Application of L-citrulline in preparation of preparation for promoting growth and metabolism of sheep sertoli cells
By using L-citrulline in the metabolic preparation for supporting cell growth of goat testicles, the problems of dead sperm and azoosperm caused by supporting cell dysfunction in goat testicles were solved, and the growth and metabolism of support cells were promoted, and the quality of semen and reproduction were improved. Function.
Patent Information
- Application Number
- CN202411821642.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-02-11
AI Technical Summary
The prior art is difficult to effectively improve the function of the supporting cells of the sheep testicles, resulting in the occurrence of dead sperm and abscess in rams, and lack of effective solutions.
In the preparation of L-citrulline to promote the growth and metabolism of cells of goat testicles, the amount used is 2.8 to 3.2 g/kg once a day by oral administration.
L-citrulline helps maintain the normal morphology of the trichosperm in the goat testicles, promotes support cell proliferation, improves support cell viability, regulates the structure of the blood testicle barrier, enhances antioxidant and anti-DNA damage ability, and improves semen quality and reproductive function. .
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Figure CN119405645A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to application of L-citrulline in preparing a preparation for promoting the growth and metabolism of sheep testicular supporting cells. Background Art
[0002] Sertoli cells play a key role in the male reproductive system. They not only provide the necessary nutrition and support for sperm production, but also participate in regulating the microenvironment in the testicles and maintaining immune protection and antioxidant defense mechanisms. If the function of Sertoli cells is impaired, it will have a significant impact on the reproductive health and growth metabolic activities of male animals, specifically manifested in decreased reproductive efficiency, weakened immune protection, and weakened antioxidant defense mechanisms. These dysfunctions will not only affect the fertility of individuals, but also lead to the deterioration of the health of the overall reproductive system, thereby affecting the reproduction and genetic quality of the population.
[0003] At present, most farms and breeders lack effective solutions when excellent breeding rams have dead sperm or azoospermia, and they can only eliminate these rams. Studies have found that the main reason for dead sperm and azoospermia is that some important cells (such as supporting cells) in the testicles of rams have problems in their functions. Therefore, how to improve the function of testicular supporting cells and achieve normal growth and metabolism is the key to overcoming this problem. Summary of the invention
[0004] The main purpose of the present invention is to provide an application of L-citrulline in the preparation of a preparation for promoting the growth and metabolism of sheep testicular support cells, thereby providing a new drug option for promoting the growth and metabolism of testicular support cells.
[0005] To achieve the above object, the present invention provides an application of L-citrulline in the preparation of a preparation for promoting the growth and metabolism of sheep testicular Sertoli cells.
[0006] In one embodiment, in the preparation, the amount of L-citrulline is 2.8-3.2 g / kg.
[0007] In one embodiment, the formulation is administered orally once a day.
[0008] The technical solution of the present invention is an application of L-citrulline in the preparation of a preparation for promoting the growth and metabolism of sheep testicular supporting cells. It is disclosed that L-citrulline is beneficial to maintaining the normal morphology of sheep testicular seminiferous tubules, can promote the proliferation of sheep testicular supporting cells, enhance the vitality of sheep testicular supporting cells, regulate the blood-testis barrier structure, and can also improve the antioxidant capacity and DNA damage resistance of sheep testicular supporting cells, regulate reproductive function and DNA metabolism-related pathways, increase the number of germ cells and improve semen quality, and actively promote the growth and metabolism of supporting cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.
[0010] Figure 1 The representative images are the hematoxylin and eosin staining results of the seminiferous tubules of the sheep testes in the control group and the experimental group in Example 1 of the present invention;
[0011] Figure 2 This is a graph showing the effect of L-citrulline on the DNA cell viability of sheep testicular Sertoli cells in Example 2 of the present invention;
[0012] Figure 3 This is a graph showing the expression levels of Occludin and Claudin11 in the testicular Sertoli cells of the sheep in the control group and the experimental group in Example 3 of the present invention;
[0013] Figure 4 The results of the cell proliferation experiment of sheep testicular supporting cells in the control group and the experimental group in Example 4 of the present invention are shown in FIG.
[0014] Figure 5 This is a graph showing the effect of L-citrulline on the antioxidant capacity of sheep Sertoli cells in Example 5 of the present invention;
[0015] Figure 6 This is a graph showing the effect of L-citrulline on the expression of DNA damage-related proteins in sheep testicular Sertoli cells in Example 6 of the present invention;
[0016] Figure 7 This is a graph showing the results of single-cell sequencing analysis of sheep testicular Sertoli cells in the control group and the experimental group in Example 7 of the present invention;
[0017] Figure 8 This is a graph showing the effect of L-citrulline on semen quality in Example 8 of the present invention, wherein: Figure 8 (A) is the result graph of sperm count in the control group and experimental group; Figure 8 (B) Sperm morphology of the control group and the experimental group; Figure 8 (C) is the result graph of the percentage of abnormal sperm count in the control group and the experimental group.
[0018] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION
[0019] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.
[0020] In the process of spermatogenesis, germ cells are the "protagonists", among which supporting cells (Sertoli cells) play a leading role in maintaining this process. Support cells are considered to be the most complex cell type in organisms because of their special three-dimensional structure. On the one hand, supporting cells provide a support framework to ensure that germ cells can efficiently complete mitosis and meiosis; on the other hand, supporting cells are the mediator between germ cells and endocrine signals, which can control the spermatogenesis process through follicle-stimulating hormone and testosterone, and have a direct impact on germ cell development through paracrine signals. However, current research has not focused on the impact of supporting cells on the growth metabolism of rams.
[0021] Supporting cells play a key role in the male reproductive system. If the supporting cells are damaged, it will have a significant impact on their reproductive health, growth and metabolic activities, such as decreased reproductive efficiency, weakened immune protection, and weakened antioxidant defense mechanisms. At present, most farms and farmers do not have a good solution when excellent breeding rams have dead sperm and azoospermia, and they can only eliminate them. Studies have found that the main reason for dead sperm and azoospermia is that some important cells in the testicles of rams (such as supporting cells) have problems with their functions. Therefore, how to improve the function of testicular supporting cells and achieve normal growth and metabolism is the key to overcoming this problem.
[0022] L-citrulline is a non-proteinogenic α-amino acid with a molecular formula of C6H 13N3O3 has good pharmacological effects and is widely used in medicine, food and health care. So far, the effect of L-citrulline on sheep testicular support cells is still unclear, and there is no report on the application of L-citrulline in the recovery of testicular cell function. Therefore, how to study the new use of L-citrulline and apply it to preparations that promote the growth and metabolism of sheep testicular support cells has important scientific significance and application value.
[0023] In view of this, the present invention provides an application of L-citrulline in preparing a preparation for promoting the growth and metabolism of sheep testicular Sertoli cells.
[0024] The technical solution of the present invention proposes an application of L-citrulline in the preparation of a preparation for promoting the growth and metabolism of sheep testicular support cells, and discloses that L-citrulline is beneficial to maintaining the normal morphology of sheep testicular seminiferous tubules, can promote the proliferation of sheep testicular support cells, enhance the vitality of sheep testicular support cells, regulate the blood-testis barrier structure, and can also improve the antioxidant capacity and DNA damage resistance of sheep testicular support cells, regulate reproductive function and DNA metabolism-related pathways, increase the number of germ cells and improve semen quality. L-citrulline has the advantages of no pollution, no residue, non-toxic and harmless to the body, and low cost. The technical solution of the present application broadens the application scope of L-citrulline and can provide a new drug option for the recovery of sheep testicular support cell function.
[0025] In the embodiment of the present invention, the dosage of L-citrulline is 2.8-3.2 g / kg. Setting the dosage of L-citrulline within this range is beneficial to promoting the realization of the growth and metabolism-related functions of sheep testicular supporting cells.
[0026] In an embodiment of the present invention, the preparation is used orally once a day.
[0027] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.
[0028] Example 1
[0029] Effects of L-citrulline on the morphology of seminiferous tubules in sheep testes:
[0030] 1) Experimental design: 20 healthy male sheep aged 2 months were randomly divided into a control group and an experimental group, with 10 rams in each group. The control group was fed a basic diet, and the experimental group was treated with L-citrulline solution orally once a day on the basis of the basic diet, calculated according to body weight, once a day, at a dosage of 3g / kg, for 4 consecutive months;
[0031] The L-citrulline solution is obtained by dissolving L-citrulline in pure water, with 100g / L L-citrulline as the effective component, and the L-citrulline solution needs to be prepared and used immediately. The nutritional components of the feeding feed meet the sheep feeding standards (protein: 16.61%; fat: 3.24%; carbohydrates: 80.15%); the L-citrulline solution is injected from the corner of the ram's mouth through a 100mL sterile metal syringe, and the ram is orally treated with the L-citrulline solution. This process is completed within 1 minute to avoid stress reactions in the ram.
[0032] 2) H&E staining: Cut a 0.5×0.5×0.5 cm sample from the testicle of a ram. 3 The testicular tissue wax blocks were cut into 5 μm sections and stained with hematoxylin and eosin to observe the morphology of the testicular seminiferous tubules.
[0033] Test results: Test results are as follows Figure 1 As shown in the figure, the black arrows point to the lost and abnormal sperm. Figure 1 It can be seen that the seminiferous tubules in the control group had irregular morphology, the number of sperm in the lumen decreased sharply, the morphology appeared fragmented and fell off and remained in the center of the lumen, and abnormal sperm could be seen in the lumen; the seminiferous tubules in the experimental group were arranged neatly and had regular morphology, a large number of mature sperm could be seen in the lumen and the abnormal sperm was significantly reduced. The above results indicate that L-citrulline is beneficial to maintaining the normal morphology of the seminiferous tubules of sheep testes.
[0034] Example 2
[0035] Effects of L-citrulline on the activity of sheep testicular Sertoli cells:
[0036] 1) Isolation and purification of supporting cells; The testicular tissue of the sheep in Example 1 was collected, the tunica albuginea and blood vessels were first removed and the tissue was washed with PBS buffer; then, the remaining tissue was cut into pieces and placed in a centrifuge tube, and a DPBS buffer containing 1 mg / mL collagenase IV was added, and the mixture was gently shaken and digested at 32°C for 10 min; after the digestion was completed, 0.25% trypsin and 100 μg / mL DNase I were added, and the mixture was gently shaken and digested in a 32°C water bath for 8 min to obtain a cell suspension; then, a DMEM medium containing 5% fetal bovine serum was added to the cell suspension to terminate the digestion; then, the digested cell suspension was filtered through a 70 μm cell sieve, the sieved cells were resuspended and transferred to a DMEM medium, and cultured at 37°C and 5% CO2 for 3 h to allow the cells to adhere to the wall and grow, and the adherent cells obtained after two rounds of adherence screening were marked as supporting cells and collected for later use.
[0037] 2) Counting live cells: Collect the supporting cell suspension obtained in step 1) and add freshly prepared trypan blue dye solution, and dye at room temperature for 3-5 minutes. Then, take a drop of the stained cell suspension and place it on a glass slide, cover the slide and transfer it to a microscope for observation and counting. Among them, dead cells are light blue and swollen, dull; living cells are not colored and maintain normal morphology and are shiny; unstained cells can be counted as live cells.
[0038] Test results: Test results are as follows Figure 2 As shown, the viable cell rate in the control group was 40.79%, and the viable cell rate in the experimental group increased to 62.24%, indicating that L-citrulline can enhance the viability of sheep testicular Sertoli cells.
[0039] Example 3
[0040] Effects of L-citrulline on the expression levels of tight junction-related genes in sheep testicular Sertoli cells:
[0041] 1) Collecting the supporting cells of step 1) of Example 2, and extracting mRNA according to the following steps:
[0042] ① Centrifuge the cells, discard the supernatant, and 6 Add 1 mL of lysis buffer RZ reagent to the cells to obtain a homogenate sample;
[0043] ② Place the homogenized sample at room temperature for 5 minutes to allow the nucleic acid-protein complex to be completely separated;
[0044] ③ Add 200 μL of chloroform and shake vigorously for 15 seconds, then place at room temperature for 3 minutes; centrifuge at 4°C, 12000 rpm (~13400 × g) for 10 minutes to separate the sample into three layers: a yellow organic phase, an intermediate layer, and a colorless aqueous phase;
[0045] ④ Transfer the aqueous phase to a new tube, slowly add 0.5 times the volume of anhydrous ethanol, mix well, transfer the resulting solution and precipitate into the adsorption column CR3, centrifuge at 4°C, 12000 rpm (~13400×g) for 30 seconds, and discard the waste liquid in the collection tube;
[0046] ⑤ Add 500 μL of deproteinized solution RD to the adsorption column CR3, centrifuge at 4°C 12000 rpm (~13400 × g) for 30 s, discard the waste liquid, and place the adsorption column CR3 in a collection tube;
[0047] ⑥ Add 500 μL of rinsing solution RW to the adsorption column CR3, let stand at room temperature for 2 min, centrifuge at 4°C 12000 rpm (~13400×g) for 30 s, and discard the waste liquid;
[0048] ⑦Repeat step ⑥;
[0049] ⑧Put the adsorption column into a 2 mL collection tube and centrifuge at 12000 rpm (~13400 × g) at 4°C for 2 min to remove the residual liquid;
[0050] ⑨ Transfer the adsorption column CR3 into a new 1.5mL centrifuge tube, add 30-100μL RNase-Free ddH2O, place at room temperature for 2min, and centrifuge at 4℃12000rpm (~13400×g) for 2min to obtain the mRNA extraction solution.
[0051] 2) The extracted mRNA was reverse transcribed into cDNA for RT-PCR (the primers were Claudin11 and Occludin, respectively).
[0052] Test results: The expression levels of Occludin and Claudin11 in the testicular support cells of sheep in the control group and the experimental group are as follows Figure 3 As shown, Figure 3 (A) is the effect of L-citrulline on the expression level of Occlduin in sheep testicular Sertoli cells. Figure 3 (B) is a graph showing the effect of L-citrulline on the expression level of Claudin11 in sheep testicular Sertoli cells. Occludin and Claudin11 are important proteins in tight junctions, and they are essential for maintaining the blood-testis barrier function between cells. When the levels of these two proteins decrease, a series of effects may occur on the function of tissues and organs. Figure 3 It can be seen that compared with the control group, the mRNA expression levels of Claudin11 and Occludin in the experimental group were significantly increased, indicating that L-citrulline is beneficial to enhance the tight junction structure of sheep testicular supporting cells and regulate the blood-testis barrier structure.
[0053] Example 4
[0054] Effects of L-citrulline on proliferation of sheep testicular Sertoli cells:
[0055] 1) Isolation and purification of supporting cells: Testicular tissues from healthy male sheep aged 2 months were collected for a Percoll experiment to isolate supporting cells. For specific steps, refer to step 1) of Example 2.
[0056] 2) Cell proliferation (MTT) experiment: Two groups were set up according to the difference in culture medium components, namely the control group and the experimental group. Among them, the DMEM culture medium in the control group contained 10% fetal bovine serum (Gibco, 10099-141) and 1% penicillin-streptomycin (HyClone, SV30010); the composition of the experimental group culture medium was based on the above control group culture medium, and 5mM L-citrulline was added. Subsequently, the supporting cells were randomly added to the two groups of culture medium, and cultured in a cell culture incubator at 37°C and 5% CO2, and the culture medium was changed every 24 hours. When the cell density reached 80-90%, the cells were passaged, and a cell proliferation experiment was performed 24 hours later to detect the effect of L-citrulline on the proliferation of supporting cells.
[0057] Test results: Test results are as follows Figure 4 As shown by Figure 4 It can be seen that the proliferation rate of supporting cells in the control group was 49.5%, while the proliferation of supporting cells in the experimental group was significantly increased, and the proportion increased to 94.82%, indicating that L-citrulline can significantly promote the proliferation of sheep testicular supporting cells.
[0058] Example 5
[0059] Effect of L-citrulline on the antioxidant capacity of sheep Sertoli cells:
[0060] 1) Isolation and purification of supporting cells: Testicular tissues of healthy male sheep aged 2 months were collected for Percoll separation of supporting cells. For specific steps, refer to step 1) of Example 2.
[0061] 2) Cell culture: Three groups were set up according to the difference in the composition of the culture medium, namely the control group, the experimental group (H2O2 was added, also described as the "H2O2" group) and the rescue group (H2O2 and L-citrulline were added, also described as the "Citr+H2O2" group). Among them, the DMEM culture medium in the control group contained 10% fetal bovine serum (Gibco, 10099-141) and 1% penicillin-streptomycin (HyClone, SV30010); the composition of the culture medium in the experimental group was based on the above control group culture medium, and 10μM H2O2 was added; the composition of the culture medium in the rescue group was based on the above experimental group culture medium, and 5μM L-citrulline was added. Subsequently, the supporting cells were randomly added to the three groups of culture medium, and cultured in a cell culture incubator at 37°C and 5% CO2, and the culture medium was changed every 24 hours. When the cell density reached 80-90%, the cells were passaged, and collected after 24 hours, and used to detect the level of reactive oxygen.
[0062] 3) Reactive oxygen level test: Add the DCFH-DA probe to the serum-free culture medium (the final concentration of DCFH-DA is 10 μM). After removing the culture medium, add the appropriately diluted DCFH-DA and fully cover the cells. First, place the three groups after step 2) in a 37°C incubator for 30 minutes. After completion, remove the culture medium and repeatedly blow it with serum-free culture medium or 0.01M PBS solution until the bottom of the bottle turns from translucent (cell monolayer connected into a sheet) to transparent by naked eye observation, and the cell layer is almost completely blown into PBS. Subsequently, all the cell suspensions are collected in a 1.5mL centrifuge tube and washed twice with serum-free culture medium or PBS solution to fully remove the DCFH-DA that has not entered the cells. Finally, the above cell suspension is centrifuged (1000rpm / min, 5min), and the supernatant is discarded and PBS is added to resuspend the cells and prepare to transfer them to a microscope to observe the signal value of FITC.
[0063] Test results: Effect of L-citrulline on the antioxidant capacity of sheep Sertoli cells Figure 5 As shown, green fluorescence (FITC) represents the active oxygen signal. Figure 5 It can be seen that no FITC signal value was observed in the control group and the rescue group ("Citr+H2O2" group), and an obvious FITC signal value appeared in the experimental group ("H2O2" group). The above results indicate that L-citrulline can reduce the level of intracellular reactive oxygen species and enhance the antioxidant capacity of supporting cells.
[0064] Example 6
[0065] Effects of L-citrulline on the expression of DNA damage-related proteins in sheep testicular Sertoli cells:
[0066] 1) Supporting cell separation: The testicular tissue of the ram in Example 1 was collected, and the tissue was first washed with PBS to remove the capsule and blood vessels; after the tunica albuginea was separated, the remaining tissue was cut into pieces and placed in a centrifuge tube, and trypsin was added, and digested at 37°C for 30 minutes, and the cells were shaken several times during the digestion process; then, DMEM / F12 culture medium containing 10% fetal bovine serum was added to terminate the digestion, and the cells were filtered with a cell sieve; finally, the cells were washed with PBS 3-4 times and cultured in a 37°C, 5% CO2 incubator.
[0067] 2) Detection of anti-DNA damage indicators: Collect the supporting cells in step 1) and discard the culture medium, add ROS staining reagent to the 96-well plate, and incubate at 37°C in the dark for 30 minutes; then wash the cells twice with PBS, and observe the fluorescence intensity of the ovaries in each group with a Nikon fluorescence microscope. In addition, the supporting cell protein was extracted using RIPA lysis buffer, and then the protein was transferred to a PVDF membrane by SDS-PAGE and blocked with a TBST solution containing 5% bovine serum albumin; after 6 hours, the membrane was incubated with β-ACTIN and γH2AX at 4°C overnight; the next morning, the PVDF membrane was incubated with the secondary antibody at room temperature for 2 hours. After the incubation, the chemiluminescence detection was completed in a short time. The protein level of γH2AX was quantified using AlphaView SA software.
[0068] Test results: Effects of L-citrulline on the expression of DNA damage-related proteins in sheep testicular Sertoli cells Figure 6 As shown, Figure 6 (A) is the effect of L-citrulline on the expression levels of γH2AX and β-ACTIN proteins; Figure 6 (B) is the quantitative results of the protein level of γH2AX in the control group and the experimental group. Figure 6 It can be seen that the protein level of γH2AX in the experimental group was reduced. The above results indicate that L-citrulline can effectively improve the phenomenon of DNA damage in supporting cells.
[0069] Example 7
[0070] Effects of L-citrulline on the development of sheep testicular Sertoli cells:
[0071] The testicular tissue of sheep of embodiment 1 is collected, first removes tunica albuginea and blood vessel and uses PBS to clean tissue, and separates testicular seminiferous tubules with clean needle. Secondly, it is infiltrated in 1mL 1mg / mL collagenase IV and 1mL 0.1% hyaluronidase, and is placed in 37 ℃, 5% incubator digestion 10min. After digestion is completed, it is necessary to add an equal amount of DEME culture medium (containing 0.5% FBS) to stop digestion. The above-mentioned termination digestion suspension is centrifuged (1200r / min, 5min), retains precipitate and adds 1mL 0.25% pancreatin and 1mL 1μg / mL DNaseI, repeatedly blows and mixes after 37 ℃, 5% incubator digestion 10min. Subsequently, after adding an equal amount of DEME culture medium (containing 0.5% FBS) to stop digestion, it is necessary to obtain cell suspension by 70μm, 40μm cell sieves successively. Finally, the cell suspension obtained above was centrifuged (1200 r / min, 10 min), the precipitate was retained and DEME medium was added to resuspend the cells, ready for single-cell sequencing and analysis.
[0072] Test results: The results of single-cell sequencing analysis of sheep testicular supporting cells in the control group and experimental group are as follows Figure 7 As shown, Figure 7 (A) is a graph showing the specific distribution of germ cell marker genes and somatic cell marker genes. Figure 7 (B) is the distribution diagram of Spem2 gene in germ cell clusters. Figure 7 (C) is the UMAP visualization of germ cell clusters and somatic cell clusters. Figure 7 (D) is the number of germ cells in the control group and the experimental group. Figure 7 (E) is the result of the analysis of the pseudo-temporal development trajectory of germ cells. Figure 7 (F) shows the percentage of germ cells in cell fate 1 and cell fate 2 in the control group and the experimental group. Figure 7 (G) is the result of functional enrichment analysis based on the differentially expressed genes in the control group and the experimental group.
[0073] Depend on Figure 7 (D) It can be seen that the number of germ cells in the control group is 6066, and the number of germ cells in the experimental group is 8960, indicating that L-citrulline can increase the number of germ cells. Figure 7 (E) and Figure 7 (F) It can be seen that there are two different developmental fates in the sperm development process of the control group and the experimental group. Among them, the control group accounts for 76.6% of the cell fate 1 and the experimental group accounts for 23.4%; the control group accounts for 21.51% of the cell fate 2 and the experimental group accounts for 78.49%. Figure 7 (G) Functional enrichment analysis showed that reproductive function and pathways related to DNA metabolism were activated. The above results indicate that L-citrulline treatment will cause germ cells to develop in two different cell fates, ultimately affecting reproductive function and DNA metabolism. Therefore, the mechanism of action of L-citrulline on germ cells is crucial.
[0074] Example 8
[0075] Effects of L-citrulline on semen quality:
[0076] 1) Semen collection: Semen of the rams in Example 1 was collected before feeding in the morning of the last day of the experiment, and the sperm concentration (100 million / ml) was measured using a hemacytometer.
[0077] 2) Microscope observation: To count the number of abnormal sperm, 50 μL of semen was mixed with 950 μL of 3% NaCl solution and placed on one end of a glass slide at a 45° angle, and then stained with Giemsa stain after natural air drying;
[0078] The method for calculating sperm concentration is as follows: 10 μL of semen is mixed evenly with 990 μL of 3% NaCl solution, and the following formula is used to calculate the sperm concentration: the number of sperm in the five large squares × 5 × 10 × 1000 × 100 (dilution multiple);
[0079] The method for calculating sperm concentration is: count the number of abnormal sperm in 1000 sperm in each sample, and the calculation formula for sperm deformity rate is abnormal sperm number / 1000×100%.
[0080] Test results: Effects of L-citrulline on sperm quality Figure 8 As shown, Figure 8 (A) is the result of sperm count in the control group and the experimental group. Figure 8 (B) is the sperm morphology of the control group and the experimental group. Figure 8 (C) is the result graph showing the percentage of abnormal sperm counts in the control group and the experimental group. Figure 8 (A) It can be seen that the sperm concentration of the control group is 154.8 million / ml, and the sperm concentration of the experimental group is 851.6 million / ml. Figure 8 (B) It can be seen that the sperm in the control group had curled tails and the sperm acrosome was broken, dotted or even disappeared. On the contrary, the sperm in the experimental group had normal morphology, with stretched tails and sickle-shaped sperm acrosome. Figure 8 (C) It can be seen that the abnormal sperm ratio in the control group was as high as 43.56%, while the abnormal sperm ratio in the experimental group was reduced to 17.21%. The above results show that L-citrulline can significantly improve the semen quality of rams.
[0081] Based on the above test results, it can be seen that L-citrulline is beneficial to maintaining the normal morphology of sheep testicular seminiferous tubules, improving the vitality of sheep testicular supporting cells, regulating the blood-testis barrier structure, promoting the proliferation of sheep testicular supporting cells, and can also reduce the level of reactive oxygen, enhance the antioxidant capacity of testicular supporting cells, effectively improve the phenomenon of DNA damage in testicular supporting cells, increase the number of germ cells and improve semen quality, regulate reproductive function and DNA metabolism-related pathways. The above results show that L-citrulline has a positive promoting effect on the growth and metabolism of supporting cells.
[0082] The above description is only an exemplary embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.
Claims
1. An application of L-citrulline in the preparation of a preparation for promoting the growth and metabolism of sheep testicular supporting cells.
2. The use of L-citrulline as claimed in claim 1 in preparing a preparation for promoting the growth and metabolism of sheep testicular supporting cells, characterized in that: The dosage of the L-citrulline is 2.8-3.2 g / kg.
3. The use of L-citrulline as claimed in claim 2 in the preparation of a preparation for promoting the growth and metabolism of sheep testicular supporting cells, characterized in that: The preparation is used orally once a day.