Application of a protein composition and its detection reagent in predicting the effect of superovulation in sheep

Abstract

The present invention discloses a protein composition and its detection reagent for use in predicting the effect of superovulation in sheep. The protein composition consists of B2M, ACAA1, ADAMTS-13 and APO-F. The present invention uses a specific combination of four proteins as markers, and predicts the number of embryos produced by sheep after superovulation and fertilization by detecting the concentration of the marker in the peripheral blood of sheep before superovulation. Compared with the existing technology, it is more convenient and efficient, and has high accuracy. It has a wide range of application value in improving the reproductive efficiency of sheep and preserving and expanding the breeding of precious sheep breeds.

Description

Technical Field

[0001] The invention relates to the technical field of animal breeding, and in particular to application of a protein composition and a detection reagent thereof in predicting the superovulation effect of sheep. Background Art

[0002] Superovulation-embryo transfer (MOET) is an important method to improve reproductive efficiency. Through the use of follicle-stimulating hormone (FSH) and other reproductive hormones, the hormone changes in the natural ovulation cycle are simulated to stimulate the ovaries to produce more follicles and release eggs, so that the donor female animal can obtain several times or even dozens of times more embryos than the natural ovulation number in one estrus cycle.

[0003] In sheep farming, due to the long reproductive cycle and relatively low reproductive capacity of sheep, the superovulation technology is used to increase the number of ovulations and pregnancy rates of sheep, which helps to improve the productivity of the sheep population. However, the superovulation effect of sheep has obvious individual differences, which is closely related to their age, body condition, number of births, etc. Therefore, accurately predicting the superovulation results of sheep is of great significance for reducing the excessive use of hormones and drugs, reducing feeding and management costs, and improving reproductive efficiency. At present, B-ultrasound technology is mainly used to predict the number of ovulations by observing the development of follicles in the sheep's ovaries. However, because the technical requirements for the operator are high, and there is a high risk of misjudgment in the results of artificial observation of the maturation of follicles in the ovaries, it is urgent to develop a simpler, more accurate and efficient method for predicting superovulation results. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides an application of a protein composition and a detection reagent thereof in the preparation of a sheep superovulation product.

[0005] The first object of the present invention is to provide a protein composition.

[0006] The second object of the present invention is to provide the use of the detection reagent of the protein composition in predicting the total number of embryos produced by superovulation and insemination in sheep.

[0007] A third object of the present invention is to provide a method for predicting the number of embryos produced by superovulation and insemination in sheep.

[0008] In order to achieve the above object, the present invention is implemented by the following scheme:

[0009] The present invention first discovered that apolipoprotein F (APO-F), β2-microglobulin (B2M), von Willebrand factor cleavage enzyme (ADAMTS-13) and acetyl-CoA acyltransferase 1 (ACAA1) in the peripheral blood of sheep before superovulation have a significant correlation with the number of embryos finally produced. By detecting the concentrations of B2M, ACAA1 and ADAMTS-13 in the blood of natural estrus donor sheep and the concentrations of B2M and APO-F in the blood of sympathetic estrus sheep, the purpose of predicting the result of superovulation can be achieved. Therefore, the present invention requests protection for the following contents:

[0010] A protein composition consisting of B2M, ACAA1, ADAMTS-13 and APO-F.

[0011] The detection reagent of the protein composition is used to predict the total number of embryos produced by superovulation and fertilization of sheep. The concentrations of B2M, ACAA1 and APO-F in the peripheral blood of the sheep are positively correlated with the total number of superovulation embryos; the concentration of ADAMTS-13 in the peripheral blood of the sheep is negatively correlated with the total number of superovulation embryos. The detection reagent is used to detect the concentration of any one or several proteins in the protein composition in the peripheral blood of the sheep.

[0012] Preferably, the concentration of the B2M in the peripheral blood of the sheep is 22-43 μg / ml.

[0013] More preferably, the concentration of the B2M in the peripheral blood of the sheep is 22.96-42.24 μg / ml.

[0014] Preferably, the concentration of ACAA1 in the peripheral blood of the sheep is 5-12 ng / ml.

[0015] More preferably, the concentration of ACAA1 in the peripheral blood of the sheep is 5.04-11.68 ng / ml.

[0016] Preferably, the concentration of APO-F in the peripheral blood of the sheep is 2-9 ng / ml.

[0017] More preferably, the concentration of APO-F in the peripheral blood of the sheep is 2.71-8.49 ng / ml.

[0018] Preferably, the concentration of ADAMTS-13 in the peripheral blood of the sheep is 495-835 pg / ml.

[0019] More preferably, the concentration of ADAMTS-13 in the peripheral blood of the sheep is 498.97-834.03 pg / ml.

[0020] Preferably, before superovulation, the sheep is in estrus; the estrus is natural estrus or synchronized estrus.

[0021] More preferably, for the sheep in natural estrus, the concentrations of the B2M and the ACAA1 in their peripheral blood are positively correlated with the total number of superovulated embryos, and the concentration of the ADAMTS-13 in their peripheral blood is negatively correlated with the total number of superovulated embryos; for the sheep in synchronized estrus, the concentrations of the B2M and the APO-F in their peripheral blood are positively correlated with the total number of superovulated embryos.

[0022] More preferably, the concentration of the B2M in the peripheral blood of the naturally estrus sheep is 22.96-40.75 μg / ml.

[0023] More preferably, the concentration of ACAA1 in the peripheral blood of the naturally estrus sheep is 5.04-11.68 ng / ml.

[0024] More preferably, the concentration of ADAMTS-13 in the peripheral blood of the naturally estrus sheep is 498.97-834.03 pg / ml.

[0025] More preferably, the concentration of the B2M in the peripheral blood of the synchronized estrus sheep is 23.57-42.24 μg / ml.

[0026] More preferably, the concentration of APO-F in the peripheral blood of the synchronized estrus sheep is 2.71-8.49 ng / ml.

[0027] Preferably, the detection reagent is an ELISA reagent.

[0028] The use of the detection reagent of the protein composition in the preparation of a product for predicting the number of embryos produced by superovulation in sheep should also be within the protection scope of the present invention.

[0029] A method for predicting the number of embryos produced by superovulation and insemination in sheep comprises the following steps:

[0030] Before superovulation, detecting the concentration of the protein composition in the peripheral blood of the sheep, and determining the number of embryos produced by the sheep through superovulation and fertilization according to the detection result;

[0031] The concentrations of the B2M, ACAA1 and APO-F in the peripheral blood of the sheep are positively correlated with the total number of superovulated embryos; the concentration of the ADAMTS-13 in the peripheral blood of the sheep is negatively correlated with the total number of superovulated embryos.

[0032] Preferably, before superovulation, the sheep is in estrus; the estrus is natural estrus or synchronized estrus.

[0033] More preferably, before superovulation, the sheep is a sheep in natural estrus, and the criterion for determining the number of embryos is:

[0034] The concentration of B2M in the peripheral blood of the sheep in natural estrus is 22-27.53 μg / mL, the concentration of ACAA1 in the peripheral blood of the sheep in natural estrus is 5-6.75 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the sheep in natural estrus is 747.87-835 pg / mL, and the number of embryos is 1-10;

[0035] The concentration of B2M in the peripheral blood of the naturally estrus sheep is 27.53-32.62 μg / mL, the concentration of ACAA1 in the peripheral blood of the naturally estrus sheep is 6.75-8.65 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the naturally estrus sheep is 652.14-747.87 pg / mL, then the number of embryos is 10-20;

[0036] If the concentration of B2M in the peripheral blood of the sheep in natural estrus is 32.62-43 μg / mL, the concentration of ACAA1 in the peripheral blood of the sheep in natural estrus is 8.65-12 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the sheep in natural estrus is 495-652.14 pg / mL, the number of embryos is 20-36.

[0037] Further preferably, before superovulation, the sheep is a sheep in natural estrus, and the criterion for determining the number of embryos is:

[0038] The concentration of B2M in the peripheral blood of the sheep in natural estrus is 22.96-27.53 μg / mL, the concentration of ACAA1 in the peripheral blood of the sheep in natural estrus is 5.04-6.75 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the sheep in natural estrus is 747.87-834.03 pg / mL, and the number of embryos is 1-10;

[0039] The concentration of B2M in the peripheral blood of the naturally estrus sheep is 27.53-32.62 μg / mL, the concentration of ACAA1 in the peripheral blood of the naturally estrus sheep is 6.75-8.65 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the naturally estrus sheep is 652.14-747.87 pg / mL, then the number of embryos is 10-20;

[0040] If the concentration of B2M in the peripheral blood of the sheep in natural estrus is 32.62-40.75 μg / mL, the concentration of ACAA1 in the peripheral blood of the sheep in natural estrus is 8.65-11.68 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the sheep in natural estrus is 498.97-652.14 pg / mL, then the number of embryos is 20-36.

[0041] Further preferably, the number of embryos is calculated according to any one of Formulas 1 to 3;

[0042] Formula 1: Y=1.387*X1-26.79;

[0043] Formula 2: Y=4.325*X2-18.34;

[0044] Formula 3: Y=-0.07380*X3+66.59;

[0045] Wherein, X1 to X3 represent the concentrations of the B2M, ACAA1 and APO-F in the peripheral blood of the naturally estrus sheep respectively, and Y represents the number of embryos.

[0046] More preferably, before superovulation, the sheep are synchronously estrus sheep, and the criteria for determining the number of embryos are:

[0047] The concentration of B2M in the peripheral blood of the synchronized estrus sheep is 22-28.12 μg / mL or the concentration of APO-F in the peripheral blood of the synchronized estrus sheep is 2-4.12 μg / ml, and the number of embryos is 1-10;

[0048] The concentration of B2M in the peripheral blood of the synchronized estrus sheep is 28.12-33.16 μg / mL or the concentration of APO-F in the peripheral blood of the synchronized estrus sheep is 4.12-5.68 μg / ml, and the number of embryos is 10-20;

[0049] The concentration of B2M in the peripheral blood of the synchronized estrus sheep is 33.16-43 μg / mL or the concentration of APO-F in the peripheral blood of the synchronized estrus sheep is 5.68-9 μg / ml, and the number of embryos is 20-38.

[0050] Further preferably, the number of embryos is calculated according to any one of Formula 4 to Formula 5;

[0051] Formula 4: Y=1.347*X4-26.10;

[0052] Formula 5: Y = 5.147*X5-10.10;

[0053] Wherein, X4 and X5 represent the concentrations of the B2M and the APO-F in the peripheral blood of the synchronously estrus sheep respectively, and Y represents the number of embryos.

[0054] Preferably, the peripheral blood is serum.

[0055] Preferably, the naturally estrus sheep are sheep that are determined to be in estrus through estrus testing.

[0056] More preferably, the naturally estrus sheep undergoes superovulation on the 13th to 15th day of natural estrus, and is inseminated on the 16th day of natural estrus; the day when estrus is determined by estrus test is the 0th day of natural estrus.

[0057] Preferably, for the sheep in natural estrus, the superovulation method is: on the 13th to 15th day of natural estrus, the sheep in natural estrus are treated with a hormone composition comprising follicle stimulating hormone and cloprostenol.

[0058] More preferably, the dosage of the hormone composition is: 210-270 μg / animal of follicle-stimulating hormone and 120-170 μg / animal of cloprostenol.

[0059] Further preferably, the use of the hormone composition to treat the naturally estrus sheep comprises the following steps: first injecting the synchronized estrus sheep with 210-270 μg / head follicle stimulating hormone and 200-260 IU / head pregnant mare serum gonadotropin, and then injecting the synchronized estrus sheep with 120-170 μg / head cloprostenol.

[0060] Still further preferably, the use of the hormone composition to treat the naturally estrus sheep comprises the following steps: first injecting the naturally estrus sheep with 250 μg / sheep of follicle stimulating hormone, and then injecting the naturally estrus sheep with 150 μg / sheep of cloprostenol.

[0061] Preferably, the method of estrus synchronization is a hormone aphrodisiac method.

[0062] More preferably, the hormonal aphrodisiac method is to treat the sheep with 250-330 mg / sheep of progesterone.

[0063] Further preferably, the method for inducing estrus in sheep is to treat the sheep with 300 mg / sheep of progesterone.

[0064] Still further preferably, the progesterone is a progesterone vaginal suppository (CIDR).

[0065] Preferably, the synchronized estrus sheep undergo superovulation on the 10th to 12th day of synchronized estrus, and are inseminated on the 13th day of synchronized estrus; the day of synchronized estrus treatment is the 0th day of synchronized estrus.

[0066] Preferably, for the synchronized estrus sheep, the superovulation method is: on the 10th to 12th day of synchronized estrus, the sheep are treated with a hormone composition comprising follicle stimulating hormone, pregnant mare serum gonadotropin and cloprostenol.

[0067] More preferably, the dosage of the hormone composition is: 210-270 μg / head of follicle-stimulating hormone, 200-260 IU / head of pregnant mare serum gonadotropin and 120-170 μg / head of cloprostenol.

[0068] Further preferably, the use of the hormone composition to treat the synchronized estrus sheep comprises the following steps: first injecting the synchronized estrus sheep with 210-270 μg / head follicle stimulating hormone and 200-260 IU / head pregnant mare serum gonadotropin, and then injecting the synchronized estrus sheep with 120-170 μg / head cloprostenol.

[0069] Still further preferably, the use of the hormone composition to treat the synchronized estrus sheep comprises the following steps: first injecting the synchronized estrus sheep with 250 μg / head follicle stimulating hormone and 250 IU / head pregnant mare serum gonadotropin, and then injecting the synchronized estrus sheep with 150 μg / head cloprostenol.

[0070] Preferably, the method of insemination comprises artificial insemination and / or mating.

[0071] More preferably, the naturally estrus sheep are mated and artificially inseminated.

[0072] More preferably, the estrus-synchronized sheep are artificially inseminated.

[0073] Preferably, embryos are collected on day 7 of insemination; the day of the first insemination is day 1 of insemination.

[0074] The present invention has no particular limitation on the breed of sheep, including but not limited to East Friesian sheep, Darryl sheep, Suffolk sheep and Dexer sheep.

[0075] The East Friesian sheep is a multi-purpose, high-fertility breed, mainly used to improve the fertility of the herd. This sheep breed uses milk as its main product and is a dairy and meat breed. The East Friesian sheep is large in size and usually has white fur. It has good milk production performance and is known as the "Holstein of the sheep world". Therefore, it is widely welcomed by the global breeding industry and is an important tool for the "revitalization of the dairy industry". The introduction of the original East Friesian sheep is extremely expensive, so it is particularly necessary to use existing individuals through modern breeding technology to quickly expand the breeding.

[0076] Preferably, the sheep are East Fleming sheep.

[0077] The applications and methods involved in the present invention are all aimed at non-disease treatment diagnosis and detection.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] The present invention uses a specific combination of four proteins as markers, and predicts the number of embryos produced by the sheep after superovulation and fertilization by detecting the concentration of the markers in the peripheral blood of the sheep before superovulation. Compared with the existing technology, the present invention is more convenient, efficient and accurate, and has broad application value in improving the reproductive efficiency of sheep and the preservation and expansion of precious sheep breeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 The results of the correlation analysis between the ALDOB concentration and the total number of embryos after natural estrus superovulation in Example 2.

[0081] Figure 2 The results of the correlation analysis between the B2M concentration and the total number of embryos after natural estrus superovulation in Example 2.

[0082] Figure 3 The results of the correlation analysis between the ADAMTS-13 concentration and the total number of embryos after natural estrus superovulation in Example 2.

[0083] Figure 4 The results of the correlation analysis between the ACAA1 concentration and the total number of embryos after natural estrus superovulation in Example 2.

[0084] Figure 5 The results of the correlation analysis between the GSN concentration and the total number of embryos after synchronous estrus superovulation in Example 2.

[0085] Figure 6 The results of the correlation analysis between the APO-F concentration and the total number of embryos after synchronous estrus superovulation in Example 2.

[0086] Figure 7 The results of the correlation analysis between the B2M concentration and the total number of embryos after synchronous estrus superovulation in Example 2.

[0087] Figure 8 These are the results of correlation analysis between the B2M concentration in the donor ewe population of Example 3 and the total number of embryos after natural estrus superovulation.

[0088] Fig. 9 The results of the correlation analysis between the ACAA1 concentration in the donor ewe group of Example 3 and the total number of embryos after natural estrus superovulation.

[0089] Fig.10 The results of the correlation analysis between the ADAMTS-13 concentration in the donor ewe population of Example 3 and the total number of embryos after natural estrus superovulation.

[0090] Fig.11 The results of the correlation analysis between the B2M concentration in the donor ewe group of Example 3 and the total number of embryos after synchronous estrus superovulation.

[0091] Fig.12 The results of the correlation analysis between the APO-F concentration in the donor ewe group of Example 3 and the total number of embryos after synchronized estrus superovulation. DETAILED DESCRIPTION

[0092] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0093] The Dongfuli sheep used in the embodiments of the present invention are all from Inner Mongolia Fengdong Zhiying Animal Husbandry Technology Co., Ltd. The selected sheep are all normally developed, in good condition, healthy and disease-free.

[0094] Example 1 Superovulation and artificial insemination of sheep

[0095] 1. Feeding and management of donor ewes

[0096] Multiparous East Fleming ewes aged 1 to 2 years and weighing 67.5±2.5 kg were used as donor ewes.

[0097] Based on the nutritional requirements of the donor ewes, they were uniformly managed in the barn and fed a conventional mixed diet with a crude to concentrate ratio of 70:30. The specific formula was as follows: in terms of mass percentage, 13.60% alfalfa hay, 27.30% sheep grass, 4.60% corn grass, 15.20% whole corn silage, 15.20% corn kernels and 24.10% ewe concentrate.

[0098] 2. Superovulation of donor ewes

[0099] Table 1 Superovulation scheme of donor ewes

[0100]

[0101] Note: "Early" means 6:00-7:00 in the morning; "Late" means 6:00-7:00 in the evening; Due to the different estrus cycles of sheep in synchronized estrus and natural estrus, different superovulation schemes are used; "Estrus breeding" is the direct mating of the breeding ram and the donor ewe.

[0102] In this embodiment, the donor ewe is superovulated by combining estrus synchronization and natural estrus with hormone injection. The process arrangement is shown in Table 1. The specific operation steps are as follows:

[0103] (1) Synchronous estrus

[0104] A progesterone sustained-release vaginal suppository (CIDR, 300 mg / goat) was inserted into the vagina of the donor ewe in the anestrus period, and the day was recorded as the 0th day of synchronous estrus. The donor ewe was injected with follicle-stimulating hormone (FSH) for 6 times in a row, with an interval of 12 hours each time. The injection doses of FSH each time were 53.4 μg / goat, 48 μg / goat, 43.2 μg / goat, 38.9 μg / goat, 35 μg / goat and 31.5 μg / goat, respectively. When the first injection of FSH was given, 250 IU of pregnant mare serum gonadotropin (PMSG) was injected simultaneously. When the fifth injection of FSH was given, cloprostenol (PG) was injected simultaneously, with an injection dose of 150 μg / goat. After the last injection was completed, the progesterone vaginal suppository was removed.

[0105] (2) Natural estrus

[0106] The donor ewes were treated with estrus test rams, and the donor ewes showing estrus on the test day were selected as natural estrus donor ewes, and the day of selection was recorded as the 0th day of natural estrus; starting from the 13th day of natural estrus, the donor ewes were continuously injected with follicle-stimulating hormone for a total of 6 times, with an interval of 12 hours each time, and the injection dose of follicle-stimulating hormone each time was 53.4μg / goat, 48μg / goat, 43.2μg / goat, 38.9μg / goat, 35μg / goat and 31.5μg / goat respectively; when the 5th injection of follicle hormone was given, cloprostenol (PG) was injected simultaneously, and the injection dose was 150μg / goat.

[0107] 3. Artificial insemination of donor ewes

[0108] Healthy East Frisian rams around 2 years old were used as breeding rams, and semen was collected for artificial insemination.

[0109] As shown in Table 1, the estrus status of the donor ewes was detected on the 13th day of synchronous estrus and the 16th day of natural estrus, and the donor ewes in estrus were selected for artificial insemination, and the semen of the breeding ram was injected into the cervix of the donor ewes; the artificial insemination operation was repeated after an interval of 12 hours.

[0110] 4. Embryo Collection

[0111] As shown in Table 1, during artificial insemination on the morning of the 13th day of synchronized estrus and the 16th day of natural estrus, 100 IU of luteinizing hormone (LH) was injected into each donor ewe; on the 19th day of synchronized estrus and the 22nd day of natural estrus, the donor ewes were anesthetized with lidocaine hydrochloride, and the uterus was surgically flushed to collect embryos.

[0112] The results of the number of embryos collected are shown in Table 2. A total of 1190 embryos were collected from 64 donor ewes treated with natural estrus, with an average of 18.6±12.4 embryos per donor ewe, a maximum of 38 embryos recovered from a single individual, and a minimum of 0 embryos; a total of 2044 embryos were collected from 142 donor ewes treated with synchronized estrus, with an average of 14.4±8.7 embryos per donor ewe, a maximum of 45 embryos recovered from a single individual, and a minimum of 0 embryos. The overall effect of superovulation of donor ewes by synchronized estrus and natural estrus combined with hormone injection was good, but there were significant differences between individuals.

[0113] Table 2 Statistical results of superovulation embryo numbers

[0114]

[0115]

[0116] Example 2 Determination and analysis of protein in peripheral blood of donor ewes

[0117] 1. Serum collection

[0118] For the 64 donor ewes treated in the natural estrus mode and the 142 donor ewes treated in the synchronized estrus mode in Example 1, before the first injection of exogenous follicle-stimulating hormone stimulation, serum was collected from all the donor ewes for later use.

[0119] 2. Protein concentration detection

[0120] The inventors have previously obtained six differentially expressed proteins in different total embryo number groups through proteomic analysis: fructose bisphosphate aldolase B (ALDOB), gelsolin (GSN), apolipoprotein F (APO-F), β2-microglobulin (B2M), von Willebrand factor lyase (ADAMTS-13) and acetyl-CoA acyltransferase 1 (ACAA1). Among them, four proteins (ALDOB, B2M, ADAMTS-13 and ACAA1) may be correlated with the number of embryos produced by natural estrus superovulation, and three proteins (GSN, APO-F and B2M) may be correlated with the number of embryos produced by synchronized estrus superovulation.

[0121] According to the number of embryos finally counted in Example 1, 20 donor ewes with different embryo numbers (1 to 38) and 20 donor ewes with different embryo numbers (3 to 36) were selected for natural estrus. The concentrations of the above six proteins in serum were detected using ELISA kits (Enzyme Immunity Industrial Co., Ltd., Jiangsu, China), and the ELISA plates were read with microplate reader (Thermo Scientific, Shanghai, China) to record the absorbance. Each standard curve was drawn to calculate the concentration of each protein.

[0122] 3. Correlation analysis between peripheral blood protein concentration and total number of embryos recovered from superovulation

[0123] like Figure 1 As shown, the average concentration of ALDOB in the peripheral blood of donor ewes before the first exogenous hormone treatment in natural estrus was 19.98 ng / mL, ranging from 10.53 ng / ml to 28.53 ng / ml. Linear regression analysis showed that there was no correlation between the concentration of ALDOB in the peripheral blood of donor ewes before the first exogenous hormone treatment and the total number of embryos finally recovered (regression equation: Y=0.5064*X+5.732, R=0.086, P>0.05).

[0124] like Figure 2 As shown, the average concentration of B2M in the peripheral blood of donor ewes before the first exogenous hormone treatment in natural estrus was 30.65 μg / ml, ranging from 21.46 μg / mL to 46.72 μg / mL. Linear regression analysis showed that the concentration of B2M in the peripheral blood of donor ewes before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered (regression equation: Y=0.8350*X-9.739, R=0.506, P<0.05).

[0125] like Figure 3As shown, the average concentration of ADAMTS-13 in the peripheral blood of donor ewes before the first exogenous hormone treatment in natural estrus was 695.22 pg / ml, ranging from 488.18 pg / ml to 956.23 pg / mL. Linear regression analysis showed that the concentration of ADAMTS-13 in the peripheral blood of donor ewes before the first exogenous hormone treatment was significantly negatively correlated with the total number of embryos finally recovered (regression equation: Y=-0.05707*X+55.43, R=0.685, P<0.05).

[0126] like Figure 4 As shown, the average concentration of ACAA1 in the peripheral blood of donor ewes before the first exogenous hormone treatment in natural estrus was 7.96 ng / ml, ranging from 5.24 ng / ml to 12.98 ng / mL. Linear regression analysis showed that the concentration of ACAA1 in the peripheral blood of donor ewes before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered (regression equation: Y=3.397*X-11.18, R=0.701, P<0.05).

[0127] like Figure 5 As shown, the average concentration of GSN in the peripheral blood of donor ewes before the first exogenous hormone treatment in synchronized estrus was 2.04 ng / mL, ranging from 1.50 ng / mL to 3.00 ng / mL. Linear regression analysis showed that there was no correlation between the concentration of GSN in the peripheral blood of donor ewes before the first exogenous hormone treatment and the total number of embryos finally recovered (regression equation: Y=-8.105*X+31.82, R=0.083, P>0.05).

[0128] like Figure 6 As shown, the average concentration of APO-F in the peripheral blood of donor ewes before the first exogenous hormone treatment in synchronized estrus was 4.87 μg / mL, ranging from 1.34 μg / ml to 8.00 μg / mL. Linear regression analysis showed that the concentration of APO-F in the peripheral blood of donor ewes before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered (regression equation: Y=4.327*X-5.834, R=0.682, P<0.05).

[0129] like Figure 7 As shown, the average concentration of B2M in the peripheral blood of donor ewes before the first exogenous hormone treatment in synchronized estrus was 30.59 μg / ml, ranging from 18.89 μg / mL to 41.25 μg / mL. Linear regression analysis showed that the concentration of B2M in the peripheral blood of donor ewes before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered (regression equation: Y=1.065*X-17.31, R=0.565, P<0.05).

[0130] In summary, B2M and ACAA1 in the peripheral blood of naturally estrus donor ewes before the first exogenous hormone treatment were significantly positively correlated with the total number of embryos finally recovered, ADAMTS-13 was significantly negatively correlated with the total number of embryos finally recovered, and ALDOB was not correlated with the total number of embryos finally recovered; APO-F and B2M in the peripheral blood of estrus donor ewes at the same time were significantly positively correlated with the total number of embryos finally recovered, and GSN was not correlated with the total number of embryos finally recovered. Therefore, APO-F, B2M, ACAA1 and ADAMTS-13 in the peripheral blood of donor ewes before the first exogenous hormone treatment can be used as protein markers to predict the total number of embryos recovered by superovulation of donor ewes.

[0131] Example 3 Evaluation of Population Prediction Performance of Peripheral Blood Protein Markers

[0132] 1. Experimental methods

[0133] (1) Feeding and grouping of donor ewes

[0134] 120 multiparous East Fleming ewes aged 1 to 2 years and weighing 67.5±2.5 kg were randomly selected as donor ewe groups, and randomly divided into two groups, one group was subjected to synchronized estrus, and the other group was subjected to natural estrus. Feeding management, superovulation, artificial insemination and embryo collection were performed on each group according to the method of Example 1, and the number of embryos recovered from each group of donor ewes was counted.

[0135] (2) Serum collection

[0136] Before the first injection of exogenous follicle-stimulating hormone stimulation, serum was collected from all donor ewes for later use.

[0137] (3) Protein concentration detection

[0138] APO-F, B2M, ADAMTS-13 and ACAA1 obtained in Example 2 were used as protein markers, and ELISA kits (Enzyme Immunity Industrial Co., Ltd., Jiangsu, China) were used to detect the concentrations of the above four proteins in the serum of each donor ewe, and the correlation between the concentration of each protein and the number of embryos was analyzed.

[0139] 2. Experimental results

[0140] (1) In the group of naturally estrus donor ewes

[0141] like Figure 8As shown, the concentration of B2M in the peripheral blood of donor sheep before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered by linear regression analysis (Y=1.387*X1-26.79, Y represents the number of embryos, X1 represents the concentration of B2M in the serum of naturally estrus sheep, R=0.7053, P<0.05). When the concentration of B2M was between 22.96 and 27.53 μg / ml, the prediction accuracy of B2M was 76.5% (13 / 17); when the concentration of B2M was between 27.53 and 32.62 μg / ml, the prediction accuracy of B2M was 81.3% (13 / 16); when the concentration of B2M was between 32.62 and 40.75 μg / ml, the prediction accuracy of B2M was 61.1% (11 / 18).

[0142] like Fig. 9 As shown, the concentration of ACAA1 in the peripheral blood of donor sheep before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered by linear regression analysis (Y=4.325*X2-18.34, Y represents the number of embryos, X2 represents the concentration of ACAA1 in the serum of naturally estrus sheep, R=0.8201, P<0.05). When the concentration of ACAA1 was between 5.04 and 6.75 ng / ml, the prediction accuracy of ACAA1 was 85.7% (18 / 21); when the concentration of ACAA1 was between 6.75 and 8.65 ng / ml, the prediction accuracy of ACAA1 was 100% (20 / 20); when the concentration of ACAA1 was between 8.65 and 11.68 ng / ml, the prediction accuracy of ACAA1 was 78.6% (11 / 14).

[0143] like Fig.10 As shown, linear regression analysis showed that the concentration of ADAMTS-13 in the peripheral blood of donor sheep before the first exogenous hormone treatment was significantly negatively correlated with the total number of embryos finally recovered (Y = -0.07380*X3+66.59, Y represents the number of embryos, X3 represents the concentration of ADAMTS-13 in the serum of naturally estrus sheep, R = 0.7064, P < 0.05). When the ADAMTS-13 concentration was between 747.87 and 834.03 pg / ml, the prediction accuracy of ADAMTS-13 was 86.7% (13 / 15); when the ADAMTS-13 concentration was between 652.14 and 747.87 pg / ml, the prediction accuracy of ACAA1 was 95% (19 / 20); when the ADAMTS-13 concentration was between 498.97 and 652.14 pg / ml, the prediction accuracy of ACAA1 was 68.8% (11 / 16).

[0144] (2) Synchronous estrus donor ewes

[0145] like Fig.11 As shown, the concentration of B2M in the peripheral blood of donor sheep before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered by linear regression analysis (Y=1.347*X4-26.10, Y represents the number of embryos, X4 represents the concentration of B2M in the serum of synchronous estrus sheep, R=0.6798, P<0.05). When the concentration of B2M was between 23.57 and 28.12 μg / ml, the prediction accuracy of B2M was 82.4% (14 / 17); when the concentration of B2M was between 28.12 and 33.16 μg / ml, the prediction accuracy of B2M was 92.9% (13 / 14); when the concentration of B2M was between 33.16 and 42.24 μg / ml, the prediction accuracy of B2M was 87.0% (20 / 23).

[0146] like Fig.12 As shown, the concentration of APO-F in the peripheral blood of donor sheep before the first exogenous hormone treatment was significantly positively correlated with the total number of embryos finally recovered by linear regression analysis (Y=5.147*X5-10.10, Y represents the number of embryos, X5 represents the concentration of APO-F in the serum of synchronous estrus sheep, R=0.8045, P<0.05). When the concentration of APO-F was between 2.71 and 4.12 μg / ml, the prediction accuracy of APO-F was 93.3% (14 / 15); when the concentration of APO-F was between 4.12 and 5.68 μg / ml, the prediction accuracy of APO-F was 100% (15 / 15); when the concentration of APO-F was between 5.68 and 8.49 μg / ml, the prediction accuracy of APO-F was 83.3% (20 / 24).

[0147] In summary, APO-F, B2M, ADAMTS-13 and ACAA1 can be used as protein markers to predict the number of embryos produced by superovulation in sheep, with a high prediction accuracy in large groups, meeting practical needs.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The use of a protein composition in predicting the total number of embryos produced by superovulation and insemination in sheep, It is characterized in that The protein composition consists of B2M, ACAA1, ADAMTS-13 and APO-F; Before superovulation, the sheep is in estrus; the estrus is natural estrus or synchronized estrus; For the naturally estrus sheep, the concentrations of B2M and ACAA1 in their peripheral blood are positively correlated with the total number of embryos, and the concentration of ADAMTS-13 in their peripheral blood is negatively correlated with the total number of embryos; For the synchronized estrus sheep, the concentrations of the B2M and APO-F in their peripheral blood were positively correlated with the total embryo number.

2. A method for predicting the number of embryos produced by superovulation and insemination in sheep, It is characterized in that The following steps are involved: Before superovulation, detecting the concentration of the protein composition of claim 1 in the peripheral blood of the sheep, and determining the number of embryos produced by the sheep through superovulation and fertilization according to the detection result; Before superovulation, the sheep is in estrus; the estrus is natural estrus or synchronized estrus; For the naturally estrus sheep, the concentrations of B2M and ACAA1 in their peripheral blood are positively correlated with the number of embryos, and the concentration of ADAMTS-13 in their peripheral blood is negatively correlated with the number of embryos; For the synchronized estrus sheep, the concentrations of the B2M and APO-F in their peripheral blood are positively correlated with the number of embryos.

3. The method according to claim 2, It is characterized in that Before superovulation, the sheep is a sheep in natural estrus, and the criteria for determining the number of embryos are: The concentration of B2M in the peripheral blood of the sheep in natural estrus is 22-27.53 μg / mL, the concentration of ACAA1 in the peripheral blood of the sheep in natural estrus is 5-6.75 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the sheep in natural estrus is 747.87-835 pg / mL, and the number of embryos is 1-10; The concentration of B2M in the peripheral blood of the naturally estrus sheep is 27.53-32.62 μg / mL, the concentration of ACAA1 in the peripheral blood of the naturally estrus sheep is 6.75-8.65 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the naturally estrus sheep is 652.14-747.87 pg / mL, then the number of embryos is 10-20; If the concentration of B2M in the peripheral blood of the sheep in natural estrus is 32.62-43 μg / mL, the concentration of ACAA1 in the peripheral blood of the sheep in natural estrus is 8.65-12 ng / mL, or the concentration of ADAMTS-13 in the peripheral blood of the sheep in natural estrus is 495-652.14 pg / mL, the number of embryos is 20-36.

4. The method according to claim 3, It is characterized in that Calculate the number of embryos according to any one of Formulas 1 to 3; Formula 1: Y=1.387*X1-26.79; Formula 2: Y=4.325*X2-18.34; Formula 3: Y=-0.07380*X3+66.59; Wherein, X1 to X3 represent the concentrations of the B2M, ACAA1 and ADAMTS-13 in the peripheral blood of the naturally estrus sheep, respectively, and Y represents the number of embryos.

5. The method according to claim 2, It is characterized in that Before superovulation, the sheep are in estrus at the same time, and the criteria for determining the number of embryos are: The concentration of B2M in the peripheral blood of the synchronized estrus sheep is 22-28.12 μg / mL or the concentration of APO-F in the peripheral blood of the synchronized estrus sheep is 2-4.12 μg / ml, and the number of embryos is 1-10; The concentration of B2M in the peripheral blood of the synchronized estrus sheep is 28.12-33.16 μg / mL or the concentration of APO-F in the peripheral blood of the synchronized estrus sheep is 4.12-5.68 μg / ml, and the number of embryos is 10-20; The concentration of B2M in the peripheral blood of the synchronized estrus sheep is 33.16-43 μg / mL or the concentration of APO-F in the peripheral blood of the synchronized estrus sheep is 5.68-9 μg / ml, and the number of embryos is 20-38.

6. The method according to claim 5, It is characterized in that Calculate the number of embryos using any one of Formulas 4 to 5; Formula 4: Y=1.347*X4-26.10; Formula 5: Y=5.147*X5-10.10; Wherein, X4 and X5 represent the concentrations of the B2M and the APO-F in the peripheral blood of the synchronously estrus sheep respectively, and Y represents the number of embryos.

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