A method for identifying and selecting musk deer germplasm suitable for plateau breeding

Abstract

The present invention discloses a method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding, and belongs to the field of biotechnology and animal breeding technology. The method comprises the following steps: collecting fresh feces of musk deer; extracting fecal hormones and immunoglobulin A; determining the concentrations of fecal hormones and immunoglobulin A; dividing the three physiological and immune parameters into a high-content group, a medium-content group, and a low-content group, and calculating the proportion of each group; selecting musk deer individuals with low sensitivity, high energy metabolism, and high intestinal immunity; and selecting musk deer individuals suitable for plateau breeding. The present invention discloses a method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding. By adopting a non-invasive fresh feces sampling method to determine the three physiological and immune parameters in musk deer feces, the scientific identification and selection of the germplasm quality of musk deer suitable for plateau breeding is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and animal breeding technology, and particularly relates to a method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding. Background Art

[0002] Although studies have examined physiological parameters in musk deer feces, these studies are limited to measuring and evaluating physiological status and have not yet been applied to identifying and selecting population germplasm quality. Musk deer germplasm quality is a combination of desirable traits, encompassing behavior, morphology, physiology, and immunity, with the ability to adapt to specific environments being a crucial component. The musk deer's ability to adapt to the plateau environment has not been fully studied.

[0003] Musk deer are characterized by timidity, vigilance, and high physiological stress levels, making them extremely sensitive to environmental changes. The hypoxic and cold conditions at high altitudes pose a significant challenge. Currently, there is a lack of research examining the adaptive capacity of musk deer through physiological approaches, and simple behavioral observations and morphological characteristics cannot accurately reflect their adaptation to the plateau environment. Summary of the Invention

[0004] The present invention aims to provide a method for identifying and selecting the germplasm quality of musk deer larvae raised in plateaus. By adopting a non-invasive fresh feces sampling method, three physiological immune parameters, namely cortisol, thyroxine and immunoglobulin A, in the feces of musk deer larvae are measured, thereby achieving scientific identification and selection of the germplasm quality of musk deer larvae raised in plateaus.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] A method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding comprises the following steps:

[0007] S1. Collect fresh feces: Collect fresh feces from musk deer at high and low altitude musk deer breeding bases;

[0008] S2. Extraction of fecal hormones and immunoglobulin A: Extraction of cortisol, thyroxine, and immunoglobulin A from feces;

[0009] S3. Concentration determination: Measure the concentrations of cortisol, thyroxine, and immunoglobulin A in feces using enzyme-linked immunosorbent assay (ELISA). Simultaneously, measure the water content of the fecal sample and convert the concentration results into the amount of cortisol, thyroxine, and immunoglobulin A per gram of feces based on the water content.

[0010] S4. Group comparison: The three physiological immune parameters of cortisol, thyroxine and immunoglobulin A of musk deer reared at high altitude and low altitude were divided into high content group, medium content group and low content group, and the proportion of each group was calculated;

[0011] S5. Identification: Based on the levels of cortisol, thyroxine, and immunoglobulin A, select individuals with low sensitivity, high energy metabolism, and high intestinal immunity;

[0012] S6. Select musk deer individuals that are suitable for the plateau.

[0013] Preferably, in step S1, fresh feces of musk deer are collected at musk deer breeding bases at high altitude and low altitude, 55 and 34 musk deer are collected respectively, once every two days, for a total of 10 times, and stored at -20°C.

[0014] Preferably, in step S2, the extracting solution for extracting cortisol and thyroxine is a 90% ethanol solution, and the immunoglobulin A extracting solution is a 1×PBS solution.

[0015] Preferably, in step S4, an iterative method is used to calculate the base values ​​of the content of cortisol, thyroxine and immunoglobulin A in the feces of each musk deer, and the measured values ​​of the three physiological immune parameters of cortisol, thyroxine and immunoglobulin A in the feces are expressed as mean ± standard error.

[0016] Preferably, in step S5, selecting low-sensitivity musk deer individuals based on cortisol content includes the following steps:

[0017] A1. Independent sample t-test was used to verify the difference in the overall cortisol levels of musk deer at high and low altitudes.

[0018] A2. One-way analysis of variance was used to verify the differences between the high-altitude and low-altitude forest musk deer cortisol high-content group, medium-content group, and low-content group;

[0019] A3. Chi-square test was used to test the differences in the proportions of high, medium, and low cortisol levels in musk deer at high and low altitudes.

[0020] A4. Raise a group of forest musk deer at low altitudes, and use the mean ± standard error of medium and low cortisol levels as the identification criteria to select low-sensitivity forest musk deer individuals, which are numbered H c 1. H c 2. H c 3…H c N,H c represents low to moderate cortisol levels, and N represents an individual.

[0021] Preferably, in step S5, selecting musk deer individuals with high energy metabolism based on thyroxine content includes the following steps:

[0022] B1. Independent sample t-test was used to verify the difference in the overall thyroid hormone levels between musk deer at high altitude and low altitude.

[0023] B2. One-way analysis of variance was used to verify the differences between the high-altitude and low-altitude thyroxine groups of forest musk deer;

[0024] B3. Chi-square test was used to test the differences in the proportions of high, medium and low thyroxine levels in musk deer at high and low altitudes;

[0025] B4. Raise a group of forest musk deer at low altitudes, and use the mean ± standard error of high and medium thyroxine levels as the identification criteria to select forest musk deer individuals with high energy metabolism, which are numbered H t 1. H t 2. H t 3…H t N,H t represents high and medium thyroxine levels, and N represents an individual.

[0026] Preferably, in step S5, selecting musk deer individuals with high intestinal immunity based on immunoglobulin A content includes the following steps:

[0027] C1. Independent sample t-test was used to verify the difference in the overall immunoglobulin A levels between musk deer at high altitude and low altitude.

[0028] C2. One-way ANOVA was used to verify the differences between the high-altitude and low-altitude forest musk deer immunoglobulin A high-content group, medium-content group, and low-content group;

[0029] C3. Chi-square test was used to test the differences in the proportions of high, medium and low immunoglobulin A levels in musk deer at high and low altitudes;

[0030] C4. Raise a group of musk deer at low altitudes, and use the mean ± standard error of high and medium immunoglobulin A levels as identification criteria to select musk deer individuals with high intestinal immunity, which are numbered H a 1. H a 2. H a 3…H a N,H a represents high school immunoglobulin A level, and N represents an individual.

[0031] Preferably, in step S6, the musk deer individuals suitable for the plateau are selected as follows: musk deer individuals suitable for the plateau = (H c 1. H c 2…H c N)∩(H t 1. H t 2…H t N)∩(H a 1. H a 2…Ha N), Hc represents low to moderate cortisol levels, H t Represents high and medium thyroid hormone levels, H a represents high school immunoglobulin A level, and N represents an individual.

[0032] Compared with the prior art, the present invention has the following advantages and technical effects:

[0033] The present invention discloses a method for identifying and selecting the germplasm quality of musk deer reared in plateaus. By adopting a non-invasive fresh feces sampling method, the three physiological immune parameters of cortisol, thyroxine and immunoglobulin A in the feces are measured, and the germplasm quality of musk deer reared in plateaus is scientifically identified and selected. This method avoids physiological damage to musk deer, improves the success rate and efficiency of sample collection, and makes the evaluation process more objective and fair. By comparing the physiological and immune characteristics of musk deer reared in plateaus and low altitudes, the present invention quantitatively evaluated the physiological state of musk deer reared in plateaus, and found that the cortisol and thyroxine levels of musk deer reared at high altitudes were significantly higher than those of musk deer reared at low altitudes, while the difference in immunoglobulin A levels was not significant, which provides a scientific basis for selecting musk deer individuals that adapt to plateau environments.

[0034] The application of this invention significantly reduced the mortality rate of musk deer transported from low-altitude areas to high-altitude areas, reduced economic losses, and promoted the stable growth of the plateau musk deer population. In addition, this study established for the first time a method for identifying musk deer germplasm quality and selecting musk deer populations suitable for breeding in plateau areas based on physiological and immune parameters. This provides a scientific basis for the scientific management and industrial development of musk deer breeding in plateaus, and has important practical application value and broad market prospects.

[0035] The technical solution of the present invention is further described in detail below through examples. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further illustrated by the following examples.

[0037] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0038] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0039] Example 1 This example provides a method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding, comprising the following steps:

[0040] S1. Collection of fresh feces: Fresh feces of musk deer were collected from musk deer breeding bases at high altitudes in Bianba County, Tibet (3700 meters above sea level) and low altitudes in Weinan, Shaanxi (400 meters above sea level). 55 musk deer were collected from musk deer, and 34 musk deer were collected from musk deer, respectively. Feces were collected every two days for a total of 10 times and stored at -20°C.

[0041] S2. Extraction of fecal hormones and immunoglobulin A: The extraction solution for fecal cortisol and thyroxine was 90% ethanol solution. After thorough shaking, the solution was centrifuged at 3000 r / min for 20 min. The supernatant (I) was removed and placed in a clean centrifuge tube. The precipitate was then re-extracted with 90% ethanol solution. The solution was centrifuged at 3000 r / min for 20 min. The supernatant (II) was removed. The two supernatants (I and II) were mixed evenly and 1 mL of the supernatant was taken. After drying, 1× PBS solution was added and mixed evenly. The mixture was frozen at -20°C for further analysis.

[0042] Extraction of fecal immunoglobulin A: Use 1× PBS extraction solution, shake thoroughly, and centrifuge at 3000 rpm for 20 min. Take 1 mL of the supernatant and freeze at -20°C for further analysis.

[0043] S3. Concentration determination: Measure the concentrations of cortisol, thyroxine, and immunoglobulin A in feces using enzyme-linked immunosorbent assay (ELISA). Simultaneously, measure the water content of the fecal sample and convert the concentration results into the amount of cortisol, thyroxine, and immunoglobulin A per gram of feces based on the water content.

[0044] S4. Group comparison: The iterative method was used to calculate the baseline values ​​of cortisol, thyroxine, and immunoglobulin A in the feces of each forest musk deer. The measured values ​​of the three physiological immune parameters of cortisol, thyroxine, and immunoglobulin A in feces were expressed as mean ± standard error. The high-altitude and low-altitude reared forest musk deer were divided into high-content group, medium-content group, and low-content group using the systematic clustering method, and the proportion of each group was calculated. The formula is as follows:

[0045] The ratio of cortisol in musk deer reared at high altitude = the number of individuals with low cortisol content / the total number of animals tested at high altitude 100% + medium-content individuals / total number of high-altitude test animals 100%+number of high-content individuals / total number of high-altitude test animals 100%

[0046] The ratio of cortisol in musk deer reared at low altitude = the number of individuals with low cortisol content / the total number of animals tested at low altitude 100% + medium content individuals / total number of low altitude test animals 100%+number of high-content individuals / total number of low-altitude test animals 100%;

[0047] The grouping calculations for thyroxine and immunoglobulin A are the same.

[0048] S5. Identification: Based on the cortisol levels, select hyposensitive musk deer individuals, including the following steps:

[0049] A1. Independent sample t-test was used to verify the difference in the overall cortisol levels of musk deer at high and low altitudes.

[0050] A2. One-way analysis of variance was used to verify the differences between the high-altitude and low-altitude forest musk deer cortisol high-content group, medium-content group, and low-content group;

[0051] A3. Chi-square test was used to test the differences in the proportions of high, medium, and low cortisol levels in musk deer at high and low altitudes.

[0052] A4. Raise a group of forest musk deer at low altitudes, and use the mean ± standard error of medium and low cortisol levels as the identification criteria to select low-sensitivity forest musk deer individuals, which are numbered H c 1. H c 2. H c 3…H c N,H c represents low to moderate cortisol levels, and N represents an individual.

[0053] Based on the thyroxine content, the selection of musk deer individuals with high energy metabolism includes the following steps:

[0054] B1. Independent sample t-test was used to verify the difference in the overall thyroid hormone levels between musk deer at high altitude and low altitude.

[0055] B2. One-way analysis of variance was used to verify the differences between the high-altitude and low-altitude thyroxine groups of forest musk deer;

[0056] B3. Chi-square test was used to test the differences in the proportions of high, medium and low thyroxine levels in musk deer at high and low altitudes;

[0057] B4. Raise a group of forest musk deer at low altitudes, and use the mean ± standard error of high and medium thyroxine levels as the identification criteria to select forest musk deer individuals with high energy metabolism, which are numbered H t 1. H t 2. H t 3…H t N,H t represents high and medium thyroxine levels, and N represents an individual.

[0058] Based on the immunoglobulin A content, the selection of musk deer individuals with high intestinal immunity includes the following steps:

[0059] C1. Independent sample t-test was used to verify the difference in the overall immunoglobulin A levels between musk deer at high altitude and low altitude.

[0060] C2. One-way ANOVA was used to verify the differences between the high-altitude and low-altitude forest musk deer immunoglobulin A high-content group, medium-content group, and low-content group;

[0061] C3. Chi-square test was used to test the differences in the proportions of high, medium and low immunoglobulin A levels in musk deer at high and low altitudes;

[0062] C4. Raise a group of musk deer at low altitudes, and use the mean ± standard error of high and medium immunoglobulin A levels as identification criteria to select musk deer individuals with high intestinal immunity, which are numbered H a 1. H a 2. H a 3…H a N,H a represents high school immunoglobulin A level, and N represents an individual.

[0063] S6. Select the musk deer individuals suitable for the plateau. The method is as follows: musk deer individuals suitable for the plateau = (H c 1. H c 2…H c N)∩(H t 1. H t 2…H t N)∩(H a 1. H a 2…H a N), Hc represents low to moderate cortisol levels, H t Represents high and medium thyroid hormone levels, H a represents high school immunoglobulin A level, and N represents an individual.

[0064] Table 1 Cortisol levels of musk deer reared at high and low altitudes

[0065]

[0066] As shown in Table 1, the cortisol levels of musk deer reared at high and low altitudes were 3553.09±82.74 ng / g and 2905.81±164.02 ng / g, respectively. The difference between the two was highly significant (P=0.001), indicating that plateau hypothermia and hypoxia significantly impacted the physiological homeostasis of these animals. Compared to low altitudes, the combined proportion of moderate and low cortisol levels at high altitudes accounted for 79.25%, indicating that these individuals exhibited better adaptability to the plateau environment.

[0067] Table 2 Thyroxine levels of musk deer reared at high and low altitudes

[0068]

[0069] As shown in Table 2, thyroxine levels in musk deer reared at high and low altitudes were 75.38±1.62 ng / g and 29.86±1.08 ng / g, respectively. The difference between the two groups was highly significant (P=0.000), indicating that plateau hypothermia and hypoxia significantly impacted energy metabolism in musk deer reared at high altitudes. Compared to low altitudes, high and medium thyroxine levels accounted for 78.18% of the total at high altitudes, indicating that these individuals exhibited better adaptability to the plateau environment.

[0070] Table 3 Grouping of immunoglobulin A levels in musk deer reared at high and low altitudes

[0071]

[0072] As shown in Table 3, the immunoglobulin A levels of musk deer reared at high and low altitudes were 385.14±6.90 ng / g and 373.25±6.00 ng / g, respectively, with no significant difference (P=0.197). This suggests that plateau hypothermia and hypoxia have no significant impact on the intestinal immunity of musk deer reared at high altitudes. Compared with low altitudes, high and medium levels of immunoglobulin A combined accounted for 68.51% of the total at high altitudes, indicating that these individuals have relatively good adaptability to the plateau environment.

[0073] The medium and low cortisol content groups were used to select individuals who were better adapted to the plateau environment, the high and medium thyroxine content groups were used to select individuals who were better adapted to the plateau environment, and the high and medium immunoglobulin A content groups were used to select individuals who were better adapted to the plateau environment. Individuals with all three good adaptation values ​​were selected.

[0074] Based on the performance of three physiological indicators of musk deer populations raised at high altitudes and low altitudes, this invention can quantitatively evaluate the physiological state of musk deer raised on the plateau, identify and select musk deer populations suitable for plateau breeding, and make the evaluation process more objective and fair. Therefore, the research results are targeted and reliable, providing scientific and technological support for selecting musk deer seed sources and expanding high-altitude musk deer populations.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding, characterized in that: The steps include: S1. Collect fresh feces: Collect fresh feces from multiple musk deer at high and low altitude musk deer breeding bases; S2. Extraction of fecal hormones and immunoglobulin A: Extraction of cortisol, thyroxine, and immunoglobulin A from feces; S3. Concentration determination: Measure the concentrations of cortisol, thyroxine, and immunoglobulin A in feces using enzyme-linked immunosorbent assay (ELISA). Simultaneously, measure the water content of the fecal sample and convert the concentration results into the amount of cortisol, thyroxine, and immunoglobulin A per gram of feces based on the water content. S4. Group comparison: The iterative method was used to calculate the baseline values ​​of cortisol, thyroxine, and immunoglobulin A in the feces of each forest musk deer. The measured values ​​of the three physiological immune parameters of cortisol, thyroxine, and immunoglobulin A in feces were expressed as mean ± standard error. The three physiological immune parameters of cortisol, thyroxine, and immunoglobulin A of forest musk deer reared at high altitude and low altitude were divided into high content group, medium content group, and low content group, and the proportion of each group was calculated; S5. Identification: Based on the levels of cortisol, thyroxine, and immunoglobulin A, select individuals with low sensitivity, high energy metabolism, and high intestinal immunity; Including: raising musk deer groups at low altitudes, using the average ± standard error of medium and low cortisol levels as identification criteria, selecting low-sensitivity musk deer individuals, numbered H c 1. H c 2. H c 3…H c N,H c represents low to moderate cortisol levels; In a musk deer population raised at low altitude, the average ± standard error of high and medium thyroxine levels was used as the identification standard to select musk deer individuals with high energy metabolism and number them as H t 1. H t 2. H t 3…H t N,H t represents high school thyroxine level; Musk deer were reared at low altitudes, and the average values ​​of high and medium immunoglobulin A contents ± standard error were used as identification criteria to select individuals with high intestinal immunity, which were numbered H a 1. H a 2. H a 3…H a N,H a represents the high school immunoglobulin A level, and N represents a certain individual; S6. Select the musk deer individuals suitable for the plateau. The method is as follows: musk deer individuals suitable for the plateau = (H c 1. H c 2…H c N)∩(H t 1. H t 2…H t N)∩(H a 1. H a 2…H a N).

2. The method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding according to claim 1, characterized in that: In step S1, fresh feces of musk deer are collected at musk deer breeding bases at high altitude and low altitude, respectively, 55 and 34 musk deer are collected, once every two days, for a total of 10 times, and stored at -20°C.

3. The method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding according to claim 1, characterized in that: In step S2, the extracting solution for extracting cortisol and thyroxine is a 90% ethanol solution, and the immunoglobulin A extracting solution is a 1×PBS solution.

4. The method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding according to claim 1, wherein: In step S5, based on the cortisol content, low-sensitivity musk deer individuals are selected, which includes the following steps: A1. Independent sample t-test was used to verify the difference in the overall cortisol levels of musk deer at high and low altitudes. A2. One-way analysis of variance was used to verify the differences between the high-altitude and low-altitude forest musk deer cortisol high-content group, medium-content group, and low-content group; A3. Chi-square test was used to test the differences in the proportions of high, medium and low cortisol groups in musk deer at high and low altitudes.

5. The method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding according to claim 1, characterized in that: In step S5, based on the thyroxine content, individuals of musk deer with high energy metabolism are selected, which includes the following steps: B1. Independent sample t-test was used to verify the difference in the overall thyroid hormone levels between musk deer at high altitude and low altitude. B2. One-way analysis of variance was used to verify the differences between the high-altitude and low-altitude thyroxine groups of forest musk deer; B3. Chi-square test was used to test the differences in the proportions of high, medium and low thyroxine groups in musk deer at high and low altitudes.

6. The method for identifying and selecting the germplasm quality of musk deer suitable for plateau breeding according to claim 1, characterized in that: In step S5, based on the immunoglobulin A content, the musk deer individuals with high intestinal immunity are selected, which includes the following steps: C1. Independent sample t-test was used to verify the difference in the overall immunoglobulin A levels between musk deer at high altitude and low altitude. C2. One-way ANOVA was used to verify the differences between the high-altitude and low-altitude forest musk deer immunoglobulin A high-content group, medium-content group, and low-content group; C3. Chi-square test was used to test the differences in the proportions of high, medium and low content groups of musk deer immunoglobulin A at high and low altitudes.

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