Application of Radix Isatidis and Green Vegetables in Improving Animal Semen Quality

Abstract

This invention provides the use of Radix Isatidis Greens for improving animal semen quality. By adding freeze-dried Radix Isatidis Greens powder to the basal diet of mice, the researchers found that feeding them significantly improved semen quality and body weight. This research provides a solid foundation and theoretical guidance for the development and utilization of Radix Isatidis Greens in areas such as health foods and feeds. It can further enhance the added value of Radix Isatidis Greens and has significant practical significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of Radix Isatidis and Green Vegetables in improving animal semen quality. Background Art

[0002] With the accelerated pace of modern life and increasing environmental pollution, declining sperm quality in men has become a significant issue. Poor sperm quality can prevent successful fertilization of the egg, increasing the risk of early and missed miscarriages. Sperm undergo three processes in the body: production, growth, and maturation. A decrease in sperm count and partial or complete loss of sperm motility due to various factors are major contributing factors to male infertility. Improving sperm quality is crucial for male reproductive health and overall well-being. In livestock breeding, semen quality also plays a crucial role in improving breed quality and enhancing herd quality. For example, bull semen quality directly impacts cow conception rates and breeding efficiency, and is a key factor influencing production profitability. Excellent boar semen quality not only improves the economic benefits of artificial insemination stations but also plays a crucial role in ensuring the reproductive performance of pig herds. Therefore, exploring effective methods to enhance semen quality is of great practical significance.

[0003] Isatis root greens (also known as Brassica napus No. 1) are derived from somatic hybridization of Isatis indigotica and Brassica napus, resulting from multiple generations of selection. Rapeseed is widely cultivated worldwide, boasting a wide variety of sources, high yields, and a rich nutritional value. Rapeseed has multiple uses and functions. Its leaves and flower stalks can be grown as vegetables, its seeds can be used for oil extraction, its meal and green fodder can be consumed by animals, and its flowers are also ornamental. As an important and excellent oilseed and green fodder crop, rapeseed is widely cultivated in my country, with an annual planting area exceeding 100 million mu (approximately 10 million mu) and a rapeseed yield exceeding 10 million tons, ranking first in the world in both area and total production. Isatis indigotica, a key medicinal plant in my country, possesses antibacterial, anti-endotoxin, anti-cancer, immunomodulatory, blood-activating, and antiviral properties. Its roots are called Isatis root and its leaves are called Daqingye (Isatis indigotica), but their sources and yields are far lower than those of rapeseed. Isatis indigotica is a new functional vegetable with broad-spectrum antiviral properties, derived from the somatic hybridization of Brassica napus and Isatis indigotica through multiple generations of backcrossing. The resulting somatic hybrid was then introduced into the rapeseed plant through the introduction of genes associated with Isatis indigotica's antiviral active ingredients. Its plant morphology and growth habits are similar to those of rapeseed, and it is rich in Isatis indigotica G monomer-derived active ingredients, including 17 amino acids and eight trace elements, including selenium, calcium, and zinc.

[0004] Studies have shown that isatis root greens have significant anti-influenza virus H5N6 activity, with an inhibitory effect comparable to that of the commonly used anti-influenza drug osamivir (Tamiflu) and significantly stronger than isatis root alone. They can improve weight loss and lung tissue lesions caused by influenza virus infection in mice, demonstrating a significant anti-influenza virus effect. Furthermore, studies have found that isatis root greens also have a significant inhibitory effect on the SARS-CoV-2 virus, reducing weight loss caused by SARS-CoV-2 infection in a mouse infection model and improving mouse survival. This suggests that they have a significant anti-novel coronavirus effect and reduce lung tissue damage caused by the novel coronavirus in mice. However, as a new functional vegetable, whether isatis root greens can improve semen quality has not been reported. Summary of the Invention

[0005] In view of this, the present invention provides the use of Radix Isatidis Greens in improving the quality of animal semen.

[0006] The present invention conducted a mouse feeding experiment, added freeze-dried powder of Radix Isatidis and cabbage to the mouse feed, and conducted a sperm quality detection experiment after continuous feeding. It was found that adding freeze-dried powder to the feed to feed mice had a significant effect on improving the sperm motility, quantity and effective sperm count of mice and reducing the sperm deformity rate.

[0007] Furthermore, in a specific embodiment, the addition amount of the isatis root and greens freeze-dried powder is 5% to 20% of the basic diet mass.

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

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] (1) The present invention found that adding 5% to 20% by mass of freeze-dried Radix Isatidis and Cabbage powder to the basal diet for 5-7 weeks can significantly improve the semen quality of mice. Specifically, 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 deformity rate decreased by 15.8% to 28.6%. In addition, while improving semen quality, the body weight of mice also increased significantly within 1-5 weeks.

[0011] (2) The research results 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 attributes of Isatis indigotica. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The results of the study were as follows: The effect of adding freeze-dried powder of Radix Isatidis and Greens to the basal diet on the sperm volume and density of mice.

[0013] Figure 2 The results of the study were as follows: the effect of adding freeze-dried powder of Radix Isatidis and Green Vegetables to the basal diet on sperm motility and effective count in mice.

[0014] Figure 3 The results of the study were as follows: The effect of adding freeze-dried powder of Radix Isatidis and Greens to the basal diet on the sperm deformity rate of mice.

[0015] Figure 4 The results of the effect of adding freeze-dried powder of Radix Isatidis and Greens to the basal diet on the body weight of mice. DETAILED DESCRIPTION

[0016] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0017] Sources and physical and chemical parameters of key test materials:

[0018] Experimental animals: 4-week-old SPF male mice were obtained from Liaoning Changsheng Biotechnology Co., Ltd.

[0019] The freeze-dried powder of Radix Isatidis and greens used in the experiment was provided by Hubei Xiangxiangqie Ecological Agriculture Co., Ltd.

[0020] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0021] Example 1

[0022] This embodiment provides a method for improving the semen quality of mice using freeze-dried Radix Isatidis and Green Cabbage powder, and the specific steps are as follows:

[0023] 1. Experimental Design

[0024] A single-factor experimental design was used: 40 SPF-grade male Liaoning Changsheng mice aged about 4 weeks were selected and randomly divided into 4 groups, with 10 mice in each group; the control group was fed a basal diet, and the experimental groups were fed a basal diet supplemented with 5%, 10% and 20% freeze-dried isatis root and greens powder, respectively; the experimental period was 7 weeks.

[0025] Phase 1: Before August 13, 2024, the initial feeding period. All 40 male rats were fed the same conventional diet for 7 days.

[0026] Phase II: From August 13, 2024, to October 1, 2024, the 40 mice were randomly divided into four groups, each with 10 mice. The control group was fed a basal diet, while the experimental groups were fed a basal diet supplemented with 5%, 10%, and 20% freeze-dried Isatis root and greens powder, respectively.

[0027] The weight of mice was measured regularly in the first and second stages, and the semen of mice was collected to measure key quality indicators.

[0028] 2. Feeding and management

[0029] All mice were housed in individual cages with free access to food and water. The environment was maintained at a temperature of 22-26°C, a relative humidity of 50%-60%, and artificial light with 12-hour light and dark cycles. The basic diet formula is shown in Table 1 below:

[0030] Table 1 Detailed formula of basic diet for mice

[0031]

[0032] Furthermore, in order to understand the semen quality of mice in the control group and the experimental groups with different amounts of Isatis Radix and Green Vegetable freeze-dried powder added, the following tests were also conducted.

[0033] 1. Testing of Mouse Sperm Volume and Density

[0034] Mice were quickly killed by cervical dislocation. The epididymis was isolated, weighed, cleaned, and minced. The sperm was then placed on a 37°C hotplate for 3 minutes. Semen was then aspirated with a 10μL pipette for measurement. The diluted semen was dropped onto a hemocytometer, covered with a coverslip, and counted under a microscope at 200-400x magnification. The sperm count (X) was then inserted into the following equation to obtain the density:

[0035] X / 80×400×10×dilution factor×1000=number of sperm per milliliter of semen

[0036] The results are as follows Figure 1 Figure (A) shows the effect of adding freeze-dried Isatis root greens powder to the basal diet on sperm count in mice. The results showed that after 5 weeks of feeding, the sperm counts of mice fed a basal diet supplemented with 5% and 10% freeze-dried Isatis root greens powder were not significantly different from those in the control group. However, the sperm counts of mice fed a 20% freeze-dried Isatis root greens powder were significantly higher than those in the control group. After 7 weeks of feeding, the sperm counts of mice in all experimental groups were significantly higher than those in the control group. Figure (B) shows the effect of adding freeze-dried Isatis root greens powder to the basal diet on sperm density in mice. The results showed that after 5 weeks of feeding, the sperm density of mice fed a basal diet supplemented with 10% and 20% freeze-dried Isatis root greens powder was 40% and 57% higher than that in the control group, respectively. These results demonstrate that Isatis root greens can increase sperm count and density in mice. In the figure, * indicates the results of 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.

[0037] 2. Test of Mouse Sperm Motility and Effective Count

[0038] Observe the percentage of sperm motility (motility) and non-motility (motility) under a microscope. Calculate: number of sperm motility (number / density - dead / abnormal sperm) / density x 100%. Observe 200-400 sperm under a microscope (400x magnification). Red sperm indicate dead sperm, while colorless sperm indicate live sperm. Record the number of viable sperm.

[0039] The results are as follows Figure 2 Figure (A) shows the effect of adding freeze-dried Isatis root greens powder to the basal diet on sperm motility in mice. The results showed that after five weeks of feeding, sperm motility in the experimental groups increased by 6.8%, 10.1%, and 11.5%, respectively, compared to the control group, with significant differences. After seven weeks of feeding, sperm motility in the experimental groups increased by 4.4%, 5.8%, and 13.3%, respectively, compared to the control group. Figure (B) shows the effect of adding freeze-dried Isatis root greens powder to the basal diet on effective sperm count in mice. The results showed that after five weeks of feeding, there was no significant difference in effective sperm count between the control group and all experimental groups. However, after seven weeks of feeding, mice fed the basal diet containing 10% and 20% freeze-dried Isatis root greens powder had significantly higher effective sperm counts than the control group, increasing by 92.9% and 175.5%, respectively. These results demonstrate that Isatis root greens can improve sperm motility and effective sperm count 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 very significant when the significance level is P<0.01.

[0040] 3. Testing of Mouse Sperm Deformity Rate

[0041] Place the prepared smear under a microscope (400x), count 200 sperm in different fields of view, and calculate the percentage of abnormal sperm: abnormality rate = number of abnormal sperm / total number of sperm examined × 100%.

[0042] The results are as follows Figure 3 As shown in the figure, after 5 weeks of feeding, the sperm deformity 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 deformity 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 greens can reduce the sperm deformity rate. In the figure, * indicates the results of the comparison of significant differences between the control group and the experimental group: * indicates a significant difference at a significance level of P < 0.05; ** indicates a very significant difference at a significance level of P < 0.01.

[0043] 4. Mouse Body Weight Testing

[0044] The mice were weighed before and after the experiment and every Monday on an empty stomach for 5 consecutive weeks. The average weight of each group of mice was calculated and the weight changes of the mice during the experiment were statistically analyzed. The results are shown in the figure. Figure 4 As shown, the weight of mice in the control group and each experimental group increased to varying degrees. Specifically:

[0045] After one week of feeding, the weight of mice in the control group increased by 3.48g. The weight of mice whose basal diet contained 5% and 20% freeze-dried powder of Isatis indigotica increased by 4.09g and 4g respectively, with no significant difference. The weight of mice whose basal diet contained 10% freeze-dried powder increased by 5.36g, which was significantly different from the control group.

[0046] After two weeks of feeding, the weight of mice in the control group increased by 8.15g. The weight of mice whose basal diet was supplemented with 5% and 20% freeze-dried powder of Isatis indigotica increased by 8.84g and 8.1g respectively, with no significant difference. The weight of mice whose basal diet was supplemented with 10% increased by 10.32g, which was significantly different from the control group.

[0047] After three weeks of feeding, the weight of mice in the control group increased by 10.59g, and the weight of mice added with 5% freeze-dried powder of Isatis indigotica indica in the basic diet increased by 11.64g, which was not significantly different from the control group; while the weight of mice added with 10% and 20% increased by 13.93g and 12.05g respectively, which was significantly different from the control group.

[0048] After four weeks of feeding, the weight of mice in the control group increased by 12.16g, and the weight of mice added with 5% freeze-dried powder of Isatis indigotica indica in the basic diet increased by 12.66g, which was not significantly different from the control group; while the weight of mice added with 10% and 20% increased by 13.63g and 13.2g respectively, which was significantly different from the control group.

[0049] After five weeks of feeding, the weight of mice in the control group increased by 14.58g. The weight gain of mice fed with 5% and 20% freeze-dried isatis root and greens powder in the basic diet had no significant difference compared with the control group; while the weight of mice fed with 10% freeze-dried isatis root and greens powder increased by 15.25g, which was significantly different from the control group.

[0050] The above results show that Radix Isatidis Greens has a promoting effect on weight gain in mice. In the figure, * indicates the results of the comparison of significant differences between the control group and the experimental group, and * indicates that the difference is significant when the significance level is P<0.05.

[0051] The above test results show that adding freeze-dried Isatis root greens powder to the basal diet of mice significantly improved sperm count, density, motility, and effective sperm count, and reduced sperm abnormalities. This provides a solid foundation and theoretical guidance for the development and utilization of Isatis root greens in health foods and feeds.

[0052] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of Isatis root greens in the preparation of feed for improving animal semen quality.

2. The use according to claim 1, characterized in that The indicators of semen quality 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 semen quality is: adding isatis root and green vegetable freeze-dried powder to the mouse's basic daily diet and continuously feeding the mouse, thereby improving the mouse's semen quality.

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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