaches 4℃ for long-term storage.

[0066] Example 2

[0067] This invention provides a method for preparing a liquid preservation diluent for animal sperm, comprising the following steps:

[0068] (1) Weigh 20g of tris(hydroxymethyl)aminomethane, 10g of citric acid, 10g of glucose and 10g of fructose using an analytical balance, measure 100ml of sterile distilled water using a graduated cylinder, mix the components to obtain mixture 1;

[0069] (2) Measure 50ml of edible blended vegetable oil;

[0070] (3) Mix the mixture from step (1) and the blended vegetable oil from step (2), stir on a magnetic stirrer for 60 minutes to obtain mixture 2, let stand at room temperature, discard the upper layer of oil, and refrigerate at 4°C to obtain a novel liquid preservation diluent for animal sperm that is 10 times concentrated.

[0071] (4) When using, dilute the new animal sperm liquid preservation diluent, which is concentrated 10 times, with sterile distilled water 10 times, that is, the volume ratio of the new animal sperm liquid preservation diluent to sterile distilled water is 1:9.

[0072] This invention provides a method for preserving sperm, comprising the following steps:

[0073] (1) Place the collected animal semen into a thermos and keep the temperature at 20°C, then bring it to the laboratory;

[0074] (2) Measure the density of the animal semen in step (1) using a spectrophotometer. Filter the novel animal sperm liquid preservation diluent prepared by the above method using a 0.25um filter membrane. The liquid preservation diluent can easily pass through a 0.25um needle filter. Dilute the fresh semen with the filtered diluent to make the sperm count density reach 3 million / ml. Aspirate the diluted semen into a 0.25ml capillary tube or fill the capillary tube with a filling machine and seal it.

[0075] (3) Place the thin tube semen or the filled semen from step (2) into a cooling cabinet and cool it down at a rate of 0.1℃ / min until it reaches 0℃ for long-term storage.

[0076] Example 3

[0077] This invention provides a method for preparing a liquid preservation diluent for animal sperm, comprising the following steps:

[0078] (1) Weigh 30g of tris(hydroxymethyl)aminomethane, 20g of citric acid, 20g of glucose and 20g of fructose using an analytical balance, and measure 80ml of sterile distilled water using a graduated cylinder. Mix the components to obtain mixture 1.

[0079] (2) Measure 50ml of edible blended vegetable oil;

[0080] (3) Mix the mixture from step (1) and the blended vegetable oil from step (2), stir on a magnetic stirrer for 60 minutes to obtain mixture 2, let stand at room temperature, discard the upper layer of oil, and refrigerate at 4°C to obtain a novel liquid preservation diluent for animal sperm that is 10 times concentrated.

[0081] (4) When using, dilute the new animal sperm liquid preservation diluent, which is concentrated 10 times, with sterile distilled water 10 times, that is, the volume ratio of the new animal sperm liquid preservation diluent to sterile distilled water is 1:9.

[0082] This invention provides a method for preserving sperm, comprising the following steps:

[0083] (1) Place the collected animal semen into a thermos and keep the temperature at 28°C, then bring it to the laboratory;

[0084] (2) Measure the density of the animal semen in step (1) using a spectrophotometer. Filter the novel animal sperm liquid preservation diluent prepared by the above method using a 0.25um filter membrane. The liquid preservation diluent can easily pass through a 0.25um needle filter. Dilute the fresh semen with the filtered diluent to make the sperm count density reach 10 million / ml. Aspirate the diluted semen into a 0.25ml capillary tube or fill the capillary tube with a filling machine and seal it.

[0085] (3) Place the thin tube semen or the filled semen from step (2) into a cooling cabinet and cool it down at a rate of 0.2℃ / min until it reaches 17℃ for long-term storage.

[0086] Example 4

[0087] The novel animal sperm liquid preservation diluent was prepared in Example 1 to preserve deer sperm. In the process of preserving deer sperm, the temperature was maintained at 35°C in step (1), and in step (2), the fresh sperm was diluted with the filtered diluent to achieve a sperm count density of 5 million / ml. All other steps were the same as in Example 1.

[0088] Using the above methods, red deer sperm and sika deer sperm were preserved respectively. After testing, the conception rate of red deer sperm preserved in the laboratory for 7 days was 95%, and that of sika deer sperm was 72%.

[0089] Sperm motility of red deer preserved for 1-10 days was detected by microscopic examination, and the results are shown in Table 1. Table 1 shows that, using the method provided by this invention, the sperm motility of deer sperm preserved for 5 days was still greater than 0.6; after 7 days of preservation, the usable motility was 0.55 (greater than 0.3); and sperm motility was still detectable in red deer sperm after 10 days of preservation.

[0090] Table 1. Effects of different storage days on sperm motility in red deer.

[0091]

[0092] Note: Sperm motility refers to the percentage of sperm that are moving forward in the semen. For example, sperm stored for 1 day has a motility of 0.9, which means that 90% of the sperm are moving forward.

[0093] This invention also conducted a comparative experiment on sperm survival time. Comparative Example 1 used the animal sperm dilution solution described in the invention patent "A Method for Cold Storage of Animal Sperm" (patent application number CN201910990863, publication number CN111066775A) to preserve sperm, with all other aspects remaining the same. Testing showed that the method provided by this invention (Example 4) increased the sperm survival time by 3 days compared to Comparative Example 1.

[0094] Example 5

[0095] Dog sperm was preserved using the novel animal sperm liquid preservation diluent prepared in Example 1. In the process of preserving dog sperm, the temperature was maintained at 28°C in step (1), and the fresh sperm was diluted with the filtered diluent in step (2) to achieve a sperm count density of 10 million / ml. All other steps were the same as in Example 1.

[0096] The semen from dogs that had been preserved for one day was transported to the site for application. Using a canine insemination device connected to a 5ml syringe, 4ml of semen was infused into each dog, resulting in a 100% conception rate.

[0097] A comparative experiment was conducted between the diluent of this invention and the diluent disclosed in a previous patent. Comparative Example 2 used the diluent disclosed in a previous patent to preserve animal sperm. The previous patent was entitled "A Method for Cold Preservation of Animal Sperm," patent application number CN201910990863, publication number CN111066775A. Except for the diluent, everything else was the same as in Example 5.

[0098] The sperm motility of dogs preserved for 1-10 days was detected by microscopic examination, and the results are shown in Table 2. Table 2 shows that the sperm motility of dogs preserved using the method provided by this invention is significantly higher than that of the diluent used in previous patents, starting from the second day of preservation.

[0099] Table 2. Effects of different storage days on sperm motility in dogs.

[0100]

[0101] Example 6

[0102] The effects of different formulations on fox sperm motility were compared. Fresh fox semen was collected, and after motility testing, it was divided into four equal portions. Motility was observed every 24 hours for 10 consecutive days.

[0103] Part 1: Fox sperm was preserved using the method described in Example 4.

[0104] Part 2: This is Comparative Example 3, which uses the animal sperm dilution solution described in the invention patent "A Method for Cold Storage of Animal Sperm" (patent application number CN201910990863, publication number CN111066775A) to preserve fox sperm. Everything else is the same as in Example 4.

[0105] Part 3: This is Comparative Example 4, in which the edible multi-vegetable blended oil in the formula is replaced with an equal amount of Amway plant protein powder, and everything else is the same as in Example 4.

[0106] Part 4: This is Comparative Example 5, in which the blended edible vegetable oils in the formula are replaced with an equal amount of lecithin, and everything else is the same as in Example 4.

[0107] The sperm motility test results after using different formulas for sperm preservation are shown in Table 3. Table 3 shows that the shortest preservation time was achieved with Amway plant protein powder (Comparative Example 4), which allowed sperm to be used for insemination after 3 days, but sperm motility was zero after 4 and 5 days. Next was the formula using lecithin (Comparative Example 5), which allowed sperm to be used for insemination after 4 days, but sperm motility was zero after 5 and 6 days. The preservation method in Comparative Example 3, which used multiple plant protein powders and ultrasonic emulsification, allowed sperm to be used for insemination after 7 days, but sperm motility was zero after 9 and 10 days. Compared to Comparative Example 3, the animal sperm liquid preservation diluent provided by this invention, when refrigerated at 4°C, increased sperm motility by 5% after 5 days of preservation, and extended the sperm survival time from 9 to 10 days after refrigeration at 4°C.

[0108] The preservation method provided by this invention is the most effective compared to the other three methods, allowing sperm to be used for insemination after 8 days and surviving for up to 10 days. The inventors speculate that the preservatives added to Amway's plant protein powder may be detrimental to sperm, and that lecithin is easily oxidized, resulting in a significant decrease in sperm motility after 2 days and poor sustained effectiveness. In contrast, the non-GMO blended plant oils used in this invention contain various nutrients, including vitamin E, unsaturated fatty acids, and some undetected nutritional factors, which can extend the sperm preservation time.

[0109] Table 3. Effects of different formulations on fox sperm motility

[0110]

[0111] Example 7

[0112] The frozen bovine semen was preserved using the method described in Example 4. The sperm motility of the bovine semen was then tested from 1 to 10 days, and the results are shown in Table 4.

[0113] As can be seen from Table 4, when bovine sperm is preserved using the method provided by this invention, the usable motility is 0.35 (greater than 0.3) after 3 days of preservation; and live sperm are still present after 7 days of preservation.

[0114] Table 4. Effects of different storage days on bovine sperm motility.

[0115]

[0116] Note: The bovine semen is the semen from frozen semen that has been thawed and centrifuged to remove the original frozen semen preservation diluent. Therefore, its motility is lower than that of fresh bovine semen. However, it can still be seen that the preservation method of this invention can maintain the motility of the semen as much as possible.

[0117] Example 8

[0118] Sperm from Hu sheep was preserved using the method described in Example 4. The sperm motility of the Hu sheep was then tested from day 1 to day 10, and the results are shown in Table 5.

[0119] As can be seen from Table 5, the sperm motility of fresh sheep is 0.8. When sheep sperm is preserved using the method provided by this invention, the usable motility is 0.35 (greater than 0.3) after 8 days of preservation; sperm motility can still be detected after 10 days of preservation.

[0120] Table 5. Effects of different storage days on sheep sperm motility.

[0121]

[0122] Furthermore, a comparative experiment was conducted regarding sperm survival time. Comparative Example 6 used the animal sperm dilution solution described in the invention patent "A Method for Cold Storage of Animal Sperm" (patent application number CN201910990863, publication number CN111066775A) to preserve Hu sheep sperm. Testing showed that after 5 days of storage, the Hu sheep sperm motility in Comparative Example 6 was 0.39, while the Hu sheep sperm motility in this invention was 0.59, indicating that the method of this invention has a motility 0.2 higher than that of Comparative Example 6. After 6 days of storage, the Hu sheep sperm motility in Comparative Example 6 was 0.37, while the Hu sheep sperm motility in this invention was 0.47, indicating that the method of this invention has a motility 0.1 higher than that of Comparative Example 6. Regarding the longest storage time, the sperm preserved by this invention remained usable after 8 days, while the longest storage time for Comparative Example 6 was 6 days, and the sperm in Comparative Example 6 became unusable after 7 days. Therefore, this invention extends the storage time by 2 days compared to Comparative Example 5.

[0123] Example 9

[0124] The effects of adding different types of vegetable oil on fox sperm motility were compared. The experimental method was the same as in Example 6, except that the types of vegetable oil were different.

[0125] The experimental results are shown in Table 6. As can be seen from Table 6, compared to non-GMO soybean oil, peanut oil, and cold-pressed olive oil, the addition of various edible vegetable blends in this invention is more beneficial for improving sperm motility and extending shelf life.

[0126] Table 6. Effects of adding different vegetable oils on fox sperm motility

[0127]

[0128] In summary, the method provided by this invention has a wide range of applications and is highly practical, and can be widely used for the preservation of sperm from animals such as cattle, deer, foxes, raccoons, dogs, and sheep. This invention uses a 0.25µm needle filter to thoroughly remove microorganisms from the diluent, improving its effectiveness, and is simple to operate. Experimental verification shows that, compared to existing diluents, sperm preserved using the diluent of this invention exhibits higher sperm clarity under a microscope, and its survival rate can be extended by 1-3 days.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a liquid preservation diluent for animal sperm, characterized in that, Used for preserving animal sperm; the liquid preservative diluent for animal sperm is prepared using the following method: (1) Mix glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid and water to obtain mixture 1; the mass-volume ratio of glucose, fructose, tris(hydroxymethyl)aminomethane, citric acid and water is (10-20) g: (10-20) g: (20-30) g: (10-20) g: (50-100) ml; (2) Mix the blended vegetable oil with the mixture 1 from step (1), stir, and the volume ratio of the blended vegetable oil to water is 50:(50-100) to obtain mixture 2; let stand, discard the upper layer of oil, refrigerate, and dilute with sterile distilled water; The ingredients of this blend of edible vegetable oils include: 65% soybean oil, 21% rapeseed oil, 6% peanut oil, 4% sunflower seed oil, 2% corn oil, 1% rice bran oil, 0.6% sesame oil, and 0.4% flaxseed oil. All of the above are percentages by weight.

2. The application according to claim 1, characterized in that, Used to maintain the viability of animal sperm and / or extend the preservation time of animal sperm.

3. The application according to claim 1 or 2, characterized in that, The animals are selected from any one or more of the following: deer, dog, fox, cow, and sheep.

4. The application according to claim 3, characterized in that, The deer in question is either a red deer or a sika deer.

5. The application according to claim 3, characterized in that, The sheep in question is a Hu sheep.

6. The application according to claim 1 or 2, characterized in that, When preserving animal sperm, the animal sperm is diluted with filtered animal sperm liquid preservation diluent to obtain a solution containing sperm, which is then placed in a container, sealed, cooled, and preserved.

7. The application according to claim 6, characterized in that, Animal sperm liquid preservation diluent was filtered using a 0.25µm filter membrane. The collected animal sperm was diluted with the filtered animal sperm liquid preservation diluent at a sperm count of 3 million / ml to 10 million / ml.

8. The application according to claim 7, characterized in that, Cool at a rate of 0.1-0.5℃ / min until it reaches 0-17℃, then store.

9. The application according to claim 7, characterized in that, Animal sperm should be kept at a temperature of 20-35℃ from collection to storage.

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