Method for improving timed insemination efficiency of dairy cow

Abstract

The invention provides a method for improving the timed insemination efficiency of cows, which comprises the following steps: injecting GnRH for the first time on the nth (n is 33-39) day after delivery of cows, and embedding CIDR at the same time; on the (n + 7) th day, PG is injected for the first time, and CIDR is withdrawn at the same time; injecting GnRH for the second time on the (n + 7 + 3) th day; injecting GnRH for the third time on the (n + 7 + 3 + 7) th day; injecting PG again on the (n + 7 + 3 + 7 + 5) th day; after PG is injected again for 56 h, GnRH injection is conducted for the fourth time; after 14-16 h of the fourth GnRH injection, artificial semen deposition is carried out; and carrying out fifth GnRH injection on the seventh day after artificial insemination, and respectively injecting ketoprofen on the fifteenth night and the sixteenth morning after artificial insemination. According to the method, the timing insemination pregnancy rate of the dairy cow can be remarkably increased, the timing insemination pregnancy rate of the dairy cow under the heat stress condition can also be increased, and the method has important economic value and social value for guaranteeing healthy development of dairy cow breeding enterprises.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry and breeding, and in particular relates to a method for improving the efficiency of timed insemination of dairy cows. Background Art

[0002] In recent years, the total raw milk production of major dairy producing countries in the world has continued to decline. With the increase in costs and the tight supply and demand of raw milk, the global dairy product prices will generally rise in 2022. The production capacity of the domestic dairy breeding industry will continue to grow in 2022, with a total milk output of 39.32 million tons, a year-on-year increase of 3.8%, and a cow's single yield of 9.2 tons, an increase of 0.5 tons from 2021, but the problem of tight milk sources in my country still exists. With the continuous development of the dairy breeding industry, the improvement of the single yield of dairy cows has also brought many problems, and the decline in the reproductive capacity of dairy cows after delivery is the most significant. High-yielding dairy cows have problems such as no estrus, quiet estrus and decreased conception rate after delivery. The estrus detection rate is less than 50%, and the conception rate has also dropped from more than 50% in the 1950s to about 35%, and even dropped to about 25% in summer. As a result, the number of offspring in high-yielding dairy cows has decreased, the spacing between dairy cows has increased, and the breeding cost has increased. Therefore, how to improve the estrus rate and estrus disclosure rate of dairy cows has become a crucial issue for the development of my country's dairy industry.

[0003] In recent decades, the Ovsynch and timed Artificial Insemination (Ovsynch-TAI) technology using gonadotropin-releasing hormone (GnRH) and prostaglandin F2α (PGF2α) has played a significant role in improving the estrus rate, estrus disclosure rate and conception rate of cows. The Ovsynch-TAI technology uses exogenous reproductive hormones or analogs to program large-scale cows, which can make these cows synchronize estrus and ovulate in a short period of time, and artificial insemination can be performed at a relatively fixed time without estrus identification. This technology is not only suitable for cows with normal estrus cycles, but also for some cows with abnormal estrus cycles, and can have a therapeutic effect.

[0004] Although researchers have conducted a lot of research, the efficiency of timed insemination is still not high, and the conception rate during estrus still needs to be improved. Previous studies have shown that CIDR can inhibit the development of the current dominant follicle and help the formation of a new follicle wave. GnRH can promote luteal cells to produce more progesterone, and ketoprofen inhibits cyclooxygenase (COX-1, COX-2) involved in the synthesis of PGF 2α and thus prevents luteal lysis. Summary of the invention

[0005] The purpose of the present invention is to provide a method for improving the efficiency of timed insemination of dairy cows, which specifically adopts CIDR, GnRH, ketoprofen and the like to improve the fertility of dairy cows under timed insemination conditions.

[0006] In order to achieve the purpose of the present invention, the present invention provides a method for improving the efficiency of timed insemination of dairy cows. The method comprises the following steps: GnRH (gonadotropin-releasing hormone) is injected for the first time on the nth day after the cow gives birth, and CIDR (cattle progesterone vaginal suppository) is buried at the same time; PG (prostaglandin) is injected for the first time on the nth day after the cow gives birth, and CIDR is withdrawn at the same time; GnRH is injected for the second time on the nth day after the cow gives birth; GnRH is injected for the third time on the nth day after the cow gives birth; PG is injected again on the nth day after the cow gives birth; PG is injected again on the nth day after the cow gives birth; artificial insemination (AI) is performed 14-16 hours after the fourth GnRH injection; the fifth GnRH is injected on the 7th day after the artificial insemination, and ketoprofen is injected respectively on the evening of the 15th day and the morning of the 16th day after the artificial insemination.

[0007] In the present invention, n is 33-39, preferably 39.

[0008] Furthermore, the cattle are cows that have given birth 2-4 times.

[0009] Furthermore, the cattle are dairy cows, preferably Holstein cows.

[0010] Furthermore, the amount of GnRH injected each time is 100ug / head.

[0011] Furthermore, the amount of PG injected each time was 0.6 mg / head.

[0012] Furthermore, the injection volume of ketoprofen per time is 10 mL / head.

[0013] Furthermore, the insemination volume is 0.25 mL of semen, and the effective sperm volume infused each time is not less than 12 million.

[0014] The time for cattle pregnancy examination is 28 days after artificial insemination.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention provides a method for improving the efficiency of timed insemination of dairy cows. Compared with the double synchronous timed insemination procedure, the method can improve the pregnancy rate of dairy cows by 18%. Under heat stress conditions, the pregnancy rate of dairy cows can be improved by 15%. The method can greatly improve the breeding efficiency of large-scale cattle farms and increase the economic and social benefits of ranches. DETAILED DESCRIPTION

[0016] The present invention provides a method for improving the efficiency of timed insemination of dairy cows. On the 33rd to 39th day after the cows give birth, the following double-synchronous timed insemination procedure is adopted, specifically: injecting GnRH (gonadotropin-releasing hormone) and simultaneously burying CIDR. Days 40-46: that is, on the 7th day (7D) after the injection of GnRH, prostaglandin (PG) is injected, and CIDR is withdrawn at the same time. Days 43-49: that is, on the 3rd day (3D) after the injection of PG, GnRH is injected again. Days 50-56: that is, on the 7th day (7D) after the second GnRH injection, GnRH is injected again. Days 57-63: that is, on the 5th day (5th) after the third GnRH injection, prostaglandin (PG) is injected. Timed insemination: that is, artificial insemination (AI) is performed 14-16 hours (14-16h) after the last PG injection.

[0017] Among them, the injection volume of GnRH each time is 100ug / head; the injection volume of PG each time is 0.6mg / head; the injection volume of ketoprofen each time is 10mL / head.

[0018] Furthermore, the insemination volume is 0.25 mL of semen, and the effective sperm volume infused each time is not less than 12 million.

[0019] Furthermore, the cattle are cows that have given birth 2-4 times.

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0021] The GnRH and PG used in the following examples were obtained from Ningbo Sansheng Biotechnology Co., Ltd., ketoprofen was obtained from Foshan Nanhai Dongfang Aolong Pharmaceutical Co., Ltd. (each mL contained 0.15 g ketoprofen), and CIDR was obtained from Zoetis Biopharmaceutical Co., Ltd.

[0022] Terms used in this invention: Double synchronization: two injections are given according to the sequence of "GnRH-PG-GnRH".

[0023] Example 1 1. Experimental Methods 1. Cattle selection and grouping The experimental cattle were healthy Chinese Holstein cows with normal body condition and estrus cycle from 2 to 4 parities. Each batch of cattle to be treated was randomly divided into three groups for operation, and the experiment was repeated more than 3 times.

[0024] 2. Experimental treatment The experimental treatments were as follows (five groups in total): (1) Double synchronization: 39 days after calving: Double synchronization timed insemination procedure, specifically: injection of GnRH (gonadotropin-releasing hormone). 46 days: 7 days after GnRH injection (7D), prostaglandin (PG) injection. 49 days: 3 days after PG injection (3D), GnRH injection again. 56 days: 7 days after the second GnRH injection (7D), GnRH injection again. 63 days: 5 days after the third GnRH injection (5th), prostaglandin (PG) injection. Timed insemination: Artificial insemination (AI) 14-16 hours (14-16h) after PG injection.

[0025] Among them, the injection volume of GnRH each time is 100ug / head; the injection volume of PG each time is 0.6mg / head; the injection volume of ketoprofen each time is 10mL / head.

[0026] (2) Double synchronization + GnRH: GnRH (100ug / head) is injected on the 7th day after scheduled insemination.

[0027] (3) Double synchronization + ketoprofen: Inject ketoprofen (10 ml / time / head) on the evening of the 15th day and the morning of the 16th day after scheduled insemination.

[0028] (4) Double synchronization + GnRH + ketoprofen: GnRH (100ug / head) is injected on the 7th day after scheduled insemination, and ketoprofen (10ml / time / head) is injected on the evening of the 15th day and the morning of the 16th day after scheduled insemination.

[0029] (5) CIDR+bisynchronous+GnRH+ketoprofen: During the bisynchronous process, the CIDR is implanted at the same time as the first injection of GnRH, and then the CIDR is withdrawn at the same time as the first injection of PG. Other procedures proceed normally. GnRH (100ug / head) is injected on the 7th day after scheduled insemination, and ketoprofen (10ml / time / head) is injected on the evening of the 15th day and the morning of the 16th day after scheduled insemination.

[0030] For the above five groups, the insemination volume of each group was 0.25 mL of semen, and the effective sperm volume infused each time was not less than 112 million.

[0031] 3. Pregnancy test On the 28th day after insemination, B-ultrasound was used to detect pregnancy in cattle.

[0032] 4. Blood collection On days 0, 7, 14, 17, and 21 after insemination, 10 ml of blood was collected from each animal, and serum was collected and stored at −80°C for hormone and growth factor detection.

[0033] 5. Hormone detection in serum Progesterone test: Take out the required strips from the aluminum foil bag (Jiangsu Enzyme Free Industrial Co., Ltd.), and seal the remaining strips with a ziplock bag and put them back in the refrigerator. Set up standard wells, blank wells, and sample wells. Add 50μL of different concentrations of standard wells, no standard wells, and 50μL of the sample to be tested to the sample wells. In addition to the blank wells, add 100μL of the detection antigen labeled with horseradish peroxidase (HRP) to the standard wells and sample wells. Cover the reaction plate with a sealing film and incubate in a 37℃ water bath or incubator for 60min. Remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 20s, shake off the washing solution, pat dry on absorbent paper, and repeat this 5 times. If using an automatic plate washer, please wash the plate according to the operating procedures of the plate washer. Adding a soaking procedure for 30s can improve the accuracy of the test. After washing the plate, before adding the substrate, pat the reaction plate dry on a clean, non-chipping paper. Mix substrate A and B in a 1:1 volume ratio and add 100μL of the substrate mixture to all wells. Cover the reaction plate with a sealing film and incubate in a 37℃ water bath or incubator for 15min. Add 50μL of the stop solution to all wells and read the absorbance (OD value) of each well on a microplate reader. Use the standard concentration as the horizontal axis and the corresponding absorbance (OD value) as the vertical axis. Use computer software and four-parameter logistic curve fitting (4-pl) to create a standard curve equation. Use the absorbance (OD value) of the sample to calculate the concentration value of the sample using the equation.

[0034] Estrogen detection: The operation steps are carried out according to the ELISA kit (Jiangsu ELISA Industrial Co., Ltd.). Specifically, take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a self-sealing bag and return them to 4°C. Set up standard wells and sample wells, add 50μL of different concentrations of standard wells to each standard well; add 50μL of the sample to be tested to the sample well; do not add to the blank well. Except for the blank well, add 100μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and incubate in a 37°C water bath or thermostat for 60min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing 5 times (you can also use a plate washer to wash the plate). Add 50μL of substrate A and B to each well and incubate at 37°C in the dark for 15min. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes. Use the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis to draw a standard curve on coordinate paper or using relevant software, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.

[0035] IGF-1 (insulin-like growth factor I) detection: Take out the required strips (Jiangsu Enzyme-Free Industrial Co., Ltd.) from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a ziplock bag and return them to 4°C. Set up standard wells and sample wells, add 50μL of different concentrations of standard wells to each standard well; add 50μL of the sample to be tested to the sample well; and do not add blank wells. Except for the blank wells, add 100μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 60min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate). Add 50μL of substrate A (hydrogen peroxide) and B (tetramethylbenzidine) to each well, and incubate at 37°C in the dark for 15min. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes. Use the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis to draw a standard curve on coordinate paper or using relevant software, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.

[0036] 2. Experimental Results As shown in Table 1, the pregnancy rate in the CIDR+bisynchronous+GnRH+ketoprofen group (55.20±5.32%) was significantly higher than that in the bisynchronous+GnRH+ketoprofen group (48.18±4.81%), bisynchronous+ketoprofen group (43.68±2.57), bisynchronous+GnRH group (42.13±3.45%), and bisynchronous group (37.62±2.46%; P<0.05).

[0037] Table 1 Effects of GnRH, ketoprofen and CIDR on the efficiency of double synchronous timed insemination

[0038] Note: Different lowercase letters / shoulder letters indicate significant differences. The same below.

[0039] Studies have shown that progesterone (P4) in serum after mating is helpful for embryo pregnancy. As shown in Table 2, at each sampling time, the content of P4 in the CIDR+double synchronization+GnRH+ketoprofen group was significantly higher than that in the double synchronization group.

[0040] Table 2 Effects of GnRH, ketoprofen, and CIDR on P4 levels (ng / ml)

[0041] Studies have shown that excessive levels of E2 (estradiol) in serum after mating are not conducive to embryonic pregnancy. As shown in Table 3, at each sampling time, there was no significant difference in the E2 content between the CIDR+bi-synchronous group and the bi-synchronous group.

[0042] Table 3 Effects of GnRH, ketoprofen and CIDR on E2 levels (ng / ml)

[0043] Studies have confirmed that P4 / E2 in bovine serum after mating is positively correlated with pregnancy. As shown in Table 4, at each sampling time, P4 / E2 in the CIDR+bisynchronous+GnRH+ketoprofen group was significantly higher than that in the bisynchronous group. On the 21st day, P4 / E2 in the CIDR+bisynchronous+GnRH+ketoprofen group was 2.18 times that of the control group. This result helps to explain the increased pregnancy rate in the CIDR+bisynchronous+GnRH+ketoprofen group.

[0044] Table 4 Effect of GnRH injection on P4 / E2 on the 7th day after double synchronous timed insemination

[0045] IGF-1 can promote the proliferation and differentiation of embryonic cells and is essential for the early development of the embryo. IGF-1 plays a role in the formation and function of the placenta, contributing to the normal development and functional maintenance of the placenta. IGF-1 is also involved in regulating maternal and fetal metabolism, including the metabolism of sugars and lipids, which is essential for maintaining energy balance during pregnancy and healthy fetal development. As shown in Table 5, at each sampling time point, the concentration of IGF-1 in the CIDR+bisynchronous+GnRH+ketoprofen group was significantly higher than that in the control group.

[0046] Table 5 Effect of IGF-1 concentration (ng / ml) on day 7 after double synchronous timed insemination

[0047] As shown in Table 6, when the THI index was >72 in August, the CIDR+bisynchronous+GnRH+ketoprofen group and the bisynchronous group showed that the pregnancy rate in the CIDR+bisynchronous+GnRH+ketoprofen group was significantly increased. This provides an effective solution to the problem of dairy cow reproduction under summer heat stress conditions.

[0048] Table 6 Comparison of timed insemination efficiency under heat stress conditions

[0049] Except for the experiment in Table 6, all other experiments were conducted in autumn, winter or spring; the experiment in Table 6 was conducted under heat stress conditions in summer.

[0050] Example 2 This example compares the effects of different doses of ketoprofen on the effect of timed insemination. As shown in Table 7, the pregnancy rate of the double synchronous group + 10ml ketoprofen (42.53±3.47%) was significantly higher than that of the other groups (30.49±2.48%, 35.29±4.15%) and the double synchronous group (35.00±2.87%; P<0.05).

[0051] Table 7 Optimization of ketoprofen injection amount

[0052] The effects of different doses of GnRH and PG on the effect of timed insemination were further compared. As shown in Table 8, the pregnancy rate of the double synchronous group (100ug / head GnRH + 0.6 mg / head PG) (34.74±2.62%) was significantly higher than that of the other groups (28.41±2.47%, 26.67±3.47%; P<0.05).

[0053] Table 8 Optimization of ketoprofen injection volume

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for improving the efficiency of timed insemination of dairy cows, characterized in that: On the nth day after calving, the cows were injected with GnRH for the first time and CIDR was buried at the same time; on the n+7th day, PG was injected for the first time and CIDR was withdrawn at the same time; on the n+7+3th day, GnRH was injected for the second time; on the n+7+3+7th day, GnRH was injected for the third time; on the n+7+3+7+5th day, PG was injected again; 56h after the second PG injection, the fourth GnRH injection was performed; 14-16h after the fourth GnRH injection, artificial insemination was performed; the fifth GnRH injection was performed on the 7th day after artificial insemination, and ketoprofen was injected on the evening of the 15th day and the morning of the 16th day after artificial insemination respectively; Among them, n is 33-39.

2. The method according to claim 1, characterized in that The cattle are cows that have given birth 2 to 4 times.

3. The method according to claim 1, characterized in that The cattle are dairy cows.

4. The method according to claim 1, characterized in that: The cattle are Holstein cows.

5. The method according to claim 1, characterized in that The amount of GnRH injected each time is 100ug / head.

6. The method according to claim 1, characterized in that The injection volume of PG is 0.6 mg / head each time.

7. The method according to claim 1, characterized in that The injection volume of ketoprofen was 10 mL per head.

8. The method according to claim 1, characterized in that n is 39.

9. The method according to claim 1, characterized in that: The insemination volume is 0.25 mL of semen, and the effective sperm volume in each insemination is not less than 12 million.

10. The method according to any one of claims 1 to 9, characterized in that: The time for checking cattle pregnancy was 28 days after artificial insemination.

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