A method for screening of dairy cow oocyte donors

Abstract

The application provides a screening method of a dairy cow oocyte donor, comprising the following steps: investigating the correlation between the body condition score, age, parity, postpartum days and milk yield of the donor and the development potential of the dairy cow oocyte; determining the body condition score, age, parity and postpartum days as the screening indexes of the donor; and screening the donor based on the screening criteria of the screening indexes. The screening method can screen the donor with high oocyte development potential, which will help to improve the selection and management strategy of the live oocyte donor cow, and provide a theoretical basis and practical guidance for improving the reproductive efficiency of the dairy cow, promoting the breeding process and improving the economic benefit of the industry.

Description

Technical Field

[0001] This application belongs to the field of animal reproductive technology, specifically, it relates to a method for screening bovine oocyte donors. Background Technology

[0002] In modern animal husbandry, the rapid propagation of high-yielding dairy cows plays a crucial role in promoting my country's dairy cattle breeding process, directly impacting the coverage rate of superior breeds, genetic progress, and the enhancement of dairy industry competitiveness. Therefore, optimizing reproductive management strategies and improving propagation efficiency have become core tasks of the current dairy cattle breeding system. In recent years, OPU-IVP (Oviposition for In Vitro Embryo Production) technology, as a highly efficient biological breeding method, has provided a new pathway for accelerating the genetic dissemination and herd improvement of superior cows. This technology not only significantly shortens the generation interval but also improves the reproductive potential of superior female animals. The quality and developmental potential of oocytes directly determine the successful application of OPU-IVP technology; therefore, clarifying its influencing factors is of significant practical importance. Summary of the Invention

[0003] The purpose of this application is to provide a method for screening bovine oocyte donors.

[0004] Specifically, this application relates to the following aspects: 1. A method for screening bovine oocyte donors, comprising the following steps: To investigate the correlation between donor body condition score, age, parity, postpartum days, and milk yield and the developmental potential of bovine oocytes; Physical condition score, age, parity, and postpartum days were determined as selection criteria for donors; Donors are selected based on screening criteria and screening indicators.

[0005] 2. According to the screening method described in item 1, the screening criterion for the physical condition score is 2.75-3.25 points.

[0006] 3. The screening method according to item 1 or 2, wherein the screening criterion for donor age is less than or equal to 80 months.

[0007] 4. The screening method according to any one of items 1-3, wherein the screening criterion for donor parity is less than or equal to 4 parities.

[0008] 5. The screening method according to any one of items 1-4, wherein the screening criterion for the number of days postpartum for the donor is 50-70 days.

[0009] 6. The screening method according to any one of items 1-5, wherein the donor milk production includes the donor milk production on the day of egg retrieval, the donor's average weekly milk production, and the donor's total milk production over 305 days.

[0010] 7. The screening method according to any one of items 1-6, wherein the developmental potential of bovine oocytes is reflected in one or more of the following indicators: the percentage of AB grade cumulus-oocyte complexes, oocyte maturation rate, fertilization rate, cleavage rate, and blastocyst rate.

[0011] 8. The screening method according to any one of items 1-7, wherein the test donor is a Holstein multiparous cow.

[0012] Beneficial effects of this application This application uses Holstein multiparous cattle as the research subject, systematically analyzing the effects of multiple donor factors, such as body condition score, age, parity, postpartum days, and milk yield, on oocyte quality and its in vitro developmental potential. Through scientific grouping and multifactorial statistical analysis of donor cattle participating in OPU (Oviposition for Purification) procedures, this application reveals the developmental patterns of oocytes under different physiological conditions, clarifies the key factors affecting the effectiveness of OPU-IVP, and establishes a donor cattle selection and evaluation system based on phenotypic data. The methods in this application will contribute to improving the selection and management strategies for live oocyte donor cattle, providing theoretical basis and practical guidance for improving dairy cow reproductive efficiency, advancing breeding processes, and enhancing the economic benefits of the industry. Attached Figure Description

[0013] Figure 1 The angle of insertion of the egg retrieval needle is shown, where A: vertical insertion (90°), bar = 4 mm; B: non-vertical insertion, bar = 4 mm.

[0014] Figure 2 The grading of cumulus-oocyte complexes (COCs) is shown, where (a) grade A COCs, bar = 120 μm; (b) grade B COCs, bar = 120 μm; (c) grade C COCs, bar = 120 μm; and (d) grade D COCs, bar = 120 μm. Detailed Implementation

[0015] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0016] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0017] To address the problems existing in the prior art, this application provides a method for screening bovine oocyte donors, comprising the following steps: Step 1: Examine the correlation between donor body condition score, age, parity, postpartum days and milk yield and the developmental potential of dairy cow oocytes; Step 2: Determine the physical condition score, age, parity, and postpartum days as the screening criteria for donors; Step 3: Select donors based on screening criteria and screening indicators.

[0018] In step 1, the body condition of the cows is scored on a 5-point scale (minimum unit 0.25 points), where 1 point represents extreme emaciation and 5 points represents excessive obesity. The assessment focuses on eight key areas: the pelvic region, hip joint, ischial tuberosity, ribs, tail root ligament, sacral ligament, hip joint, and dorsal ribs. As the score increases, the body characteristics show systematic changes: ① Pelvic shape: ≤ 3 points, “V” shape; > 3 points, “U” shape; ② Skeletal prominence: In low-scoring individuals, the hip bones and ischial tuberosities are clearly defined, gradually becoming covered by fat as the score increases, and are no longer visible at 5 points; ③ Intercostal groove: ≤ 2.25 points, visible; > 2.25 points, invisible; ④ Ligaments and joints: Structures such as the tail root ligament are not assessed below 3 points, while these structures gradually become blurred between 3 and 5 points, and the hip joint tends to flatten.

[0019] In practice, raters need professional training to conduct assessments using a combination of visual and tactile methods: ① Visual observation: Observe the pelvic region from the side (the line connecting the hip bone, sacrum, and coccyx), and check the degree of coccyx root depression from the rear; ② Tactile assessment: Use the short ribs as the key palpation site. 1 point: Short ribs protrude at an acute angle, without fat coverage; 2 points: Partially covered by fat; 3 points: Requires forceful pressure to palpate; 4 points: Still difficult to palpate even with forceful pressure; 5 points: The body is barrel-shaped, with ribs, thighs, etc., covered by fat, and abnormal gait. To improve scoring consistency, a folding goniometer can be placed above the coccyx root, and objective correction can be made by the color of the pointer (yellow for obesity, green for moderate obesity).

[0020] The donor's milk production can include the milk production on the day of egg retrieval, the donor's average weekly milk production, and the donor's total milk production over 305 days.

[0021] The developmental potential of dairy cow oocytes is reflected in one or more of the following indicators: the percentage of grade A and B cumulus-oocyte complexes, oocyte maturity rate, fertilization rate, cleavage rate, and blastocyst rate.

[0022] In some implementations, the test donor is a Holstein multiparous cow. Those skilled in the art will understand that other breeds of dairy cows are also suitable for the screening methods of this application.

[0023] Following step 1, this application found that donor body condition score, age, parity, and postpartum days are all correlated with oocyte developmental potential. However, milk yield is not a direct factor affecting the oocyte developmental potential of dairy cows. Therefore, in step 2, body condition score, age, parity, and postpartum days were determined to be used as donor screening criteria.

[0024] In step 3, donors are selected based on screening criteria including physical condition score, age, parity, and postpartum days. The screening criteria for donor physical condition score are 2.75-3.25 points. The screening criteria for donor age are 80 months or less. The screening criteria for donor parity are 4 or less. The screening criteria for donor postpartum days are 50-70 days.

[0025] In some implementations, the method for screening bovine oocyte donors includes the following steps: Step 1: Examine the correlation between the donor's body condition score, age, parity, postpartum days, milk yield and the developmental potential of dairy cow oocytes; Step 2: Determine the physical condition score, age, parity, and postpartum days as the screening criteria for donors; Step 3: Select donors based on the screening criteria; the screening criteria for donor physical condition score is 2.75-3.25, the screening criteria for donor age is less than or equal to 80 months, the screening criteria for donor parity is less than or equal to 4 parities, and the screening criteria for donor postpartum days is 50-70 days.

[0026] The screening method proposed in this application can identify donors with high oocyte development potential, which will help improve the selection and management strategy of live oocyte donor cattle, and provide theoretical basis and practical guidance for improving dairy cow reproductive efficiency, advancing the breeding process, and enhancing the economic benefits of the industry.

[0027] Example 1. Experimental Materials 1.1 Experimental Ranch This experiment was conducted on three large-scale dairy farms. The first farm, located in Lingwu City, Ningxia Hui Autonomous Region, currently has 8,065 Holstein dairy cows, of which 4,215 are lactating. The average parity is 2.88, the average lactation days are 146 days, and the average annual milk yield is 11.28 tons per cow. The second farm, located in Jiayuguan City, Gansu Province, has 8,300 high-quality dairy cows, including 3,500 adult cows, with an average annual milk yield of 10.8 tons per cow. The third farm, located in Huayin City, Weinan City, Shaanxi Province, currently has over 3,750 high-quality dairy cows, with an average annual milk yield of 11.5 tons per cow in 2023. All lactating cows on the three farms were grouped according to their lactation days. The experiment followed a standardized stall management process: the cows were allowed free movement and grazing in the barns, and three TMR feedings and three milkings were performed daily (8-hour intervals). The "Yimuyun" system was used for digital management. Throughout the trial, the ranch's facilities, diet, personnel, and herd structure remained stable, and no outbreaks or infectious diseases occurred.

[0028] 1.2 Experimental Animals From March 2022 to December 2024, Holstein multiparous cows (30-70 days postpartum) receiving OPUs at a large-scale dairy farm in Ningxia, Gansu, and Shaanxi were all in a voluntary waiting period (VWP). During the 30-70 days postpartum period, each donor cow received 2-3 OPUs; after the last OPU, the cows underwent a 10-15 day recovery period before entering the farm's synchronization program and AI process.

[0029] To analyze the impact of a single donor factor on oocyte developmental potential, this embodiment employs a controlled variable design. Specifically, when assessing any donor factor (such as body condition score, age, parity, postpartum days, and milk production), a strictly paired design is used to ensure that all groups remain substantially consistent across other background factors.

[0030] 2. Test Methods 2.1 Physical condition score Body condition scoring standards for dairy cows vary regionally. In my country, a 5-point scale (minimum unit 0.25 points) is generally used, where 1 point represents extreme emaciation and 5 points represents excessive obesity. The assessment mainly targets eight key areas: pelvic region, hip joint, ischial tuberosity, ribs, tail root ligament, sacral ligament, hip joint, and dorsal ribs. As the score increases, body characteristics show systematic changes: ① Pelvic shape: ≤ 3 points, "V" shape; > 3 points, "U" shape; ② Skeletal prominence: In low-scoring individuals, the hip bones and ischial tuberosities are clearly defined, gradually becoming covered by fat as the score increases, and are no longer visible at 5 points; ③ Intercostal groove: ≤ 2.25 points, visible; > 2.25 points, invisible; ④ Ligaments and joints: Below 3 points, structures such as the tail root ligament are not assessed; from 3-5 points, these structures gradually become blurred, and the hip joint tends to flatten.

[0031] In practice, raters need professional training to conduct assessments using a combination of visual and tactile methods: ① Visual observation: Observe the pelvic region from the side (the line connecting the hip bone, sacrum, and coccyx), and check the degree of coccyx root depression from the rear; ② Tactile assessment: Use the short ribs as the key palpation site. 1 point: Short ribs protrude at an acute angle, without fat coverage; 2 points: Partially covered by fat; 3 points: Requires forceful pressure to palpate; 4 points: Still difficult to palpate even with forceful pressure; 5 points: The body is barrel-shaped, with ribs, thighs, etc., covered by fat, and abnormal gait. To improve scoring consistency, a folding goniometer can be placed above the coccyx root, and objective correction can be made by the color of the pointer (yellow for obesity, green for moderate obesity).

[0032] 2.2 Live egg retrieval 2.2.1. Pre-retrieval treatment Based on the team's previous research results (Xiao et al. 2025), and adjusted according to the dosage recommended by the pharmaceutical manufacturer, the ovarian stimulation protocol for this trial was modified.

[0033] (1) If a corpus luteum is present on the ovary, the following protocol shall be followed: ① Using a veterinary OPU ultrasound diagnostic instrument, follicles with a diameter ≥ 5 mm were aspirated on any day of the donor dairy cow's estrous cycle (denoted as 0 d) to achieve follicular wave synchronization; ② 24 h after aspiration, glycerol PGF2α (25 mg) was injected intramuscularly; ③ Subsequently, FSH (100 IU) was injected intramuscularly every 12 h for 5 consecutive injections; ④ OPU operation was performed 36 h after the last FSH injection.

[0034] (2) If there is no corpus luteum on the ovary, the following procedure shall be followed: ① Use a veterinary OPU ultrasound diagnostic instrument to aspirate follicles with a diameter ≥ 5 mm on any day of the donor dairy cow's estrous cycle (referred to as 0 d) to achieve follicular wave synchronization; ② 36 h after follicle aspiration, inject the first dose of FSH (100 IU); ③ Inject FSH (100 IU) intramuscularly every 12 h for 5 consecutive times; ④ 12 h after the last FSH injection, inject glycerol PGF2α (25 mg) intramuscularly to eliminate possible interference from early corpus luteum on the OPU procedure; ⑤ Administer OPU 24 h after PGF2α injection.

[0035] 2.2.2 Egg Retrieval Process The OPU procedure in this study was performed in a dedicated oocyte collection room with controlled ambient temperature, using standardized procedures executed by designated technicians. The specific steps were as follows: ① After the donor cow was restrained in a six-post pen, epidural anesthesia (5 mL of 2% lidocaine hydrochloride) was administered at the sacral junction, followed by perineal cleaning and disinfection. ② The OPU system was assembled, with a negative pressure of 20-80 mmHg, and the oocyte collection fluid preheated to 37°C. ③ Using a veterinary OPU ultrasound diagnostic system, a vaginal probe equipped with a sterile oocyte collection needle was inserted, and the ovary was brought into close contact with the probe through rectal manipulation. Under ultrasound guidance, visible follicles larger than 2 mm in diameter were aspirated, and follicular fluid was collected in centrifuge tubes containing preheated oocyte collection fluid. ④ After bilateral ovarian collection, the samples were transported to the laboratory within 1 hour under constant temperature conditions of 37.5-38.5°C for further processing.

[0036] The angle of insertion of the egg retrieval needle is as follows: Figure 1 As shown.

[0037] 2.3 In vitro maturation 2.3.1 Classification of COCs The grading of COCs (cow embryos) is based on the national standard (GB / T 45202 Technical Specifications for Bovine In Vitro Embryo Production and Transfer). Under a stereomicroscope, COCs are morphologically classified into the following four grades: Grade A: Homogeneous cytoplasm, enclosing ≥5 layers of dense and intact cumulus cells; Grade A oocytes show... Figure 2 Figure a. Grade B: Uniform cytoplasm, enclosing 3-5 layers of cumulus cells; Grade B oocytes are shown in Figure a. Figure 2 Figure b. Grade C: Cytoplasm is unevenly homogeneous, enclosing 1-3 layers of cumulus cells; Grade C oocytes are shown in Figure b. Figure 2 Figure c. Grade D: Loose cumulus cells or naked oocytes; Grade D oocytes are shown in Figure c. Figure 2 The d-graph.

[0038] 2.3.2 In vitro maturation of COCs Prepare 100 μL droplets of oocyte maturation (OM) in a cell culture dish, cover with mineral oil, and equilibrate in a CO2 incubator for more than 2 hours.

[0039] The collection and grading of follicular cells (COCs) were performed under a thermostatic stereomicroscope. First, the follicular fluid containing COCs was filtered through a 75μm oocyte collection cup and rinsed until the filtrate was clear. Then, the cup was placed on a 37℃ heated stage, and all COCs were collected under the microscope using an oocyte collection needle for morphological grading (Grades A and B COCs were used for IVM). The selected COCs were washed three times in preheated PBS, followed by a rinse in OM (oxygenated precipitate), and then transferred to preheated, equilibrated droplets containing OM. Finally, the cells were placed in a CO2 incubator for maturation for 22-24 hours. The maturation rate was calculated (maturation rate = number of PB1 cells / (number of COCs - dead oocytes)).

[0040] 2.4 In vitro fertilization 2.4.1 Sperm purification To prepare the Percoll density gradient system, place 2 mL of 80% lower solution in a 15 mL centrifuge tube, and then slowly place 2 mL of 40% upper solution on top of the 80% lower solution to form an osmotic gradient, taking care not to generate air bubbles, and cover the tube. Add 1 mL of TL-Hepes solution (sealed) to a 1.5 mL centrifuge tube, and preheat both centrifuge tubes in a CO2 incubator for 0.5 h in advance.

[0041] Next, prepare to thaw the sperm. Quickly remove 1-3 vials of frozen sperm from the liquid nitrogen tank using tweezers and immediately place them in 37°C warm water for thawing. Perform the procedure in a clean bench, using sterile surgical scissors to cut both ends of the frozen sperm straw (cut the top first, then the bottom), and place the semen in a 30 mm dish. Use a sperm needle to aspirate the semen from the dish and inject it into the upper liquid (be careful to do this quickly). Centrifuge at 1470 rpm for 20 min, then discard the supernatant (discard as completely as possible, as Percoll is toxic to sperm). Aspirate the sperm from the bottom of the centrifuged sediment and transfer it directly into TL-Hepes solution, mix well, and then centrifuge at 1380 rpm for 5 min. Discard the supernatant again, leaving approximately 100 μL of liquid.

[0042] 2.4.2 Measurement of sperm motility and density (1) Sperm motility assay The test was performed using a hemocytometer. The thawed sperm sample was rediluted with sterile saline. 10 μL of the diluted thawed sperm sample was slowly pipetted into the counting chamber of the hemocytometer. Sperm motility was then calculated by counting the sperm that moved in a straight line and those that oscillated in place in the five squares (including the four outer corners and the center square) on the hemocytometer.

[0043] (2) Sperm density measurement Place the cleaned hemocytometer on the operating table. First, moisten the outer edge of the groove of the hemocytometer with a small amount of ultrapure water, then cover it with a coverslip. Use a pipette to draw a small amount of the thawed semen to be tested, and then dilute it 10 times with ultrapure water. Take 10 μL of the diluted sample, wipe the pipette tip, and slowly place the tip on the edge of the coverslip to allow the sample to flow out slowly, allowing it to flow into the counting chamber of the hemocytometer through capillary action. Place the hemocytometer on a culture dish that has been pre-placed with moistened filter paper, and cover the dish. After standing for 5 minutes, gently remove the hemocytometer and place it on the microscope stage for 2-3 minutes. After the sperm stops moving, begin counting. Under low magnification, first locate the large central square of the counting chamber of the hemocytometer, then adjust to high magnification to count, and finally calculate the sperm density.

[0044] Sperm motility and density can also be calculated using a computer-aided semen analysis system (Sperm Class Analyzer, Microptic SL, Spain).

[0045] 2.4.3 Sperm and egg incubation Centrifuge tubes containing TL-Hepes solution and microdroplets of in vitro fertilization (IVF) solution were preheated in a CO2 incubator 2 hours in advance.

[0046] Gently pipette COCs with a 1 mL pipette tip until their overall structure becomes loose. Then, carefully remove excess granulosa cells using a retrieval needle. Transfer mature MII stage oocytes to two TL-Hepes containers, washing each once. Discard naked and dead oocytes. Transfer mature oocytes into IVF containers and wash twice more before finally transferring them to IVF droplets. Sperm (1.5-2 × 10⁻⁶) is then added. 7 (number per mL), fertilization time is 10-12 h.

[0047] 2.5 In vitro culture Prepare 100 μL microdroplets of in vitro embryo culture medium in cell culture dishes, cover with mineral oil, and equilibrate in a CO2 incubator for at least 3 hours. After fertilization, transfer COCs to hyaluronidase working solution, gently aspirate repeatedly with a pipette to remove cumulus cells and adhering sperm, wash three times with in vitro embryo culture medium, transfer to the microdroplets of in vitro embryo culture medium, and culture in a tri-gas incubator. Each microdroplet should contain 10-20 embryos. Observe cleavage after 48 hours and observe blastocysts on day 7. Calculate the fertilization rate (fertilization rate = number of PB2 cells / number of PB1 cells), cleavage rate (cleavage rate = number of cleavages / number of PB1 cells), and blastocyst rate (blastocyst rate = number of blastocysts / number of cleavages).

[0048] Embryo quality assessment was conducted in accordance with the national standard (GB / T 25881 Bovine Embryos). Grade A and Grade B embryos were selected for ET (Embryo Transfer), as shown in Table 1.

[0049] Table 1 Embryo Quality Assessment Standards

[0050] 2.6 Data Statistical Analysis The data were organized and statistically analyzed using Microsoft Excel. GraphPad Prism 10.1.3 and SPSS Statistics 25 were used for data analysis and visualization. All data are expressed as "means ± SEM". One-way ANOVA (post-hoc multiple test: Tukey HSD) was used for statistical analysis of differences. Different letters indicate significant differences between groups. P <0.05).

[0051] 3. Experimental Results 3.1 Correlation analysis between donor body condition score and developmental potential of Holstein multiparous bovine oocytes This study aimed to analyze the correlation between donor body condition scores and the developmental potential of oocytes from Holstein multiparous cattle. Donor cattle participating in the OPU were scored and divided into 9 groups (2-4 points, in 0.25 increments). The results are shown in Table 2. Donor body condition scores significantly affected the proportion of COCs at grade AB and subsequent developmental indicators. The proportion of COCs at grade AB initially increased and then decreased with increasing donor body condition scores, remaining at the highest level (59.72%–59.96%) within the 2.75–3.25 score range, significantly higher than the low-score group (e.g., 2 points: 51.44%). P <0.05) and high-scoring groups (e.g., 4 points: 44.59%) P <0.05). Regarding subsequent developmental potential, maturity rate, fertilization rate, cleavage rate, and blastocyst rate all showed similar patterns, with each indicator reaching its peak within the 2.75–3.25 score range (maturation rate: 83.56%–84.38%). P >0.05; Fertilization rate: 81.88%–83.10%, P >0.05; Cleavage rate: 82.79%–84.13%, P >0.05; Blastocyst rate: 37.32%–41.95%, P >0.05), with no significant difference between groups, but all were significantly better than those with excessively low physical condition scores (e.g., 2 points). P <0.05) or too high (3.75 or 4 points, P Groups with scores <0.05. In summary, maintaining the body condition score of Holstein multiparous cattle between 2.75 and 3.25 can significantly improve oocyte collection efficiency and subsequent developmental potential, which is a key management strategy for optimizing OPU effects.

[0052] Table 2. Correlation analysis between donor physical condition score and developmental potential of Holstein multiparous bovine oocytes.

[0053] The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences. P <0.05, one-way ANOVA).

[0054] 3.2 Correlation analysis between donor age and developmental potential of Holstein multiparous bovine oocytes This study aimed to analyze the correlation between donor age and the developmental potential of oocytes from Holstein multiparous cattle. Donor cattle participating in OPU were divided into four groups based on age (20-40 months, 40-60 months, 60-80 months, and 80-100 months). The results are shown in Table 3. Donor age had a significant impact on oocyte quality and subsequent developmental indicators. In groups with an age not exceeding 80 months, oocyte quality and developmental potential remained at a high and stable level. In the 20-40 month, 40-60 month, and 60-80 month age groups, the percentage of grade AB COCs obtained was 59.45% vs 62.23% vs 59.03%. P >0.05), maturity rate (82.66% vs 82.30% vs 81.34%) P >0.05), fertilization rate (83.30% vs 81.63% vs 81.96%) P >0.05), cleavage rate (85.67 vs 83.22% vs 86.31%) P >0.05) and blastocyst rate (39.80% vs 40.35% vs 39.80%) P There were no significant differences in any of the indicators (>0.05). However, when the age increased to 80-100 months, all indicators showed a significant decline. The proportion of grade AB COCs decreased to 44.61%, and the maturity rate, fertilization rate, and cleavage rate decreased to 71.75% ( P <0.05), 71.18% P <0.05) and 75.53% ( P <0.05%, and the blastocyst rate decreased significantly to 30.10% ( P <0.05). In summary, Holstein cows under 80 months of age can consistently provide high-quality oocytes with good developmental potential. Age is a key factor affecting oocyte quality and subsequent embryonic development efficiency, and 80 months of age may be an important time point for maintaining high-quality reproductive performance.

[0055] Table 3. Correlation analysis between donor age and developmental potential of oocytes from Holstein multiparous cattle.

[0056] The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences. P <0.05, one-way ANOVA).

[0057] 3.3 Correlation analysis between donor parity and developmental potential of Holstein multiparous bovine oocytes This study aimed to analyze the correlation between donor parity and the developmental potential of oocytes from Holstein multiparous cows. Donor cows participating in the OPU were divided into 6 groups (parities 1-6). The results are shown in Table 4. Donor parity significantly affected oocyte quality (indicated by the percentage of COCs in grade AB) and its in vitro maturation and fertilization capacity, while its effect on cleavage rate and blastocyst rate was not significant. During parities 1 to 4, oocyte quality and early developmental indicators remained at high and stable levels. The percentages of COCs in grade AB across parities were 60.29% vs 65.16% vs 63.42% vs 61.68%. P >0.05), maturity rate (81.64% vs 85.30% vs 85.60% vs 82.64%) P >0.05), fertilization rate (87.21% vs 85.52% vs 86.46% vs 87.02%) P >0.05) Cleavage rate (83.59% vs 90.43% vs 80.74% vs 86.39%) P >0.05) and blastocyst rate (38.74% vs 37.39% vs 40.06% vs 39.69%) P There were no significant differences (>0.05). However, from the 5th pregnancy onwards, oocyte quality and early development efficiency showed a significant decline; the proportion of grade AB oocytes in this pregnancy significantly decreased to 54.60% ( P <0.05%, the maturity rate and fertilization rate decreased to 71.09% ( P <0.05) and 73.54% ( P <0.05). By the sixth pregnancy, the percentage of AB grade pregnancies had further decreased to 45.78% ( P <0.05), and the maturation rate and fertilization rate were also significantly reduced (69.67% and 69.57%, respectively). P <0.05); Although the cleavage rate of the 5th and 6th parities was not significantly different from that of the previous parities, the blastocyst rate showed a decreasing trend (34.53% for the 6th parity). P>0.05). In summary, Holstein multiparous cattle can sustainably provide high-quality oocytes with good developmental potential during the first to fourth parities, with the fifth parity potentially being a critical point where reproductive performance declines significantly. Parity is a key factor affecting oocyte quality and in vitro fertilization efficiency, and it has important guiding significance for optimizing the selection and management of live oocyte donor cattle.

[0058] Table 4. Correlation analysis between donor parity and developmental potential of oocytes from multiparous Holstein cows.

[0059] The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences. P <0.05, one-way ANOVA).

[0060] 3.4 Correlation analysis between donor postpartum days and developmental potential of Holstein multiparous bovine oocytes This study aimed to analyze the correlation between Days in Milk (DIM) and oocyte developmental potential in Holstein multiparous cattle. Donor cattle participating in the OPU were divided into four groups based on DIM (30-40 days, 40-50 days, 50-60 days, and 60-70 days). The results are shown in Table 5. Days in Milk had a highly significant impact on oocyte quality (percentage of COCs AB grade) and all subsequent developmental indicators (maturation rate, fertilization rate, cleavage rate, and blastocyst rate), with all indicators showing a significant upward trend with increasing postpartum days. Oocyte quality and developmental efficiency were at a low level in the early postpartum period (30-50 days); at 30-40 days postpartum, the percentage of COCs AB grade was only 40.99%, and the maturity rate, fertilization rate, and blastocyst rate were at their lowest values ​​(69.72%, 68.56%, and 32.59%, respectively). At 40–50 days postpartum, the cleavage rate significantly increased to 82.42% ( P Apart from <0.05, the percentage of COCs in grade AB (44.67%) and other early developmental indicators were not significantly different from the 30–40 d group. P >0.05). From 50-60 days postpartum, all indicators showed significant improvement compared to the 30-40 day group; the proportion of COCs AB grade significantly increased to 54.45% ( P <0.05%, the maturation rate, fertilization rate and cleavage rate reached 81.04% ( P <0.05), 79.67% P <0.05) and 82.61% ( P <0.05, the blastocyst rate significantly increased to 35.89% ( P<0.05). At 60-70 days postpartum, oocyte quality and developmental potential reached their peak, with AB grade oocytes accounting for 56.70%, and maturation, fertilization, and cleavage rates reaching 83.61%, 82.69%, and 87.00%, respectively. The blastocyst rate significantly increased to 39.71%. In summary, the collection quality and developmental potential of oocytes from Holstein multiparous cattle significantly increased with increasing postpartum days; compared to 30-50 days, 50-70 days postpartum is the ideal period for obtaining high-quality, high-development-potential oocytes. This example clarifies that postpartum days are a key factor affecting oocyte quality, providing important scientific basis for optimizing the selection and reproductive management of live oocyte donor cattle.

[0061] Table 5. Correlation analysis between donor postpartum days and oocyte developmental potential of Holstein multiparous bovines.

[0062] The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences. P <0.05, one-way ANOVA).

[0063] 3.5 Correlation analysis of milk production on the day of donor oocyte retrieval and the developmental potential of oocytes from Holstein multiparous cattle This experiment aimed to analyze the correlation between milk production on the day of oocyte retrieval and the developmental potential of oocytes from Holstein multiparous cattle. Donor cattle participating in the OPU were divided into five groups based on their milk production on the day of oocyte retrieval (20-30 kg, 30-40 kg, 40-50 kg, 50-60 kg, and 60-70 kg). The results are shown in Table 6. Within the milk production range of 20–70 kg, different milk production levels had no significant effect on the percentage of COCs AB grade, maturity rate, fertilization rate, cleavage rate, and blastocyst rate of the retrieved oocytes. P >0.05). Looking at the data trends, most assessment indicators showed a slow downward trend as daily milk production increased. When daily milk production reached 60-70 kg, the percentage of COCs AB grade (50.96%), maturity rate (78.01%), fertilization rate (75.82%), cleavage rate (77.72%), and blastocyst rate (31.31%) all dropped to the lowest levels among the groups, but the differences compared to low-yield groups (such as the 20-30 kg group) were still not significant. P>0.05). In summary, under the conditions of this experiment, milk yield on the day of oocyte collection was not a direct factor affecting the quality of oocyte collection and early developmental potential in Holstein multiparous cattle. This study suggests that a wider range of daily milk yield (20–70 kg) may not significantly restrict oocyte developmental capacity in this breed. However, the systematic downward trend in various indicators under extremely high milk yields warrants attention, and its biological significance may require further verification through larger sample sizes or more refined experimental designs. This finding can provide a more flexible reference for the practical selection of lactating donor cattle.

[0064] Table 6. Correlation analysis between milk production on the day of oocyte retrieval and the developmental potential of oocytes from Holstein multiparous cattle.

[0065] The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences. P <0.05, one-way ANOVA).

[0066] 3.6 Correlation analysis between donor weekly average milk yield and the developmental potential of Holstein multiparous bovine oocytes This experiment aimed to analyze the correlation between the average weekly milk yield of donors and the developmental potential of oocytes from Holstein multiparous cattle. Donor cattle participating in the OPU were divided into five groups based on their average weekly milk yield (20-30 kg, 30-40 kg, 40-50 kg, 50-60 kg, and 60-70 kg). The results are shown in Table 7. Within the average weekly milk yield range (20-70 kg) set in this example, different milk yield levels had no significant effect on the proportion of COCs AB grade, maturity rate, fertilization rate, cleavage rate, and blastocyst rate of the obtained oocytes. Specifically, the core indicators for each group, including the percentage of COCs grade AB (49.34%–53.84%), maturity rate (78.72%–81.62%), fertilization rate (76.64%–81.79%), cleavage rate (76.92%–82.10%), and blastocyst rate (34.63%–37.36%), fluctuated slightly, but the differences between groups were not significant. P>0.05). Although the values ​​of various indicators in the 60-70 kg group decreased slightly, they did not reach a statistically significant level, indicating that the oocyte developmental potential of donor cows remained stable even under high milk production conditions. In summary, the average weekly milk yield in the 20-70 kg range did not have a significant adverse effect on the OPU-IVP efficiency of Holstein multiparous cows. This result indirectly reflects the feasibility of high-producing dairy cows as OPU donors, and their oocyte quality and embryonic developmental potential were not reduced due to increased milk production load. In the breeding process, the selection of OPU donor cows should focus more on overall health and nutritional status rather than short-term milk production fluctuations. Therefore, individuals with excellent milk production performance can be selected as donor cows to achieve a synergistic improvement in production performance and reproductive expansion, providing an important basis for the construction of an efficient breeding system.

[0067] Table 7. Correlation analysis between donor weekly average milk yield and oocyte developmental potential of Holstein multiparous cattle.

[0068] The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences. P <0.05, one-way ANOVA).

[0069] 3.7 Correlation analysis of donor milk production at 305 days and oocyte developmental potential in Holstein multiparous cattle This study aimed to analyze the correlation between 305-day milk production in donor cattle and the developmental potential of oocytes in Holstein multiparous cattle. Donor cattle participating in the OPU were divided into five groups based on their 305-day milk production (11–12 t, 12–13 t, 13–14 t, 14–15 t, and 15–16 t). The results are shown in Table 8. Within the 305-day milk production range of 11–16 t, different milk production levels had no significant effect on the proportion of COCs AB grade, maturity rate, fertilization rate, cleavage rate, and blastocyst rate of oocytes. P >0.05). Looking at the data trends, when milk production was in the 11–14 t range, all indicators remained at a relatively stable and high level. However, when milk production rose to 15–16 t, all indicators showed a slight decrease, with the blastocyst rate (33.47%) being the lowest among all groups, but the difference compared to the low milk production group was still not significant. P>0.05). In summary, under the conditions of this experiment, the 305-day milk yield of donor cattle was not a direct factor affecting the quality of oocyte collection and in vitro developmental potential of Holstein multiparous cattle. This result indicates that a 305-day milk yield range of 11–16 t may not significantly restrict the developmental capacity of oocytes in this breed. This finding has significant practical implications for breeding: 305-day milk yield, as a key genetic indicator for evaluating the lifetime lactation performance of dairy cows, can be used for screening OPU donor cattle. In actual breeding and reproductive management, more attention should be paid to other physiological or genetic indicators more closely related to oocyte quality, thus providing a more scientific basis for donor cattle selection. However, the systematic downward trend of various indicators under extremely high milk yields still warrants further investigation, and its potential impact may need to be verified through larger-scale population studies.

[0070] Table 8. Correlation analysis between donor milk production at 305 days and oocyte developmental potential in Holstein multiparous cattle.

[0071] The data in the table are expressed as mean ± standard error. Different letters in the same column indicate significant differences. P <0.05, one-way ANOVA).

[0072] 4. Discussion This embodiment evaluated the effects of several key donor factors, including body condition score, age, parity, postpartum days, and milk yield, on the developmental potential of oocytes from Holstein multiparous cattle. The results showed that body condition score, age, parity, and postpartum days all had significant or highly significant effects on oocyte quality and embryonic development, while milk yield did not show a significant effect within the set range. Significant differences in oocyte quality and in vitro embryonic production efficiency were observed among donor cattle with different physiological states and production performance. These results are consistent with recent research trends in dairy bovine reproductive physiology and oocyte development regulation mechanisms, particularly providing new evidence for practice in areas such as energy balance, oxidative stress, and epigenetic regulation. The findings of this embodiment will be analyzed and discussed in depth below, combining reproductive physiology and energy metabolism theories.

[0073] 4.1 Appropriate body condition score is the energy basis for ensuring oocyte quality and developmental potential. This study found that maintaining the body condition score of donor cows within the range of 2.75-3.25 yielded optimal AB grade oocyte percentage, maturity rate, and blastocyst rate. This result profoundly reveals the intrinsic link between the body's energy reserves and reproductive cell quality. Body condition score in dairy cows assesses their body fat percentage and is considered a crucial factor in dairy cow management by animal scientists and producers. Standards for measuring body condition scores vary by country, but low values ​​generally reflect emaciation, while high values ​​are equivalent to obesity. Studies by Roche et al. have shown that the ideal body condition score for dairy cows is 3.00 ≤ body condition score ≤ 3.50; within this range, their health and reproduction are unaffected. Ovarian tissue directly responds to metabolic signals in response to changes in body condition score, influencing follicle development, steroid production, and oocyte and embryo development. Proper nutritional management before and after calving is essential for successful reproduction; changes in body condition score are closely related to the processes leading to ovulation, estrus, and conception.

[0074] From a reproductive physiology perspective, body condition score is a direct indicator of an organism's energy balance. A low body condition score (e.g., 2 points in this example) indicates that the cow is in a negative energy balance. In this state, to maintain higher-priority physiological functions such as life activities, the function of the HPO axis is inhibited, leading to reduced GnRH secretion, which in turn affects the normal development of follicles and the final maturation of oocytes. Simultaneously, energy deficiency also directly affects the nutrient composition and hormone levels in the follicular fluid, resulting in insufficient reserves of energy substances such as lipids and glycogen in the oocyte cytoplasm, ultimately manifesting as maturation disorders and decreased developmental potential.

[0075] Conversely, excessive obesity, such as a body condition score of 4 in this example, is also detrimental to reproductive performance. Excessive body fat exacerbates insulin resistance in peripheral tissues and disrupts the normal metabolic patterns of gonadal steroid hormones, potentially leading to an increased rate of ovarian follicle atresia. Furthermore, obesity-induced chronic inflammation and elevated oxidative stress levels can directly damage sensitive oocytes. An excessively high body condition score can easily trigger lipid metabolism disorders and insulin resistance, altering the metabolome composition of follicular fluid (such as increased free fatty acids and decreased glucose utilization), thereby interfering with meiosis and embryonic genome activation.

[0076] Therefore, the optimal range of 2.75-3.25 points determined in this embodiment represents the best balance between sufficient energy supply and excessive metabolic burden. This provides a direct and quantifiable theoretical basis for ranches to improve OPU-IVP efficiency through precise nutrition management.

[0077] 4.2 Age in months and parity together revealed the impact of reproductive senescence on the developmental potential of oocytes. The results of this embodiment show that donor cattle under 80 months of age and before the fourth parity can consistently provide high-quality oocytes with stable developmental potential; thereafter, all indicators show a significant decline. This finding clearly outlines the decline curve of Holstein cattle reproductive performance with age and parity, consistent with the general pattern of reproductive aging. When the age exceeds 80 months or the parity is higher than the fourth, the quality of oocytes and blastocyst rate decrease significantly, which is closely related to ovarian reserve depletion and the accumulation of epigenetic errors, consistent with the conclusions of Du et al. in Japanese Black cattle.

[0078] Lunar age and parity are essentially two distinct dimensions of reproductive aging. With increasing age and repeated pregnancies and lactation cycles, the quantity and quality of the ovarian follicle pool naturally decline, a phenomenon known as decreased ovarian reserve. More importantly, oocytes, as long-lived cells, accumulate oxidative damage over their decades-long lifespan, leading to problems such as mitochondrial DNA mutations and telomere shortening. This molecular-level damage weakens the oocyte's ability to support fertilization and early embryonic development, ultimately resulting in a decreased blastocyst formation rate. The age of the oocyte donor can influence the developmental capacity of oocytes retrieved by OPU through the effects of steroid hormone balance on follicle development.

[0079] This embodiment found that the fifth calving is a critical turning point, which coincides with the physiological stage of older cows (80 months old, approximately 6.5 years old). At this time, the cow has not only experienced the physiological stress of multiple calvings and lactations, but her reproductive system may also have accumulated damage. Therefore, in the selection of OPU donors, young multiparous cows under 80 months of age and with no more than four calvings should be given priority to ensure the best embryo production results.

[0080] 4.3 The number of postpartum days is a key time window reflecting the recovery process of uterine-ovarian function. This embodiment clearly demonstrates the significant impact of postpartum days on oocyte quality; OPU performed 50-70 days postpartum yields oocytes of optimal quality with the highest developmental potential. This dynamic process accurately reflects the physiological pattern of postpartum reproductive function recovery.

[0081] In the early postpartum period, cows are in a critical stage of transition from pregnancy and calving to a new reproductive cycle. At this time, uterine involution is not yet complete, and the ovarian cycle is in the recovery period. Early follicular wave development is often asynchronous and unstable, resulting in uneven quality of recruited oocytes. In addition, the strong negative energy balance in the early postpartum period also directly inhibits the function of the reproductive axis.

[0082] As the postpartum period progresses, the uterine environment gradually improves, ovarian function stabilizes, and energy balance is alleviated. By 50-70 days postpartum, the cow has essentially completed its physiological adjustment and entered its optimal reproductive state, at which point the quality of mature follicles and oocytes is naturally at its highest. This conclusion provides precise guidance for determining the scientific timing of OPU initiation, suggesting that major OPU procedures be scheduled after 50 days postpartum. However, for donor cows in the VWP stage (30-70 days postpartum), considering maximizing the number of oocytes retrieved and the number of COCs obtained, the OPU program may begin as early as 40 days postpartum.

[0083] 4.4.4 Correlation analysis between milk yield and oocyte quality One of the most practically significant findings of this embodiment demonstrates that, within a broad range (daily milk yield 20-70 kg, weekly average milk yield 20-70 kg, 305-day milk yield 11-16 t), milk production levels have no significant negative impact on oocyte developmental potential. This challenges the traditional understanding that high milk production inevitably leads to low fertility, providing strong support for selecting high-producing dairy cows as OPU donors. Studies have reported that the demand for high milk production primarily impairs reproductive performance through delayed ovarian activity and reduced conception rates. Rearte reported that the negative correlation between daily milk production and pregnancy risk in dairy cows increases with increasing postpartum days; the relationship between milk production and reproductive performance is statistically significant, but the effect size is actually small and influenced by the herd's production level. This phenomenon can be understood through the body's nutrient allocation priorities and metabolic adaptation mechanisms. Under modern high-producing dairy cow breeding and nutritional management, the body possesses a certain ability to coordinate resource allocation between high milk production and basic reproductive functions. Provided the nutrition supply is sufficient and the management is proper, high-yielding dairy cows can still allocate the necessary resources to their reproductive system to maintain core reproductive functions.

[0084] However, it must be noted that in the highest milk production group (e.g., average weekly milk production of 60-70 kg, 305-day milk production of 15-16 t), all assessment indicators showed a consistent downward trend. This suggests that there may be a physiological threshold for milk production; exceeding this threshold, the metabolic stress brought about by high production (such as oxidative stress and accumulation of metabolic waste) will eventually overwhelm the body's compensatory capacity and begin to erode oocyte quality. Although this trend did not reach a significant level in the sample size of this embodiment, it serves as an important risk warning for practitioners pursuing ultimate breeding efficiency.

[0085] This embodiment has certain limitations: it does not directly detect cellular and molecular indicators such as follicular fluid metabolites, mitochondrial function, or chromosomes; milk yield analysis can be extended to ultra-high-yielding populations (>70 kg / d); despite efforts to standardize management conditions, diet and OPU operations may still introduce confounding effects. Future research can incorporate multi-omics technologies, such as establishing a correlation model between body condition score, follicular fluid metabolites, and embryonic development; analyzing epigenetic aging markers in advanced-age / high-parity oocytes; developing an oocyte quality prediction system based on metabolic markers (such as succinate); and exploring the effects of nutrient interventions such as antioxidants on improving the quality of oocytes from older donors.

[0086] 5. Summary This embodiment, based on a systematic analysis of OPU-IVP results, clarifies the impact of donor factors on the developmental potential of Holstein multiparous bovine oocytes in the VMP stage. The main conclusions are as follows: (1) Body condition score significantly affects oocyte quality and embryonic development, with the optimal range being 2.75–3.25; (2) When the age exceeds 80 months or the parity is higher than 4, the quality of oocytes and the ability of early embryos to develop are significantly reduced; (3) The number of days after calving significantly affects the developmental potential of bovine oocytes, and the ideal time for oocyte retrieval is 50–70 days postpartum; (4) Milk production has no significant impact within the set range, but there is a downward trend under extremely high milk production.

Claims

1. A method for screening bovine oocyte donors, comprising the following steps: To investigate the correlation between donor body condition score, age, parity, postpartum days and milk yield and the developmental potential of bovine oocytes; Physical condition score, age, parity, and postpartum days were determined as selection criteria for donors; Donors are selected based on screening criteria and screening indicators.

2. The screening method according to claim 1, wherein the screening criterion for the donor's physical condition score is 2.75-3.25 points.

3. The screening method according to claim 1 or 2, wherein the screening criterion for donor age is less than or equal to 80 months.

4. The screening method according to any one of claims 1-3, wherein the screening criterion for donor parity is less than or equal to 4 parities.

5. The screening method according to any one of claims 1-4, wherein the screening criterion for the number of days postpartum of the donor is 50-70 days.

6. The screening method according to any one of claims 1-5, wherein the donor milk production includes the donor milk production on the day of egg collection, the donor's average weekly milk production, and the donor's total milk production over 305 days.

7. The screening method according to any one of claims 1-6, wherein the developmental potential of bovine oocytes is reflected in one or more of the following indicators: the percentage of AB grade cumulus-oocyte complexes, oocyte maturation rate, fertilization rate, cleavage rate, and blastocyst rate.

8. The screening method according to any one of claims 1-7, wherein the test donor is a Holstein multiparous cow.

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