Method for screening ewes with high egg laying donors and application of method

Abstract

The invention discloses a method for screening ewes with high egg laying donors and application of the method. According to the method, a standardized method for screening the ewes with high egg laying donors is established, and the problems that the number of ovulation of the ewes with the donors is small and the difference is large due to subjective judgment only in a conventional method are solved. According to the method, donor ewes can be effectively screened, the embryo production efficiency can be improved, the manpower and material resource investment can be reduced, the breeding cost can be greatly saved, the economic benefit can be further improved, and the method has great significance in promoting industrial application of a livestock embryo production technology. In addition, the method has the advantages of simplicity in operation, high accuracy, high efficiency, convenience in popularization and use and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of livestock breeding, and in particular relates to a method for screening high-egg-producing donor ewes and an application thereof. Background Art

[0002] Embryo engineering refers to modern biotechnology that involves various micromanipulations and manipulations of early animal embryos or gametes (sperm and eggs). It encompasses in vitro fertilization, embryo transfer, embryo splitting, embryo cryopreservation, sex control, and nuclear transfer. Through techniques such as embryo production and transfer, the reproductive potential of high-quality breeding ewes can be fully realized, increasing their annual offspring production by 30-40 times, thereby improving their reproductive efficiency. Furthermore, embryo engineering can shorten the reproductive cycle of ewes, accelerate the breeding process, and increase the efficiency of developing new sheep breeds by 50-70%. Embryo engineering has become an essential supporting technology for my country's efforts to become a leading seed industry powerhouse.

[0003] The selection of oocyte donor animals is a critical step in embryonic engineering technology. Oocytes are the starting point of embryonic development, and their quantity and quality directly affect the embryo's developmental potential and the success rate of embryonic engineering. In actual production, the number of ovulations after hormone induction varies greatly among different donor ewes, and some even fail to ovulate. This increases breeding costs, reduces economic benefits, and wastes human resources. Conventional methods for selecting oocyte donor animals rely on subjective evaluations, mainly including body condition, disease presence, age, parity, breed, pedigree, and other information, and lack standardized procedures.

[0004] Therefore, establishing a more efficient, accurate and standardized method to screen donor animals is an urgent problem to be solved in the field of livestock breeding. Summary of the Invention

[0005] To solve at least some of the technical problems in the above-mentioned prior art, the present invention provides a method for screening high-egg-producing donor ewes and its application. Specifically, the present invention includes the following contents.

[0006] A first aspect of the present invention provides a method for screening high-egg-producing donor ewes, comprising the following steps:

[0007] (1) Prepare ewe blood samples;

[0008] (2) detecting γ-glutamyl transpeptidase activity, alkaline phosphatase activity, and / or platelet count in the blood sample to obtain respective detection values;

[0009] (3) High egg-producing donor ewes are identified based on the respective detection values.

[0010] In certain embodiments, according to the method for screening high egg-producing donor ewes of the present invention, step (3) includes the step of comparing the respective detection values with their corresponding threshold values.

[0011] In certain embodiments, according to the method for screening high-egg-producing donor ewes of the present invention, when the detection value of γ-glutamyl transpeptidase activity is ≥ its corresponding threshold value, the detection value of alkaline phosphatase activity is ≥ its corresponding threshold value and / or the detection value of platelet count is ≤ its corresponding threshold value, the ewe is determined to be a high-egg-producing donor ewe.

[0012] In certain embodiments, in the method for screening high-egg-producing donor ewes according to the present invention, the threshold value is a value statistically obtained from a sample group of the same age as the donor ewe.

[0013] In certain embodiments, in the method for screening high egg-producing donor ewes according to the present invention, the threshold value is a value statistically obtained from a sample group equivalent to the donor ewe population.

[0014] In certain embodiments, according to the method for selecting high egg-producing donor ewes of the present invention, the ewes include sheep or goats.

[0015] In certain embodiments, the method for screening high-egg-producing donor ewes according to the present invention further comprises the step of separating serum before detecting the activity of γ-glutamyl transpeptidase and alkaline phosphatase.

[0016] In certain embodiments, in the method for screening high-egg-producing donor ewes according to the present invention, the blood sample is obtained by collecting venous blood from the ewe.

[0017] In certain embodiments, according to the method for screening high-egg-producing donor ewes of the present invention, the venous blood includes venous blood collected from the jugular vein, the tail vein, or the udder vein.

[0018] The second aspect of the present invention provides the use of the method according to the present invention in sheep breeding or in vitro embryo production.

[0019] The present invention uses γ-glutamyl transpeptidase activity, alkaline phosphatase activity or platelet count as evaluation indicators to establish a standardized method for confirming and screening high-oviposition donor ewes, avoiding the problem of a small number of ovulations and a large gap in the number of donor ewes ovulated by subjective judgment alone, which is caused by conventional methods. The method of the present invention can effectively screen donor ewes, improve embryo production efficiency, reduce manpower and material resources, greatly save breeding costs, and thus improve economic benefits, which is of great significance to the development of livestock embryo production technology. In addition, the method of the present invention has the advantages of simple operation, high accuracy, high efficiency, and ease of promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shown are the AUC evaluation results for γ-glutamyl transpeptidase activity, alkaline phosphatase activity, or platelet count as criteria for screening donor ewes.

[0021] Figure 2 Shown is the technical roadmap of the present invention. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0025] Method for selecting high egg-producing donor ewes

[0026] One aspect of the present invention provides a method for screening high-egg-producing donor ewes, the method comprising the following steps:

[0027] (1) Prepare ewe blood samples;

[0028] (2) detecting γ-glutamyl transpeptidase activity, alkaline phosphatase activity, and / or platelet count in the blood sample to obtain respective detection values;

[0029] (3) High egg-producing donor ewes are identified based on the respective detection values.

[0030] In a preferred embodiment, step (3) of the present invention includes the step of comparing the respective detection values with their corresponding threshold values, and when the detection value of γ-glutamyl transpeptidase activity is ≥ its corresponding threshold value, the detection value of alkaline phosphatase activity is ≥ its corresponding threshold value and / or the detection value of platelet count is ≤ its corresponding threshold value, the ewe is confirmed to be a high egg-producing donor ewe.

[0031] In the present invention, the threshold value is a value statistically obtained from a sample group of the same age as the donor ewe, or the threshold value is a value statistically obtained from a sample group of the same breed as the donor ewe.

[0032] In a preferred embodiment, the threshold value of the γ-glutamyl transpeptidase activity is 61.85 U / L, the threshold value of the alkaline phosphatase activity is 73.85 U / L, and the threshold value of the platelet count is 280×10 9 / L. When the detection value of γ-glutamyl transpeptidase activity is ≥61.85U / L, the detection value of alkaline phosphatase activity is ≥73.85U / L and / or the detection value of platelet count is ≤280×10 9 / L, the ewe is confirmed to be a high egg-producing donor ewe.

[0033] In the present invention, serum γ-glutamyl transpeptidase activity, serum alkaline phosphatase activity and blood platelet count can be used as separate evaluation indicators, or a combination of any two or three thereof can be used to screen high-egg-producing donor ewes, without particular limitation.

[0034] In a specific embodiment, the method for selecting high egg-producing donor ewes comprises the following steps:

[0035] (1) Collecting ewe blood samples, allowing them to stand and centrifuging them to obtain serum;

[0036] (2) detecting serum γ-glutamyl transpeptidase activity and obtaining a test value;

[0037] (3) When the detected value of the γ-glutamyl transpeptidase activity is ≥61.85 U / L, the ewe is confirmed to be a high-egg-producing donor ewe.

[0038] In another specific embodiment, the method for screening high egg-producing donor ewes comprises the following steps:

[0039] (1) Collecting ewe blood samples, allowing them to stand and centrifuging them to obtain serum;

[0040] (2) detecting serum alkaline phosphatase activity and obtaining a test value;

[0041] (3) When the detection value of the alkaline phosphatase activity is ≥73.85 U / L, the ewe is confirmed to be a high-egg-producing donor ewe.

[0042] In another specific embodiment, the method for selecting high egg-producing donor ewes comprises the following steps:

[0043] (1) Collect blood samples from ewes;

[0044] (2) detecting the number of platelets in the blood sample to obtain a detection value;

[0045] (3) When the platelet count is ≤280×10 9 / L, the ewe is confirmed to be a high egg-producing donor ewe.

[0046] In another specific embodiment, the method for selecting high egg-producing donor ewes comprises the following steps:

[0047] (1) Collecting ewe blood samples, allowing them to stand and centrifuging them to obtain serum;

[0048] (2) detecting serum γ-glutamyl transpeptidase activity and serum alkaline phosphatase activity to obtain test values;

[0049] (3) When the detection value of the γ-glutamyl transpeptidase activity is ≥61.85 U / L and the detection value of the alkaline phosphatase activity is ≥73.85 U / L, the ewe is confirmed to be a high-egg-producing donor ewe.

[0050] In another specific embodiment, the method for selecting high egg-producing donor ewes comprises the following steps:

[0051] (1) Collecting ewe blood samples, allowing them to stand and centrifuging them to obtain serum;

[0052] (2) detecting serum γ-glutamyl transpeptidase activity and blood platelet count in the blood sample to obtain test values;

[0053] (3) When the detection value of the γ-glutamyl transpeptidase activity is ≥61.85 U / L, and the detection value of the platelet count is ≤280×10 9 / L, the ewe is confirmed to be a high egg-producing donor ewe.

[0054] In another specific embodiment, the method for selecting high egg-producing donor ewes comprises the following steps:

[0055] (1) Collecting ewe blood samples, allowing them to stand and centrifuging them to obtain serum;

[0056] (2) detecting serum alkaline phosphatase activity and blood platelet count in the blood sample to obtain test values;

[0057] (3) When the detection value of alkaline phosphatase activity is ≥73.85 U / L and the detection value of platelet count is ≤280×10 9 / L, the ewe is confirmed to be a high egg-producing donor ewe.

[0058] In the present invention, the breed of the ewe is not particularly limited, and examples thereof include but are not limited to sheep, goats, and the like.

[0059] In the present invention, the blood sample is obtained by collecting venous blood from ewes, wherein the source of venous blood is not particularly limited, and examples thereof include but are not limited to venous blood collected from the jugular vein, the tail vein, or the mammary vein.

[0060] In the above steps involving standing and centrifugation, the standing time is 20-70 min, preferably 25-65 min, and more preferably 30-60 min, for example, 30, 35, 40, 45, 50, 55, or 60 min. The centrifuge speed is 2000-5000 r / min, preferably 2200-4800 r / min, and more preferably 2400-4600 r / min, more preferably 2600-4400 r / min, more preferably 2800-4200 r / min, and more preferably 3000-4000 r / min, for example, 3000, 3200, 3400, 3600, 3800, or 4000 r / min.

[0061] In the present invention, the method for detecting the activities of γ-glutamyl transpeptidase and alkaline phosphatase is not particularly limited and can be performed using methods known in the art, examples of which include but are not limited to colorimetry, fluorescent probe method, enzyme-linked immunosorbent assay, etc. The method for detecting platelet count is not particularly limited and can be performed using methods known in the art, examples of which include but are not limited to microscopic counting, flow cytometry, impedance spectroscopy, light scattering method, etc.

[0062] application

[0063] One aspect of the present invention provides the use of the method according to the present invention in sheep breeding or embryo production.

[0064] The present invention selects high-quality donor ewes through blood indicators for subsequent breeding or embryo production, which can quickly increase the number of high-quality breeding sheep, greatly improve the production efficiency of high-quality embryos, and thus enhance breeding benefits.

[0065] Example

[0066] The following shows the process of establishing a method for selecting high-producing egg donor ewes.

[0067] 1. Experimental Materials

[0068] Unless otherwise specified, the methods used in this example are conventional methods known to those skilled in the art. All reagents, consumables, and other materials used are commercially available products unless otherwise specified.

[0069] 2. Experimental Methods

[0070] 2.1 Selection of breeding ewes

[0071] 400 individuals with clear pedigree, good health, good body condition, normal sexual cycle and no reproductive disorders were selected from the large breeding ewe herd. They were 2-4 years old and weighed about 45-55kg.

[0072] 2.2 Sheep jugular vein blood sample collection

[0073] Grasp the sheep's horns or ears with both hands, tilting the head upward and toward the side opposite the blood collection site. Clip and disinfect the blood collection site. Use your left thumb to press the lower middle portion of the jugular groove, attempting to distend the jugular vein. Use your right thumb or index finger to palpate the vein in the groove. Quickly penetrate the skin and blood vessels at a 30-45° angle to the skin at the jugular groove insertion point. If blood is seen returning, advance the needle parallel to the skin 1-2 cm. After collecting 10 mL of blood, disinfect with 5% iodine tincture.

[0074] 2.3 Routine blood test

[0075] A fully automatic blood cell analyzer was used to measure routine blood parameters, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet count (PLT).

[0076] 2.4 Serum sample preparation and collection

[0077] 5 mL of jugular vein blood sample from a donor ewe was placed at room temperature for 30-60 minutes, centrifuged at 3500 r / min for 10 minutes to separate the serum, dispensed into 1.5 mL centrifuge tubes, and stored frozen at -20°C.

[0078] 2.5 Serum biochemical index detection

[0079] An automatic biochemical analyzer was used to measure serum biochemical parameters, including γ-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), globulin (GLB), creatinine (Cr), urea (Urea), potassium ion (K+), serum calcium (sCa), serum phosphorus (sP), serum magnesium (sMg), triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), glucose (Glu), β-hydroxybutyrate (β-HB), creatine kinase (CK), and lactate dehydrogenase (LDH).

[0080] 2.6 Serum antioxidant index detection

[0081] Kits produced by Nanjing Jiancheng Bioengineering Institute were used to detect serum antioxidant-related indicators, including serum total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) content, and total superoxide dismutase (T-SOD) activity.

[0082] 2.7 Serum hormone index detection

[0083] ELISA kits were used to detect serum hormone levels, including follicle-stimulating hormone (FSH), estrogen (E2), and progesterone (P4), and the OD value of each serum sample was measured at a wavelength of 450 nm using a microplate reader.

[0084] 2.8 Follicle-stimulating hormone induction in donor ewes

[0085] On any day of the estrous cycle, progesterone suppositories were implanted into the vagina for 10 days. On the 10th day, hormone induction was initiated with the following procedure: 1 injection of FSH per day for 2 consecutive days, 240 IU per injection.

[0086] 2.9 Live egg collection from donor ewes

[0087] After the second FSH injection, the ewes were deprived of food and water. Forty-eight hours later, the donor ewes were anesthetized and secured in a restraining frame for laparoscopic oocyte retrieval. Following oocyte retrieval, the progesterone suppository was removed, and the total number of eggs retrieved from each donor ewe was recorded.

[0088] According to the total number of eggs recovered, the donor ewes were divided into a high egg recovery group (total number of eggs>15) and a low egg recovery group (total number of eggs<10).

[0089] 2.10 Statistics

[0090] The experimental data were recorded using Excel and statistical software was used for difference analysis.

[0091] 3. Test results

[0092] 3.1 Analysis of blood routine indicators of donor ewes in high and low egg retrieval groups

[0093] As shown in Table 1, the platelet count of the donor ewes in the high egg retrieval group was 182.53±8.16, which was significantly lower than the platelet count of the donor ewes in the low egg retrieval group (363.11±11.47) (P<0.01). There were no differences in other indicators between the two groups (P>0.05).

[0094] Table 1 Routine blood parameters of donor ewes in high and low egg retrieval groups

[0095]

[0096] Note: ** indicates extremely significant difference (P<0.01).

[0097] 3.2 Analysis of serum antioxidant indexes in donor ewes of high and low oocyte retrieval groups

[0098] As shown in Table 2, there was no difference in various antioxidant indicators in the serum of donor ewes between the high and low egg retrieval groups (P>0.05).

[0099] Table 2 Antioxidant indexes in serum of donor ewes in high and low egg retrieval groups

[0100]

[0101] 3.3 Analysis of serum biochemical parameters in donor ewes of high and low egg retrieval groups

[0102] As shown in Table 3, the serum γ-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) activities of donor ewes in the high-egg retrieval group were 74.78±2.15 and 95.23±2.06, respectively, significantly higher than those in the low-egg retrieval group (52.14±1.38, 51.46±1.67) (P<0.01). No significant differences were found in other serum biochemical parameters between the two groups.

[0103] Table 3 Serum biochemical parameters of donor ewes in high and low egg retrieval groups

[0104]

[0105] Note: ** indicates extremely significant difference (P<0.01).

[0106] 3.4 Analysis of serum hormone indicators in donor ewes in high and low egg retrieval groups

[0107] As shown in Table 4, there was no difference in the serum hormone levels of donor ewes in the high and low egg retrieval groups (P>0.05).

[0108] Table 4 Serum hormone levels of donor ewes in high and low egg retrieval groups

[0109]

[0110] 3.5 Serum γ-glutamyl transpeptidase, alkaline phosphatase activity and blood platelet count as donor ewe screening criteria

[0111] The receiver operating characteristic curve (ROC) was used to evaluate the ability of serum γ-glutamyl transpeptidase, alkaline phosphatase activity and blood platelet count as the distinguishing criteria for donor ewes screening, and then the area under the curve (AUC) of the ROC curve was used to describe the ability of γ-glutamyl transpeptidase, alkaline phosphatase activity and blood platelet count to distinguish donor ewes. When AUC>0.7, it can be shown that the target index has a certain accuracy for the screening criteria results. This embodiment uses 0.8 as a consideration value to improve the accuracy of the standard. Analysis of the ROC curve found that the AUC of serum γ-glutamyl transpeptidase activity reached 0.92, the AUC of serum alkaline phosphatase activity reached 0.94, and the AUC of blood platelet count reached 0.92 ( Figure 1 , Table 5). This suggests that serum γ-glutamyl transpeptidase, alkaline phosphatase activity, and platelet count can be used as high-quality criteria for selecting donor ewes. Specific data are shown in Table 5.

[0112] Table 5. Threshold data of serum γ-glutamyl transpeptidase, alkaline phosphatase activities and platelet count as screening criteria for donor ewes

[0113]

[0114]

[0115] Choosing any threshold involves a trade-off between sensitivity and specificity.

[0116] As shown in Table 5, the sensitivity of γ-glutamyl transpeptidase activity was 75.78%, the specificity was 93.75%, and the Youden index was 0.701; the sensitivity of alkaline phosphatase activity was 96.27%, the specificity was 86.25%, and the Youden index was 0.813; the sensitivity of platelet count was 95.65%, the specificity was 82.5%, and the Youden index was 0.824. In other words, the serum γ-glutamyl transpeptidase activity of the donor ewe was greater than 61.85 U / L, the serum alkaline phosphatase activity was greater than 73.85 U / L, or the platelet count was less than 280×10 9 / L, the donor ewe can be screened out for production use.

[0117] 3.6 Validation of serum γ-glutamyl transpeptidase, alkaline phosphatase activity, and platelet count as screening criteria

[0118] In order to detect the accuracy of serum γ-glutamyl transpeptidase, alkaline phosphatase activity and blood platelet count as screening criteria, 100 sheep were selected for prediction verification. The verification results are shown in Table 6. The accuracy of serum γ-glutamyl transpeptidase, alkaline phosphatase activity and blood platelet count as screening criteria was 91.01%, 96.47% and 93.41%, respectively. The above results show that the indicators proposed in this example are very accurate in screening donor ewes.

[0119] Table 6 Validation of serum γ-glutamyl transpeptidase, alkaline phosphatase activities and blood platelet count as screening criteria

[0120]

[0121] In summary, the present invention has obtained a method for screening donor ewes through analysis of a large amount of experimental data, that is, detecting the serum γ-glutamyl transpeptidase activity, serum alkaline phosphatase activity or blood platelet count of the donor ewes, and screening high-quality donor ewes according to the standard values.

[0122] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for screening high-egg-producing donor ewes, characterized in that: The method comprises the following steps: (1) Prepare ewe blood samples; (2) detecting γ-glutamyl transpeptidase activity, alkaline phosphatase activity, and / or platelet count in the blood sample to obtain respective detection values; (3) High egg-producing donor ewes are identified based on the respective detection values.

2. The method for selecting high egg-producing donor ewes according to claim 1, characterized in that: Step (3) includes the step of comparing the respective detection values with their corresponding threshold values.

3. The method for selecting high egg-producing donor ewes according to claim 1, characterized in that: When the detection value of γ-glutamyl transpeptidase activity is ≥ its corresponding threshold value, the detection value of alkaline phosphatase activity is ≥ its corresponding threshold value and / or the detection value of platelet count is ≤ its corresponding threshold value, the ewe is confirmed to be a high egg production donor ewe.

4. The method for selecting high egg-producing donor ewes according to claim 2, characterized in that: The threshold value is a value statistically obtained from a sample group of the same age as the donor ewe.

5. The method for selecting high egg-producing donor ewes according to claim 2, characterized in that: The threshold values are statistically obtained from a sample group comparable to the donor ewe population.

6. The method for selecting high egg-producing donor ewes according to claim 1, characterized in that: The ewe includes sheep or goats.

7. The method for selecting high egg-producing donor ewes according to claim 1, characterized in that: The method further comprises the step of separating serum before detecting the γ-glutamyl transpeptidase activity or alkaline phosphatase activity.

8. The method for selecting high egg-producing donor ewes according to claim 1, characterized in that: The blood samples were obtained by collecting venous blood from ewes.

9. The method for selecting high egg-producing donor ewes according to claim 8, characterized in that: The venous blood includes venous blood collected from the jugular vein, the tail vein or the mammary vein.

10. Use of the method according to any one of claims 1 to 9 in sheep breeding or embryo production.

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