Application of spermidine in preparation of medicine for improving semen quality of heat stress substance

Abstract

The invention discloses application of spermidine in preparation of a medicine for improving semen quality of a heat stress substance. The spermidine is adopted to improve the sperm motility of a heat stress agonist and relieve testis injury, is an important substance required in the normal physiological process of mammals, and widely participates in the processes of cell growth, proliferation, differentiation and the like, so that the influence of heat stress on germ cells is relieved by utilizing the spermidine, and the effect of preventing and treating testis injury is achieved. The potential side effect is small or even no theoretically, and the production and application value is high. According to the invention, spermidine is adopted, so that sperm movement parameters of a heat stress mouse can be improved, the integrity of testis tissues is obviously recovered, the repair of a seminiferous tubule structure is promoted, and cell layer arrangement tends to be normal, which indicates that spermidine can relieve mouse testis dysfunction caused by heat stress.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of spermidine in the preparation of a medicament for improving the semen quality of heat-stressed animals. Background Art

[0002] With the development of the global industrial economy, the accumulation of greenhouse gases (such as water vapor, carbon dioxide, methane, nitrous oxide, and ozone, etc.) has been continuously rising, the average temperature of the earth has been increasing year by year, and global warming has intensified. Especially in summer, continuous high temperature causes heat stress (HS) in male livestock, increasing the sperm malformation rate and decreasing the motility. This effect often exceeds one spermatogenic cycle, meaning that there is a relatively long empty window period for breeding in summer, and the production efficiency is severely affected. Therefore, how to reduce the adverse effects of HS on the spermatogenic ability of male livestock testis and semen quality, analyze the sperm damage mechanism of HS animals and provide corresponding solutions is a research hotspot in this field.

[0003] Since the establishment of mouse HS models is similar at present, the reproductive ability of HS mice is mainly improved by increasing the antioxidant capacity of tissues or cells through injection, gavage or drinking water, etc., to improve heat tolerance and improve the reproductive damage of HS mice. Common antioxidants include melatonin, vitamin C, vitamin E, quercetin, etc. [1-4] The addition of these substances can improve the reproductive performance of HS male livestock to a certain extent, but the effects are not significant. For example, melatonin significantly improved the testicular index of bisphenol A-treated mice (0.34 ± 0.08‰ vs 0.33 ± 0.07‰), but did not improve the testicular index of HS mice (0.34 ± 0.08‰ vs 0.33 ± 0.07‰) [5] ; The addition of vitamin C and vitamin E in the diet significantly increased the sperm motility of yaks (60.83 ± 2.66% vs 66.72 ± 1.31%) [6] and mice (58.73 ± 4.50 % vs 66.61 ± 3.58%) [7] while vitamin C (11.01 ± 2.51‰ vs 12.36 ± 2.04‰) and vitamin E (11.01 ± 2.51‰ vs 12.61 ± 2.08‰) did not improve the testicular index of HS chickens, and continuous feeding of vitamin C (77.95 ± 1.95 % vs 79.90 ± 0.79%) and vitamin E (77.95 ± 1.95% vs 80.45 ± 1.37%) for 21 days did not significantly improve the sperm motility of HS chickens [2]Quercetin significantly improved the effects of benzo[a]pyrene on the testicular index of rats (2.68 ± 0.16‰ vs 2.99 ± 0.11‰) and the total sperm motility (27.65 ± 2.05% vs 31.28 ± 2.29%). [8] , while continuous feeding of quercetin for 56 days did not significantly change the total sperm motility of HS rabbits (69.9 ± 9.3% vs 80.6 ± 14.9%). In addition, the use of these antioxidants may have potential side effects.

[0004] Spermidine (SPD) is a bioactive substance naturally present in animals and plants, with a variety of biological functions, including antioxidant, anti-aging, anti-inflammatory, anti-tumor, etc. It can react with free radicals, reduce the oxidative damage of free radicals to cells, and enhance the antioxidant capacity of the body by promoting the synthesis and activity of antioxidant enzymes. At the same time, spermidine can also promote the hypusination modification of the translation regulatory factor eIF5A and inhibit the activation of the protein acetyltransferase EP300, thereby playing an anti-aging role. HS causes a decline in the semen quality of male animals, which is quite similar to the premature aging of male animals. However, whether SPD can alleviate the decline in the semen quality of male animals triggered by HS like anti-aging has not been reported yet.

[0005] It should be noted that the information disclosed in the above background art section is only used to enhance the understanding of the background of the present disclosure, and thus may include information that does not constitute the prior art known to those of ordinary skill in the art.

[0006] References: 10. Effects of melatonin and heat stress on sperm quality and embryonic development in mice, Master's thesis of Hebei Agricultural University, 2013; 2. Effects of adding vitamin C and vitamin E on the reproductive performance of male Lueyang black-bone chickens in summer high-temperature season, Master's thesis of Northwest A&F University, 2015; 3. Research on the effects of flaxseed, γ-aminobutyric acid, and vitamin C on heat stress resistance in breeding bulls, [J]. Chinese Journal of Animal Science, 2025, 61(01): 351-6; 4. Dietary quercetin maintains the semen quality in rabbits under summer heat stress, [J]. Theriogenology, 2018, 122: 88-93; 5. Melatonin alleviates oxidative stress damage in mouse testes induced by bisphenol A [J]. Front Cell Dev Biol, 2024, 12: 1338828; 6. Effects of dietary vitamin C supplementation levels on semen quality and antioxidant function of Datong yaks, [J]. Chinese Journal of Veterinary Science, 2017, 37(01): 172 - 6; 7. Study on the effect of vitamin E on sperm quality in mice, [J]. Heilongjiang Journal of Animal Reproduction, 2021, 29(04): 6 - 10; 8. Mechanism study on quercetin improving reproductive function damage in male rats induced by benzo(a)pyrene, [J]. Acta Chinese Medicine, 2024, 39(02): 375 - 82. Summary of the Invention

[0007] The object of the present invention is to provide the application of spermidine in the preparation of a drug for improving the semen quality of heat - stressed animals, which solves the problem of the impact of heat stress on animal semen quality, and can improve the sperm motility of heat - stressed animals and alleviate testicular damage.

[0008] To achieve the above object, the present invention provides the application of spermidine in the preparation of a drug for improving the semen quality of heat - stressed animals.

[0009] Preferably, the spermidine can improve the sperm motility parameters of heat - stressed animals.

[0010] Preferably, the sperm motility parameters include: total sperm motility and / or progressive motility.

[0011] Preferably, the spermidine can improve testicular damage.

[0012] Preferably, the spermidine can restore the integrity of testicular tissue, promote the repair of seminiferous tubule structure, and the arrangement of cell layers tends to be normal.

[0013] Preferably, the drug includes: mice.

[0014] Preferably, the dosage form of the drug includes: injection.

[0015] Preferably, the injection is an intraperitoneal injection.

[0016] Preferably, the dosage of the spermidine is 5 mg / kg.

[0017] More preferably, a physiological saline solution containing spermidine is used.

[0018] The application of spermidine in the preparation of a drug for improving the semen quality of heat-stressed animals solves the problem of the impact of heat stress on animal semen quality and has the following advantages: (1) In the present invention, spermidine (SPD) is used to improve the sperm motility of heat-stressed animals and relieve testicular injury. SPD is an important substance required in the normal physiological processes of mammals and is widely involved in processes such as cell growth, proliferation, and differentiation. Therefore, using SPD to relieve the impact of HS on germ cells has theoretically little or no potential side effects and great production application value; (2) In the present invention, SPD (5 mg / kg) is first used by intraperitoneal injection to improve the semen quality and testicular morphological changes of HS mice, which belongs to a new use of SPD and expands the potential application value; (3) In the present invention, spermidine can improve the sperm motility parameters of mice after HS and significantly restore the integrity of the testicular tissue of heat-stressed animals, promoting the repair of the seminiferous tubule structure and the arrangement of cell layers tending to be normal, indicating that SPD can relieve the testicular dysfunction caused by HS. Description of the Drawings

[0019] Figure 1 It is a graph showing the effect of SPD injection on the testicular index of HS mice in Experimental Example 1 of the present invention.

[0020] Figure 2 It is a H&E bright-field image of the testis of HS mice after SPD injection in Experimental Example 1 of the present invention; scale bar = 100, 50 μm, and black asterisks indicate luminal vacuoles.

[0021] Figure 3 It is a graph showing the effect of SPD injection on the proportion of severely damaged seminiferous tubules (B) and the number of germ cell layers (A) in the testis of HS mice in Experimental Example 1 of the present invention.

[0022] Figure 4 It is a H&E bright-field image of the epididymis of HS mice after SPD injection in Experimental Example 1 of the present invention; scale bar = 50 μm.

[0023] Figure 5 It is a graph showing the effect of SPD injection on the sperm morphology of HS mice in Experimental Example 1 of the present invention.

[0024] Figure 6 It is a graph showing the effect of SPD injection on the total sperm motility (A) and progressive motility (B) of HS mice in Experimental Example 1 of the present invention.

[0025] Note: Figure 1 and Figure 2 In, the results are expressed as mean ± standard error (Mean ± SE), and the experiment was repeated at least 3 times. Different letters indicate significant differences ( p <0.05). Detailed implementation manners

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part rather than all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] It should be noted that: for those not specified with specific conditions in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the instruments not specified with the manufacturer, they are all conventional products that can be obtained through commercial purchase. For the raw materials and reagents not specified with the manufacturer, they are all commercially available products or can be prepared by known methods.

[0028] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of the numerical range or percentage range should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0029] The features mentioned in the present invention can be combined arbitrarily as long as there is no contradiction in the combination of these features. All possible combinations should be considered as within the scope described in this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0030] Experimental Example 1 Effect of Spermidine on Semen Quality of Heat-Stressed Mice 1. Experimental treatment Twenty-one 10-week-old ICR male mice were randomly divided into 3 groups, namely a control group, a heat stress treatment group, and a heat stress + spermidine intraperitoneal injection group, with 7 mice in each group.

[0031] Blank control group (CON group): On the 0th day of the experiment, the recipient mice were anesthetized (the day of the water bath treatment was recorded as the 0th day of the experiment). After the testicles descended into the scrotum, the posterior half of 1 / 3 of the body (ensuring that the testicles were completely immersed in water) was immersed in a 30 °C water bath for 20 min. After the treatment was completed, 1 mL / kg of normal saline was immediately injected intraperitoneally and continuously injected for 14 days; Heat stress treatment group (HS group): On the 0th day of the experiment, the recipient mice were anesthetized. After the testicles descended into the scrotum, the posterior half of 1 / 3 of the body (ensuring that the testicles were completely immersed in water) was immersed in a 42 °C water bath for 20 min. After the treatment was completed, 1 mL / kg of normal saline was immediately injected intraperitoneally and continuously injected for 14 days; Heat stress + spermidine intraperitoneal injection group (HS + SPD group): On the 0th day of the experiment, the recipient mice were anesthetized. After the testes descended into the scrotum, the posterior half of 1 / 3 of the body (ensuring that the testes were completely immersed in water) was immersed in a 42 °C water bath for 20 min. Immediately after the treatment, 5 mg / kg of SPD (SPD dissolved in physiological saline) was intraperitoneally injected for 14 consecutive days.

[0032] 2. Index determination The above three groups of experimental mice were intraperitoneally injected with physiological saline and SPD daily respectively. On the 14th day of the experiment, the body weights were measured and the epididymides and testes were collected to detect the following indexes: (1) Determination of total sperm motility and forward motility The mice were quickly sacrificed by cervical dislocation. One-sided epididymis was separated and placed in 500 μL of M2 medium (Sigma, M7167). The epididymis was minced and incubated on a 37 °C constant temperature stage for 15 min. The CASA software was used to detect and analyze the sperm motility parameters. (2) Determination of sperm malformation rate Preparation of sperm smear: 20 μL of sperm suspension was aspirated using a micropipette, and a uniform smear was prepared on a clean glass slide by the "drop-drawing method" and air-dried at room temperature.

[0033] Staining treatment: Drop gentian violet staining solution to cover the smear area, stain at room temperature for 1 min, slowly rinse with distilled water to remove the excess staining solution, and air-dry at room temperature.

[0034] Microscopic examination and analysis: First, select an area with clear background, uniform sperm distribution and no overlapping area under a 100× low-power microscope, and then switch to a 200× high-power microscope for observation. At least 300 sperm were counted, and the number of abnormal sperm was recorded.

[0035] Calculation of malformation rate: Malformation rate (%) = (number of abnormal sperm / total number of counted sperm) × 100%.

[0036] (3) Determination of gross morphology of testis and testis index After the experiment, the body weights of the mice were measured, the mice were sacrificed by cervical dislocation, the testes were removed, observed and weighed. The testis index was calculated according to the formula: Testis index = testis weight (mg) / mouse body weight (g);

[0037] (4) Histopathological observation of testis and epididymis The mouse testis and epididymis tissues were placed in 4% PFA and fixed at 4 °C for 24 h. They were dehydrated successively in 30%, 50%, 75%, 95%, 100% and 100% ethanol for 1 h each time, cleared in xylene for 15 min, soaked in paraffin for 6 h, then paraffin-embedded. The paraffin-embedded blocks were cut into 8-μm thick continuous sections with a microtome, and the paraffin sections were spread on glass slides in water at 45 °C and baked on a baking machine at 37 °C for 4 h. The sections were immersed in xylene twice for 10 min each; in absolute ethanol twice for 10 min each; in 95% ethanol for 5 min, in 75% ethanol for 5 min; in 50% ethanol for 5 min; rinsed with ddH2O for 5 min; stained with hematoxylin for 30 s; differentiated with 1% hydrochloric acid aqueous solution for 5 s, rinsed with running water for 10 min; stained with eosin for 10 s; sealed with neutral resin, and the pathological changes of testicular tissues in each group were observed under an optical microscope.

[0038] 3. Experimental results As Figure 1 shown, it is the result graph of the effect of injecting SPD on the testicular index of HS mice in Experimental Example 1 of the present invention. Compared with the HS group, the testicular index of mice in the HS+SPD group (4.18 ± 0.34‰ vs 5.23 ± 0.37‰) increased significantly on the 14th day after heat stress treatment ( p < 0.05) ( Figure 1 ). The H&E results showed that compared with the CON group, HS could damage the structure of testicular tissues, destroy the structure of seminiferous tubules, and disorder the arrangement of cell layers; after intraperitoneal injection of SPD, the integrity of testicular tissues was significantly restored, and the proportion of damaged seminiferous tubules decreased significantly (33.35 ± 16.37 vs 3.89 ± 2.15, Figure 3 ), promoting the repair of the structure of seminiferous tubules and the arrangement of cell layers tending to be normal, indicating that SPD can relieve the testicular dysfunction caused by HS ( Figure 2 ).

[0039] Meanwhile, the H&E results showed that compared with the CON group, after HS, the lumen of the epididymal head of mice was severely vacuolated, and there were many abnormal sperm in the epididymal tail ( Figure 4 ), and the sperm abnormality rate increased (36.93 ± 2.41 vs 61.35 ± 3.64, Figure 5 ). However, intraperitoneal injection of SPD could effectively prevent this phenomenon from occurring, and the sperm abnormality rate (61.35 ± 3.64 vs 50.99 ± 2.23%), total sperm motility (54.93 ± 1.72% vs 60.79 ± 2.09 %), and forward motility (45.55 ± 1.57% vs 50.70 ± 2.13%) increased significantly ( p < 0.05) (Figure 6 ), indicating that SPD can significantly improve the sperm quality of mice after HS.

[0040] The above results indicate that injecting SPD into HS mice, SPD alleviates testicular injury to a certain extent, protects sperm from the influence of HS, promotes the repair of sperm structure after HS injury, and finally significantly improves semen quality, indicating that SPD has a new function of promoting the recovery of spermatogenic ability of the testis and improving sperm quality in HS mice.

[0041] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. Application of spermidine in the preparation of a drug for improving semen quality of heat-stressed animals.

2. The application according to claim 1, wherein The spermidine can improve sperm motility parameters of heat-stressed animals.

3. The application according to claim 2, characterized in that, The sperm motility parameters include: total sperm motility and / or progressive motility.

4. The application according to claim 1, characterized in that The spermidine can improve testicular injury.

5. The application according to claim 4, wherein The spermidine can restore the integrity of testicular tissue, promote the repair of seminiferous tubule structure, and the arrangement of cell layers tends to be normal.

6. The application according to claim 1, characterized in that, The drug includes: mice.

7. The application according to claim 1, characterized in that The dosage form of the drug includes: injection.

8. The application according to claim 7, wherein The injection is an intraperitoneal injection.

9. The application according to any one of claims 1 to 8, characterized in that, The dosage of the spermidine is 5 mg / kg.

10. The application according to claim 9, wherein Use a physiological saline solution containing spermidine.

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