CN117770234A - Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution - Google Patents
Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution Download PDFInfo
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- CN117770234A CN117770234A CN202311845686.XA CN202311845686A CN117770234A CN 117770234 A CN117770234 A CN 117770234A CN 202311845686 A CN202311845686 A CN 202311845686A CN 117770234 A CN117770234 A CN 117770234A
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- Prior art keywords
- donkey
- milk
- diluent
- sperm
- nutrient solution
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of livestock breeding, in particular to donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution; the method comprises the following steps of: firstly, measuring and mixing the treated room-temperature donkey milk and yolk, adding penicillin and streptomycin, and shaking to obtain a suspension; step two, centrifuging the suspension to obtain a centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, the required equipment is simple, the types of medicines required for preparation are few, the preparation procedure is simple, the preparation difficulty is low, no higher professional skill is required for operators, and the work can be independently completed in a common farm or a basic-level artificial insemination point; is suitable for popularization and application in large-scale farms and artificial insemination spots.
Description
Technical Field
The invention relates to the technical field of livestock breeding, in particular to donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution.
Background
The research on the low-temperature and cryopreservation of equine sperm cells at home and abroad has been long-lived. At present, research reports and patent literature on equine animal sperm preservation diluents are available at home and abroad.
Foreign report: 20% of the donkey colostrum diluent reported by the agricultural institute of Spain Sarogosa Ala in 2019 and the center of the military for horse reproduction, cristina Á lvarez, victoria Lu ñ o, noelia Gonz lez and the like has good effect on donkey sperm freezing (Criolog, 2019, 87:110-114), 20% of the donkey colostrum diluent reported in 7 months in the same year has better protection effect on donkey sperm freezing than 20% of the horse colostrum and 20% of the yolk diluent, and various indexes such as total sperm viability, movement speed and the like are obviously improved after frozen and thawed sperm (Reproduction in Domestic Animals,2019, 7:94-97). The addition of egg yolk centrifugate to sperm freezing dilutions was reported by Argentina Ailisi university, 2018, alejandro Ferrante, clara Baca Castex, santiago Bruno et al to have a detrimental effect on sperm motility, membrane function and acrosome status (Journal of Equine Veterinary Science,2018, 65:12-18). Sorbitol was reported by the university of Spanish, coladow, veterinary drug institute J.Dorado, M.Hidalgo, D.Acha et al in 2019 as an alternative to improving sperm quality (Animal Reproduction Science,2019, 208:1-9).
Foreign patent products: foreign equine dilutions products have become popular such as the IRA series of IMV in france, gent A in Germany, E-Z Mixin dilutions in the U.S. INRA82 and INRA96 dilutions in France have been commercially sold by the French IMV Kasu company, and are formulated primarily with sugar salts and other additional ingredients.
Domestic reports: after semen centrifugation, the semen was stored at 5℃with INRA82 (manufactured by Kasu technologies Co., ltd., france) for an effective storage time of not more than 72 hours. The INRA82 dilution contains saccharides (glucose, lactose and raffinose), buffer substances (sodium citrate, potassium citrate and hydroxyethyl piperazine ethyl sulfate), anti-freezing agent (skimmed milk), antibiotics (penicillin and streptomycin) and other substances (Heilongjiang animal doctor, 2020, (38) 20:32-35). The addition of resveratrol and chlorogenic acid in semen diluent can significantly improve sperm quality and improve low-temperature preservation effect of donkey semen (Chinese journal of livestock, 2022, (58) 5:153-157). Adding 40 mu m resveratrol and 5 mu m MnCl into the refrigerating fluid 2 Can improve the frozen semen quality of donkey, remarkably improve the sperm motility, plasma membrane integrity and acrosome integrity after freezing and thawing, reduce the ROS content and improve the antioxidant enzyme activity (Jilin livestock veterinarian, 2021, 12:1-3). The addition amounts of the citric acid, the glucose and the yolk are respectively 17.0 g/L, 15.0g/L and 15 percent, and the donkey semen preservation effect at normal temperature is better (Heilongjiang livestock veterinarian, 2018, 13:102-104). The Tris-based diluent has better preservation and freezing effects on donkey sperm at normal temperature (Heilongjiang animal doctor, 2017, (09 below): 83-86). The filtration mode of adding yolk after the filtration of the base solution, the addition of 200 mu g/100 ml or 300 mu g/100 ml VC diluent, the addition of 5% VE diluent and the addition of 30% graphene oxide diluent have good low-temperature preservation effect on donkey sperms (Tianjin academy of agriculture, 2020, (27) 1:53-57). Zhang Wenqian, wang Lu, zhang Jiang et al report that the addition of 0.06g/L grape seed procyanidins to the diluent helps to improve the cryopreservation quality of donkey semen (Heilongjiang livestock veterinarian, 2022, (140) 17:52-55). Han Yingxue, gao Peng, li Nan and the like report that the addition of vitamin E with concentration of 1%, 2%, 4% and 6% in the diluent can improve the kinetic parameters, mitochondrial membrane potential, acrosome integrity and DNA integrity after donkey sperm are frozen and thawed and reduce the deformity rate, wherein 2% of vitamin E has the best effect (Chinese journal of livestock, 2022, (58) 9:179-183). Zhao Juan, li Jiandong report that arginine and BSA have a certain degree of improvement on sperm motility, acrosome integrity and plasma membrane integrity of donkey semen when added togetherHas a certain synergistic effect, and the optimal combination concentration is 1.0mg/mL of arginine and 2mg/mL of BSA (Heilongjiang livestock veterinarian, 2022, (141) 18:68-72).
Domestic patent literature: 1) The donkey sperm diluent, the preparation method and the application thereof (CN 111493062A) applied by Taidong (Zhenjiang) biotechnology Co-Ltd in 2020, comprises the following components in mass: 30.4+/-0.5 g of Tris, 17.0+/-0.2 g of citric acid, 7.5+/-0.1 g of glucose, 12.5+/-0.2 mg of taurine, 36.3+/-0.5 mg of sodium pyruvate, 1.5+/-0.1 g of fructose, 2+/-0.1 g of modified lecithin, 1.5+/-0.1 g of pentoxifylline and 25.4+/-0.3 mg of soybean isoflavone. 2) A donkey frozen semen diluent formula and a preparation method thereof (CN 111374124A) applied by a modern agricultural production base construction engineering center of Liaoning province in 2020 comprises 1.2g of glucose, 1.3g of citric acid, 2.4g of Tris2.4g of glycerol, 5ml of double distilled water, 100ml of yolk, 20ml of penicillin 1X 105IU and 1X 105IU of streptomycin. 3) In 2019, shandong livestock veterinary professional college applied for a donkey semen diluent and a preparation method thereof (CN 110583630A), the donkey semen diluent comprises glucose, lactose, raffinose, sodium citrate pentahydrate, potassium citrate, HEPES, EDTA, egg yolk, green streptomycin, sterile distilled water, defatted donkey milk, and BSA and A solution, wherein the A solution comprises taurine, L-lysine hydrochloride, inositol, vitamin B6, vitamin B12, citric acid, sodium citrate, lemon yellow and purified water. 4) In 2017 Tianjin agricultural institute application of a method for preserving donkey semen at low temperature (CN 107183013A), a base solution is prepared according to the mass parts of 3.04 parts of Tris, 1.70 parts of citric acid, 1.50 parts of glucose, 1.50 parts of fructose, 0.06 part of penicillin, 0.10 part of streptomycin and 100 parts of double distilled water, and 85 parts of the base solution and 15 parts of egg yolk solution are used for preparing a semen diluent.
Besides the above foreign and domestic reports and patents, some of the prior reported and declared donkey sperm diluent patent literature and horse sperm diluent patent literature commonly adopt the above sugar salts, skimmed milk, egg yolk and bioactive substances and other additive components as main components for preparing the diluent.
However, the donkey sperm low-temperature preservation diluent in the prior art has short preservation time, more instruments and equipment are needed for preparation, higher expertise is needed for operators, the preparation procedure is complicated, the preparation difficulty is high, the cost is high, and the work cannot be independently completed at a farm or a basic artificial insemination point, so that the requirements cannot be met.
Disclosure of Invention
The invention provides a donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution, which overcomes the defects of the prior art, and can effectively solve the problems that the donkey sperm low-temperature preservation diluent in the prior art has short preservation time, needs more instruments and equipment for preparation, has higher professional skills for operators, has complicated preparation procedures, large preparation difficulty and high cost, and cannot independently complete the work at a plant or a basic artificial insemination point, and cannot meet the requirements.
The technical scheme of the invention is realized by the following measures: a donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution is obtained by the following steps: firstly, weighing 80ml to 400ml of treated room temperature donkey milk, mixing with 20ml to 100ml of yolk, adding 10 to 50 ten thousand IU of penicillin and 10 to 50 ten thousand IU of streptomycin, and repeatedly oscillating for 30 to 50 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 to 3 times, wherein each time lasts for 10 to 20 minutes, and sucking the centrifugate after each treatment and repeating the operation to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution.
The following are further optimizations and/or improvements to the above-described inventive solution:
in the preparation method of the donkey sperm low-temperature preservation diluent, in the second step, a 50ml centrifuge tube is used for subpackaging the suspension and centrifuging the suspension at a rotating speed of 2200 rpm; in the third step, packaging according to the dosage of 100 ml/bottle, and freezing and preserving after packaging.
The treated room temperature donkey milk is obtained by the following steps: firstly, scrubbing and cleaning the nipple of the donkey, sterilizing, milking, and filtering the extruded fresh donkey milk; secondly, placing the filtered fresh donkey milk into a sterilized container, boiling, skimming the swelled foam, and continuously 2 to 4 times until no foam swells, and covering a cover for cooling; and thirdly, cooling to room temperature, and filtering to obtain the treated room temperature donkey milk.
In the preparation method of the treated room temperature donkey milk, in the first step, the nipple of the donkey with lactation is scrubbed cleanly by a towel, and is milked after being disinfected by a special disinfectant, and the extruded fresh donkey milk is filtered by 6 layers of sterilizing gauze; in the second step, the filtered fresh donkey milk is placed in a sterilized stainless steel container, and the swelled foam is skimmed by boiling in an electromagnetic oven; and in the third step, cooling to room temperature, and filtering with 6 layers of sterilized gauze to obtain the treated room temperature donkey milk.
The yolk is obtained by the following steps: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
The donkey milk is secreted by the donkey after delivery for more than 15 days; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
The preparation process of the donkey sperm low-temperature preservation diluent is carried out under aseptic conditions.
The donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, compared with the existing donkey sperm low-temperature preservation diluent, the preparation method has the advantages of simple equipment, few types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and independent completion of the work in general farms or basic-level artificial insemination points; is suitable for popularization and application in large-scale farms and artificial insemination spots.
Description of the embodiments
The present invention is not limited by the following examples, and specific embodiments can be determined according to the technical scheme and practical situations of the present invention.
Example 1, the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, weighing 80ml to 400ml of treated room temperature donkey milk, mixing with 20ml to 100ml of yolk, adding 10 to 50 ten thousand IU of penicillin and 10 to 50 ten thousand IU of streptomycin, and repeatedly oscillating for 30 to 50 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 to 3 times, wherein each time lasts for 10 to 20 minutes, and sucking the centrifugate after each treatment and repeating the operation to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, compared with the existing donkey sperm low-temperature preservation diluent, the preparation method has the advantages of simple equipment, few types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and independent completion of the work in general farms or basic-level artificial insemination points; is suitable for popularization and application in large-scale farms and artificial insemination spots. The donkey sperm low-temperature preservation diluent has long low-temperature preservation time and good effect on donkey sperm, is preserved for 5 to 6 days in a refrigerator at the temperature of 2 to 7 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; sperm motility decreased significantly from day 7 and the rate of motility slowed until day 14 with swinging sperm. The conception rate during estrus is about 60% on average due to factors such as the fatness of the donkey, the foal time, the insemination dosage and the like when the ovulation-promoting hormone is matched for use.
Example 2, the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, 80ml of treated room temperature donkey milk is measured and mixed with 20ml of yolk, 10 ten thousand IU of penicillin and 10 ten thousand IU of streptomycin are added, and the mixture is repeatedly vibrated for 30 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 times, each for 10 minutes, and sucking the centrifugate after each treatment to repeatedly operate to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent in the embodiment 2 has long low-temperature preservation time and good effect on donkey sperm, is preserved for 6 days in a refrigerator at 7 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; the conception rate of the female donkey can reach 65% due to factors such as the fatness of the female donkey, the time of foal production, the insemination dosage and the like when the female donkey is matched with the ovulation-promoting hormone.
Example 3, the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, weighing 400ml of treated room temperature donkey milk and 100ml of yolk, mixing, adding 50 ten thousand IU of penicillin and 50 ten thousand IU of streptomycin, and repeatedly oscillating for 35 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 3 times, 15 minutes each time, and sucking the centrifugate after each treatment to repeatedly operate to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent in the embodiment 3 has long low-temperature preservation time and good effect on donkey sperm, and the donkey sperm has little reduction of sperm motility after being preserved in a refrigerator at 5 ℃ for 5 days, and still keeps a faster movement speed; the conception rate of the female donkey can reach 68% due to factors such as the fatness of the female donkey, the time of foal production, the insemination dosage and the like when the female donkey is matched with the ovulation-promoting hormone.
Example 4 the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, weighing 240ml of treated room temperature donkey milk and 60ml of yolk, mixing, adding 30 ten thousand IU of penicillin and 30 ten thousand IU of streptomycin, and repeatedly oscillating for 40 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 3 times, 15 minutes each time, and sucking the centrifugate after each treatment to repeatedly operate to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent in the embodiment 4 has long low-temperature preservation time and good effect on donkey sperm, is preserved for 5 days in a refrigerator at the temperature of 2 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; the conception rate of the female donkey can reach 69% due to factors such as the fat condition of the female donkey, the foal time, the insemination dosage and the like when the female donkey is matched with the ovulation-promoting hormone.
Example 5 as an optimization of the above example, in the preparation method of donkey sperm cryopreservation diluent, in the second step, the suspension was dispensed with 50ml centrifuge tubes and centrifuged at 2200 rpm; in the third step, packaging according to the dosage of 100 ml/bottle, and freezing and preserving after packaging.
Example 6 as an optimization of the above example, the treated room temperature donkey milk was obtained as follows: firstly, scrubbing and cleaning the nipple of the donkey, sterilizing, milking, and filtering the extruded fresh donkey milk; secondly, placing the filtered fresh donkey milk into a sterilized container, boiling, skimming the swelled foam, and continuously 2 to 4 times until no foam swells, and covering a cover for cooling; and thirdly, cooling to room temperature, and filtering to obtain the treated room temperature donkey milk.
Example 7, as an optimization of the above example, in the preparation method of the treated room temperature donkey milk, in the first step, the nipple of the lactating donkey is scrubbed clean with a towel, sterilized with a special sterilizing liquid and then milked, and the extruded fresh donkey milk is filtered with 6 layers of sterilizing gauze; in the second step, the filtered fresh donkey milk is placed in a sterilized stainless steel container, and the swelled foam is skimmed by boiling in an electromagnetic oven; and in the third step, cooling to room temperature, and filtering with 6 layers of sterilized gauze to obtain the treated room temperature donkey milk.
Example 8, as an optimization of the above example, the yolk was obtained as follows: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
Example 9, as an optimization of the above example, donkey milk was secreted by the donkey for 15 or more days post partum; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
Example 10 as an optimization of the above example, donkey milk is fresh donkey milk.
The invention relates to a method for using donkey sperm low-temperature preservation diluent
40min before semen collection by a pseudo-vagina method, adjusting the water temperature of a water bath kettle to 37 ℃, and putting the donkey sperm low-temperature preservation diluent into low-temperature preservation. The microscope is started to carry a heating plate, and the cover glass, the glass slide and the beaker to be filled with semen are preheated.
After semen collection and filtration, a 50ml centrifuge tube is used for split charging and centrifuging for 10 minutes at the rotating speed of 2200 rpm, the supernatant is discarded, the donkey sperm of the invention with the volume equal to that of the original semen is added into the low-temperature preservation diluent, and after the donkey sperm is subjected to microscopic examination, the donkey sperm is wrapped in a plurality of layers by towels and is preserved in a refrigerator at the temperature of 2 ℃. During artificial insemination, the inhalation injector is connected with the insemination jacket for donkey to perform artificial insemination on oestrus female donkey, and 5ml to 10ml of diluted semen is input into each donkey uterus.
Drug and device sources
The donkey milk and yolk used for storing the diluent at low temperature are self-made, and the green streptomycin is produced by Sichuan Dingjian pharmaceutical industry Limited company; human chorionic gonadotrophin (hCG) is produced by Ningbo Sansheng biotechnology Co.
An electric heating constant temperature water bath (model DZKW-D-2) produced by Yongguangming medical instrument Co., ltd; a bench type low-speed centrifuge (model TDZ 4-WS), manufactured by Hunan Instrument laboratory instruments development Co., ltd; the B-type ultrasonic diagnostic apparatus for animals is produced by Zhengzhou Boxiang electronic technology Co., ltd; the model of the biological microscope is XSP-T36B-1600X.
Technical effect verification of the invention
The donkey sperm low-temperature preservation diluent has long low-temperature preservation time and good effect on donkey sperm, is preserved for 5 to 6 days in a refrigerator at the temperature of 2 to 7 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; sperm motility decreased significantly from day 7 and the rate of motility slowed until day 14 with swinging sperm. The conception rate during estrus is about 60% on average due to factors such as the fatness of the donkey, the foal time, the insemination dosage and the like when the ovulation-promoting hormone is matched for use. The specific mating method comprises the following steps: the ovaries on both sides of the oestrus donkey are scanned through rectal examination and B ultrasonic scanning, the follicles develop to more than 3.0cm, the human chorionic gonadotrophin is injected into 5000IU for the first time after 12 hours, the seed is bred for the second time after 36 hours, and then the ovaries can be supplemented for 1 time at 48 hours or 60 hours according to the development condition of the follicles until ovulation.
From the production cost, the cost of the donkey sperm low-temperature preservation diluent per 100ml is about 10 yuan RMB according to the market price, and the market price of the donkey sperm low-temperature preservation diluent with the same volume is more than 100 yuan, so that the cost of the diluent can be saved by more than 10 times for donkey farms and artificial insemination points.
Compared with the existing donkey sperm low-temperature preservation diluent, the donkey sperm low-temperature preservation diluent provided by the invention has the advantages of simple equipment, less types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and capability of independently completing the work in a general farm or a basic-layer artificial insemination spot; the cost per unit volume is far lower than the selling price of donkey sperm diluent in the existing market; the most important is that the sperm obtains higher conception rate after low-temperature preservation and hybridization, and the conception rate is superior to or equivalent to the prior art; is suitable for popularization and application in large-scale farms and artificial insemination spots.
In conclusion, the donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, compared with the existing donkey sperm low-temperature preservation diluent, the preparation method has the advantages of simple equipment, few types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and independent completion of the work in general farms or basic-level artificial insemination points; is suitable for popularization and application in large-scale farms and artificial insemination spots.
The technical characteristics form the embodiment of the invention, have stronger adaptability and implementation effect, and can increase or decrease unnecessary technical characteristics according to actual needs so as to meet the requirements of different situations.
Claims (10)
1. A donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution is characterized by being prepared by the following steps: firstly, weighing 80ml to 400ml of treated room temperature donkey milk, mixing with 20ml to 100ml of yolk, adding 10 to 50 ten thousand IU of penicillin and 10 to 50 ten thousand IU of streptomycin, and repeatedly oscillating for 30 to 50 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 to 3 times, wherein each time lasts for 10 to 20 minutes, and sucking the centrifugate after each treatment and repeating the operation to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution.
2. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 1, wherein in the preparation method of the donkey sperm cryopreservation diluent, in the second step, a 50ml centrifuge tube is used for subpackaging the suspension and centrifuging at a rotation speed of 2200 rpm; in the third step, packaging according to the dosage of 100 ml/bottle, and freezing and preserving after packaging.
3. Donkey sperm cryopreservation diluent with donkey milk as nutritional liquid according to claim 1 or 2, characterized in that the treated room temperature donkey milk is obtained by the following steps: firstly, scrubbing and cleaning the nipple of the donkey, sterilizing, milking, and filtering the extruded fresh donkey milk; secondly, placing the filtered fresh donkey milk into a sterilized container, boiling, skimming the swelled foam, and continuously 2 to 4 times until no foam swells, and covering a cover for cooling; and thirdly, cooling to room temperature, and filtering to obtain the treated room temperature donkey milk.
4. A donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 3, wherein in the preparation method of the donkey milk at room temperature after treatment, in the first step, the nipple of the donkey which is used for lactation is scrubbed cleanly by a towel, is disinfected by a special disinfectant, is milked, and the extruded fresh donkey milk is filtered by 6 layers of sterile gauze; in the second step, the filtered fresh donkey milk is placed in a sterilized stainless steel container, and the swelled foam is skimmed by boiling in an electromagnetic oven; and in the third step, cooling to room temperature, and filtering with 6 layers of sterilized gauze to obtain the treated room temperature donkey milk.
5. Donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 1 or 2, characterized in that the yolk is obtained by the following method: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
6. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 4, wherein the yolk is obtained by the following method: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
7. The donkey sperm cryopreservation diluent taking donkey milk as a nutrient solution according to claim 1 or 2, which is characterized in that donkey milk is secreted by the donkey for more than 15 days after delivery; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
8. The donkey sperm cryopreservation diluent taking donkey milk as a nutrient solution according to claim 6, which is characterized in that donkey milk is secreted by the donkey for more than 15 days after delivery; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
9. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 1 or 2, wherein the donkey sperm cryopreservation diluent is prepared under aseptic conditions.
10. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 8, wherein the donkey sperm cryopreservation diluent is prepared under aseptic conditions.
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