Screening method and application of ewes with high egg laying donors

Abstract

The invention discloses a screening method and application of ewes with high egg laying donors. 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

[0001] This application is a divisional application of application number 202510584403.3 filed on May 7, 2025, entitled "A method for screening high-producing ewe donors and its application", which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention belongs to the field of livestock breeding technology, and in particular relates to a method for screening high-producing egg donor ewes and its application. Background Technology

[0003] Embryo engineering technology refers to modern biotechnology that involves various micromanipulations and treatments of early animal embryos or gametes (sperm and eggs). It includes in vitro fertilization, embryo transfer, embryo splitting, embryo cryopreservation, sex control, and nuclear transfer. Through embryo production and transfer techniques, the reproductive potential of superior breeding ewes can be fully realized, increasing their annual offspring production by 30-40 times, thereby improving ewe reproductive efficiency. Furthermore, embryo engineering technology can shorten the ewe reproductive cycle, accelerate the breeding process, and increase the efficiency of developing new sheep breeds by 50%-70%.

[0004] The selection of oocyte donor animals is a crucial step in embryo engineering, as oocytes are the starting point of embryonic development, and their quantity and quality directly affect the developmental potential of the embryo and the success rate of embryo engineering. In actual production, the number of ovulations varies significantly among different donor ewes after hormone induction, with some even failing to ovulate. This leads to increased breeding costs, reduced economic benefits, and a waste of human resources. Conventional methods for selecting oocyte donor animals rely on subjective evaluation, with evaluation indicators mainly including body condition, disease presence, age, parity, breed, and pedigree, lacking standardized procedures.

[0005] Therefore, establishing more efficient, accurate, and standardized methods for screening donor animals is an urgent problem to be solved in the field of livestock breeding. Summary of the Invention

[0006] To address at least some of the technical problems in the prior art, the present invention provides a method for screening high-producing donor ewes and its application. Specifically, the present invention includes the following:

[0007] A first aspect of the present invention provides a method for screening high-producing donor ewes, comprising the following steps: (1) Prepare blood samples from ewes; (2) Detect each serum biochemical index in the blood sample and obtain their respective detection values, wherein the serological index is selected from at least one of γ-glutamyl transpeptidase activity, alkaline phosphatase activity and platelet count; (3) Based on the respective detection values, identify high-producing egg donor ewes.

[0008] In some embodiments, the method for screening high-producing donor ewes according to the present invention includes step (3) of comparing the respective detection values ​​with their corresponding thresholds.

[0009] In some embodiments, according to the screening method for high-producing donor ewes of the present invention, a ewe is identified as a high-producing donor ewe when the detected value of γ-glutamyl transferase activity is ≥ its corresponding threshold, the detected value of alkaline phosphatase activity is ≥ its corresponding threshold, and / or the detected value of platelet count is ≤ its corresponding threshold.

[0010] In some embodiments, according to the method for screening high-producing donor ewes of the present invention, the threshold is a value statistically obtained from a sample group of ewes of similar age to the donor ewes.

[0011] In some embodiments, according to the method for screening high-producing donor ewes according to the present invention, the threshold is a value statistically obtained from a sample group equivalent to the donor ewe population.

[0012] In some embodiments, the method for screening high-producing donor ewes according to the present invention includes sheep or goats.

[0013] In some embodiments, the method for screening high-producing donor ewes according to the present invention further includes a step of separating serum before detecting γ-glutamyl transpeptidase activity and alkaline phosphatase activity.

[0014] In some embodiments, according to the method for screening high-producing oviparous donor ewes of the present invention, the blood sample is obtained by collecting venous blood from the ewe.

[0015] In some embodiments, according to the method for screening high-producing donor ewes of the present invention, the venous blood includes venous blood obtained from jugular vein collection, tail vein collection, or mammary vein collection.

[0016] A second aspect of the invention provides the application of the method described according to the invention in the propagation of superior sheep breeds or in vitro embryo production.

[0017] This invention utilizes γ-glutamyl transferase activity, alkaline phosphatase activity, or platelet count as evaluation indicators to establish a standardized method for identifying and screening high-ovulation donor ewes. This avoids the problem of conventional methods relying solely on subjective judgment, which can lead to low and inconsistent ovulation rates among donor ewes. The method of this invention can effectively screen donor ewes, improve embryo production efficiency, reduce labor and material inputs, significantly save on breeding costs, and thus improve economic benefits. It is of great significance for advancing the development of livestock embryo production technology. Furthermore, the method of this invention has the advantages of simple operation, high accuracy, high efficiency, and ease of promotion and application. Attached Figure Description

[0018] Figure 1 The AUC assessment results are shown, using γ-glutamyl transpeptidase activity, alkaline phosphatase activity, or platelet count as criteria for selecting donor ewes.

[0019] Figure 2 A technical roadmap of the present invention is shown. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Screening methods for high-producing egg donor ewes One aspect of the present invention provides a method for screening high-producing donor ewes, the method comprising the following steps: (1) Prepare blood samples from ewes; (2) Detect each serum biochemical index in the blood sample and obtain their respective detection values, wherein the serological index is selected from at least one of γ-glutamyl transpeptidase activity, alkaline phosphatase activity and platelet count; (3) Based on the respective detection values, identify high-producing egg donor ewes.

[0024] In a preferred embodiment, step (3) of the present invention includes comparing the respective detection values ​​with their corresponding thresholds. When the detection value of γ-glutamyl transferase activity is ≥ its corresponding threshold, the detection value of alkaline phosphatase activity is ≥ its corresponding threshold, and / or the detection value of platelet count is ≤ its corresponding threshold, the ewe is confirmed as a high-producing egg donor ewe.

[0025] In this invention, the threshold is a value obtained statistically from a sample group with an age equivalent to that of the donor ewe, or the threshold is a value obtained statistically from a sample group with a population equivalent to that of the donor ewe.

[0026] In a preferred embodiment, the threshold values ​​for γ-glutamyl transferase activity are 61.85 U / L, for alkaline phosphatase activity are 73.85 U / L, and for platelet count are 280 × 10⁻⁶. 9 / L. When the detected value of γ-glutamyl transferase activity is ≥61.85 U / L, the detected value of alkaline phosphatase activity is ≥73.85 U / L, and / or the detected value of platelet count is ≤280×10⁻⁶. 9 When / L, the ewe is confirmed to be a high-producing egg donor ewe.

[0027] In this invention, serum gamma-glutamyl transferase activity, serum alkaline phosphatase activity, and blood platelet count can be used as individual evaluation indicators, or any combination of two or three of them can be used to screen high-producing egg donor ewes, without any particular limitation.

[0028] In one specific implementation, the method for selecting high-producing egg-producing donor ewes includes the following steps: (1) Collect blood samples from ewes, let them stand and centrifuge to obtain serum; (2) Detect serum γ-glutamyl transpeptidase activity and obtain the detection value; (3) When the detected value of the γ-glutamyl transpeptidase activity is ≥61.85 U / L, the ewe is confirmed to be a high-producing egg donor ewe.

[0029] In another specific implementation, the method for selecting high-producing egg-producing donor ewes includes the following steps: (1) Collect blood samples from ewes, let them stand and centrifuge to obtain serum; (2) Detect serum alkaline phosphatase activity and obtain the detection value; (3) When the alkaline phosphatase activity is ≥73.85 U / L, the ewe is confirmed as a high-producing egg donor ewe.

[0030] In yet another specific implementation, the method for selecting high-producing egg-producing donor ewes includes the following steps: (1) Collect blood samples from ewes; (2) Detect the platelet count in the blood sample and obtain the detection value; (3) When the platelet count is ≤280×10 9 If / L, then the ewe is confirmed to be a high-producing egg donor ewe.

[0031] In yet another specific implementation, the method for selecting high-producing egg-producing donor ewes includes the following steps: (1) Collect blood samples from ewes, let them stand and centrifuge to obtain serum; (2) Detect serum γ-glutamyl transpeptidase activity and serum alkaline phosphatase activity, and obtain the detection values; (3) When the detected value of the γ-glutamyl transpeptidase activity is ≥61.85 U / L and the detected value of the alkaline phosphatase activity is ≥73.85 U / L, the ewe is confirmed as a high-producing egg donor ewe.

[0032] In yet another specific implementation, the method for selecting high-producing egg-producing donor ewes includes the following steps: (1) Collect blood samples from ewes, let them stand and centrifuge to obtain serum; (2) Detect the serum γ-glutamyl transferase activity and blood platelet count in the blood sample to obtain the detection values; (3) When the detected value of the γ-glutamyl transferase activity is ≥61.85 U / L and the detected value of the platelet count is ≤280×10⁻⁶. 9 When / L, the ewe is confirmed to be a high-producing egg donor ewe.

[0033] In yet another specific implementation, the method for selecting high-producing egg-producing donor ewes includes the following steps: (1) Collect blood samples from ewes, let them stand and centrifuge to obtain serum; (2) Detect the serum alkaline phosphatase activity and blood platelet count in the blood sample to obtain the detection values; (3) When the alkaline phosphatase activity is ≥73.85 U / L and the platelet count is ≤280×10⁻⁶. 9 When / L, the ewe is confirmed to be a high-producing egg donor ewe.

[0034] In this invention, the breed of the ewe is not particularly limited, and examples include, but are not limited to, sheep and goats.

[0035] In this invention, the blood sample is obtained by collecting venous blood from ewes. The source of the venous blood is not particularly limited, and examples include, but are not limited to, venous blood obtained by collecting blood from the jugular vein, tail vein, or mammary vein.

[0036] In the above steps involving settling and centrifugation, the settling time is 20-70 min, preferably 25-65 min, and even more preferably 30-60 min, for example 30, 35, 40, 45, 50, 55, or 60 min. The centrifugation speed is 2000-5000 r / min, preferably 2200-4800 r / min, even more preferably 2400-4600 r / min, more preferably 2600-4400 r / min, even more preferably 2800-4200 r / min, and even more preferably 3000-4000 r / min, for example 3000, 3200, 3400, 3600, 3800, or 4000 r / min.

[0037] In this invention, the methods for detecting the activities of γ-glutamyl transferase and alkaline phosphatase are not particularly limited, and methods known in the art can be used, including but not limited to colorimetric methods, fluorescent probe methods, and enzyme-linked immunosorbent assays. The methods for detecting platelet count are not particularly limited, and methods known in the art can be used, including but not limited to microscopic counting, flow cytometry, impedance spectroscopy, and light scattering.

[0038] application One aspect of the present invention provides the application of the method according to the invention in the propagation of superior sheep breeds or in embryo production.

[0039] This invention uses blood indicators to screen for high-quality donor ewes for subsequent breeding or embryo production, which can rapidly increase the number of high-quality breeding sheep, greatly improve the production efficiency of high-quality embryos, and thus enhance the breeding benefits.

[0040] Example The following illustrates the process of establishing a method for screening high-producing egg donor ewes.

[0041] 1. Experimental Materials Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art. Unless otherwise specified, all reagents, consumables, and other materials used are commercially available products.

[0042] 2. Experimental Methods 2.1 Selection of breeding ewes Select 400 individuals from a large flock of breeding ewes that have a clear pedigree, are healthy, in good condition, have normal estrous cycles, and are free from reproductive disorders. These individuals should be 2-4 years old and weigh approximately 45-55 kg.

[0043] 2.2 Collection of blood samples from the jugular vein of sheep Grasp the sheep's horns or ears with both hands, tilting its head upwards and towards the opposite side of the blood collection site. Clip the wool and disinfect the collection site. With your left thumb, press down on the lower part of the jugular groove to make the jugular vein as engorged and distended as possible; with your right thumb or forefinger, palpate for the blood vessel in the jugular groove. Quickly insert the needle into the skin and blood vessel at a 30-45° angle at the insertion point in the jugular groove. If blood return is observed, insert the needle forward 1-2 cm parallel to the skin. After collecting 10 mL of blood, disinfect with 5% iodine tincture.

[0044] 2.3 Complete blood count (CBC) A fully automated 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).

[0045] 2.4 Serum Sample Preparation and Collection Use 5 mL of jugular vein blood sample from a donor ewe, leave at room temperature for 30-60 minutes, centrifuge at 3500 r / min for 10 minutes to separate the serum, aliquot into 1.5 mL centrifuge tubes, and freeze at -20℃.

[0046] 2.5 Serum Biochemical Indicators Detection Serum biochemical indicators, including gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), globulin (GLB), creatinine (Cr), urea, potassium (K+), serum calcium (sCa), serum phosphorus (sP), serum magnesium (sMg), triglycerides (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), were measured using a fully automated biochemical analyzer.

[0047] 2.6 Detection of serum antioxidant indicators Serum antioxidant-related indicators, including total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) content, and total superoxide dismutase (T-SOD) activity, were detected using reagent kits produced by Nanjing Jiancheng Biotechnology Research Institute.

[0048] 2.7 Serum Hormone Level Detection Serum hormone levels, including follicle-stimulating hormone (FSH), estrogen (E2), and progesterone (P4), were detected using an ELISA kit. The OD value of each serum sample was measured at a wavelength of 450 nm using an ELISA reader.

[0049] 2.8 Follicle-stimulating hormone induction in donor ewes On any day of the ewe's estrus cycle, administer progesterone suppositories into the vagina for 10 days. Begin hormone induction on the 10th day, following the procedure: inject FSH once daily for 2 consecutive days, with each injection containing 240 IU.

[0050] 2.9 Live egg collection from donor ewes Following the second FSH injection, the ewes were fasted and deprived of food and water. Forty-eight hours later, the donor ewes were anesthetized and restrained in a sling for laparoscopic oocyte retrieval. After oocyte retrieval, the progesterone suppositories were removed, and the total number of oocytes retrieved from each donor ewe was recorded.

[0051] Based on the total number of eggs recovered, the donor ewes were divided into a high-egg-retrieval group (total number of eggs > 15) and a low-egg-retrieval group (total number of eggs < 10).

[0052] 2.10 Data Statistics Experimental data were recorded using Excel, and statistical software was used for difference analysis.

[0053] 3. Experimental Results 3.1 Analysis of routine blood parameters of donor ewes in high and low oocyte retrieval groups As shown in Table 1, the platelet count in the blood of donor ewes in the high oocyte retrieval group was 182.53±8.16, which was significantly lower than that in the low oocyte retrieval group (363.11±11.47) (P<0.01). There were no differences in other indicators between the two groups (P>0.05).

[0054] Table 1. Blood routine indicators of donor ewes in high and low oocyte retrieval groups Note: ** indicates a highly significant difference. P <0.01).

[0055] 3.2 Analysis of serum antioxidant indicators in donor ewes of high and low oocyte retrieval groups As shown in Table 2, there were no differences in various antioxidant indicators in the serum of donor ewes in the high and low oocyte retrieval groups (P>0.05).

[0056] Table 2. Serum antioxidant indices of donor ewes in high and low oocyte retrieval groups 3.3 Analysis of serum biochemical indicators of donor ewes in high and low oocyte retrieval groups Table 3 shows that the serum γ-glutamyl transferase (GGT) and alkaline phosphatase (ALP) activities of donor ewes in the high oocyte retrieval group were 74.78±2.15 and 95.23±2.06, respectively, which were significantly higher than those in the low oocyte retrieval group (52.14±1.38 and 51.46±1.67). P <0.01). There were no significant differences in other serum biochemical indicators between the two groups.

[0057] Table 3 Serum biochemical indicators of donor ewes in high and low oocyte retrieval groups Note: ** indicates a highly significant difference. P <0.01).

[0058] 3.4 Analysis of serum hormone levels in donor ewes in high and low oocyte retrieval groups As shown in Table 4, there were no differences in the serum levels of various hormones in donor ewes between the high and low oocyte retrieval groups (P>0.05).

[0059] Table 4. Serum hormone levels of donor ewes in high and low oocyte retrieval groups 3.5 Serum gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) activity, and blood platelet count were used as screening criteria for donor ewes. Receiver operating characteristic (ROC) curves were used to assess the discriminative power of serum gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) activity, and blood platelet count as screening criteria for donor ewes. The area under the ROC curve (AUC) was then used to describe the ability of these indicators to differentiate donor ewes. An AUC > 0.7 indicates that the target indicators have a certain degree of accuracy in the screening criteria; in this example, 0.8 was used as a benchmark to improve the accuracy of the criteria. Analysis of the ROC curves revealed that the AUC for serum GGT activity reached 0.92, the AUC for serum ALP activity reached 0.94, and the AUC for blood platelet count reached 0.92. Figure 1 (See Table 5). This indicates that serum gamma-glutamyl transferase, alkaline phosphatase activity, and blood platelet count can be used as high-quality standards for screening donor ewes. Specific data are shown in Table 5.

[0060] Table 5. Threshold data for serum gamma-glutamyl transferase, alkaline phosphatase activity, and platelet count as screening criteria for donor ewes. Choosing any of these thresholds involves a trade-off between sensitivity and specificity.

[0061] As shown in Table 5, the sensitivity of γ-glutamyl transferase 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, donor ewes with serum γ-glutamyl transferase activity greater than 61.85 U / L, serum alkaline phosphatase activity greater than 73.85 U / L, or blood platelet count less than 280 × 10⁻⁶ U / L are considered to have these criteria. 9 When the ratio is / L, the donor ewe can be selected for production use.

[0062] 3.6 Validation of serum gamma-glutamyl transferase, alkaline phosphatase activity, and blood platelet count as screening criteria To test the accuracy of serum gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) activity, and blood platelet count as screening criteria, 100 ewes were selected for predictive validation. The validation results are shown in Table 6. The accuracy of serum GGT, ALP activity, and blood platelet count as screening criteria were 91.01%, 96.47%, and 93.41%, respectively. These results demonstrate that the indicators proposed in this embodiment are highly accurate for screening donor ewes.

[0063] Table 6. Validation of serum gamma-glutamyl transferase, alkaline phosphatase activity, and blood platelet count as screening criteria. In summary, through the analysis of a large amount of experimental data, this invention has yielded a method for screening donor ewes, namely, detecting serum γ-glutamyl transferase activity, serum alkaline phosphatase activity, or blood platelet count in donor ewes, and screening for high-quality donor ewes based on standard values.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening high-producing egg-producing donor ewes, characterized in that, The method includes the following steps: (1) Prepare blood samples from ewes; (2) Detect each serum biochemical index in the blood sample and obtain their respective detection values, wherein the serological index is selected from at least one of γ-glutamyl transpeptidase activity, alkaline phosphatase activity and platelet count; (3) Based on the respective detection values, identify high-producing egg donor ewes.

2. The method for screening high-producing donor ewes according to claim 1, characterized in that, Step (3) includes comparing the respective detection values ​​with their corresponding thresholds.

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

4. The method for screening high-producing donor ewes according to claim 2, characterized in that, The threshold is a value obtained statistically from a sample group of donor ewes of similar age.

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

6. The method for screening high-producing donor ewes according to claim 1, characterized in that, The ewes include sheep or goats.

7. The method for screening high-producing donor ewes according to claim 1, characterized in that, The procedure of separating serum is further included before detecting γ-glutamyl transpeptidase activity or alkaline phosphatase activity.

8. The method for screening high-producing donor ewes according to claim 1, characterized in that, The blood sample was obtained by collecting venous blood from the ewe.

9. The method for screening high-producing donor ewes according to claim 1, characterized in that, The threshold values ​​for the γ-glutamyl transferase activity are 61.85 U / L, the threshold values ​​for the alkaline phosphatase activity are 73.85 U / L, and the threshold values ​​for the platelet count are 280 × 10⁻⁶. 9 / L.

10. The application of the method according to any one of claims 1-9 in the propagation of superior sheep breeds or embryo production.

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