Application of indigowoad root green vegetables in improvement of animal semen quality

Abstract

According to the application of the indigowoad root green vegetables in improving the semen quality of the animals, the indigowoad root green vegetable freeze-dried powder is added into basic daily ration of mice for feeding, and it is found that the semen quality and the weight of the mice can be remarkably improved. The research result provides a solid foundation and theoretical guidance for development and utilization of the isatis root green vegetables in the fields of health food, feed and the like; the added value attribute of the indigowoad root green vegetables can be further improved, and great practical significance is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of Isatis indigotica in improving the quality of animal semen. Background Technology

[0002] With the fast pace of modern life and increasing environmental pollution, declining sperm quality in men has become a significant problem. Poor sperm quality can prevent successful fertilization, increasing the risk of early miscarriage and missed abortion. Sperm undergoes three processes in the body: production, growth, and maturation. Reduced sperm count and loss of motility due to various reasons are major factors contributing to male infertility. Improving sperm quality is crucial for male reproductive health and overall health. In livestock breeding, semen quality also plays a vital role in breed improvement and herd quality enhancement. For example, bull semen quality directly affects cow conception rates and breeding efficiency, and is a key factor influencing production efficiency. Excellent boar semen quality not only improves the economic benefits of artificial insemination stations but also ensures the reproductive performance of pig herds. Therefore, exploring effective methods to enhance semen quality is of significant practical importance.

[0003] Isatis root vegetable (also known as Isatis oil No. 1) is derived from somatic cell hybridization of Isatis root and Brassica napus, followed by multiple generations of selection. Rapeseed is widely cultivated globally, with diverse sources, high yields, and rich nutritional value. Rapeseed has multiple functions and uses: its leaves and flower stalks can be used as vegetables, its seeds can be pressed for oil, its seed meal and green fodder can be used as animal feed, and its flowers also have ornamental value. As an important and high-quality oilseed and green fodder crop, rapeseed is widely cultivated in my country, with an annual planting area exceeding 100 million mu (approximately 6.67 million hectares) and a rapeseed yield exceeding 10 million tons, ranking first in the world in both area and total output. Isatis root, on the other hand, is an important medicinal plant in my country, possessing antibacterial, anti-endotoxin, anticancer, immunomodulatory, blood-activating, and antiviral effects. Its root is called Isatis root, and its leaves are called Daqingye (Big Green Leaf), but its sources and yields are far lower than those of rapeseed. Isatis root green vegetable is a novel functional vegetable with broad-spectrum antiviral effects, obtained through somatic cell hybridization of rapeseed and Isatis indigotica. The resulting hybrid is backcrossed with rapeseed for multiple generations to introduce genes related to the antiviral active components of Isatis indigotica into rapeseed. Its plant morphology and growth habits are similar to rapeseed, and it is rich in the active components of the Isatis indigotica G monomer addition line, containing 17 amino acids and 8 trace elements including selenium, calcium, and zinc.

[0004] Previous studies have shown that Isatis root (Banlangen var. sarcodactylis) has significant anti-influenza virus H5N6 activity, with inhibitory effects comparable to the commonly used anti-influenza drug Osdavir (Tamiflu), and significantly stronger than Isatis root alone. It can improve weight loss and lung tissue lesions caused by influenza virus infection in mice, demonstrating a significant anti-influenza virus infection effect. Furthermore, studies have found that Isatis root (Banlangen var. sarcodactylis) also has a significant inhibitory effect on SARS-CoV-2 virus. In mouse infection models, it can reduce weight loss caused by SARS-CoV-2 infection, improve mouse survival rate, and has a significant anti-novel coronavirus effect and reduces lung tissue damage caused by the novel coronavirus in mice. However, as a novel functional vegetable, whether Isatis root (Banlangen var. sarcodactylis) can improve semen quality has not yet been reported. Summary of the Invention

[0005] In view of this, the present invention provides the application of Isatis indigotica in improving the quality of animal semen.

[0006] This invention, through mouse feeding experiments, added freeze-dried powder of Isatis indigotica to mouse feed and conducted sperm quality testing experiments after continuous feeding. It was found that adding freeze-dried powder to the feed of mice has a significant effect on improving sperm motility, quantity and effective sperm count and reducing sperm abnormality rate.

[0007] Furthermore, in a specific embodiment, the amount of freeze-dried Isatis indigotica powder added is 5% to 20% of the basal diet weight.

[0008] Furthermore, in a specific embodiment, the continuous feeding period is 5 to 7 weeks.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention found through research that adding 5% to 20% by mass of Isatis indigotica freeze-dried powder to the basal diet for 5 to 7 weeks can significantly improve the semen quality of mice. Among them, sperm count increased by 9.8% to 53.7%, sperm density increased by 13.6% to 57.1%, sperm motility increased by 4.5% to 13.3%, effective sperm count increased by 20.5% to 175.5%, and sperm abnormality rate decreased by 15.8% to 28.6%; in addition, while improving semen quality, the weight of mice also increased significantly within 1 to 5 weeks.

[0010] (2) The results of the study on mice provide a solid foundation and theoretical guidance for the development and utilization of Isatis indigotica in the fields of health food and feed; and further enhance the added value of Isatis indigotica. Attached Figure Description

[0011] Figure 1 The results show the effects of adding Isatis indigotica freeze-dried powder to the basal diet on sperm count and density in mice.

[0012] Figure 2 The results show the effects of adding Isatis indigotica freeze-dried powder to the basal diet on sperm motility and effective count in mice.

[0013] Figure 3 The results show the effect of adding Isatis indigotica freeze-dried powder to the basal diet on the sperm abnormality rate of mice.

[0014] Figure 4 The results show the effect of adding Isatis indigotica freeze-dried powder to the basal diet on the body weight of mice. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0016] Key experimental material sources and physicochemical parameters: Laboratory animals: SPF-grade 4-week-old male rats, sourced from Liaoning Changsheng Biotechnology Co., Ltd.

[0017] The freeze-dried powder of Isatis indigotica and green vegetables used in the experiment was provided by Hubei Xiangqie Ecological Agriculture Co., Ltd.

[0018] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0019] Example 1 This embodiment provides a method for improving mouse semen quality using freeze-dried Isatis indigotica powder. The specific steps are as follows: 1. Experimental Design A single-factor experimental design was adopted: 40 male SPF-grade Liaoning Changsheng mice around 4 weeks old were randomly divided into 4 groups of 10 mice each; the control group was fed a basal diet, while the experimental groups were fed a basal diet supplemented with 5%, 10%, and 20% of Isatis indigotica freeze-dried powder, respectively; the experimental period was 7 weeks.

[0020] Phase 1: Before August 13, 2024, was the initial feeding period. All 40 male rats participating in the experiment were fed the same routine diet for 7 days.

[0021] Phase 2: From August 13, 2024 to October 1, 2024, the feeding period was conducted. The 40 mice participating in the experiment were randomly divided into 4 groups of 10 mice each. The control group was fed a basal diet, while the experimental groups were fed a basal diet supplemented with 5%, 10%, and 20% of Isatis indigotica freeze-dried powder, respectively.

[0022] Mouse body weight was measured periodically in the first and second phases, and mouse semen was collected to determine key quality indicators.

[0023] 2. Feeding and Management All mice were housed individually in cages with free access to food and water. The rearing environment was maintained at a temperature of 22–26℃ and a relative humidity of 50%–60%, with 12 hours of artificial light and 12 hours of darkness each. The basal diet formula is shown in Table 1 below: Table 1. Detailed basal diet formulation for mice

[0024] Furthermore, in order to understand the semen quality of mice in the control group and experimental groups with different amounts of Isatis indigotica freeze-dried powder, the following tests were also conducted.

[0025] 1. Testing of sperm count and density in mice Mice were euthanized quickly by cervical dislocation. The epididymis was isolated, weighed, cleaned, and chopped. It was then placed on a 37°C hot plate for 3 minutes. 10 μL of semen was aspirated using a sampling needle for measurement. The diluted semen was dropped onto a hemocytometer, covered with a coverslip, and observed and counted under a 200–400x microscope. The sperm count (X) was then substituted into the following equation to obtain the sperm density: X / 80 × 400 × 10 × Dilution factor × 1000 = Number of sperm per milliliter of semen The results are as follows Figure 1 As shown in Figure A, the effect of adding Isatis indigotica freeze-dried powder to the basal diet on sperm count in mice was observed. The results showed that after 5 weeks of feeding, there was no significant difference in sperm count between mice with 5% and 10% Isatis indigotica freeze-dried powder in their basal diet and the control group, while the sperm count of mice with 20% Isatis indigotica freeze-dried powder was significantly higher than the control group. After 7 weeks of feeding, the sperm count of all experimental groups was significantly higher than the control group. Figure B shows the effect of adding Isatis indigotica freeze-dried powder to the basal diet on sperm density in mice. The results showed that after 5 weeks of feeding, there was no significant difference in sperm density between the control group and all experimental groups. After 7 weeks of feeding, the sperm density of mice with 10% and 20% Isatis indigotica freeze-dried powder in their basal diet increased by 40% and 57% respectively compared to the control group. These results demonstrate that Isatis indigotica can improve sperm count and density in mice. In the figure, * indicates the results of the comparison of significant differences between the control group and the experimental group: * indicates that the difference is significant when the significance level is P<0.05; ** indicates that the difference is extremely significant when the significance level is P<0.01.

[0026] 2. Testing of mouse sperm motility and effective count The percentage of sperm motility, including those with forward and non-forward movement, was observed under a microscope. The calculation method was: (number of sperm with forward movement (quantity / density - dead / abnormal sperm) / density) × 100%. 200-400 sperm were observed under a microscope (400x magnification); red sperm were considered dead, and colorless sperm were considered live. The number of viable sperm was recorded.

[0027] The results are as follows Figure 2 As shown in Figure A, the effect of adding Isatis indigotica freeze-dried powder to the basal diet on sperm motility in mice was observed. The results showed that after 5 weeks of feeding, compared with the control group, sperm motility in the experimental groups increased by 6.8%, 10.1%, and 11.5%, respectively, with significant differences. After 7 weeks of feeding, sperm motility in the experimental groups increased by 4.4%, 5.8%, and 13.3% compared with the control group, respectively. Figure B shows the effect of adding Isatis indigotica freeze-dried powder to the basal diet on the effective sperm count in mice. The results showed that after 5 weeks of feeding, there was no significant difference in the effective sperm count between the control group and all experimental groups. However, after 7 weeks of feeding, the effective sperm count in mice with 10% and 20% Isatis indigotica freeze-dried powder in their basal diet was significantly higher than that in the control group, increasing by 92.9% and 175.5%, respectively. These results demonstrate that Isatis indigotica can improve sperm motility and effective sperm count in mice. In the figure, * indicates the results of the comparison of the significance of the difference between the control group and the experimental group: * indicates that the difference is significant when the significance level is P<0.05; ** indicates that the difference is highly significant when the significance level is P<0.01.

[0028] 3. Testing of sperm abnormality rate in mice Place the prepared smear under a microscope (400x) and count 200 sperm in different fields of view to calculate the percentage of abnormal sperm: Abnormal sperm rate = number of abnormal sperm / total number of sperm examined × 100%.

[0029] The results are as follows Figure 3 As shown, after 5 weeks of feeding, the sperm abnormality rate in the experimental group decreased by 18.2%, 29.9%, and 24.6% compared with the control group, respectively, with significant differences. After 7 weeks of feeding, the sperm abnormality rate in the experimental group also decreased significantly compared with the control group, by 15.8%, 12.3%, and 28.6%, respectively; indicating that Isatis root and green vegetables can reduce sperm abnormality rate. * in the figure indicates the results of comparing the significance of differences between the control and experimental groups: * indicates a significant difference at the P-level (P < 0.05); ** indicates a highly significant difference at the P-level (P < 0.01).

[0030] 4. Mouse weight test Mice were weighed on an empty stomach before and after the experiment, and every Monday for 5 consecutive weeks. The average body weight of each group was calculated, and the changes in body weight during the experiment were recorded. The results are as follows: Figure 4 As shown, the body weight of mice in both the control group and each experimental group increased to varying degrees. Specifically: After one week of feeding, the control group mice gained 3.48g in weight. The basal diet with 5% freeze-dried Isatis root and green vegetable powder added to 20% of the mice resulted in weight increases of 4.09g and 4g respectively, with no significant difference. However, the mice with 10% of the mice added to the diet gained 5.36g in weight, which was significantly different from the control group.

[0031] After two weeks of feeding, the control group mice gained 8.15g in weight. The basal diet with 5% freeze-dried Isatis root and green vegetable powder added to 20% of the mice increased in weight by 8.84g and 8.1g respectively, with no significant difference. However, the mice with 10% added to the diet gained 10.32g in weight, which was significantly different from the control group.

[0032] After three weeks of feeding, the mice in the control group gained 10.59g in weight, and the mice in the basal diet with 5% freeze-dried Isatis indigotica powder gained 11.64g in weight, with no significant difference compared to the control group; while the mice with 10% and 20% of the powder gained 13.93g and 12.05g in weight, respectively, with significant differences compared to the control group.

[0033] After four weeks of feeding, the mice in the control group gained 12.16g in weight, and the mice in the basal diet with 5% freeze-dried Isatis indigotica powder gained 12.66g in weight, which was not significantly different from the control group; while the mice with 10% and 20% of the powder gained 13.63g and 13.2g in weight, respectively, which were significantly different from the control group.

[0034] After five weeks of feeding, the control group mice gained 14.58g in weight. The mice with 5% freeze-dried Isatis root and green vegetable powder in their basal diet (20%) showed no significant difference in weight gain compared to the control group. However, the mice with 10% freeze-dried Isatis root and green vegetable powder gained 15.25g in weight, which was significantly different from the control group.

[0035] The results above indicate that Isatis root and leafy greens promote weight gain in mice. In the figure, * indicates a significant difference between the control and experimental groups; * indicates a significant difference at a P<0.05 level.

[0036] The test results above show that adding freeze-dried Isatis indigotica root and leafy greens powder to the basal diet of mice can significantly improve sperm count, density, motility, effective sperm count, and reduce sperm abnormalities. This provides a solid foundation and theoretical guidance for the development and utilization of Isatis indigotica root and leafy greens in health foods, feed, and other fields.

[0037] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of Isatis indigotica L. in improving animal semen quality.

2. The use according to claim 1, characterized in that: The semen quality indicators include sperm volume, sperm density, sperm motility, effective sperm count and sperm deformity rate.

3. The use according to claim 1, characterized in that: When the animal is a mouse, the specific method for improving the semen quality is: adding isatis root and green vegetables freeze-dried powder to the basic daily diet of the mouse, and continuously feeding the mouse, so as to improve the semen quality of the mouse.

4. The use according to claim 3, characterized in that: The addition amount of Isatis root and green vegetable freeze-dried powder is 5%~20% of the basic diet weight.

5. The use according to claim 3, characterized in that: The feeding period is 5 to 7 weeks.

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