Application of L-glutamine in preparation of livestock semen refrigerant

Abstract

The invention relates to the technical field of biology, in particular to application of L-glutamine in preparation of a livestock semen refrigerant. The invention provides an application of L-glutamine in preparation of a livestock semen refrigerant, and aims to overcome the defects in the existing semen cryopreservation technology. The L-glutamine can effectively reduce the damage of sperm cells in the freezing process, reduce the formation of ice crystals, protect the integrity of cell membranes and improve the survival rate, exercise ability and fertilization ability of frozen sperms through the excellent antioxidant effect and low toxicity of the L-glutamine. Meanwhile, the L-glutamine has better biocompatibility and stronger adaptability, can be widely applied to semen cryopreservation of different animal varieties, and provides a safe, efficient and low-toxicity novel cryoprotectant choice.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of L-glutamine in preparing livestock semen cryopreservative. Background Art

[0002] Semen cryopreservation technology is an important and indispensable technology in animal reproduction and genetic resource protection. With the continuous development of animal husbandry and animal breeding, semen cryopreservation technology has been widely used in the fields of breeding of breeding stock, protection of germplasm resources and genetic breeding. The core purpose of semen cryopreservation is to preserve sperm samples at low temperatures, maintain their vitality and genetic properties for a long time, and provide available semen for subsequent artificial insemination. However, during the process of semen cryopreservation, sperm cells will experience extreme temperature changes of freezing and thawing. This process causes serious physical and chemical damage to sperm, especially the freezing of cell water and the formation of ice crystals during the freezing process, which leads to a series of irreversible damages such as cell membrane rupture and membrane protein inactivation, thereby significantly affecting the cryopreservation effect of semen.

[0003] In order to reduce damage during sperm freezing, the use of cryoprotectants is crucial. Existing protectants also have some shortcomings. Permeable protectants may be toxic to cells when protecting sperm, resulting in reduced sperm motility after thawing; non-permeable protectants have poor freezing effects and it is difficult to effectively improve sperm survival rates. It can be seen that existing semen cryoprotectants have certain limitations in improving the quality of semen preservation, especially under freezing conditions, sperm vitality and motility are often greatly affected. How to improve the protective effect during semen freezing, reduce sperm damage, and ensure the quality and survival rate of semen is still a technical problem for semen cryogens. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the use of L-glutamine in preparing livestock semen cryopreservative.

[0005] The first aspect of the present invention provides the use of L-glutamine in preparing livestock semen cryopreservative.

[0006] The L-glutamine in the present invention can effectively reduce the damage of sperm cells during the freezing process, reduce the formation of ice crystals, protect the integrity of the cell membrane, and improve the survival rate, motility and fertilization ability of sperm after freezing. In addition, L-glutamine has good biocompatibility, antioxidant effect and low toxicity, and can effectively solve the problems in the prior art that permeable protective agents are toxic to sperm and that non-permeable protective agents cause a decrease in sperm survival rate.

[0007] In another preferred embodiment, the livestock semen freezing agent contains L-glutamine as the only active ingredient.

[0008] In another preferred embodiment, the concentration of L-glutamine in the livestock semen cryopreservant is 0.3 g / L~1.2 g / L, which can effectively improve the quality of livestock frozen semen. If the concentration is too low, the purpose of improving the survival rate, motility and fertilization ability of sperm after freezing cannot be achieved, while if the concentration is too high, the metabolic burden of cells will be increased, and the osmotic pressure of sperm cells will be changed, thereby affecting the quality of sperm after freezing.

[0009] In another preferred embodiment, the livestock semen cryopreservative is obtained by diluting L-glutamine with sterile water.

[0010] In another preferred embodiment, the livestock semen cryopreservation agent is used to improve the quality of livestock frozen semen.

[0011] In another preferred embodiment, the livestock frozen semen quality refers to sperm motility, sperm motility, sperm plasma membrane integrity, sperm total antioxidant capacity and sperm ATP content.

[0012] In another preferred embodiment, the livestock is cattle.

[0013] A second aspect of the present invention provides a method for improving the quality of frozen semen of livestock, comprising the following steps: The livestock semen cryogen is added into the semen, balanced in a 4°C to 5°C environment, and cryopreserved in a liquid nitrogen environment.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses L-glutamine as a semen cryopreservative for the first time. Through its excellent biocompatibility, antioxidant effect and low toxicity, it effectively reduces the damage of sperm cells during freezing and thawing, becoming a safe and effective new cryopreservative for improving the problems of reduced sperm motility and low survival rate after thawing of existing semen cryopreservatives.

[0015] L-glutamine, through its excellent antioxidant effect and low toxicity, can effectively reduce the damage of sperm cells during the freezing process, reduce ice crystal formation, protect the integrity of cell membranes, and improve the survival rate, motility and fertilization ability of sperm after freezing. At the same time, L-glutamine has good biocompatibility and strong adaptability, and can be widely used in the cryopreservation of semen of different animal species, providing a safe, efficient and low-toxic new cryoprotectant option. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the effects of different cryogens on sperm motility.

[0017] Figure 2 This is the result of the linear motion rate of sperm under different cryogens.

[0018] Figure 3 The results of the sperm plasma membrane integrity rate under different cryogens are shown in the figure.

[0019] Figure 4 This is the result graph of the total antioxidant capacity of sperm under different cryogens.

[0020] Figure 5 The results of sperm ATP content under different cryogens are shown in the figure. DETAILED DESCRIPTION

[0021] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0022] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0023] In the existing semen cryopreservation technology, the commonly used cryoprotectants have certain limitations. Traditional permeable protective agents, such as glycerol and dimethyl sulfoxide, can effectively enter the sperm cells for protection, but their toxicity has adverse effects on sperm, which may lead to reduced sperm survival rate and motility after freezing and thawing. Non-permeable protective agents, such as glucose and sucrose, although they form a protective layer outside the cells and slow down the formation of ice crystals, their effects are limited and it is difficult to prevent damage to the cell membrane. Existing protective agents have poor adaptability to different animals or breeds, and the quality of sperm after freezing and thawing is difficult to guarantee. Therefore, there is an urgent need for a cryoprotectant that can effectively protect sperm and has low toxicity to cells.

[0024] L-glutamine is an important non-essential amino acid that is widely present in the human body and animals, especially playing a key role in the immune system and energy metabolism. As a precursor of glutathione, L-glutamine promotes the synthesis of glutathione. Glutathione is a powerful antioxidant that can directly remove oxygen free radicals and reactive oxygen in the body. L-glutamine also regulates the expression of various antioxidant enzymes by activating the antioxidant transcription factor Nrf2, thereby significantly enhancing the antioxidant capacity of cells and enhancing the antioxidant capacity of cells. In addition, as one of the main nitrogen sources for cells, L-glutamine plays a vital role in amino acid metabolism. At the same time, L-glutamine also participates in the tricarboxylic acid cycle, provides key energy support for mitochondria, and plays an important role in maintaining cell membrane stability and function.

[0025] The present invention provides the use of L-glutamine in the preparation of livestock semen cryopreservation agents, aiming to overcome the deficiencies in existing semen cryopreservation technologies. L-glutamine, through its excellent antioxidant effect and low toxicity, can effectively reduce the damage to sperm cells during the freezing process, reduce ice crystal formation, protect the integrity of cell membranes, and improve the survival rate, motility and fertilization ability of sperm after freezing. At the same time, L-glutamine has good biocompatibility and strong adaptability, and can be widely used in the cryopreservation of semen of different animal species, providing a safe, efficient and low-toxic semen cryopreservation agent option.

[0026] 1. Experimental reagents and instruments Experimental reagents: diluent (IMV, France); fructose (Solebao, China); sodium citrate (Solebao, China); fructose and sodium citrate are used to prepare hypotonic solution to detect plasma membrane integrity. L-glutamine (Solebao, China, purity 99%); ATP content detection kit (Solebao, China); total antioxidant capacity detection kit (Solebao, China).

[0027] Instruments: fully automatic sperm quality analyzer (SQA-Vb, Israel), frozen sperm tubes (IMV, France); fully automatic straw tube filling machine (IMV, France).

[0028] 2. Effect of L-glutamine on frozen semen The specific experimental process is as follows: L-glutamine is mixed with double distilled water to prepare a 25 mg / mL L-glutamine solution; the 25 mg / mL L-glutamine solution is mixed with a diluent to obtain a mixed solution, and the final concentration of L-glutamine in the mixed solution is 0 mg / mL, 0.3 mg / mL, 0.6 mg / mL, and 1.2 mg / mL, respectively, to obtain livestock semen cryopreservatives of different concentrations.

[0029] Fresh semen was mixed with livestock semen cryopreservation agent of the same concentration at a volume ratio of 1:20 for subsequent experimental determination. Among them, the sperm motility in fresh semen was 51.3%~57.8%.

[0030] Each group of bovine semen was packaged, frozen and stored in liquid nitrogen. After one week, each group of sperm was thawed in a 37°C water bath, and the sperm motility and energy indicators were tested using a fully automatic sperm quality analyzer and related test kits.

[0031] 1. Determination of sperm motility and sperm linear motion speed.

[0032] Sperm motility refers to the ability of sperm to move in liquid. High sperm motility means that the sperm can swim better after thawing and has a stronger fertilization potential. Sperm linear motion speed refers to the speed at which the sperm moves along a straight path. The faster the linear motion speed, the faster the sperm can swim to the egg cell, increasing the chance of fertilization. Sperm motility and sperm linear motion speed are tested by a fully automatic sperm quality analyzer, and the test results are input into the graph prism software for plotting and analyzing significance. The results are as follows: Figure 1 and Figure 2 shown.

[0033] from Figure 1 It can be seen that with the increase of L-glutamine concentration, sperm motility first increases and then decreases. When the L-glutamine concentration is 0.6 mg / mL, sperm motility is the highest. Figure 2 It can be seen that with the increase of L-glutamine content, the linear motion speed of sperm increases first and then stabilizes in a certain area.

[0034] 2. Determination of sperm plasma membrane integrity Sperm plasma membrane integrity refers to whether the structure and function of the sperm cell membrane are intact. The sperm membrane is the contact barrier between sperm cells and the external environment, and is crucial to the survival, movement and fertilization ability of sperm. The integrity of the plasma membrane is counted after taking pictures under a microscope, and the counting results are input into the graph prism software for graphing and analysis of significance. Different letters indicate significant differences (P < 0.05), and the same letters indicate insignificant differences (P > 0.05). The results are shown in Figure 3 shown.

[0035] from Figure 3 It can be seen that when the L-glutamine concentration is 0.6 mg / mL, the plasma membrane integrity is the best. When the L-glutamine concentration is further increased to 1.2 mg / mL, the plasma membrane integrity decreases, indicating that too high L-glutamine concentration will affect the integrity of the plasma membrane. This is mainly because high concentrations of L-glutamine will cause osmotic pressure changes. This change in osmotic pressure may cause sperm cell edema or dehydration, thereby destroying the stability of the plasma membrane.

[0036] 3. Determination of total sperm antioxidant capacity and sperm ATP content.

[0037] The total antioxidant capacity of sperm refers to the sum of antioxidant substances in sperm. Oxidative stress can lead to damage to sperm membranes, DNA breakage and decreased motility. Therefore, the antioxidant capacity of sperm is essential to maintaining its vitality and fertilization ability. Sperm ATP content refers to the concentration of adenosine triphosphate (ATP) in sperm. ATP is the main energy source for cells. Higher ATP content is usually associated with excellent sperm motility, better quality and stronger fertilization potential. The total antioxidant capacity and sperm ATP content are tested using a test kit, and the test results are input into the graph prism software for plotting and analyzing significance. The results are as follows: Figure 4 and Figure 5 shown.

[0038] from Figure 4 and Figure 5 The results show that when the L-glutamine concentration is 0.6 mg / mL, the total antioxidant capacity and sperm ATP content reach the highest, and when the L-glutamine concentration further increases to 1.2 mg / mL, the total antioxidant capacity and sperm ATP content both decrease. This is mainly because too high a concentration may cause cell stress response. In addition, too high a L-glutamine concentration may increase the metabolic burden of cells, thereby affecting the total antioxidant capacity of sperm and sperm ATP content.

[0039] From the above results, it can be seen that the total sperm motility in bovine frozen semen without the addition of livestock semen cryopreservation agent is about 42%, which only meets the minimum requirement in the national standard (GB 4143-2022) (i.e., sperm motility ≥ 40%). When livestock semen cryopreservation agent is added, especially when the concentration of L-glutamine in the livestock semen cryopreservation agent is 0.6 mg / mL, the total sperm motility in bovine frozen semen can reach 55%, far exceeding the minimum requirement of sperm motility ≥ 40% in GB 4143-2022, and the sperm motility is significantly improved (P<0.001); after adding 0.6 mg / mL of L-glutamine, the linear motility of sperm is also significantly improved (P<0.01), and the sperm plasma membrane integrity, sperm total antioxidant capacity and sperm ATP content are significantly greater than those of the control group (P<0.001). It can be seen that the livestock semen cryopreservative prepared by L-glutamine can effectively reduce the damage of sperm cells during the freezing process, reduce the formation of ice crystals, protect the integrity of the cell membrane, and improve the survival rate, motility and fertilization ability of sperm after freezing. In addition, L-glutamine has good biocompatibility, antioxidant effect and low toxicity, which can effectively solve the problems of toxicity of permeable protective agents to sperm and survival rate of sperm with non-permeable protective agents in the prior art.

[0040] The above are only preferred embodiments of the present invention and are 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 in the protection scope of the present invention.

Claims

1. Application of L-glutamine in the preparation of livestock semen cryopreservative.

2. The use of L-glutamine in the preparation of livestock semen cryopreservative according to claim 1, characterized in that: The livestock semen freezing agent uses L-glutamine as the only active ingredient.

3. The use of L-glutamine in the preparation of livestock semen cryopreservative according to claim 1, characterized in that: The concentration of L-glutamine in the livestock semen cryopreservative is 0.3 g / L to 1.2 g / L.

4. The use of L-glutamine in the preparation of livestock semen cryopreservative according to claim 3, characterized in that: The livestock semen freezing agent is obtained by diluting L-glutamine with sterile water.

5. The use of L-glutamine in preparing livestock semen cryopreservative according to claim 1, characterized in that: The livestock semen freezing agent is used for improving the quality of livestock frozen semen.

6. The use of L-glutamine in preparing a livestock semen cryopreservative according to claim 5, characterized in that: The livestock frozen semen quality refers to sperm motility, sperm movement speed, sperm plasma membrane integrity, sperm antioxidant capacity and sperm ATP content.

7. The use of L-glutamine in preparing livestock semen cryopreservative according to claim 1, characterized in that: The livestock is cattle.

8. A method for freezing livestock semen, characterized in that: The following steps are involved: The livestock semen cryogen according to any one of claims 1 to 7 is added to livestock semen to obtain a mixed solution, which is equilibrated at 4°C to 5°C and cryopreserved in a liquid nitrogen environment.

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