Method and system for improving cryopreservation effect of donkey sperms

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for improving the cryopreservation effect of donkey sperms, which comprises the following steps: selecting a healthy breeding male donkey with vigorous sexual desire, and collecting the sperm by using a strictly disinfected false vagina; seminal fluid quality detection: immediately evaluating sperm motility of the collected seminal fluid under a 37 DEG C constant-temperature phase contrast microscope; measuring the sperm density by using a sperm density instrument; diluting the semen, and freezing the semen by adopting a programmed cooling method; unfreezing the seminal fluid, taking out the thin tube from the liquid nitrogen, immediately putting the thin tube into a constant-temperature water bath at 38 DEG C, slightly shaking, taking out the thin tube after the seminal fluid in the thin tube is completely unfrozen, wiping water drops on the surface of the thin tube by using sterile gauze, and transferring the seminal fluid into a sterile test tube for later use. According to the invention, sperm motility and fertilization capability are improved: sperm collection and treatment processes are optimized, a proper freezing diluent formula is screened, and freezing is carried out by adopting a programmed cooling method, so that damage of sperms in freezing and unfreezing processes is effectively reduced, and sperm motility and fertilization capability after cryopreservation are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method and system for improving the effect of frozen storage of donkey sperm. BACKGROUND

[0002] Donkeys, as an important domestic animal, play an important role in agricultural production, transportation, and production of meat, skin, and milk products. However, the reproductive efficiency of donkeys is relatively low, the reproductive cycle of female donkeys is long, the gestation period is about 12 months, and the estrus time is irregular, making it difficult to accurately grasp the ovulation time, which greatly limits the development of the donkey industry. Sperm freezing and preservation technology is one of the effective means to solve the above problems. Through sperm freezing and preservation, the long-term preservation and long-distance transportation of excellent male donkey sperm can be realized, the limitations of time and space can be broken through, the utilization rate of male donkey can be improved, and the process of donkey breed improvement can be accelerated. However, the current donkey sperm freezing and preservation technology still faces many challenges. Donkey sperm is extremely sensitive to low temperature, and during freezing and thawing, the sperm membrane, acrosome and nucleus are easily damaged, resulting in significant decrease in sperm motility and fertilization ability. In the prior art, the sperm motility of thawed donkey frozen semen is generally low, and the pregnancy rate is not ideal, which seriously restricts the application of the technology in actual production. At present, a large number of researches on donkey sperm freezing and preservation technology have been carried out at home and abroad. Some researches focus on the screening and optimization of antifreeze agents, and try to use different types of antifreeze agents such as glycerol, dimethyl sulfoxide, ethylene glycol, etc., and adjust the concentration of antifreeze agents to reduce freezing damage, but the effect is not satisfactory. Some researches are committed to improving the freezing process, such as using programmed cooling, vitrification freezing, etc. However, due to the special biological characteristics of donkey sperm, there is still a large space for improvement in these methods to improve the effect of sperm freezing and preservation. SUMMARY

[0003] In view of the problems existing in the prior art, the present application provides a method for improving the effect of donkey sperm freezing and preservation.

[0004] The present application is implemented as follows: a method for improving the effect of donkey sperm freezing and preservation, the method comprising: S1: semen collection, selecting healthy and sexually vigorous male donkeys, collecting semen in an environment with a temperature of 20-25℃ and a relative humidity of 60-70%, using a strictly sterilized artificial vagina; S2: semen quality detection, immediately evaluating the sperm motility of the collected semen under a constant temperature phase contrast microscope at 37℃, requiring the sperm motility to be above 0.7; at the same time, measuring the sperm density using a sperm density instrument, and the density needs to be above 100 million / ml, and the semen with normal sperm morphology and no odor can be used for subsequent processing; S3: semen dilution, including preparation of basic diluent, preparation of freezing diluent and semen dilution operation; S4: semen freezing, using programmed cooling method for semen freezing; S5: semen thawing, taking the thin tube out of liquid nitrogen, immediately putting it into a 38 DEG C constant temperature water bath, gently shaking, taking out after the semen in the thin tube is completely thawed, wiping the water droplets on the surface of the thin tube with sterile gauze, cutting off the two ends of the thin tube, and transferring the semen to a sterile test tube for standby.

[0005] Further, in S1, before semen collection, the external genitalia of the male donkey is washed with warm water at 38-40 DEG C and dried, the temperature of the false vagina is kept at 38-42 DEG C, and an appropriate amount of sterile lubricant is applied; the collected semen is filtered with 8 layers of sterile gauze and then quickly placed in a 37 DEG C constant temperature container for detection. Further, the preparation of the basic diluent specifically comprises the following steps: weighing Tris 3.5g, glucose 2g, citric acid 2g, egg yolk 20ml, penicillin 500,000 IU and streptomycin 50,000 IU, adding distilled water to make up to 100ml, fully stirring and dissolving, filtering and sterilizing with a 0.22μm filter membrane, adjusting the pH value to 7.2-7.4, and placing in a 4 DEG C refrigerator for standby.

[0006] Further, the preparation of the freezing diluent specifically comprises the following steps: adding glycerol 6ml to the basic diluent and fully mixing to obtain the freezing diluent.

[0007] Further, the semen dilution operation specifically comprises the following steps: slowly adding the qualified semen to the pre-warmed 37 DEG C basic diluent at a volume ratio of 1:1, gently shaking, and balancing for 30min at 37 DEG C; then slowly adding the balanced semen to the pre-cooled 4 DEG C freezing diluent at a volume ratio of 1:1, gently shaking, and continuing to balance for 60min at 4 DEG C.

[0008] Further, S4 specifically comprises the following steps: dispensing the balanced semen into 0.5ml thin tubes, sealing the thin tubes with an ultrasonic sealing machine, placing the thin tubes into a programmed cooling instrument, reducing the temperature from 4 DEG C to -20 DEG C at a speed of 1 DEG C per minute, then reducing the temperature to -80 DEG C at a speed of 5 DEG C per minute, and finally quickly putting the thin tubes into liquid nitrogen for storage. Another object of the present application is to provide a system for improving the freezing preservation effect of donkey semen based on the method for improving the freezing preservation effect of donkey semen. The semen collection module selects a healthy and sexually active male donkey, and collects semen in a strictly sterilized false vagina in an environment with a temperature of 20-25 DEG C and a relative humidity of 60-70%; The semen quality testing module is connected to the semen collection module. The collected semen is immediately evaluated for sperm motility under a 37°C constant temperature phase contrast microscope, requiring a sperm motility of 0.7 or higher. At the same time, a sperm density meter is used to measure sperm density, which must be above 100 million / ml. Only semen with normal sperm morphology and no odor can be used for subsequent processing. The semen dilution module, connected to the semen quality testing module, includes basic diluent preparation, frozen diluent preparation, and semen dilution operation. The semen freezing module is connected to the semen dilution module and uses a programmed cooling method to freeze the semen. The semen thawing module is connected to the semen freezing module. The capillary tube is taken out from the liquid nitrogen and immediately placed in a 38°C constant temperature water bath. It is gently shaken and removed after the semen in the capillary tube is completely thawed. The water droplets on the surface of the capillary tube are wiped dry with sterile gauze, and the two ends of the capillary tube are cut off. The semen is then transferred to a sterile test tube for later use.

[0009] Another object of the present invention is to provide a computer device, the computer device including a memory and a processor, the memory storing a computer program, which, when executed by the processor, causes the processor to perform the steps of the method for improving the cryopreservation effect of donkey sperm.

[0010] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0011] Improving sperm motility and fertilization capacity: By optimizing semen collection and processing procedures, selecting suitable cryodiluent formulations, and employing programmed cooling for freezing, damage to sperm during freezing and thawing is effectively reduced, significantly improving sperm motility and fertilization capacity after cryopreservation. Experimental results show that donkey sperm cryopreserved using this method exhibits a motility of over 0.45 and an acrosome integrity rate of over 85% after thawing, representing a substantial improvement compared to traditional methods. Improving the utilization rate of breeding bulls: The method of this invention enables the long-term effective preservation and long-distance transportation of donkey semen, allowing the semen of excellent breeding bulls to be used in a wider range, breaking through the limitations of time and space, greatly improving the utilization rate of breeding bulls, and accelerating the process of donkey breed improvement. Simple to operate and moderately costly: The equipment and reagents used in this invention are commonly used in the field of livestock breeding. The operation steps are relatively simple and easy to master, and the cost is moderate, making it suitable for promotion and application in actual production. Attached Figure Description

[0012] Figure 1 This is a flowchart of a method for improving the cryopreservation effect of donkey sperm provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of semen dilution provided in an embodiment of the present invention; Figure 3 This is a system structure diagram of the embodiment of the present invention for improving the cryopreservation effect of donkey sperm; Figure 4 This is a schematic diagram of the basic diluent composition provided in an embodiment of the present invention; In the diagram: 1. Semen collection module; 2. Semen quality testing module; 3. Semen dilution module; 4. Semen freezing module; 5. Semen thawing module. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0014] like Figure 1 As shown, a method for improving the cryopreservation effect of donkey sperm includes: S1: Semen collection. Healthy, sexually active male donkeys are selected. Semen is collected using a strictly sterilized artificial vagina in an environment with a temperature of 20-25℃ and a relative humidity of 60-70%. S2: Semen quality testing. The collected semen will be immediately assessed for sperm motility under a 37°C constant temperature phase contrast microscope. The sperm motility must be 0.7 or higher. At the same time, the sperm density will be measured using a sperm density meter. The density must be 100 million / ml or higher. Only semen with normal sperm morphology and no odor can be used for subsequent processing. S3: Semen dilution, including preparation of basal diluent, preparation of cryopreservative diluent, and semen dilution procedures, such as... Figure 2 As shown; S4: Semen freezing, using programmed cooling method for semen freezing; S5: Thaw the semen. Remove the capillary tube from the liquid nitrogen and immediately place it in a 38°C constant temperature water bath. Gently shake it until the semen in the capillary tube is completely thawed. Remove the capillary tube, wipe the water droplets off the surface of the capillary tube with sterile gauze, cut off both ends of the capillary tube, and transfer the semen to a sterile test tube for later use.

[0015] Current donkey semen cryopreservation technologies largely draw upon equine systems. However, significant differences exist in the lipid composition, osmotic pressure tolerance, and antioxidant enzyme activity of donkey sperm cell membranes. Conventional dilution solutions are prone to causing membrane damage, mitochondrial dysfunction, and DNA fragmentation during freezing and thawing, severely impacting the motility and fertilization capacity of the revived sperm. Therefore, it is urgent to optimize cryopreservation systems specifically for donkey characteristics to overcome the technical bottlenecks of cold shock damage and osmotic rupture in traditional methods.

[0016] This invention introduces a multi-component balanced buffer system into the traditional Tris-citric acid-fructose basal dilution system, adjusting the pH between 6.8 and 7.2, and controlling the osmotic pressure to 300–340 mOsm / kg to maximize the simulation of the physicochemical environment of donkey seminal plasma. This improvement significantly reduces the intracellular and extracellular osmotic pressure difference before and after freezing, lowers the probability of sperm volume mutation, and provides physical protection for membrane stability during the freezing process.

[0017] While traditional single glycerol protectants can prevent ice crystal formation, they are toxic to donkey sperm membrane proteins. This solution employs a composite protection system of glycerol, hydroxyethyl starch, chitosan oligosaccharide, and bovine serum albumin (BSA). Glycerol regulates the freezing point balance inside and outside the cell, hydroxyethyl starch provides an external colloidal protective layer, chitosan oligosaccharide stabilizes the glycolipid complex structure on the sperm membrane, and BSA adsorbs cryo-induced free radicals, achieving dual protection inside and outside the cell.

[0018] Oxidative stress is a significant cause of membrane damage during freezing and thawing. This protocol introduces a synergistic antioxidant system of vitamin E and glutathione. Vitamin E is a lipid-soluble antioxidant that can embed itself in the sperm membrane to prevent lipid peroxidation, while glutathione is a water-soluble antioxidant that can scavenge free radicals in the cytoplasm. This dual-antioxidant system works simultaneously during freezing stress, effectively preventing sperm membrane peroxidation and nucleic acid breakage.

[0019] Traditional cooling processes often employ a fixed cooling rate, neglecting the thermal inertia and water migration dynamics of sperm cells. This invention proposes a "three-stage programmed cooling curve," controlling the cooling rate at different temperature zones to match the rates of intracellular and extracellular dehydration with ice crystal formation. This gradient cooling method effectively prevents membrane rupture and frostbite caused by uneven cooling, maintains membrane permeability and mitochondrial activity, and significantly improves motility after cryopreservation and thawing.

[0020] During the thawing process, the thin tubes were directly placed in a 38°C constant-temperature water bath, supplemented with a resuscitation diluent (secondary diluent) containing fructose, vitamin C, and trace amounts of lecithin. This system rapidly restores energy metabolism and rebuilds membrane lipid fluidity during the resuscitation phase, reducing cryolysis-induced osmotic rupture. Experimental results show that this method can significantly improve the overall viability and acrosome integrity rate of resuscitated sperm.

[0021] Through the synergistic effect of composite protective agents, antioxidant systems, and programmed cooling curves, this invention achieves a multi-layered protective mechanism for donkey sperm cryopreservation, encompassing "preservation of membrane integrity, stability of mitochondrial function, and safety of genetic material." The revived sperm motility is increased by an average of over 20%, the abnormality rate decreases by 30%, and the conception rate is increased by approximately 25% compared to traditional methods. This technology is not only applicable to the long-term preservation and transportation of male donkey semen but also provides technical reference and industrialization value for the cryopreservation of semen from equines and other large herbivores.

[0022] In step S1, before semen collection, the external genitalia of the male donkey are rinsed with warm water at 38-40℃ and dried. The temperature inside the artificial vagina is maintained at 38-42℃. An appropriate amount of sterile lubricant is applied. The collected semen is filtered through 8 layers of sterile gauze and then quickly placed in a 37℃ constant temperature container for testing. like Figure 4 As shown, the preparation of the basic diluent specifically includes: weighing 3.5g of Tris, 2g of glucose, 2g of citric acid, 20ml of egg yolk, 500,000 IU of penicillin, and 50,000 IU of streptomycin, adding distilled water to a final volume of 100ml, stirring thoroughly to dissolve, filtering through a 0.22μm filter membrane for sterilization, adjusting the pH value to 7.2-7.4, and storing at 4℃ for later use.

[0023] The preparation of the cryo-diluent specifically includes: adding 6 ml of glycerol to the base diluent and mixing thoroughly to obtain the cryo-diluent.

[0024] The semen dilution procedure specifically includes: slowly adding qualified semen to a pre-warmed basal diluent at a 1:1 volume ratio, gently shaking, and equilibrating at 37°C for 30 minutes. Then, slowly adding the equilibrated semen to a pre-cooled 4°C cryogenic diluent at a 1:1 volume ratio, gently shaking while adding, and continuing to equilibrate at 4°C for 60 minutes.

[0025] S4 specifically includes: dispensing the balanced semen into 0.5ml capillary tubes and sealing them with an ultrasonic sealer. The capillary tubes are then placed in a programmed cooling system and cooled from 4℃ to -20℃ at a rate of 1℃ per minute, then cooled to -80℃ at a rate of 5℃ per minute, and finally rapidly immersed in liquid nitrogen for preservation. like Figure 3 As shown, this embodiment of the invention provides a system for improving the cryopreservation effect of donkey sperm based on the method for improving the cryopreservation effect of donkey sperm. The system includes: Semen collection module 1: Healthy, sexually active male donkeys are selected and semen is collected using a strictly sterilized artificial vagina in an environment with a temperature of 20-25℃ and a relative humidity of 60-70%. Semen quality testing module 2 is connected to semen collection module 1. The collected semen is immediately evaluated for sperm motility under a 37℃ constant temperature phase contrast microscope, requiring a sperm motility of 0.7 or higher. At the same time, a sperm density meter is used to measure sperm density, which must be above 100 million / ml. Only semen with normal sperm morphology and no odor can be used for subsequent processing. Semen dilution module 3, connected to semen quality testing module 2, includes basic diluent preparation, frozen diluent preparation and semen dilution operation; Semen freezing module 4 is connected to semen dilution module 3 and uses a programmed cooling method to freeze semen. Semen thawing module 5 is connected to semen freezing module 4. The capillary tube is taken out from liquid nitrogen and immediately placed in a 38°C constant temperature water bath. It is gently shaken and removed after the semen in the capillary tube is completely thawed. The water droplets on the surface of the capillary tube are wiped dry with sterile gauze, the two ends of the capillary tube are cut off, and the semen is transferred to a sterile test tube for later use.

[0026] The system for improving the cryopreservation effect of donkey sperm provided by this invention adopts a modular structure design as its core concept, covering the entire process of collection, detection, dilution, freezing, and thawing, and realizing intelligent control of the entire process from obtaining biological samples to restoring sperm motility. Each module is linked with the control unit through sensors to achieve automated management of temperature, time, dilution ratio, and freezing curve. This system not only achieves standardized operation but also reduces human error, making the donkey sperm cryopreservation process repeatable and traceable.

[0027] The semen collection module consists of a temperature control device, a prosthetic vagina assembly, and sterile collection tubes. The system maintains the temperature of the prosthetic vagina's inner wall at 42±1℃ via a temperature control unit, while a humidity sensor maintains a relative humidity of 60-70% to prevent cold shock stress. After collection, the sample is automatically transferred to the detection module, avoiding external contamination and temperature fluctuations. Through precise temperature control and a sterile collection environment, this module ensures the stability of the initial semen quality, which is fundamental to improving the success rate of freezing.

[0028] The detection module consists of a microscopic imaging unit, a motility analysis submodule, and a density detection device. The system uses a computer-aided sperm analysis system (CASA) to evaluate collected samples in real time, with parameters including motility (VSL, VCL), forward motility rate, and abnormality rate. Data is automatically uploaded to a central control unit, and a threshold screening mechanism is set, allowing samples to be screened only when motility ≥ 0.7 and density ≥ 1 × 10⁻⁶. 8 Only samples with a density of / ml can proceed to the dilution process. This module's automatic judgment and screening mechanism significantly improves the success rate of cryopreservation and data consistency.

[0029] The dilution module consists of a multi-channel liquid mixing system, a stirring and temperature control unit, and an automatic dilution control device. The system automatically prepares the basal diluent and cryodiluent according to a set ratio, containing a complex cryoprotectant (glycerol, hydroxyethyl starch, chitosan oligosaccharide, BSA) and antioxidant components (vitamin E, glutathione). The control unit precisely controls the dilution rate to ≤1 ml / min via a peristaltic pump to ensure osmotic balance. This module effectively reduces sperm cold shock damage and osmotic rupture, improving cell stability before freezing.

[0030] The freezing module employs a multi-stage programmed cooling control system, consisting of a temperature sensor, a speed-controlled refrigerator, and a liquid nitrogen storage tank. The programmed cooling curve is divided into three stages: +5℃ to -5℃ (0.25℃ / min), -5℃ to -40℃ (0.5℃ / min), and -40℃ to -120℃ (1.0℃ / min), before finally transferring to liquid nitrogen for storage. The system uses a closed-loop temperature control algorithm to ensure that the temperature difference between the sample center and the outer layer does not exceed 3℃, preventing uneven ice crystal formation. Experimental results show that this module can significantly improve sperm membrane integrity and mitochondrial activity.

[0031] The thawing module includes a constant-temperature water bath and a rapid transfer mechanism. The system automatically identifies liquid nitrogen samples, controls the water bath temperature within the range of 38±0.5℃, and uses micro-vibration to assist thawing, ensuring uniform heat transfer. After thawing, a resuscitation diluent (containing fructose and vitamin C) is automatically added to rebuild the energy metabolism system. This module achieves dynamic matching between the thawing rate and osmotic pressure recovery, effectively avoiding membrane rupture and acrosome damage induced by cold thawing.

[0032] Through the coordinated operation of the aforementioned modules, this invention's system achieves standardized, visualized, and intelligent control of the entire process of donkey sperm cryopreservation. Extensive experimental verification has shown that, compared to traditional artificial methods, sperm resuscitation motility is increased by 20%–30%, membrane integrity by 25%, acrosome integrity by approximately 18%, and conception rate is significantly improved. The system can operate long-term in animal breeding centers, research institutions, and stud donkey stations, providing efficient technical support for donkey breeding, genetic improvement, and germplasm resource preservation, and has broad application value.

[0033] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for improving the cryopreservation effect of donkey sperm, characterized in that, Includes the following steps: S1: Semen collection. Healthy, sexually active male donkeys are selected and semen is collected using a sterilized artificial vagina in an environment with a temperature of 20 to 25 degrees Celsius and a relative humidity of 60 to 70%. S2: Semen quality testing. Sperm motility will be assessed immediately under a 37°C constant-temperature phase-contrast microscope. The motility must be no less than 0.

7. Semen density will also be measured using a sperm density meter, with a density no less than 1 × 10⁻⁶. 8 One per milliliter; S3: Semen dilution, the qualified semen is added to the basic diluent and the freezing diluent in proportion for secondary dilution; S4: Programmed cooling freezing, using a multi-stage programmed cooling method to cool the balanced semen to liquid nitrogen temperature; S5: Thawing and resuscitation: The frozen semen is placed in a constant temperature water bath at 38 degrees Celsius for rewarming and then treated with sterile diluent to obtain highly viable resuscitated semen.

2. The method according to claim 1, characterized in that, In step S1, before semen collection, the external genitalia of the male donkey are rinsed with warm water at 38 to 40 degrees Celsius and dried. The temperature of the inner wall of the artificial vagina is maintained at 38 to 42 degrees Celsius. The inner wall of the artificial vagina is pre-coated with lubricant. The collected semen is filtered through multiple layers of sterile gauze and placed in a constant temperature container at 37 degrees Celsius.

3. The method according to claim 1, characterized in that, The basic diluent consists of 3.5 g Tris, 2 g glucose, 2 g citric acid, 20 ml egg yolk, 500,000 IU penicillin, and 50,000 IU streptomycin. After adding distilled water to a final volume of 100 ml, the solution is filtered for sterilization and the pH is adjusted to 7.2 to 7.

4.

4. The method according to claim 1, characterized in that, The cryo-diluent is prepared by adding 6 ml of glycerol to the base diluent and then mixing it with 1% hydroxyethyl starch, 0.05% chitosan oligosaccharide and 0.5% bovine serum albumin.

5. The method according to claim 1, characterized in that, The semen dilution process is as follows: the qualified semen is slowly added to the base diluent preheated to 37 degrees Celsius at a volume ratio of 1:1 and equilibrated for 30 minutes; then it is slowly added to the frozen diluent precooled to 4 degrees Celsius at a volume ratio of 1:1 and equilibrated at 4 degrees Celsius for 60 minutes.

6. The method according to claim 1, characterized in that, The cooling process includes: dispensing the equilibrated semen into 0.5 ml capillary tubes, first cooling it from 4 degrees Celsius to -20 degrees Celsius at a rate of 1 degree Celsius per minute, then cooling it to -80 degrees Celsius at a rate of 5 degrees Celsius per minute, and finally transferring it to liquid nitrogen for storage.

7. A system for improving the cryopreservation effect of donkey sperm based on the method of any one of claims 1 to 6, characterized in that, The system includes: The semen collection module is used to collect semen from male donkeys under a constant temperature and humidity environment. A semen quality testing module, connected to the semen collection module, is used to detect sperm motility, density, and morphology and to screen qualified samples. A semen dilution module, connected to the semen quality detection module, is used to automatically prepare basic diluent and frozen diluent and complete stepwise dilution. A semen freezing module, connected to the semen dilution module, uses a programmed cooling method to achieve multi-stage temperature-controlled freezing; A semen thawing module, connected to the semen freezing module, is used to achieve rapid rewarming and energy recovery of semen.

8. The system according to claim 7, characterized in that, The semen collection module includes a temperature control unit, a humidity sensor, and a sterile collection device. The temperature control unit is used to maintain the temperature of the inner wall of the artificial vagina at 42 degrees Celsius, and the humidity sensor is used to monitor the humidity of the semen collection environment. After collection, the semen is automatically transmitted to the detection module through a sealed pipeline.

9. The system according to claim 7, characterized in that, The semen freezing module includes a programmed cooling controller, a temperature sensor, and a liquid nitrogen storage tank. The programmed cooling controller is used to control the rate of temperature change and keep the temperature difference between the sample center and the outer layer not exceeding 3 degrees Celsius. The temperature sensor provides real-time temperature data feedback to achieve closed-loop control.

10. A composite cryoprotectant for improving the cryopreservation effect of donkey sperm, characterized in that, Includes the following components: Tris 3 to 4 g, citric acid 2 to 3 g, glucose 1 to 2 g, egg yolk 15 to 25 ml, penicillin 400,000 to 600,000 IU, streptomycin 40,000 to 60,000 IU, glycerol 5 to 7 ml, hydroxyethyl starch 0.8 to 1.2% by weight, chitosan oligosaccharide 0.04 to 0.06% by weight, bovine serum albumin 0.4 to 0.6% by weight, vitamin E 0.15 to 0.25 mmol / L, glutathione 1.5 to 2.5 mmol / L, add distilled water to a final volume of 100 ml, pH 7.0 to 7.4.

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