A method for predicting true pregnancy of giant pandas

Abstract

This invention relates to the field of giant panda reproductive technology, and in particular to a method for predicting true pregnancy in giant pandas. Based on the physiological characteristic that the mother's immunity gradually decreases during pregnancy due to reproductive stress, this invention predicts true pregnancy in female giant pandas. It can accurately and scientifically determine whether a mated giant panda is truly pregnant, and the prediction results can be obtained 1-2 weeks before the actual delivery date. This is significant for making early changes to the feeding and management of truly pregnant giant pandas, the formulation and implementation of newborn panda delivery procedures, and the optimization of reproductive management in captive giant pandas. Furthermore, the prediction method provided by this invention enriches the methods for distinguishing between true and false pregnancies in giant pandas by examining pregnancy metabolites. Because it uses the ELISA method, it eliminates the need for professional training in ultrasound diagnosis and the need to acclimatize mated giant pandas, saving significant manpower and resources while simplifying the operational procedures.

Description

Technical Field

[0001] The invention relates to the technical field of giant panda reproduction, in particular to a method for predicting true pregnancy of giant pandas. Background Art

[0002] There are two significant characteristics of giant panda (Ailuropoda melanoleuca) pregnancy. On the one hand, it is prolonged implantation. The occurrence of prolonged implantation makes the gestation period of giant pandas generally 85-185 days. On the other hand, giant pandas will also experience pseudopregnancy. Pseudopregnant giant pandas show behavioral, physiological and sex hormone changes similar to those of true pregnant giant pandas. Among them, behavioral and physiological phenomena include loss of appetite, nesting, breast enlargement, etc.; in terms of sex hormones, there is no difference in the duration and content of urine progesterone between true pregnancy and pseudopregnancy giant pandas. Pseudopregnant individuals are basically diagnosed as pregnancy failure, and pregnancy failure is speculated to be related to unobserved embryonic or fetal death. At present, the prediction methods for true pregnancy of giant pandas generally include molecular biology and B-ultrasound.

[0003] The patent document with application number "CN202210794976.5" discloses a molecular method for testing early pregnancy in giant pandas. The innate and adaptive immune genes that are significantly differentially expressed between pregnant and non-pregnant giant pandas are used as markers, and the markers in the test samples are quantitatively detected by real-time fluorescence quantitative PCR. This patent document tests the pregnancy of giant pandas at the molecular biological level. Although it can predict whether the giant panda is really pregnant to a certain extent, the test samples used are relatively difficult to obtain, the operation is relatively difficult, and the professionalism is relatively strong.

[0004] The method of using B-ultrasound diagnosis technology to determine whether a giant panda is truly pregnant, on the one hand, because the fetus is only about 1 / 1000 of the mother's weight, the diagnostic operation requires high requirements for B-ultrasound detection technicians, otherwise it is easy to misjudge intestinal food as a fetus. On the other hand, giant pandas that undergo B-ultrasound detection need long-term domestication before they can cooperate with B-ultrasound detection according to instructions. Among the giant pandas participating in breeding every year, only a small number of individuals are domesticated and cooperate with B-ultrasound detection, which is difficult to implement, and the detection process requires considerable manpower and material resources. Therefore, there are certain limitations to conducting B-ultrasound detection on every giant panda participating in breeding.

[0005] Therefore, the present invention provides a method for predicting true pregnancy of giant pandas, which is used to solve the above-mentioned technical problems. Summary of the invention

[0006] The purpose of the present invention is to provide a prediction method for true pregnancy of giant pandas, which can not only accurately and scientifically judge whether the mating giant panda is truly pregnant, but also predict the result in advance 1-2 weeks before the birth of the truly pregnant giant panda, which is of great significance for changing the feeding management of truly pregnant giant pandas in advance, formulating and implementing the delivery of newborn giant pandas, and optimizing the breeding management of captive giant pandas. Furthermore, the prediction method provided by the present invention not only simplifies the operation process and saves a lot of manpower and material resources, but also enriches the method for distinguishing true pregnancy from false pregnancy of giant pandas.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for predicting true pregnancy of a giant panda comprises the following steps:

[0009] The first step is to collect urine samples from female giant pandas after mating, and then test the progesterone concentration and neopterin concentration in the urine; the measured progesterone concentration and neopterin concentration are divided by the creatinine concentration in the urine sample to obtain the progesterone correction value and neopterin correction value, and (ng / mL Cr) is used as the measurement unit of the progesterone correction value and neopterin correction value;

[0010] The second step is to calculate the average value of neopterin correction from the time when the progesterone correction value in the urine of female giant pandas first exceeds 100ng / mL Cr to the time when the progesterone correction value rises to the peak value as N1; the average value of neopterin correction from the peak value to the time when the progesterone correction value first drops below 200ng / mL Cr is N2; when the N2 / N1 value is between 1.39-2.08 (95%CI), it indicates that the breeding giant panda is truly pregnant, otherwise it is a false pregnancy.

[0011] Furthermore, the urine sample collection frequency of female giant pandas is once a week; and the urine collection time is 7:00-9:00 in the morning.

[0012] Furthermore, the specific operation of urine sample collection is as follows: after the female giant panda urinates, 2 ml of urine is sucked from the clean floor of the animal house with a disposable syringe and placed in a sampling tube, which is then marked with the corresponding giant panda's name and sampling date.

[0013] Furthermore, the urine samples collected from female giant pandas were centrifuged at 3000 r / min for 10 min at room temperature, and then the urine samples after centrifugation were taken for later use.

[0014] Furthermore, the concentrations of progesterone and neopterin in urine samples of female giant pandas were determined by enzyme-linked immunosorbent assay.

[0015] Furthermore, a commercial ELISA kit was used to measure the concentration of neopterin in urine samples of female giant pandas.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention predicts true pregnancy of female giant pandas based on the physiological characteristic that the mother is in a state of reproductive stress during pregnancy and her immunity will gradually decrease as the pregnancy progresses. It can not only accurately and scientifically judge whether the mating giant panda is truly pregnant, but also predict the result 1-2 weeks before the birth of the truly pregnant giant panda, which is of great significance for changing the feeding management of truly pregnant giant pandas in advance, formulating and implementing the delivery of newborn giant pandas, and optimizing the breeding management of captive giant pandas. Furthermore, the prediction method provided by the present invention enriches the method for distinguishing true pregnancy from false pregnancy of giant pandas from the perspective of pregnancy metabolites.

[0018] 2. The present invention starts from the direction of pregnancy metabolites, and adopts the Elisa method, which does not require professional training for B-ultrasound diagnosis, nor does it require the domestication of breeding giant pandas, saving a lot of manpower and material resources. In addition, compared with the molecular biology method, the present invention uses giant panda urine as the sample to be tested, and the sample is easier to obtain. At the same time, the detection method of progesterone and neopterin concentration is also relatively simple and easy to implement. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The present invention is based on the fact that the mother is in a state of reproductive stress during pregnancy, and the mother's immunity will gradually decrease as the pregnancy progresses. After delivery, the mother's immunity has the physiological characteristics of self-recovery. At the same time, the small molecule metabolite neopterin in the animal body can reflect the activation state of T cells and macrophages, and is a sensitive indicator indicating the body's immune level. Neopterin exists in blood samples and urine, has good biological stability, and its content is negatively correlated with the cellular immunity level. In view of the similar change pattern of progesterone in true pregnancy and false pregnancy giant pandas after mating, that is, the progesterone level after mating gradually rises from less than 100ng / mL Cr to 100ng / mL Cr, and then continues to rise to a peak value and then begins to continuously decline and fall back to below 100ng / mL Cr. We used urine samples of true and false pregnant giant pandas stored during the breeding season at the Chengdu Research Base of Giant Panda Breeding, and selected urine samples with progesterone-corrected values ​​from the first time higher than 100ng / mLCr to before reaching the peak (counted as the pre-peak group); and urine samples with progesterone-corrected values ​​from the peak to the first time lower than 200ng / mLCr (counted as the post-peak group) to measure the neopterin-corrected value of urine samples. The urine sampling frequency was selected as once a week. Data analysis showed that the neopterin-corrected value of the post-peak group of true pregnant giant pandas was significantly higher than that of the pre-peak group (p=0.001), indicating that the neopterin-corrected value of true pregnant giant pandas increased after the progesterone peak, corresponding to a decrease in the level of immunity. It is speculated that the embryo implantation of true pregnant giant pandas occurred after the progesterone peak, which is consistent with the physiological characteristics of the maternal immunity gradually decreasing as the pregnancy progresses. The changes in the corrected neopterin value of pseudopregnant giant pandas showed two situations. One was that the corrected neopterin value of the post-peak group was significantly lower than that of the pre-peak group (p=0.05), indicating that the corrected neopterin value of the pseudopregnant giant pandas decreased after the progesterone peak, indicating that the immunity level gradually increased and recovered; the other was that there was no difference in the corrected neopterin value between the post-peak group and the pre-peak group (p=0.059).

[0021] Based on the above invention concept, a method for predicting true pregnancy of giant pandas comprises the following steps:

[0022] The first step is to collect urine samples from female giant pandas after mating, and then test the progesterone and neopterin concentrations in urine. Because the expression units of hormone content in feces and blood are different, the progesterone and neopterin concentrations in urine are affected by the water content of the giant panda's drinking water and food. Depending on the water the animal drinks, the urine is either diluted or concentrated, which causes different progesterone and neopterin concentrations in urine, and cannot reflect the actual progesterone and neopterin concentration changes in the giant panda's body. Creatinine is a metabolic product of muscle in the animal's body. After being excreted in urine, the creatinine concentration has a good correlation with the urine concentration. Therefore, in order to correct the differences caused by changes in water content in urine samples, the progesterone concentration and neopterin concentration (ng / mL) measured in each urine sample were divided by the creatinine concentration (mg / mL) in the urine sample. The ratios obtained are the progesterone correction value and neopterin correction value, and (ng / mL Cr) is used as the measurement unit of the progesterone correction value and neopterin correction value;

[0023] Step 2: From the time when the progesterone-corrected value in the urine of female giant pandas first exceeded 100 ng / mL Cr to the time when the progesterone-corrected value rose to the peak, the average value of the neopterin-corrected value corresponding to the period from the peak value to the time when the progesterone-corrected value first dropped below 200 ng / mL Cr was calculated as N2; when the N2 / N1 value was between 1.39-2.08 (95% CI), it indicated that the mating giant panda was truly pregnant, otherwise it was a false pregnancy;

[0024] The “95% CI” mentioned above refers to the confidence interval, which is the estimated interval of the population parameter constructed by the sample statistic. In statistics, the confidence interval (Confidence interval) of a probability sample is an interval estimate of a population parameter of this sample.

[0025] In the present invention, the urine sample collection frequency of female giant pandas is once a week; and the urine collection time is 7:00-9:00 in the morning; after the female giant panda urinates, 2 ml of urine is sucked from the clean floor of the animal house with a disposable syringe and loaded into a sampling tube, and centrifuged at a rate of 3000r / min for 10 minutes at room temperature, and then the urine sample after centrifugation is taken, and the corresponding giant panda name and sampling date are marked.

[0026] In the present invention, the concentration of progesterone and neopterin in urine samples of female giant pandas is determined by enzyme-linked immunosorbent assay, and the progesterone content is determined by using a commercial progesterone antibody (Smithsonian Conservation Biology Institute, item number CL425, United States, sensitivity 2.5 μg / L, intra-batch coefficient of variation less than 10%, and inter-batch coefficient of variation less than 12%). A commercial ELISA kit (IBL, item number RE59321, Germany, sensitivity 0.2 μg / L, intra-batch coefficient of variation less than 11%, and inter-batch coefficient of variation less than 14%) is used to determine the concentration of neopterin in urine samples of female giant pandas. Progesterone and neopterin detection are performed in accordance with the reagent detection operating instructions.

[0027] The creatinine concentration was measured by the following method. The test procedure is as follows:

[0028] (1) Label the 12×75 mm test tubes as follows: 100, 50, 25, 12.5, 6.25 (μg / mL) and 0 (blank control) from high to low.

[0029] (2) Take 150 μL of the standard creatinine stock solution (100 μg / mL) and add it to the test tube marked 100; add 150 μL of the standard stock solution to the test tube marked 50, and then add 150 μL of the working solution to dilute it in equal multiples; take 150 μL of the mixed solution from the test tube marked 50 and add it to the test tube marked 25, and then add 150 μL of the working solution to dilute it in equal multiples; continue the dilution in equal multiples;

[0030] (3) Sample preparation: Take the above centrifuged giant panda urine samples (giant panda urine samples were centrifuged at 3000 r / min for 10 min at room temperature), and dilute each urine sample 20 times with the working solution;

[0031] (4) Add the standard samples of various concentrations to the 96-well ELISA plate in the order of A1→B1, from blank control to high concentration, and then add the test samples. Add 50 μL of the standard sample and the test sample to each well, and perform duplicate wells. Add the standard sample and the test sample to the bottom of the well of the ELISA plate, and try not to touch the well wall;

[0032] (5) Add 50 μL of deionized water to each well of the standard and test samples using an 8-channel pipette;

[0033] (6) Add 50 μL of 0.75 N NaOH to each well of the standard and test samples using an 8-channel pipette;

[0034] (7) Add 50 μL of 0.4N Picric Acid to each well of the standard and test samples using an 8-channel pipette;

[0035] (8) Gently shake the ELISA plate and place it at room temperature (18-25°C) for 20 min.

[0036] (9) Reading: Use blank air conditioning as zero, and use an ELISA reader to detect the optical density value at a wavelength of 490nm (reference wavelength 620nm). Draw a standard curve based on the standard readings and calculate the creatinine concentration of each test sample. When the creatinine value is lower than 0.1mg / mL, the sample is considered to be over-diluted (usually contaminated by water).

[0037] Example

[0038] A method for predicting true pregnancy of a giant panda comprises the following steps:

[0039] The first step is to collect urine samples from female giant pandas after mating. The collected urine samples are centrifuged at 3000r / min for 10 minutes at room temperature, and then the urine samples after centrifugation are taken for later use;

[0040] The urine samples of female giant pandas are collected once a week and at 7:00 a.m. The specific operation of urine collection is as follows: after the female giant panda urinates, use a disposable syringe to draw 2 ml of urine from the clean floor of the animal house and put it into the sampling tube, and then mark the corresponding giant panda name and sampling date;

[0041] Then, the progesterone concentration and neopterin concentration in urine were detected by enzyme-linked immunosorbent assay; the measured progesterone concentration and neopterin concentration were divided by the creatinine concentration in urine sample to obtain the progesterone correction value and neopterin correction value, and (ng / mL Cr) was used as the measurement unit of the progesterone correction value and neopterin correction value;

[0042] The second step is to calculate the average value of neopterin correction from the time when the progesterone correction value in the urine of female giant pandas first exceeds 100ng / mL Cr to the time when the progesterone correction value rises to the peak value as N1; the average value of neopterin correction from the peak value to the time when the progesterone correction value first drops below 200ng / mL Cr is N2; when the N2 / N1 value is between 1.39-2.08 (95%CI), it indicates that the breeding giant panda is truly pregnant, otherwise it is a false pregnancy.

[0043] Experimental Example 1

[0044] From March 29, 2023, the urine of the giant panda "Beichuan" after mating was tested according to the prediction method of true pregnancy of giant pandas provided in the embodiment. The changes of progesterone correction value and neopterin correction value in the urine samples obtained during the detection process are shown in the following table:

[0045]

[0046] 1. The changes in the corrected progesterone value in the panda's urine were monitored after mating on March 29. On May 9, the corrected progesterone value of the giant panda was higher than 100ng / mL Cr for the first time (the corresponding value was 154.2 ng / mL Cr), and the determination of the corrected neopterin value began. The corrected progesterone value of the giant panda reached its peak on June 17 (the corresponding value was 418.7ng / mL Cr); on July 6, it dropped below 200ng / mL Cr for the first time (the corresponding value was 172.1ng / mLCr).

[0047] 2. Calculated by the following method, N1=(5.2+3.7+5.6+4.5+5.3) / 5=4.86; N2= (6.2+10.0+7.8+12.8) / 4 = 9.2. N2 / N1= 9.2 / 4.86 = 1.89, which is consistent with the N2 / N1 value being between 1.39-2.08 (95%CI), indicating that the mating giant panda is truly pregnant. The giant panda "Beichuan" gave birth on July 15, 10 days after the progesterone-corrected value first dropped from the peak to below 200ng / mL Cr (corresponding to July 6).

[0048] Experimental Example 2

[0049] From April 13, 2023, the urine of the giant panda "Chuanzai" after mating was tested according to the prediction method of true pregnancy of giant pandas provided in the embodiment. The changes of progesterone correction value and neopterin correction value in the urine samples obtained during the detection process are shown in the following table:

[0050]

[0051] 1. The changes in the corrected progesterone value in the panda's urine were monitored after mating on April 13. On May 2, the corrected progesterone value of the giant panda was higher than 100ng / mL Cr for the first time (the corresponding value was 117.1ng / mL Cr), and the determination of the corrected neopterin value began. The corrected progesterone value of the giant panda reached its peak on July 4 (the corresponding value was 563.0ng / mL Cr), and on July 16, it dropped below 200ng / mL Cr for the first time (the corresponding value was 181.4ng / mLCr).

[0052] 2. Calculated by the following method, N1=(5.1+4.8+6.1+7.9+6.8+8.2+11.2+10.1) / 8 = 7.53; N2=(12.8+17.5+16.0) / 3 = 15.43. N2 / N1= 15.43 / 7.53 = 2.05, which is consistent with the N2 / N1 value being between 1.39-2.08 (95%CI), indicating that the mating giant panda is truly pregnant. Giant panda "Chuanzai" gave birth on July 22, 7 days after the progesterone correction value first dropped below 200ng / mg Cr (corresponding to July 16).

[0053] Experimental Example 3

[0054] From February 16, 2023, the urine of the giant panda "Ni Xiao" after mating was tested according to the prediction method of true pregnancy of giant pandas provided in the embodiment. The changes of progesterone correction value and neopterin correction value in the urine samples obtained during the test are shown in the following table:

[0055]

[0056] 1. The changes in the corrected progesterone value in the panda's urine were monitored after mating on February 16. On March 29, the corrected progesterone value of the giant panda was higher than 100ng / mL Cr for the first time (the corresponding value was 100.1ng / mL Cr), and the determination of the corrected neopterin value began. The corrected progesterone value of the giant panda reached its peak on April 28 (the corresponding value was 599.1ng / mL Cr), and on May 12, it dropped below 200ng / mL Cr for the first time (the corresponding value was 154.5ng / mLCr).

[0057] 2. Calculated by the following method, N1=(2.1+3.2+6.1+6.0) / 4 = 4.35; N2=(8.2+6.3+7.5) / 3 =7.3. N2 / N1= 7.3 / 4.9 = 1.68, which is consistent with the N2 / N1 value between 1.39 and 2.08 (95%CI), indicating that the mating giant panda is truly pregnant. The giant panda "Ni Xiao" gave birth on May 19, 8 days after the progesterone correction value first dropped below 200ng / mL Cr (corresponding to May 12).

[0058] Experimental Example 4

[0059] Starting from March 2, 2023, the urine of the giant panda "Chengda" after mating was tested according to the prediction method for true pregnancy of giant pandas provided in the embodiment. The changes in the progesterone correction value and neopterin correction value in the urine sample obtained during the test are shown in the following table:

[0060]

[0061] 1. The changes in the corrected progesterone value in the panda's urine were monitored after mating on March 2. On April 16, the corrected progesterone value of the giant panda was higher than 100ng / mL Cr for the first time (the corresponding value was 125.6ng / mL Cr), and the corrected neopterin value was measured. The corrected progesterone value of the giant panda reached its peak on June 13 (the corresponding value was 713.4ng / mL Cr); on July 2, it dropped below 200ng / mL Cr for the first time (the corresponding value was 161.5ng / mLCr).

[0062] 2. Calculated using the following method, N1=(8.0+8.9+6.5+11.7+12.1+10.0+13.3+7.0) / 8 = 9.69; N2=(5.4+6.7+5.1+3.6) / 4 =5.2. N2 / N1=5.2 / 9.69 =0.54, which does not meet the N2 / N1 value between 1.39-2.08 (95%CI), indicating that the breeding of giant pandas is a false pregnancy. Giant panda Chengda did not give birth after breeding in 2023.

[0063] Experimental Example 5

[0064] From April 26, 2023, the urine of the giant panda "Meibao" after mating was tested according to the prediction method of true pregnancy of giant pandas provided in the embodiment. The changes of progesterone correction value and neopterin correction value in the urine samples obtained during the test are shown in the following table:

[0065]

[0066] 1. The changes in the corrected progesterone value in the panda's urine were monitored after mating on April 26. On June 30, the corrected progesterone value of the giant panda was higher than 100ng / mL Cr for the first time (the corresponding value was 136.5ng / mL Cr), and the corrected neopterin value was measured. The corrected progesterone value of the giant panda reached its peak on July 30 (the corresponding value was 984.6ng / mL Cr), and on August 21, it dropped below 200ng / mL Cr for the first time (the corresponding value was 128.3ng / mLCr).

[0067] 2. Calculated by the following method, N1=(8.3+6.8+7.7+6.9+8.2) / 5=7.58; N2= (8.7+9.1+7.5+5.9) / 4=7.8. N2 / N1=7.8 / 7.58=1.03, which does not meet the N2 / N1 value between 1.39-2.08 (95%CI), indicating that the breeding of giant pandas is a false pregnancy. The giant panda "Meibao" did not give birth after breeding in 2023.

[0068] In summary, the prediction method for true pregnancy of giant pandas provided by the present invention can not only accurately and scientifically determine whether the mating giant panda is truly pregnant, but also predict the result 1 to 2 weeks before the birth of the truly pregnant giant panda, which is of great significance for changing the feeding management of truly pregnant giant pandas in advance, formulating and implementing the delivery of newborn giant pandas, and optimizing the breeding management of captive giant pandas. Furthermore, the prediction method provided by the present invention not only simplifies the operating procedures and saves a lot of manpower and material resources, but also enriches the method for distinguishing true pregnancy from false pregnancy of giant pandas.

[0069] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0070] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. An application of progesterone, neopterin and creatinine in the preparation of a reagent for determining true pregnancy in giant pandas, characterized in that: The application comprises the following steps: The first step is to collect urine samples from female giant pandas after mating, and then test the progesterone concentration and neopterin concentration in the urine; the measured progesterone concentration and neopterin concentration are divided by the creatinine concentration in the urine sample to obtain the progesterone correction value and neopterin correction value, and ng / mL Cr is used as the measurement unit of the progesterone correction value and neopterin correction value; The second step is to calculate the average value of neopterin correction from the time when the progesterone correction value in the urine of female giant pandas first exceeds 100ng / mL Cr to the time when the progesterone correction value rises to the peak value as N1; the average value of neopterin correction from the time when the progesterone correction value first drops from the peak value to below 200ng / mL Cr is N2; when the N2 / N1 value is between 1.39 and 2.08, it indicates that the breeding giant panda is truly pregnant, otherwise it is a false pregnancy.

2. The use of progesterone, neopterin and creatinine in the preparation of a reagent for determining true pregnancy of a giant panda according to claim 1, characterized in that: The urine samples of female giant pandas are collected once a week; and the urine collection time is 7:00-9:00 in the morning.

3. The use of progesterone, neopterin and creatinine in the preparation of a reagent for determining true pregnancy of a giant panda according to claim 1, characterized in that: The specific operation of urine sample collection is as follows: after the female giant panda urinates, 2 ml of urine is sucked from the clean floor of the animal house with a disposable syringe and placed in a sampling tube, which is then marked with the corresponding giant panda's name and sampling date.

4. The use of progesterone, neopterin and creatinine in the preparation of a reagent for determining true pregnancy of a giant panda according to claim 1, characterized in that: The urine samples collected from female giant pandas were centrifuged at 3000 r / min for 10 min at room temperature, and then the urine samples after centrifugation were taken for later use.

5. The use of progesterone, neopterin and creatinine in the preparation of a reagent for determining true pregnancy of a giant panda according to claim 1, characterized in that: The concentrations of progesterone and neopterin in urine samples of female giant pandas were determined by enzyme-linked immunosorbent assay.

6. The use of progesterone, neopterin and creatinine in the preparation of a reagent for determining true pregnancy of a giant panda according to claim 1, characterized in that: A commercial ELISA kit was used to measure the concentration of neopterin in urine samples of female giant pandas.

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