CN116559469A

CN116559469A - Application of serum factor marker in early pregnancy diagnosis of dairy cows with different reproduction modes

Info

Publication number: CN116559469A
Application number: CN202310741712.8A
Authority: CN
Inventors: 汤克琼; 张云; 靳亚平; 杨武才; 陶启兵; 柳茗茗; 王疌
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2023-06-21
Filing date: 2023-06-21
Publication date: 2023-08-08

Abstract

The invention discloses a method for auxiliary prediction of early pregnancy states of cows in different breeding modes by serum factor marker level analysis, which carries out pregnancy diagnosis on the cows in different breeding modes by detecting serum factor (P4, PAG, EPF) levels, and relates to the technical field of early pregnancy diagnosis of cows. The method has the characteristics of high sensitivity and high accuracy, can obviously improve the accuracy of early pregnancy diagnosis of cows, shortens the nonpregnant days of cattle groups, and reduces economic losses in aspects of calving, milk production, feed, labor and the like caused by nonpregnant.

Description

Application of serum factor marker in early pregnancy diagnosis of dairy cows with different reproduction modes

Technical Field

The invention relates to the technical field of early pregnancy diagnosis of cows, in particular to application of a serum factor marker in early pregnancy diagnosis of cows with different propagation modes.

Background

In recent years, the innovation of germplasm resources of cows and the healthy development of the milk industry are greatly emphasized by novel key technologies of fine breed cultivation and rapid propagation of high-yield cows. In Embryo Transfer (ET) experiments, whether or not Embryo and recipient are synchronized is critical to successful establishment of pregnancy. Therefore, the recipient cows are scientifically and reasonably screened on day 0 and day before ET, and the method has important significance for improving the gestation level of the recipient cows, further accelerating the establishment of the improved breed cow nuclear population and improving the economic benefit of pastures. At present, dairy cows are still judged to estrus by manually observing the characteristics of climbing behavior, mood swings, pudendum mucus and the like in the production process of animal husbandry, and a systematic study which can be effectively monitored by utilizing Progesterone (P4) detection is lacking.

The main factors of pregnancy failure in cows include embryonic death (days 0-42) and fetal death (days 42-280), the majority of which results in pregnancy establishment failure due to embryonic death (Rabaglino et al 2020). Notably, the loss of maternal pregnancy recognition embryonic death was greatest among them 14-24 d after mating. Over the past few decades, various early pregnancy diagnostic methods have emerged, including observation of oestrus, rectal palpation, ultrasound examination and detection of specific antigens in the blood. The former three are common clinical diagnosis methods in pastures in China at present, and are usually carried out 30-35 days after the dairy cows are bred, and the dairy cows enter the second estrus after the breeding. Unlike the first three pregnancy diagnostic methods, blood specific antigens are detected without direct contact with the female animal and without reliance on the technical expertise and practical experience of the operator, and in addition, bulk detection of body fluid samples is possible. These blood tests mainly involve the detection of blood (progestrone, P4), pregnancy related glycoproteins (PAG-Associated Glycoprotein) and Early Pregnancy factors (Early PregnancyFactor, EPF), however, a single analysis of one of the indices does not accurately determine the Pregnancy status of the cow (Szenci 2021;Wang et al.2020). At present, the immunological diagnosis methods related to progesterone, pregnancy related glycoprotein and early pregnancy factor are still to be further studied in the application to the early pregnancy diagnosis of pasture cows.

Therefore, it is a urgent need for those skilled in the art to provide an effective method for diagnosing early pregnancy of cows.

Disclosure of Invention

In view of the above, the invention provides the application of serum factors P4 (progesterone), PAG (pregnancy related glycoprotein) and EPF (early pregnancy factor) in early pregnancy diagnosis of cows with different propagation modes, and has the characteristics of high sensitivity and high accuracy, and can obviously improve the accuracy of early pregnancy diagnosis of cows.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

an application of a serum factor marker in early pregnancy diagnosis of cows with different reproduction modes, wherein the serum factor marker comprises serum P4, serum PAG and serum EPF; the different propagation modes comprise IVF, SCNT and AI.

The invention also aims to provide the application of the cow early pregnancy diagnosis serum factor marker in preparing products for predicting the cow early pregnancy.

Still another object of the present invention is to provide a method for assisting in predicting early pregnancy status of cows with different reproductive patterns by the serum factor marker level analysis, which is characterized by comprising the following steps: (1) Collection of blood samples

Collecting tail vein blood 2mL of embryo transfer cattle 21d and 28d and artificial insemination cattle 28d in a vacuum blood collection tube without anticoagulant, and standing at room temperature; the day calculation is defined as 0 day on the day of the synchronous estrus degree or the climbing behavior of the dairy cows;

(2) Collection of specimen serum

Standing at room temperature for 1h, standing at 4deg.C for about 5h, centrifuging at 3500r/min for 5min, collecting upper serum, placing in 1.5mL centrifuge tube, and storing at-20deg.C for further analysis;

(3) Detection of serum pregnancy factors

The detection of three factors, namely serum P4, PAG and EPF, is carried out by adopting an ELISA kit, the dosage of each sample hole is 50 mu L, and the detection is carried out according to the instruction of the kit;

(4) Statistical analysis

1) Serum P4, PAG and EPF factor levels

Measuring the absorbance (OD value) of each hole by using an enzyme-labeled instrument, substituting the OD value of the sample into a standard curve equation, and calculating the concentration of the sample;

2) Early pregnancy diagnosis of cows based on serum P4, PAG and EPF levels

For IVF embryo receptor cattle and SCNT embryo receptor cattle, the level of P4 in the serum of the 21d cow after estrus or the level of PAG and EPF in the serum of the 28d cow after estrus is judged to be pregnant if the levels are respectively larger than the corresponding critical values, namely cut-off values; similarly, for an artificially inseminated cow, the pregnancy is determined if the levels of PAG and EPF in the serum of the cow at 28d after estrus are respectively greater than the respective threshold values, that is, cut-off values.

As a preferred technical solution, the criteria for predicting early pregnancy ending of cows include:

(1) The threshold value of the early pregnancy outcome prediction of P4 on day 21 of IVF embryo recipient cattle is 7.93ng/mL respectively; the critical value of the early pregnancy outcome prediction of P4 on day 21 of SCNT embryo recipient cattle is 5.27ng/mL respectively;

(2) In IVF embryo recipient cattle, SCNT embryo recipient cattle and AI cattle, the 28 th day serum PAG predicted thresholds for early pregnancy outcome were 5.68ng/mL, 7.84ng/mL, 8.96ng/mL, respectively;

(3) In IVF embryo recipient cattle, SCNT embryo recipient cattle and AI cattle, the 28 th day serum EPF predicted threshold for early pregnancy outcome was 33.18ng/mL, 39.12ng/mL, 16.74ng/mL, respectively.

As a preferred technical solution, the efficacy of predicting early pregnancy ending of cows is:

a. the area under the 21 st curve of IVF embryo receptor bovine serum P4 is 0.989, so that the prediction value is high;

the area under the 21 st curve of SCNT embryo receptor bovine serum P4 is 0.993, and the method has higher predictive value;

b. the area under the 28 th day curve of the IVF embryo receptor bovine serum PAG is 0.917, so that the method has higher predictive value;

the area under the 28 th day curve of the SCNT embryo receptor bovine serum PAG is 0.824, so that the method has higher predictive value;

the area under the 28 th day curve of the AI bovine serum PAG is 0.844, and the prediction value is higher;

c. the area under the 28 th day curve of IVF embryo receptor bovine serum EPF is 0.880, and the prediction value is higher;

the area under the 28 th day curve of the SCNT embryo receptor bovine serum EPF is 0.746, and the method has higher predictive value;

the area under the 28 th day curve of the AI bovine serum EPF is 0.807, and the prediction value is higher.

According to the technical scheme, the application of the serum factors P4, PAG and EPF in early pregnancy diagnosis of cows is disclosed, and the method has the characteristics of high sensitivity and high accuracy, can obviously improve the accuracy of early pregnancy diagnosis of cows, shortens the nonpregnant days of cow groups, and reduces economic losses in aspects of calving, milk production, feed, labor and the like caused by nonpregnant.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of the subject bovine treatment, ultrasound examination and blood sample collection of the present invention; where US is ultrasound examination, ET is embryo transfer, PD is pregnancy diagnosis, BC is blood collection.

FIG. 2 shows the results of progesterone content (P4) in the serum of an in vitro fertilized embryo recipient cow of the present invention in early gestation; and (3) injection: * P <0.05, <0.01 means that pregnant (n=15) and non-pregnant (n=12) cattle are statistically different. Data are expressed as mean ± standard error.

FIG. 3 shows the results of progesterone content (P4) in the serum of a somatic cell nuclear transfer embryo recipient cow in early gestation; * P <0.05, <0.01 means that pregnant (n=18) and non-pregnant (n=24) cattle are statistically different. Data are expressed as mean ± standard error.

FIG. 4 is a ROC graph of the invention for predicting the pregnancy outcome of in vitro fertilized embryo recipient cattle using serum progesterone (P4).

FIG. 5 is a ROC graph of the predicted somatic cell nuclear transfer embryo receptor cattle gestation outcome of serum progesterone (P4) of the invention.

FIG. 6 shows pregnancy related glycoprotein (PAG) levels in day 28 serum of dairy cows of different reproductive patterns according to the invention; wherein IVF: 12 pregnant, 16 non-pregnant; SCNT: gestation 19, non-gestation 17; AI: 29 pregnant, 25 not pregnant; * P <0.05, <0.01 means that pregnant versus non-pregnant cattle are statistically different; data are expressed as mean ± standard error.

FIG. 7 is a ROC graph of serum Pregnancy Associated Glycoprotein (PAG) of the present invention for predicting pregnancy outcome in cows of different reproductive modes.

FIG. 8 shows Early Pregnancy Factor (EPF) content in day 28 serum of dairy cows with different breeding modes according to the present invention; wherein IVF: 12 pregnant, 16 non-pregnant; SCNT: gestation 19, non-gestation 17; AI: 29 pregnant, 25 not pregnant; * P <0.05, <0.01 means that pregnant versus non-pregnant cattle are statistically different; data are expressed as mean ± standard error.

FIG. 9 is a ROC graph of Early Pregnancy Factor (EPF) prediction of pregnancy outcome for cows of different reproductive modes according to the invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention discloses application of serum factors P4, PAG and EPF in early pregnancy diagnosis of cows with different breeding modes, all reagents used in the invention are commercially available, the sources of the reagents are not particularly limited, and the related methods, such as no special mention, are conventional methods and are not repeated herein.

It should be noted that, the present invention is defined as 0 day on the day of estrus characterization of cows through a synchronous estrus program or the occurrence of a climbing behavior, etc.

Example 1

1. Test object: the tested cows were selected from the pasture of the Shang-on origin of Ningxia Hui municipality, including 133 embryo transfer cows (55 in vitro fertilized embryo recipient cows+78 somatic cell nuclear transfer embryo recipient cows) and 54 artificial inseminates.

Recipient cow inclusion criteria: selecting an unprepared herd or a nonpregnant warp herd in a pasture, wherein the recipient cows should meet the following basic requirements: (1) physical development is normal, physical condition is good, and clinical examination is healthy; (2) genetic background investigation shows that the reproductive performance is excellent; (3) excluding genetic diseases and infectious diseases (such as brucellosis, BVDV, IBR, etc.); (4) immunization and insect repellent work was completed at least 1 month prior to embryo transfer; (5) ensuring the temperature and the smoothness of the cattle as much as possible and reducing the stress response.

Wherein, the non-equipped cattle should also meet the following requirements: (1) the month age is more than 13.5 months, the weight is more than 375kg, and the height is more than 130cm; (2) the development is good, and the size and shape of the genital tract and the pelvic bone are normal; (3) there was no history of long-term medication and embedded substances.

2. Test grouping: group P4 included 27 in vitro fertilized embryo recipient cows and 42 somatic cell nuclear transfer embryo recipient cows;

the PAG/EPF group includes 28 in vitro fertilized embryo recipient cows, 36 somatic cell nuclear transfer embryo recipient cows and 54 artificial inseminate cows.

3. Test method (for flow chart see fig. 1):

(1) Serum factor level analysis

P4 day tail vein blood of embryo transfer cattle is collected in anticoagulant-free vacuum blood collection tubes respectively, cattle numbers and blood collection dates are marked, the collected blood is transported to a laboratory, the blood is kept stand for 1h at room temperature, then the blood is placed in a refrigerator at 4 ℃ for about 5h, and is centrifuged for 5min by a centrifuge 3500r/min, and upper serum is taken and placed in a 1.5mL centrifuge tube and stored at-20 ℃ for further P4 analysis.

The PAG/EPF group collects tail venous blood on the 28 th day in a vacuum blood collection tube without anticoagulant, marks the number of cattle and the date of blood collection, conveys the collected blood to a laboratory, stands for 1h at room temperature, stands for about 5h at a refrigerator of 4 ℃, then uses a centrifuge 3500r/min to centrifuge for 5min, takes upper serum to place in a 1.5mL centrifuge tube, and stores at-20 ℃ for further PAG/EPF analysis.

Serum P4 analysis method:

progesterone (P) assay kit (ELISA) (Beijing northern Biotechnology research Co., ltd.)

Preparing a sample: taking out the serum to be tested from the refrigerator at the temperature of minus 20 ℃, standing for about 30min at room temperature, and after complete melting, carrying out vortex vibration to fully and uniformly mix the serum. Serum samples were prevented from repeated freeze thawing.

Balance: the detection reagent was equilibrated for at least 30min at room temperature and mixed well using a vortex shaker prior to sample addition.

Preparing liquid: taking 20 times of concentrated washing liquid, and according to the amount of the samples detected in the batch, using distilled water according to the ratio of 1: diluting 20, and uniformly mixing for later use.

Sample adding: standard, sample and blank (control) wells were placed in 96-well plates, respectively, and their positions recorded. Each standard substance is sequentially provided with two holes, and 50L of corresponding standard substance is added into each hole; the blank holes are not added with any liquid; the remaining wells were filled with 50L of serum to be tested. The sample should be added to the bottom of the reaction well to avoid contact with the inner wall and bubble generation.

Adding enzyme: in addition to the blank wells, 50L of enzyme-labeled antigen was added to each well.

Adding an antibody: in the same order as in the previous step, 50L of antibody was added to each well. The sides of the plates were gently tapped to ensure adequate mixing of the solution.

Incubation: a layer of sealing plate film is attached to prevent the solution from evaporating. Incubate in a constant temperature incubator at 37℃for 1h.

Washing the plate: removing the liquid in the holes, sucking the prepared washing liquid by a syringe or a discharge gun to fill each hole, standing for about 10s, spin-drying, repeating the process for three times, and cleaning by filling each well. After the last washing, the orifice plate is turned over and is patted dry on clean thick absorbent paper.

Color development: 50L of the developer A solution was added to each well, and then 50L of the developer B solution was added in the same order. After fully mixing, a new sealing plate film is attached for sealing, and the mixture is incubated for 15min in a constant temperature incubator at 37 ℃.

And (3) terminating: 50L of stop solution was added to each well to terminate the reaction.

And (3) measuring: absorbance (OD value) was measured at 450nm for each well using a microplate reader. The measurement should be performed within 15min after the addition of the stop solution.

Serum PAG assay method:

bovine Pregnancy Associated Glycoprotein (PAG) kit (ELISA) (Shanghai enzyme-linked Biotechnology Co., ltd.) was used

Preparing a sample: taking out the serum sample from the refrigerator at the temperature of minus 20 ℃, standing at room temperature, and fully and uniformly mixing the serum sample by using a vortex oscillator after the serum sample is completely melted.

Balance: and (3) standing the detection reagent at room temperature to enable the detection reagent to reach the room temperature of 20-25 ℃ when in use, and carrying out vortex vibration to enable the reagent to be fully and uniformly mixed.

Preparing liquid: 20-fold dilution of the wash buffer was performed with distilled water at 1: diluting 20, namely adding 19 parts of distilled water into one part of 20X washing buffer solution, and uniformly mixing for later use.

Sample adding: standard, sample and blank (control) wells were placed in 96-well plates (pre-coated with bovine PAG capture antibody) and their positions recorded. Each standard substance is sequentially provided with two holes, and 50L of standard substances with different concentrations are added; blank holes are not added; the remaining wells were filled with 50L of serum to be tested. The sample should be added to the bottom of the reaction well to avoid contact with the inner wall and bubble generation.

Adding enzyme: in addition to the blank wells, peroxidase-labeled antibody 100L was added to each of the standard wells and the sample wells.

Incubation: the reaction holes are sealed by a sealing plate film to prevent the solution from evaporating. Incubation was carried out in a constant temperature incubator at 37℃for 60min.

Washing the plate: the liquid in the holes is discarded, the prepared washing liquid is sucked by a syringe or a discharge gun to fill each hole (350L), the washing liquid is thrown off after standing for 1min, the washing liquid is beaten dry on the water absorbing paper, and the process is repeated for 5 times.

Color development: the developer A solution 50L was added to each well, and then the developer B solution 50L was added in the same order. Fully and uniformly mixing. A layer of sealing plate film is attached to the culture medium for sealing, and the culture medium is incubated in a constant temperature incubator at 37 ℃ for 15min.

And (3) measuring: absorbance (OD value) per well was measured at a wavelength of 450nm using an enzyme-labeled instrument. The measurement should be performed within 15min after the addition of the stop solution.

Serum EPF analysis method:

the detection step was performed using bovine early pregnancy factor protein (EPF) kit (ELISA) (Shanghai enzyme-linked biotechnology limited) with reference to serum PAG assay.

(2) Uterine fetal detection and diagnosis

On day 32 (corresponding to 26d after embryo transfer), carrying out uterine fetal testing by a pregnant testing professional using portable B ultrasonic for livestock through rectum, and if the endometrial thickening is detected, and black areas with different sizes, namely, images of embryos, carrying out conception on the recipient cattle; without the expansion of the uterine horn, there is no image of the embryo and other features, i.e., the recipient cow is not conception. Cows are classified into pregnant groups and non-pregnant groups according to the diagnosis result.

(3) Construction of early pregnancy prediction model for cows

And (3) counting the detection results of the receptor bovine serum P4, PAG and EPF obtained in the step (1) and the detection results of the uterine fetus obtained in the step (2) by using Excel. Data analysis was performed using GraphPad PRISM 6. P4 concentration, PAG concentration and EPF concentration in pregnant and non-pregnant groups were used to identify differences using one-way ANOVA and independent sample T-test. Results are expressed as mean ± standard error (mean ± SEM). Differences are considered statistically significant when P <0.05 or P < 0.01.

To examine the ability of serum factor P4 concentrations at days 0, 5, 12, 21 and serum factors PAG, EPF to determine the end of pregnancy at day 32 on day 28, subject work characteristic ROC curve analysis was performed, performance of cow serum P4 level prediction at early pregnancy end was evaluated, ROC curves were plotted with true positive rate (Sensitivity) as ordinate and false positive rate (1-Specificity) as abscissa, and optimal cut-off values at each time point were determined by about the Dent index, wherein cows diagnosed as gestation by ultrasonics at day 32 were true positive, and Sensitivity, specificity and accuracy were calculated.

Reliability of the predicted pregnancy outcome at each time point cut-off value was assessed by a precision logistic regression analysis using SAS 9.4 software. Regression analysis was performed by dividing each group of cattle into two groups (cut-off and > cut-off) with cut-off as a boundary, incorporating independent variable X, and B-ultrasonic diagnosis of pregnancy ending at 32 days as dependent variable Y, pregnancy=1, non-pregnancy=0. The corresponding OR (Odds ratios) value, 95% confidence interval and P value were calculated. P <0.05 indicates significant differences; when P is more than 0.05 and less than 0.1, different trends are indicated.

Example 2

Group P4

(1) P4 expression level analysis

Group P4 included 27 in vitro fertilized embryo recipient cattle (IVF) and 42 somatic cell nuclear transfer embryo recipient cattle (SCNT).

The expression level of the IVF embryo receptor cattle in the early gestation group and the non-gestation group P4 is shown in the figure 2, and the result shows that the concentration of the P4 in the gestation group cattle gradually increases with time, and the concentration of the progesterone in the non-gestation group cattle gradually increases and then decreases. Pregnant cattle had a higher average P4 concentration than the non-pregnant group on days 5, 12 and 21 and a lower average P4 concentration than the non-pregnant group on day 0. The P4 level on day 21 was found to be statistically significant in pregnant groups over non-pregnant groups (P < 0.01) by one-way ANOVA.

The expression level of SCNT embryo receptor cattle in early gestation group and non-gestation group P4 shows that the concentration of P4 in gestation group cattle gradually increases with time, the concentration of P4 in non-gestation group cattle gradually increases with time and then decreases, and the concentration reaches a peak value at 12 days. The average P4 concentration was higher in the pregnant group than in the non-pregnant group on days 5, 12 and 21 and lower in the non-pregnant group on day 0. The P4 level of the pregnant group is significantly higher than that of the non-pregnant group (P < 0.01) in 21 days by the one-factor ANOVA test, and the method has statistical significance.

(2) Evaluation of serum P4 efficacy in predicting pregnancy outcome Using ROC Curve

Serum P4 levels were evaluated for their predicted performance at early pregnancy outcome, and ROC curves were plotted for P4 levels at 0, 5, 12 and 21 days with true positive (Sensitivity) on the ordinate and false positive (1-Specificity) on the abscissa. And the corresponding P4 demarcation point when the about step index is maximum is the cut-off value.

ROC curve for early pregnancy resolution predicted by IVF embryo receptor bovine P4 levels as shown in fig. 4 and table 1, day 0P 4 levels were 1.33ng/mL cut-off in predicted pregnancy, AUC 0.700 (p=0.079), sensitivity: 87%, specificity: 58%, accuracy: 74%; day 5P 4 levels were cut-off 4.06ng/mL in predicted pregnancy, AUC 0.589 (p=0.435), sensitivity: 80%, specificity: 50%, accuracy: 67%; day 12P 4 level was 10.77ng/mL cut-off in predicted pregnancy, AUC 0.706 (p=0.071), sensitivity: 67%, specificity: 83%, accuracy: 74%; day 21P 4 levels were predicted to have a cut-off value of 7.93ng/mL, AUC of 0.989 (P < 0.001), sensitivity: 93%, specificity: 100%, accuracy: 96%. The area under the curve (AUC) of the in vitro fertilized dairy cow serum P4 on the 21 st day is 0.989, so that the prediction value is higher; the area under the curve (AUC) on the 0 th day is 0.700, and the area under the curve (AUC) on the 12 th day is 0.706, so that the method has general predictive value; area under the 5 th day curve (AUC) was 0.589, with lower predictive value.

ROC curve for SCNT embryo receptor cattle P4 level prediction early pregnancy resolution as shown in fig. 5 and table 1, day 0P 4 level was 0.63ng/mL at cut-off value in predicted pregnancy, AUC was 0.625 (p=0.170), sensitivity: 67%, specificity: 67%, accuracy: 67%; day 5P 4 levels were predicted to have a cut-off value of 1.95ng/mL, AUC of 0.660 (p=0.080), sensitivity: 89%, specificity: 46%, accuracy: 64%; day 12P 4 level was 7.93ng/mL cut-off in predicted pregnancy, AUC 0.678 (p=0.050), sensitivity: 83%, specificity: 58%, accuracy: 69%; day 21P 4 levels were predicted to have a cut-off value of 5.27ng/mL, AUC of 0.993 (P < 0.001), sensitivity: 100%, specificity: 92%, accuracy: 95%. The area under the curve of the serum P4 of the somatic cell nuclear transfer dairy cow on the 21 st day is 0.993, so that the method has higher prediction value; the area under the curve on the 0 th day is 0.625, the area under the curve on the 5 th day is 0.660, and the area under the curve on the 12 th day is 0.678, thus having general predictive value.

ROC curve analysis of P4 predictive early pregnancy outcome is shown in table 1.

TABLE 1

PAG group

(1) PAG expression level analysis

The PAG group included 28 in vitro fertilized embryo recipient cows (IVF), 36 somatic cell nuclear transfer embryo recipient cows (SCNT), and 54 artificial inseminate cows (AI).

For cows of different breeding patterns, the relationship between the serum PAG level at 28 days and the pregnancy outcome is analyzed, and the result is shown in figure 6, wherein the PAG content in the serum at 28 days of pregnant cows is higher than that of non-pregnant cows in three groups of cows, and the differences are significant through one-way ANOVA test (IVF: P <0.01, SCNT: P <0.01, AI: P < 0.01).

(2) Evaluation of serum PAG efficacy in predicting pregnancy outcome Using ROC Curve

The ROC curves of IVF embryo receptor cattle, SCNT embryo receptor cattle and AI cattle for predicting the end of pregnancy at day 32, with PAG levels on day 28, are shown in FIG. 7 and Table 2, the AUC of the three ROC curves are 0.917, 0.824 and 0.844, the cut-off values are 5.68ng/mL, 7.84ng/mL and 8.96ng/mL, the sensitivities are 100%, 89% and 79%, the specificities are 75%, 65% and 80% respectively, and the accuracies are 86%, 78% and 80% respectively. The area under the 28 th day curve of the in vitro fertilized embryo receptor bovine serum PAG is 0.917, so that the method has higher predictive value; the area under the 28 th day curve of the somatic cell nuclear transfer embryo receptor bovine serum PAG is 0.824, so that the method has higher prediction value; the area under the 28 th day curve of the serum PAG of the artificial insemination dairy cow is 0.844, and the method has higher predictive value.

TABLE 2

EPF group

(1) EPF expression level analysis

The EPF group includes 28 in vitro fertilized embryo recipient cows (IVF), 36 somatic cell nuclear transfer embryo recipient cows (SCNT) and 54 artificial inseminate cows (AI).

The relationship between EPF level of the day 28 serum and pregnancy outcome was analyzed for cows of different breeding patterns, and the results are shown in FIG. 8, wherein the EPF content of the day 28 serum of the cows is higher in the three groups of cows than in the non-pregnant cows, and the differences are significant by one-way ANOVA test (IVF: P <0.01, SCNT: P <0.01, AI: P < 0.01).

(2) Evaluation of serum EPF efficacy in predicting pregnancy outcome Using ROC Curve

ROC curves for predicting early pregnancy outcome for day 28 EPF levels in IVF embryo recipient cattle, SCNT embryo recipient cattle and AI cattle, where day 28 EPF levels in predicted pregnancy were 33.18ng/mL cut-off and AUC was 0.880 (P < 0.001), sensitivity are shown in FIG. 9 and Table 3: 67%, specificity: 100%, accuracy: 86%; in SCNT embryo recipient cattle, the EPF level on day 28 was 39.12ng/mL cut-off in predicted pregnancy, AUC 0.746 (p=0.012), sensitivity: 47%, specificity: 100%, accuracy: 72%; in AI cattle, the EPF level at day 28 was 16.74ng/mL cut-off in the predicted pregnancy, AUC was 0.807 (P < 0.001), sensitivity: 93%, specificity: 56%, accuracy: 76%. The area under the 28 th day curve of the in vitro fertilization embryo receptor bovine serum EPF is 0.880, so that the prediction value is high; the area under the 28 th day curve of the somatic cell nuclear transfer embryo receptor bovine serum EPF is 0.746, so that the method has higher prediction value; the area under the 28 th day curve of the serum EPF of the artificial insemination dairy cow is 0.807, and the method has higher predictive value.

TABLE 3 Table 3

Example 3

And according to the comparison of the pregnant groups and the non-pregnant groups of the dairy cows with different breeding modes, the value of the pregnancy ending of the dairy cows on the 32 th day is predicted by using the corresponding cut-off values of the precision logistic regression analysis P4, the PAG and the EPF. Each group of cattle was divided into two groups (cut-off and > cut-off) with cut-off as a boundary, and independent variable X was included, and the end of pregnancy diagnosed by B-ultrasonic diagnosis on day 32 was dependent variable Y, pregnancy=1, and non-pregnancy=0. The cut-off value levels of P4, PAG, EPF in cows of different breeding patterns are closely related to early pregnancy outcome by regression analysis (Table 4). It was found by analysis that IVF embryo recipient cattle with serum P4 concentrations higher than 7.93ng/mL on day 21 of the estrus cycle were diagnosed with a 105-fold lower probability of pregnancy (P < 0.001) on day 32 than 7.93ng/mL; SCNT embryo recipient cattle with P4 concentrations above 5.27ng/mL are diagnosed with a 171-fold lower probability of gestation (P < 0.001) on day 32 than 5.27 ng/mL.

IVF embryo recipient cows with serum PAG concentrations higher than 5.68ng/mL on day 28 of gestation of the cows are diagnosed with a 38-fold lower probability (P < 0.001) than 5.68ng/mL on day 32 of gestation; SCNT embryo recipient cattle with PAG concentrations above 7.84ng/mL were diagnosed with a 14-fold lower probability of gestation (p=0.002) on day 32 than 7.84 ng/mL; AI cattle with PAG concentrations above 8.96ng/mL were diagnosed with a 14-fold lower probability of gestation (P < 0.001) on day 32 than 8.96ng/mL; IVF embryo recipient cattle with serum EPF concentrations above 33.18ng/mL are diagnosed with a 33-fold lower probability of gestation (P < 0.001) on day 32 than 33.18 ng/mL; SCNT embryo recipient cattle with EPF concentrations higher than 39.12ng/mL were diagnosed with a 19-fold lower probability of gestation (p=0.002) on day 32 than 39.12 ng/mL; AI cattle with EPF concentrations higher than 16.74ng/mL were diagnosed with a 16-fold lower probability of pregnancy (P < 0.001) on day 32 than 16.74ng/mL.

TABLE 4 Table 4

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of serum factor markers in early pregnancy diagnosis of cows with different reproduction modes is characterized in that: the serum factor markers include serum P4, serum PAG, and serum EPF; the different propagation modes comprise IVF, SCNT and AI.

2. Use of the serum factor marker for early pregnancy diagnosis of cows of claim 1 in the preparation of a product for predicting early pregnancy of cows.

3. A method for assisting in predicting early pregnancy status of cows of different reproductive patterns based on the serum factor marker level analysis as claimed in claim 1, comprising the steps of:

(1) Collection of blood samples

(2) Collection of specimen serum

(3) Detection of serum pregnancy factors

(4) Statistical analysis

1) Serum P4, PAG and EPF factor levels

2) Early pregnancy diagnosis of cows based on serum P4, PAG and EPF levels

4. A method according to claim 3, wherein the criteria for predicting the early pregnancy outcome of a cow comprises:

(1) The threshold for early pregnancy outcome prediction by day 21P 4 in IVF embryo recipient cattle was 7.93ng/mL; the threshold for predicting early pregnancy outcome for P4 on day 21 in SCNT embryo recipient cattle was 5.27ng/mL;

5. The method of claim 3, wherein the efficacy of predicting early pregnancy outcome in a cow is: