Use of L-glutamine in the preparation of a cryoprotectant for livestock semen

Abstract

The present application relates to the field of biotechnology, and particularly relates to application of L-glutamine in preparation of livestock semen cryoprotectant.The present application provides application of L-glutamine in preparation of livestock semen cryoprotectant, and aims to overcome the shortcomings in the existing semen cryopreservation technology.L-glutamine can effectively reduce the damage of sperm cells in the freezing process, reduce the formation of ice crystals, protect the integrity of the cell membrane, and improve the survival rate, motility and fertilization capacity of the frozen sperm through its excellent antioxidant effect and low toxicity.Meanwhile, L-glutamine has good biocompatibility and strong adaptability, and can be widely applied to the semen cryopreservation of different animal breeds, and provides a safe, efficient and low-toxicity new cryoprotectant selection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of L-glutamine in the preparation of livestock semen cryogens. Background Technology

[0002] Semen cryopreservation is an indispensable technology in animal reproduction and genetic resource conservation. With the continuous development of animal husbandry and animal breeding, semen cryopreservation has been widely applied in breeding stock reproduction, germplasm resource conservation, and genetic breeding. The core purpose of semen cryopreservation is to preserve sperm samples at low temperatures to maintain their viability and genetic properties for long-term use, providing usable semen for subsequent artificial insemination. However, during the cryopreservation process, sperm cells undergo extreme temperature changes during freezing and thawing. This process causes severe physical and chemical damage to the sperm, especially the freezing and ice crystal formation of cell water during freezing, which leads to a series of irreversible damages such as cell membrane rupture and membrane protein inactivation, thus significantly affecting the cryopreservation effect.

[0003] The use of cryoprotectants is crucial to minimizing damage during sperm freezing. However, existing cryoprotectants have some limitations. Permeable cryoprotectants may be toxic to cells while protecting sperm, leading to reduced sperm motility after thawing; non-permeable cryoprotectants have poor freezing effects and are unlikely to effectively improve sperm survival rates. Therefore, existing semen cryoprotectants have limitations in improving semen preservation quality, especially since sperm motility and viability are often significantly affected under freezing conditions. Improving the protective effect during semen freezing, reducing sperm damage, and ensuring semen quality and survival rates remain key technical challenges for semen cryopreservation. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of L-glutamine in the preparation of livestock semen cryogens.

[0005] The first aspect of this invention provides the application of L-glutamine in the preparation of livestock semen cryogens.

[0006] The L-glutamine in this invention can effectively reduce sperm cell damage during freezing, reduce ice crystal formation, protect cell membrane integrity, and improve sperm survival rate, motility, and fertilization capacity after freezing. Furthermore, L-glutamine has good biocompatibility, antioxidant properties, and low toxicity, effectively solving the problems of toxicity to sperm from permeable preservatives and reduced sperm survival rate caused by non-permeable preservatives in existing technologies.

[0007] In another preferred embodiment, the livestock semen cryoprotectant uses L-glutamine as the sole active ingredient.

[0008] In another preferred embodiment, the concentration of L-glutamine in the livestock semen cryoprotectant is 0.3 g / L to 1.2 g / L. At this concentration, it can effectively improve the quality of frozen livestock semen. If the concentration is too low, it will not be able to improve the survival rate, motility and fertilization ability of the sperm after freezing. If the concentration is too high, it will increase the metabolic burden on the cells and cause changes in the osmotic pressure of the sperm cells, thereby affecting the quality of the frozen sperm.

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

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

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

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

[0013] A second aspect of the present invention provides a method for improving the quality of frozen semen from livestock, comprising the following steps:

[0014] The livestock semen cryogenic agent described above was added to the semen, equilibrated at 4℃~5℃, and then frozen and stored in liquid nitrogen.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention is the first to use L-glutamine as a semen cryoprotectant. Through its excellent biocompatibility, antioxidant properties and low toxicity, it effectively reduces sperm cell damage during freezing and thawing, becoming a safe and effective new cryoprotectant. It is used to improve the problems of reduced sperm motility and low survival rate after thawing of existing semen cryoprotectants.

[0017] L-Glutamine, with its excellent antioxidant properties and low toxicity, can effectively reduce sperm cell damage during freezing, decrease ice crystal formation, protect cell membrane integrity, and improve sperm survival rate, motility, and fertilization capacity after freezing. Simultaneously, L-glutamine exhibits good biocompatibility and strong adaptability, making it widely applicable for the cryopreservation of semen from various animal breeds, providing a safe, efficient, and low-toxicity novel cryoprotectant option. Attached Figure Description

[0018] Figure 1 The graph shows the effects of different cryoprotectants on sperm motility.

[0019] Figure 2 The graph shows the results of sperm linear motility rates under different cryoprotectants.

[0020] Figure 3 The graph shows the results of sperm plasma membrane integrity under different cryogenic agents.

[0021] Figure 4 This graph shows the results of total antioxidant capacity of sperm under different cryoprotectants.

[0022] Figure 5 The graph shows the results of sperm ATP content under different cryogenic conditions. Detailed Implementation

[0023] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0024] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0025] Current cryopreservation techniques for semen have limitations with commonly used cryoprotectants. Traditional permeable cryoprotectants, such as glycerol and dimethyl sulfoxide, while effectively penetrating sperm cells for protection, have toxic effects on sperm, potentially leading to reduced sperm viability and motility after freezing and thawing. Non-permeable cryoprotectants, such as glucose and sucrose, although forming a protective layer outside the cells and slowing ice crystal formation, have limited effectiveness and are insufficient to prevent cell membrane damage. Existing cryoprotectants also exhibit 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 while exhibiting low toxicity to cells.

[0026] L-Glutamine is an important non-essential amino acid widely found in humans and animals, playing a crucial role, particularly in the immune system and energy metabolism. As a precursor to glutathione, L-glutamine promotes glutathione synthesis. Glutathione is a potent antioxidant capable of directly scavenging oxygen free radicals and reactive oxygen species in the body. L-Glutamine also significantly enhances cellular antioxidant capacity by activating the antioxidant transcription factor Nrf2 and regulating the expression of various antioxidant enzymes. Furthermore, as one of the main nitrogen sources in cells, L-glutamine plays a vital role in amino acid metabolism. It also participates in the tricarboxylic acid cycle, providing crucial energy support to mitochondria and playing an important role in maintaining cell membrane stability and function.

[0027] This invention provides the application of L-glutamine in the preparation of cryopreservation agents for livestock semen, aiming to overcome the shortcomings of existing semen cryopreservation technologies. L-glutamine, through its excellent antioxidant properties and low toxicity, can effectively reduce sperm cell damage during freezing, reduce ice crystal formation, protect cell membrane integrity, and improve sperm survival rate, motility, and fertilization capacity after freezing. Simultaneously, L-glutamine exhibits good biocompatibility and strong adaptability, and can be widely applied to the cryopreservation of semen from different animal breeds, providing a safe, efficient, and low-toxicity semen cryopreservation agent option.

[0028] 1. Experimental reagents and instruments

[0029] Experimental reagents: Diluent (IMV, France); fructose (Solepro, China); sodium citrate (Solepro, China); fructose and sodium citrate were used to prepare hypotonic solutions for detecting plasma membrane integrity. L-glutamine (Solepro, China, 99% purity); ATP content assay kit (Solepro, China); total antioxidant capacity assay kit (Solepro, China).

[0030] Instruments: Fully automated sperm quality analyzer (SQA-Vb, Israel), frozen seminal tubing (IMV, France); fully automated seminal tubing filling machine (IMV, France).

[0031] 2. Effects of L-glutamine on frozen semen

[0032] The experimental procedure is as follows: L-glutamine was mixed with double-distilled water to prepare a 25 mg / mL L-glutamine solution; the 25 mg / mL L-glutamine solution was mixed with a diluent to obtain a mixture, so that the final concentrations of L-glutamine in the mixture were 0 mg / mL, 0.3 mg / mL, 0.6 mg / mL, and 1.2 mg / mL, respectively, thus obtaining different concentrations of livestock semen cryogens.

[0033] Fresh semen was mixed with a livestock semen cryogenic agent of the same concentration at a volume ratio of 1:20 for subsequent experimental determination. The sperm motility in the fresh semen ranged from 51.3% to 57.8%.

[0034] Each group of bovine semen was aliquoted, cryopreserved, and stored in liquid nitrogen. One week later, the sperm were thawed in a 37°C water bath, and sperm motility and energy levels were tested using an automated sperm quality analyzer and related reagent kits.

[0035] 1. Measurement of sperm motility and sperm linear velocity.

[0036] Sperm motility refers to the ability of sperm to move in a liquid. High sperm motility indicates that sperm can swim well after thawing and have a strong fertilization potential. Sperm linear motility speed refers to the speed at which sperm travel along a straight path. The faster the linear motility speed, the faster the sperm can swim towards the egg cell, increasing the chances of fertilization. Sperm motility and sperm linear motility speed are detected using a fully automated sperm quality analyzer. The test results are input into Graph Prism software for graphing and significance analysis. The results are as follows: Figure 1 and Figure 2 As shown.

[0037] from Figure 1 It can be seen that sperm motility initially increases and then decreases with increasing L-glutamine concentration, reaching its highest level at an L-glutamine concentration of 0.6 mg / mL. Figure 2 It can be seen that with the increase of L-glutamine content, the linear sperm motility speed first increases and then stabilizes in a certain region.

[0038] 2. Determination of sperm plasma membrane integrity

[0039] Sperm plasma membrane integrity refers to the integrity of the structure and function of the sperm cell membrane. The sperm membrane is a contact barrier between the sperm cell and the external environment, and is crucial for sperm survival, motility, and fertilization ability. Plasma membrane integrity was determined by counting sperm cells after microscopic photography. The counting results were then input into Graph Prism software for graphing and significance analysis. Different letters indicate significant differences (P < 0.05), while the same letter indicates no significant difference (P > 0.05). The results are shown below. Figure 3 As shown.

[0040] from Figure 3 It can be seen that the plasma membrane integrity is best when the L-glutamine concentration is 0.6 mg / mL. When the L-glutamine concentration is further increased to 1.2 mg / mL, the plasma membrane integrity decreases. This indicates that excessively high L-glutamine concentration will affect the integrity of the plasma membrane. This is mainly because high concentrations of L-glutamine will cause changes in osmotic pressure. Such changes in osmotic pressure may lead to sperm cell edema or dehydration, thereby damaging the stability of the plasma membrane.

[0041] 3. Determination of total antioxidant capacity and ATP content in sperm.

[0042] Total antioxidant capacity of sperm refers to the sum of antioxidants in sperm. Oxidative stress can lead to sperm membrane damage, DNA breakage, and decreased motility. Therefore, sperm antioxidant capacity is crucial for maintaining sperm motility 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 generally associated with excellent sperm motility, better quality, and stronger fertilization potential. Total antioxidant capacity and sperm ATP content were measured using a kit. The results were input into Graph Prism software for graphing and significance analysis. The results are shown below. Figure 4 and Figure 5 As shown.

[0043] from Figure 4 and Figure 5 The results showed that the total antioxidant capacity and sperm ATP content reached their highest levels when the L-glutamine concentration was 0.6 mg / mL. However, when the L-glutamine concentration was further increased to 1.2 mg / mL, both the total antioxidant capacity and sperm ATP content decreased. This is mainly because excessively high concentrations may lead to cellular stress responses. In addition, excessively high L-glutamine concentrations may increase the metabolic burden on cells, thereby affecting the total antioxidant capacity and sperm ATP content.

[0044] The results show that the total sperm motility in bovine frozen semen without the addition of a livestock semen cryoprotectant was only about 42%, meeting the minimum requirement of the national standard (GB 4143-2022) (i.e., sperm motility ≥ 40%). However, with the addition of a livestock semen cryoprotectant, especially when the L-glutamine concentration in the cryoprotectant was 0.6 mg / mL, the total sperm motility in bovine frozen semen reached 55%, far exceeding the minimum requirement of ≥ 40% in GB 4143-2022, and sperm motility was significantly improved (P < 0.001). Furthermore, the addition of 0.6 mg / mL of L-glutamine significantly improved sperm linear motility (P < 0.01), and the sperm plasma membrane integrity, total sperm antioxidant capacity, and sperm ATP content were all significantly higher than the control group (P < 0.001). This demonstrates that the livestock semen cryoprotectant prepared with L-glutamine can effectively reduce sperm cell damage during freezing, decrease ice crystal formation, protect cell membrane integrity, and improve sperm survival rate, motility, and fertilization capacity after freezing. Furthermore, L-glutamine exhibits good biocompatibility, antioxidant properties, and low toxicity, effectively addressing the issues of sperm toxicity with permeable cryoprotectants and sperm survival rate with non-permeable cryoprotectants in existing technologies.

[0045] The above are merely 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 within the protection scope of the present invention.

Claims

1. An application of L-glutamine in the preparation of a bovine semen refrigerant, characterized in that, The bovine semen cryoprotectant uses L-glutamine as its sole active ingredient. The concentration of L-glutamine in the bovine semen cryoprotectant is 0.3 mg / mL to 0.6 mg / mL.

2. The application of L-glutamine according to claim 1 in the preparation of bovine semen cryoprotectant, characterized in that, The bovine semen cryoprotectant is obtained by diluting L-glutamine with sterile water.

3. The application of L-glutamine according to claim 1 in the preparation of bovine semen refrigerant, characterized in that, The bovine semen cryoprotectant is used to improve the quality of frozen bovine semen.

4. The application of L-glutamine according to claim 3 in the preparation of bovine semen refrigerant, characterized in that, The quality of the frozen bovine semen refers to sperm motility, sperm movement speed, sperm plasma membrane integrity, sperm antioxidant capacity, and sperm ATP content.

5. A method for freezing bovine semen, characterized in that, Includes the following steps: Add the bovine semen cryoprotectant according to any one of claims 1 to 4 to the bovine semen to obtain a mixture, equilibrate it at 4°C to 5°C, and freeze it in liquid nitrogen.

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