Method suitable for identifying and selecting germplasm quality of forest musk deer raised in plateau

Abstract

The invention discloses a method suitable for identifying and selecting germplasm quality of forest musk deer bred in plateau, and belongs to the technical field of biotechnology and animal breeding. The method comprises the following steps: collecting fresh excrement of the forest musk deer; extracting excrement hormone and immune globulin A; measuring the concentration of excrement hormone and immune globulin A; dividing the three physiological immune parameters into a high-content group, a medium-content group and a low-content group, and calculating the proportion of each group; selecting forest musk deer individuals with low sensitivity, high energy metabolism and high intestinal immunity; and selecting forest musk deer individuals suitable for the plateau. The invention discloses a method suitable for identifying and selecting germplasm quality of forest musk deer raised in plateau, which adopts a non-invasive fresh excrement sampling method to measure three physiological immune parameters in excrement of the forest musk deer, and realizes scientific identification and selection of germplasm quality of the forest musk deer raised in plateau.

Description

Technical Field

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

[0002] Although there have been studies on the determination of physiological parameters of musk deer feces, these studies are limited to the determination and evaluation of physiological status and have not yet been applied to the identification and selection of population germplasm quality. The germplasm quality of musk deer is a collection of its excellent traits, involving multiple levels such as behavior, morphology, physiological immunity, etc., and the ability to adapt to a specific environment is an important component of germplasm quality. Since the establishment of the musk deer breeding base in Bianba County, Tibet (3,700 meters above sea level) in 2018, a total of 170 musk deer have been transferred from low-altitude musk deer breeding farms. The first 100 musk deer died, and the second 70 musk deer died, with a total loss of 77 musk deer. The mortality rate of the high-altitude musk deer population during the initial adaptation process of the introduced musk deer population was 45.3%, and the number of survivors (excluding newborns) was 93. The process of the captive musk deer population experiencing the high-altitude hypoxic environment and establishing initial adaptation resulted in nearly half of the losses, thus changing the germplasm structure of the high-altitude musk deer population and forming a stable population, but the adaptability of musk deer to the plateau environment has not been fully studied.

[0003] Musk deer have the biological characteristics of being timid, alert, and having high physiological stress. They are extremely sensitive to environmental changes, and the high-altitude hypoxia and cold environment pose a huge challenge to them. Currently, there is a lack of research on the physiological identification of the adaptability of musk deer, and simple behavioral observations and morphological characteristics cannot accurately reflect the adaptability of musk deer 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-destructive 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 solution adopted by the present invention is as follows: A method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding, comprising the following steps: S1. Collect fresh feces: Collect fresh feces of musk deer at musk deer breeding bases at high and low altitudes; S2. Extraction of fecal hormones and immunoglobulin A: Extraction of cortisol, thyroxine and immunoglobulin A from feces; S3. Determination of concentration: The concentration of cortisol, thyroxine and immunoglobulin A in feces was determined by enzyme-linked immunosorbent assay, and the water content of fecal samples was determined at the same time. The concentration results were converted into the content of cortisol, thyroxine and immunoglobulin A per gram of feces according to the water content; S4. Group comparison: 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 contents of cortisol, thyroxine and immunoglobulin A, select individuals with low sensitivity, high energy metabolism and high intestinal immunity; S6. Select musk deer individuals that are suitable for the plateau.

[0006] 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.

[0007] Preferably, in step S2, the extracting solution for extracting cortisol and thyroxine is an ethanol solution with a concentration of 90%, and the immunoglobulin A extracting solution is a 1×PBS solution.

[0008] 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.

[0009] Preferably, in step S5, selecting low-sensitive musk deer individuals based on the cortisol content comprises the following steps: A1. Independent sample t-test method was used to verify the difference in the overall cortisol level of musk deer at high altitude and low altitude; 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 difference in the proportion of high, medium and low cortisol groups of musk deer at high and low altitudes; A4. Raise forest musk deer in low altitudes, use the average ± standard error of medium and low cortisol levels as identification criteria, select low-sensitivity forest musk deer individuals, and number them as 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.

[0010] Preferably, in step S5, selecting musk deer individuals with high energy metabolism based on thyroxine content comprises the following steps: B1. Independent sample t-test method was used to verify the difference in the overall level of thyroxine in musk deer at high altitude and low altitude; B2. One-way ANOVA 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 difference in the proportion of high, medium and low thyroxine groups in high-altitude and low-altitude forest musk deer; B4. Raise forest musk deer populations at low altitudes, use the mean ± standard error of high and medium thyroxine content as identification criteria, select forest 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 and medium thyroxine levels, and N represents an individual.

[0011] Preferably, in step S5, selecting musk deer individuals with high intestinal immunity based on immunoglobulin A content comprises the following steps: C1. Independent sample t-test method was used to verify the difference in the overall level of immunoglobulin A between high-altitude and low-altitude musk deer; 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 levels of immunoglobulin A in musk deer at high and low altitudes; C4. Raise forest musk deer populations at low altitudes, and use the mean ± standard error of high and medium levels of immunoglobulin A as the identification criteria to select forest 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.

[0012] 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. Ha 2…H a N), Hc represents low to medium cortisol levels, H t Represents high school thyroid hormone level, H a represents high school immunoglobulin A level, and N represents an individual.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention discloses a method for identifying and selecting the germplasm quality of musk deer reared in plateaus. By adopting a non-destructive fresh feces sampling method, three physiological and immune parameters, namely cortisol, thyroxine and immunoglobulin A, in feces are measured to scientifically identify and select the germplasm quality of musk deer reared in plateaus. 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 evaluates the physiological state of musk deer reared in plateaus, and finds that the cortisol and thyroxine levels of musk deer reared at high altitudes are significantly higher than those of musk deer reared at low altitudes, while the difference in immunoglobulin A levels is not significant, which provides a scientific basis for selecting musk deer individuals adapted to plateau environments.

[0014] The application of the present invention significantly reduces the mortality of musk deer transported from low-altitude areas to high-altitude areas, reduces economic losses, and promotes the stable growth of the plateau musk deer population. In addition, this study is the first to establish a method for identifying the quality of musk deer germplasm and selecting musk deer populations suitable for plateau breeding based on physiological immune parameters, which provides a scientific basis for the scientific management and industrial development of plateau breeding musk deer, and has important practical application value and broad market prospects.

[0015] The technical solution of the present invention is further described in detail below through embodiments. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further illustrated by the following embodiments.

[0017] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0018] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0019] Example 1 This example provides a method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding, comprising the following steps: S1. Collect fresh feces: Collect fresh feces of musk deer at high altitudes in Bianba County, Tibet (3700 meters above sea level) and low altitudes in Weinan, Shaanxi (400 meters above sea level), respectively, 55 and 34 musk deer were collected every two days, for a total of 10 times, and stored at -20℃; S2. Extraction of fecal hormones and immunoglobulin A: The extract of fecal cortisol and thyroxine is selected to be a 90% ethanol solution. After sufficient oscillation, centrifuge at 3000 r / min for 20 min, take out the supernatant (I) and put it into a clean centrifuge tube. Then re-extract the precipitate with a 90% ethanol solution. Centrifuge at 3000 r / min for 20 min, take out the supernatant (II), mix the two supernatants (I, II) evenly, take 1 mL of the supernatant, dry it, add 1×PBS solution and mix well, freeze it at -20℃ for testing; Extraction of fecal immunoglobulin A: Select 1× PBS extraction solution, shake thoroughly, centrifuge at 3000 r / min for 20 min, take 1 mL of supernatant, freeze at -20℃ for testing; S3. Determination of concentration: The concentration of cortisol, thyroxine and immunoglobulin A in feces was determined by enzyme-linked immunosorbent assay, and the water content of fecal samples was determined at the same time. The concentration results were converted into the content of cortisol, thyroxine and immunoglobulin A per gram of feces according to the water content; S4. Group comparison: The iterative method was used to calculate the base 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 the feces were expressed as mean ± standard error. The system clustering method was used to divide the three physiological immune parameters of cortisol, thyroxine and immunoglobulin A of forest musk deer raised at high altitude and low altitude into high content group, medium content group and low content group, and the proportion of each group was calculated. The formula is as follows: The grouping 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%+the number of individuals with medium cortisol content / the total number of animals tested at high altitude*100%+the number of individuals with high cortisol content / the total number of animals tested at high altitude*100% The grouping ratio of cortisol in musk deer reared at low altitude = number of individuals with low cortisol content / total number of experimental animals at low altitude*100%+number of individuals with medium cortisol content / total number of experimental animals at low altitude*100%+number of individuals with high cortisol content / total number of experimental animals at low altitude*100%; The grouping calculations for thyroxine and immunoglobulin A are similar.

[0020] S5. Identification: Based on the cortisol content, low-sensitivity musk deer individuals are selected, including the following steps: A1. Independent sample t-test method was used to verify the difference in the overall cortisol level of musk deer at high altitude and low altitude; 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 difference in the proportion of high, medium and low cortisol groups of musk deer at high and low altitudes; A4. Raise forest musk deer in low altitudes, use the average ± standard error of medium and low cortisol levels as identification criteria, select low-sensitivity forest musk deer individuals, and number them as 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] Based on the thyroxine content, the selection of musk deer individuals with high energy metabolism includes the following steps: B1. Independent sample t-test method was used to verify the difference in the overall level of thyroxine in musk deer at high altitude and low altitude; B2. One-way ANOVA 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 difference in the proportion of high, medium and low thyroxine groups in high-altitude and low-altitude forest musk deer; B4. Raise forest musk deer populations at low altitudes, use the mean ± standard error of high and medium thyroxine content as identification criteria, select forest 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 and medium thyroxine levels, and N represents an individual.

[0022] Based on the immunoglobulin A content, the selection of musk deer individuals with high intestinal immunity includes the following steps: C1. Independent sample t-test method was used to verify the difference in the overall level of immunoglobulin A between high-altitude and low-altitude musk deer; 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 levels of immunoglobulin A in musk deer at high and low altitudes; C4. Raise forest musk deer populations at low altitudes, and use the mean ± standard error of high and medium levels of immunoglobulin A as the identification criteria to select forest 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.

[0023] S6. Select the individuals of musk deer suitable for the plateau. The method is as follows: The individuals of musk deer 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 medium cortisol levels, H t Represents high school thyroid hormone level, H a represents high school immunoglobulin A level, and N represents an individual.

[0024] Table 1 Cortisol levels of musk deer reared at high and low altitudes

[0025] As shown in Table 1, the cortisol levels of musk deer raised at high altitude and low altitude were 3553.09±82.74ng / g and 2905.81±164.02 ng / g, respectively, with a significant difference (P=0.001), indicating that low temperature and hypoxia at high altitude had a significant impact on the physiological homeostasis of musk deer. Compared with low altitude, the combination of medium and low levels at high altitude accounted for 79.25% in total, and these individuals had better adaptability to the plateau environment.

[0026] Table 2 Grouping of thyroxine levels in musk deer reared at high and low altitudes

[0027] As shown in Table 2, the thyroxine levels of musk deer raised at high altitude and low altitude were 75.38±1.62 ng / g and 29.86±1.08 ng / g, respectively, with a significant difference (P=0.000), indicating that low temperature and hypoxia at high altitude had a significant effect on the energy metabolism of musk deer raised. Compared with low altitude, the high and medium content groups at high altitude accounted for 78.18% in total, and these individuals had better adaptability to the plateau environment.

[0028] Table 3 Grouping of immunoglobulin A levels in musk deer reared at high and low altitudes

[0029] As shown in Table 3, the immunoglobulin A of musk deer reared at high altitude and low altitude was 385.14±6.90 ng / g and 373.25±6.00 ng / g, respectively, and the difference between the two was not significant (P=0.197), indicating that the plateau hypothermia and hypoxia had no significant effect on the intestinal immunity of musk deer reared. Compared with the low altitude, the high and medium content groups at high altitude accounted for 68.51% in total, and these individuals had relatively good adaptability to the plateau environment.

[0030] The medium and low levels of cortisol were used to select individuals who were better adapted to the plateau environment, the high and medium levels of thyroxine were used to select individuals who were better adapted to the plateau environment, and the high and medium levels of immunoglobulin A were used to select individuals who were better adapted to the plateau environment. Individuals with all three good adaptation values ​​were selected.

[0031] Based on the performance of three physiological indicators of musk deer populations raised at high altitudes and low altitudes, the present invention can quantitatively evaluate the physiological state of musk deer raised on plateaus, 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.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. 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 solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution 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 of musk deer at musk deer breeding bases at high and low altitudes; S2. Extraction of fecal hormones and immunoglobulin A: Extraction of cortisol, thyroxine and immunoglobulin A from feces; S3. Determination of concentration: The concentration of cortisol, thyroxine and immunoglobulin A in feces was determined by enzyme-linked immunosorbent assay, and the water content of fecal samples was determined at the same time. The concentration results were converted into the content of cortisol, thyroxine and immunoglobulin A per gram of feces according to the water content; S4. Group comparison: 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 contents of cortisol, thyroxine and immunoglobulin A, select individuals with low sensitivity, high energy metabolism and high intestinal immunity; S6. Select musk deer individuals that are suitable for the plateau.

2. The method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding according to claim 1, characterized in that: In step S1, fresh feces of musk deer are collected from 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.

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

4. The method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding according to claim 1, characterized in that: 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 forest musk deer. The measured values ​​of the three physiological immune parameters of cortisol, thyroxine and immunoglobulin A in the feces are expressed as mean values ​​± standard errors.

5. The method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding according to claim 1, characterized in that: In step S5, based on the cortisol content, low-sensitivity musk deer individuals are selected, including the following steps: A1. Independent sample t-test method was used to verify the difference in the overall cortisol level of musk deer at high altitude and low altitude; 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 difference in the proportion of high, medium and low cortisol groups of musk deer at high and low altitudes; A4. Raise forest musk deer in low altitudes, use the average ± standard error of medium and low cortisol levels as identification criteria, select low-sensitivity forest musk deer individuals, and number them as 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.

6. The method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding according to claim 1, characterized in that: In step S5, based on the thyroxine content, the individuals of musk deer with high energy metabolism are selected, which includes the following steps: B1. Independent sample t-test method was used to verify the difference in the overall level of thyroxine in musk deer at high altitude and low altitude; B2. One-way ANOVA 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 difference in the proportion of high, medium and low thyroxine groups in high-altitude and low-altitude forest musk deer; B4. Raise forest musk deer populations at low altitudes, use the mean ± standard error of high and medium thyroxine content as identification criteria, select forest 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 and medium thyroxine levels, and N represents an individual.

7. The method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding according to claim 1, characterized in that: In step S5, based on the immunoglobulin A content, the forest musk deer individuals with high intestinal immunity are selected, including the following steps: C1. Independent sample t-test method was used to verify the difference in the overall level of immunoglobulin A between high-altitude and low-altitude musk deer; 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 levels of immunoglobulin A in musk deer at high and low altitudes; C4. Raise forest musk deer populations at low altitudes, and use the mean ± standard error of high and medium levels of immunoglobulin A as the identification criteria to select forest 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.

8. The method for identifying and selecting the quality of musk deer germplasm suitable for plateau breeding according to claim 1, characterized in that: In step S6, the musk deer individuals suitable for the plateau are selected as follows: 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 medium cortisol levels, H t Represents high school thyroid hormone level, H a represents high school immunoglobulin A level, and N represents an individual.

Images