Rabbit semen preservation diluent containing serine

Abstract

The invention discloses a rabbit semen preservation diluent containing serine and a preparation method, the diluent comprises the following components in percentage by weight: 2-4% of serine, 3-8% of glucose, 3-5% of yolk, 0.1-0.5% of citric acid, 0.2-0.5% of monopotassium phosphate, 0.2-0.5% of sodium bicarbonate, 0.01-0.1% of ethylene diamine tetraacetic acid (EDTA) and the balance of distilled water, so that the total volume reaches 100%. The method for prolonging the sperm preservation time comprises the steps that the concentration of serine is preferably 3%, the pH value is adjusted to 6.8-7.2, collected rabbit semen and a diluent are mixed according to the proportion of 1: 2-1: 5, and then the mixture is placed in a preservation environment at the temperature of 4 DEG C to be preserved. The method is characterized in that sperms can be preserved for 7-10 days, and the sperm motility is kept above 75% during the period. The invention can prolong the preservation time of the rabbit semen and keep the sperm motility. The diluent disclosed by the invention is beneficial to improving the fertilization rate and the breeding efficiency of rabbits.

Description

Technical Field

[0001] The invention relates to the technical field of animal breeding, in particular to a serine-containing semen preservation diluent specially designed for rabbits, aiming to improve the preservation effect of rabbit semen, prolong the preservation time, and maintain high sperm motility and fertilization ability. Background Art

[0002] In the rabbit breeding industry, artificial insemination technology is one of the important means to improve reproductive efficiency and economic benefits, and can greatly improve the utilization rate of excellent male rabbits of Rex rabbits. In production, the ratio of males to females in natural mating is generally 1:4~6, and artificial insemination technology can be expanded to 1:80~120, which can save the purchase and feeding management costs of male rabbits. The use of high-quality rabbit semen can also improve the estrus conception rate and litter size of female rabbits. At the same time, artificial insemination technology can monitor the quality of semen at any time to avoid adverse effects caused by semen quality problems. Semen is easy to transport, and artificial insemination allows female rabbits to breed without geographical restrictions. In this process, the preservation of semen is crucial to the quality of semen. Although conventional rabbit semen preservation diluents have a certain effect in maintaining semen quality and extending the storage time, they still face many problems, such as decreased sperm motility and unstable fertilization rate. Therefore, developing new and efficient semen preservation diluents to optimize semen preservation conditions and improve sperm quality and fertilization ability is an urgent problem that needs to be solved in the current rabbit breeding industry.

[0003] Serine, as a naturally occurring non-essential amino acid, has a variety of important physiological functions in organisms. Especially in the field of reproductive system, serine has shown significant protective and promoting effects on sperm. It has strong antioxidant capacity, can remove free radicals generated during the storage of semen, reduce the damage of oxidative stress to sperm, and protect the integrity of sperm membrane and DNA. This is crucial for prolonging the storage time of semen and maintaining high sperm motility. In addition, serine can also provide necessary energy support for sperm and promote sperm metabolic activity. During the storage of semen, sperm needs to continuously consume energy to maintain its vitality and motility. As an important participant in energy metabolism, serine can ensure that sperm obtains sufficient energy supply during storage, thereby maintaining its optimal state. Serine is also involved in regulating the physiological functions and signal transduction processes of sperm. Studies have shown that serine can affect the physiological activities of sperm such as acrosome reaction and membrane potential changes, thereby improving the fertilization ability of sperm, which is of great significance for improving the reproductive efficiency and economic benefits of rabbits.

[0004] In view of the unique advantages of serine in sperm protection, energy supply and physiological function regulation, its introduction into rabbit semen preservation diluent is expected to significantly improve semen preservation effect, sperm vitality and fertilization ability. Therefore, the present invention proposes a rabbit semen preservation diluent containing serine, aiming to optimize the diluent formula by scientifically proportioning serine and other necessary ingredients to meet the rabbit breeding industry's demand for efficient and stable breeding technology. Summary of the invention

[0005] The present invention provides a serine-containing rabbit semen preservation diluent and a preparation method thereof. The technical solution aims to optimize the semen preservation environment, prolong the semen preservation time, and improve the survival rate, motility and fertilization ability of rabbit sperm by scientifically matching multiple ingredients, especially introducing an appropriate amount of serine. The specific technical solution is as follows: (1) Diluent ingredients and their functions Basic nutrients: including glucose (5%-10%) and sucrose (10%-15%), which serve as the main energy source for sperm metabolism and maintain the basic physiological functions of sperm in vitro.

[0006] Key ingredient - serine: The added amount is 0.3%-4.0%. As an antioxidant, it can reduce the oxidative stress damage to sperm during storage, protect the integrity of the sperm membrane, and prolong the survival time of sperm.

[0007] Buffer substances: citric acid (0.1%~0.2%) and potassium dihydrogen phosphate (0.2%~0.3%) are used to adjust the pH value of the diluent to an appropriate range (6.8-7.2), simulate the physiological environment in the rabbit body, and maintain the stability of sperm.

[0008] Protective agents: including egg yolk (3%-7%), milk extract (optional, 0%-5%) and vitamin E (0.01%-0.1%). Egg yolk is rich in phospholipids and proteins, which helps maintain the acrosome integrity and motility of sperm; milk extract provides additional nutrients and growth factors; vitamin E, as a powerful antioxidant, further enhances the antioxidant capacity of the diluent.

[0009] Antimicrobial agents: such as penicillin and streptomycin (added in conventional doses) are used to prevent microbial contamination of the diluent during preparation and storage.

[0010] (2) Preparation method a. Accurately weigh each ingredient according to the predetermined ratio.

[0011] b. Mix the basic nutrients (glucose, sucrose) and buffer substances (citric acid, potassium dihydrogen phosphate) in an appropriate amount of distilled water, heat to 40-50℃ to dissolve and stir evenly.

[0012] c. While stirring, add serine, egg yolk (pre-processed into a uniform suspension), milk extract (if used) and vitamin E in sequence, and continue stirring until all ingredients are fully blended.

[0013] d. Filter the mixed solution to remove insoluble matter or impurities.

[0014] e. Sterilize the filtered diluent, such as by ultraviolet irradiation, high temperature short-time sterilization or filtration sterilization, to ensure the sterility of the diluent.

[0015] f. Dispense the treated diluent into sterile containers and seal them for future use. Be careful to avoid contamination during the dispensing process and ensure that the container is well sealed.

[0016] (3) Mechanism of action Serine plays a major antioxidant role in the diluent, protecting sperm from oxidative damage by scavenging free radicals and reducing lipid peroxidation reactions. At the same time, serine can also work synergistically with other ingredients in the diluent to maintain the normal physiological function of sperm. For example, basic nutrients provide energy support; buffer substances adjust the pH value to maintain sperm stability; protective agents further protect sperm from physical and chemical damage; and antibacterial agents ensure the sterility of the diluent. These ingredients work together to provide an ideal storage environment for sperm, thereby significantly improving the preservation effect of semen.

[0017] (4) Beneficial effects Significantly improve sperm survival rate: Due to the antioxidant effect of serine, the diluent of the present invention can significantly reduce the lipid peroxidation level of sperm during storage, thereby prolonging the survival time of sperm.

[0018] Maintain sperm fertilization ability: The nutrients and protective agents in the diluent work together to provide a suitable storage environment for sperm, maintain its acrosome integrity and motility, and ensure that the sperm still has a high fertilization ability after storage.

[0019] Simple operation and low cost: The diluent preparation process of the present invention is simple, the raw materials are easily available and the cost is low, and it is suitable for large-scale production and application. DETAILED DESCRIPTION

[0020] The following are specific examples of the present invention, which are used to further illustrate the technical solutions and effects of the present invention. These examples are only for illustrative purposes and do not limit the scope of protection of the present invention.

[0021] Example 1 Basic formula and preparation The formula is: serine: 2% (based on the total volume of the diluent), glucose: 5%, egg yolk: 4%, citric acid: 0.2%, potassium dihydrogen phosphate: 0.3%, ethylenediaminetetraacetic acid (EDTA): 0.05%, vitamin E: 0.02%, distilled water: the remainder, so that the total volume reaches 100%.

[0022] Preparation method: According to the above formula ratio, weigh the raw materials of each component.

[0023] Heat distilled water to a suitable temperature (such as room temperature to lukewarm), add glucose, citric acid, sodium bicarbonate, and EDTA in sequence, stirring while adding to ensure full dissolution. Slowly add serine powder while stirring continuously until completely dissolved. Use a pH meter to adjust the pH of the solution to 7.0 ± 0.1. Filter the solution through a sterile filter to remove impurities and microorganisms. Dispense the filtered solution into sterile containers and seal for later use.

[0024] The preservation effect of the above-mentioned Example 1 was evaluated as follows: (1) Sperm motility Computer-Aided Semen Analysis (CASA) was used as a technical means to accurately quantify and evaluate the survival rate of sperm. The specific operation is: the semen sample is transferred to a centrifuge tube with a capacity of 1 ml, and then 10 microliters (μL) of semen sample is accurately measured with a micropipette and gently dripped onto the surface of a slide that has been pre-incubated at 37°C, and then covered with a cover glass that has also been preheated at 37°C to ensure the temperature stability and suitability of the detection environment. Subsequently, the CASA system was used for observation and analysis. Five different microscope fields of view were randomly selected as sample areas for each analysis to comprehensively and objectively evaluate the survival status and motility characteristic parameters of sperm in the serine-added group and the control group at room temperature.

[0025] The results show that: after 7 days of storage using the serine-containing diluent prepared in this embodiment to preserve rabbit semen, the test shows that sperm motility remains above 80%, which is significantly higher than the control group using the traditional diluent. The average sperm path speed is significantly higher than the control group at 1 day and 7 days. The curve and linear speed of sperm on the 1st and 7th days are also higher than the control group. On the 7th day, the sperm acrosome integrity rate and plasma membrane integrity rate of the serine-added group are significantly higher than those of the control group.

[0026] Table 1 Effects of serine-added semen extender on sperm motility and motility parameters of rabbit semen at 1 and 7 days

[0027] Table 2 Effects of serine-added semen extender on the integrity of rabbit sperm acrosome and plasma membrane

[0028] Embodiment 2: Optimizing Serine Concentration formula: Serine: optimized to 3% (based on the total volume of diluent) Other ingredients are the same as in Example 1 Preparation method: Similar to Example 1, only the concentration of serine was adjusted to 3%, and the other steps remained unchanged.

[0029] Experimental results: Through comparative experiments, it was found that increasing the serine concentration to 3% was better than adding 2%, and the storage time of semen could be extended to 10 days, and the sperm motility was still maintained at more than 75%. This shows that increasing the serine concentration within a certain range can further improve the preservation effect of the diluent.

[0030] Table 3 Effects of semen diluent supplemented with 3% serine on sperm motility and motility parameters of rabbit semen at 1, 7, 10, and 13 days

[0031] Embodiment 3: Effect verification at different storage temperatures Formula: Use the optimized formula in Example 2 Experimental design: The collected rabbit semen was diluted with the diluents in this example and dispensed into multiple sterile containers. These containers were stored at different storage temperatures (4°C, 20°C). The sperm motility and morphology at each temperature were tested regularly (1d, 7d, 10d).

[0032] Experimental results: Table 4 Effects of different storage temperatures on rabbit semen motility and motility parameters

[0033] The test results show that the sperm motility and morphology of semen stored at 4°C are the best, and they can still maintain a high level of motility even after 10 days. However, the sperm motility of semen stored at 20°C gradually decreases over time, and the morphological damage is aggravated. This shows that low-temperature storage is more conducive to maintaining sperm motility and stability.

[0034] Compared with the prior art, the present invention has the following significant effects: Prolong storage time: Due to the addition of serine, the diluent of the present invention can significantly improve the antioxidant capacity of rabbit semen, reduce the damage of oxidative stress to sperm, and thus prolong the storage time of semen. Maintain sperm motility: Serine can provide necessary energy support for sperm, promote sperm metabolism, and maintain the activity of sperm during storage. Improve fertilization ability: High-quality sperm can more easily penetrate the egg to complete the fertilization process, so the diluent of the present invention helps to improve the fertilization rate and reproductive efficiency of rabbits. Significant economic benefits: By improving reproductive efficiency and fertilization rate, the diluent of the present invention can significantly reduce the cost input of rabbit breeding industry and improve economic benefits.

[0035] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A serine-containing rabbit semen preservation diluent, characterized in that The invention comprises the following components in weight percentage: 2-4% serine, 3-8% glucose, 3-5% egg yolk, 0.1-0.5% citric acid, 0.2-0.5% potassium dihydrogen phosphate, 0.2-0.5% sodium bicarbonate, 0.01-0.1% ethylenediaminetetraacetic acid (EDTA), and the balance of distilled water, so that the total volume reaches 100%.

2. The diluent according to claim 1, characterized in that The concentration of serine is preferably 3%.

3. The diluent according to claim 1 or 2, characterized in that The step of adjusting the pH value to 6.8-7.2 is also included.

4. A method for preserving rabbit semen using the diluent according to any one of claims 1 to 3, comprising the steps of mixing the collected rabbit semen with the diluent in a ratio of 1:2 to 1:5, and then preserving the mixture in a storage environment at 4°C.

5. The method according to claim 4, characterized in that The storage time is 7-12 days, during which the sperm motility remains above 75%.

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