A method of increasing the availability of recipient cows for timed embryo transfer
Patent Information
- Application Number
- CN202611084251.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-21
- Publication Date
- 2026-09-15
Smart Images
Figure CN122744933A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of mammalian embryo transfer technology, and more particularly to a method for improving the availability of recipient cattle in timed embryo transfer. Background Technology
[0002] Embryo transfer technology, a core tool in bovine reproductive biotechnology, involves transferring early-stage embryos from a superior donor into a physiologically synchronized recipient, enabling pregnancy and delivery. It plays an irreplaceable role in accelerating genetic improvement of superior breeds, expanding the size of superior cattle herds, and enhancing the economic benefits of the livestock industry. Currently, the industrial application of bovine embryo transfer technology in my country still faces bottlenecks. Among these, the low availability of recipient cattle is one of the core issues hindering the effectiveness of technology promotion and limiting the success rate of transplantation and the improvement of reproductive benefits.
[0003] Recipient cow availability refers to the proportion of recipient cows that meet the physiological and health standards for embryo transfer, are capable of receiving embryo transfer, and whose embryo implantation and development can be guaranteed, out of the total number of candidate recipient cows. Its level directly determines the scale efficiency and ultimate profitability of embryo transfer. Recipient cow selection can be based on natural estrus or estrus synchronization via injection, and estrus synchronization programs play a crucial role in improving production efficiency.
[0004] Estrus synchronization technology is based on the physiological characteristics of the estrous cycle of cows. It uses hormones to artificially prolong or shorten the luteal phase in order to regulate follicle development and thus induce estrus and ovulation in the herd of cows. In practice, two main methods are used: progesterone to prolong the luteal phase or prostaglandin to shorten the luteal phase.
[0005] By implanting progesterone suppositories to increase progesterone levels in cows, the synthesis and secretion of gonadotropin-releasing hormone and gonadotropins are inhibited, thus hindering follicle maturation. Upon removal of the suppositories, progesterone levels decrease, follicle development resumes, and most cows exhibit estrus symptoms within a short period, achieving estrus control within the herd. Shortening the luteal phase generally involves injecting prostaglandins and their analogues. Prostaglandins dissolve the corpus luteum, thereby relieving progesterone's inhibitory effect on follicle development and inducing estrus synchronization. GnRH has a follicle-promoting effect and is widely used in estrus synchronization.
[0006] By continuously optimizing the estrus synchronization technology, the estrus rate and corpus luteum quality of a batch of cattle can be improved, thereby increasing the availability of recipient cattle.
[0007] Currently, with the widespread application of embryo transfer technology and its great value in dairy cow breeding, there is a need in this field for new methods that can further improve the synchronization of estrus and ovulation in recipient cows, in order to increase the availability of recipient cows in timed embryo transfer. Summary of the Invention
[0008] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method to improve the availability of recipient cattle in timed embryo transfer.
[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for improving the availability of recipient cattle in timed embryo transfer includes the following steps: S1: Select healthy cows with a body condition score of 3.0-3.5, and then select individuals with normal uterine morphology and normal ovarian size and morphology as recipient cows through ultrasound examination; S2: On the morning of day D0, progesterone plugs were implanted into the recipient cows, and progesterone, estradiol, and vitamins A, D, and E were injected intramuscularly. S3: On the morning of day D9, administer intramuscular injections of cloprostenol, vitamins A, D, and E to the recipient cows, and continue intramuscular injections of cloprostenol in the afternoon. S4: Remove the progesterone plug from the recipient cow on day 12 and flush the cow's vagina with saline. S5: Inject GnRH intramuscularly into recipient cows on day 13, and then observe and record estrus in recipient cows from day 13 to day 15. S6: On day D20, assess the quality of the corpus luteum in recipient cows by rectal examination, labeling grade A, grade B, grade C, and no corpus luteum, and recording whether the corpus luteum is located on the left or right side; S7: On day D20, anesthetize the recipient cow by injecting 5-7 mL of 2% procaine hydrochloride injection solution between the first and second caudal vertebrae. Then, transfer the embryo into the uterine horn on the side of the recipient cow with well-developed corpus luteum. After the transfer, administer progesterone intramuscularly. S8: After transplantation, strengthen the nutrition and feeding management of the recipient cow, conduct regular checks on the pen, pay attention to the return to estrus status of the recipient cow, and perform ultrasound pregnancy checks on the 30th and 60th days after transplantation to confirm the pregnancy status of the recipient cow.
[0010] Preferably, in step S1, after the recipient cow is restrained by a restraint frame, an ultrasound examination is performed on the recipient cow's uterus.
[0011] Furthermore: In S2, the injection volume of progesterone is 100-200 mg, the injection volume of estradiol is 2-6 mg, and the injection volume of vitamins A, D, and E is 10-15 mL.
[0012] Further preferred embodiment: In S3, the cloprostol is injected on the 9th day after thrombectomy, the injection volume of cloprostol is 0.3-0.7 mg, the injection volume of vitamins A, D, and E is 10-15 mL, and the interval between the two cloprostol injections is 12 hours.
[0013] As a preferred embodiment of the present invention: in step S4, 200-250 mL of physiological saline is injected into the vagina of the recipient cow using a vaginal irrigation tube and a large-volume syringe to irrigate and maintain the normal physiological state of the recipient cow's vagina.
[0014] As a further preferred embodiment of the present invention: in S5, the injection dose of GnRH is 100-120 μg, thereby realizing the transformation of the recipient cow from "synchronized estrus" to "synchronized ovulation".
[0015] As a further aspect of the present invention: In S5, the estrus status of the recipient cows is observed from 5:00 to 20:00 on days D13-15, and the stable acceptance of mounting behavior by the cows is used as the standard for estrus. The first estrus date and the last estrus date of each cow are accurately recorded.
[0016] Based on the aforementioned scheme: In S6, the corpus luteum quality of the recipient cow is assessed by combining rectal examination with ultrasound examination.
[0017] Based on the aforementioned scheme, the preferred method is as follows: In S7, based on the last estrus date of the recipient cow, the cows that came into estrus on D13 will undergo embryo transfer on D20; the cows that came into estrus on D14 will undergo embryo transfer on D21; the cows that came into estrus on D15 will undergo embryo transfer on D22. After the transfer, the recipient cows will receive an intramuscular injection of 450-550 mg of progesterone.
[0018] The beneficial effects of this invention are as follows: 1. After implanting progesterone vaginal suppositories into recipient cows, progesterone is injected simultaneously to immediately increase the progesterone concentration in the recipient cow's peripheral blood and maintain the optimal 12-day treatment period, thereby improving the synchronicity of follicle development and creating a stable uterine environment for embryo implantation.
[0019] 2. During thrombectomy, vitamins A, D, and E are injected to provide nutrition for follicle initiation and development, and to improve follicle quality. After thrombectomy, GnRH is injected to promote ovulation and further improve ovulation synchronicity. At the same time as embryo transfer, progesterone is injected again to strengthen corpus luteum function and uterine receptivity. This treatment method can significantly improve the estrus rate and corpus luteum quality of recipient cows, and improve the availability of recipient cows.
[0020] 3. This method improves the concentration of estrus in recipient cows and flexibly schedules embryo transfer time based on the date of the last estrus, ensuring that embryo transfer is synchronized with the corpus luteum development stage. This reduces the risk of embryo transfer delays and corpus luteum degeneration caused by individual differences in estrus time, thereby achieving a higher proportion of available recipient cows and a more stable pregnancy rate in large-scale embryo transfer. Attached Figure Description
[0021] Figure 1This is a flowchart illustrating a method for improving the availability of recipient cattle in timed embryo transfer, as proposed in this invention. Figure 2 This is a schematic diagram of the processing steps of a method for improving the availability of recipient cattle in timed embryo transfer proposed in this invention. Detailed Implementation
[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.
[0023] Example 1: A treatment method to improve the availability of recipient cattle in timed embryo transfer, such as... Figure 1-2 As shown, it includes the following steps: S1: Select healthy cows with a body condition score of 3.0-3.5, and then select individuals with normal uterine morphology and normal ovarian size and morphology as recipient cows through ultrasound examination; S2: On the morning of day D0, progesterone plugs were implanted into the recipient cows, and progesterone, estradiol, and vitamins A, D, and E were injected intramuscularly. S3: On the morning of day D9, administer intramuscular injections of cloprostenol, vitamins A, D, and E to the recipient cows, and continue intramuscular injections of cloprostenol in the afternoon. S4: Remove the progesterone plug from the recipient cow on day 12 and flush the cow's vagina with saline. S5: Inject GnRH intramuscularly into recipient cows on day 13, and then observe and record estrus in recipient cows from day 13 to day 15. S6: On day D20, assess the quality of the corpus luteum in recipient cows by rectal examination, labeling grade A, grade B, grade C, and no corpus luteum, and recording whether the corpus luteum is located on the left or right side; S7: On day D20, anesthetize the recipient cow by injecting 5-7 mL of 2% procaine hydrochloride injection solution between the first and second caudal vertebrae. Then, transfer the embryo into the uterine horn on the side of the recipient cow with well-developed corpus luteum. After the transfer, administer progesterone intramuscularly. S8: After transplantation, strengthen the nutrition and feeding management of the recipient cow, conduct regular checks on the pen, pay attention to the return to estrus status of the recipient cow, and perform ultrasound pregnancy checks on the 30th and 60th days after transplantation to confirm the pregnancy status of the recipient cow.
[0024] In step S1, after the recipient cow is restrained by a restraint frame, an ultrasound examination is performed on the recipient cow's uterus. In S2, the injection volume of progesterone is 100-200mg, the injection volume of estradiol is 2-6mg, and the injection volume of vitamins A, D, and E is 10-15mL. In S3, cloprostol is injected on the 9th day after thrombectomy, the injection volume of cloprostol is 0.3-0.7 mg, the injection volume of vitamins A, D and E is 10-15 mL, and the interval between the two injections of cloprostol is 12 hours. In step S4, 200-250 mL of physiological saline is injected into the vagina of the recipient cow using a vaginal irrigation tube and a large-volume syringe to irrigate it and maintain the normal physiological state of the recipient cow's vagina. In S5, the injection dose of GnRH is 100-120 μg, which realizes the transformation of the recipient cow from "synchronized estrus" to "synchronized ovulation". In S5, the estrus status of the recipient cows is observed from 5:00 to 20:00 on days D13-15. The stable acceptance of mounting behavior by the cows is used as the standard for estrus. The first estrus date and the last estrus date of each cow are accurately recorded. In S6, the corpus luteum quality of the recipient cow is assessed by combining rectal examination with ultrasound examination; In S7, based on the last estrus date of the recipient cow, the cows that came into estrus on D13 will undergo embryo transfer on D20; the cows that came into estrus on D14 will undergo embryo transfer on D21; and the cows that came into estrus on D15 will undergo embryo transfer on D22. After the transfer, the recipient cows will receive an intramuscular injection of 450-550 mg of progesterone.
[0025] By injecting progesterone, a high concentration of progesterone can be provided immediately, forcibly inhibiting follicle development and ensuring the initiation of estrus at the same time. After successful embryo transfer, progesterone injection can maintain progesterone levels and corpus luteum function required for pregnancy, providing a stable hormonal environment for embryo implantation and early development, and improving the success rate of embryo transfer. Injecting vitamins A, D, and E can maintain the health of the reproductive tract mucosa, enhance ovarian function, improve follicle quality, ensure reproductive function, promote normal estrus in cattle, and ensure subsequent conception rate. Extending the embolization time to no more than 12 days allows for the continuous use of exogenous progesterone to maintain the uterus in a resting state, improve endometrial receptivity, and create a stable environment for embryo implantation. At the same time, it avoids the risk of uterine over-maturation and infection caused by prolonged progesterone embolization. Injecting cloprostenol a few days before embolization removal can promote the early dissolution of the corpus luteum. Combined with GnRH injection after embolization removal, it can induce concentrated estrus in the recipient cow after embolization removal.
[0026] In this embodiment, a 1-5 point scale (GB / T41194-2021) was used to score the body condition of the recipient cows; The grading criteria for Grade A corpus luteum are: the corpus luteum is clearly protruding from the surface of the ovary, with a diameter of about 2-2.5 cm and good elasticity; The grading criteria for Grade B corpus luteum are: protruding from the surface of the ovary, with a diameter of 1-1.9 cm and moderate elasticity; The grading criteria for Grade C corpus luteum are: the corpus luteum is not prominent, the diameter is less than 1 cm, the texture is soft, and the elasticity is poor.
[0027] Application Example 1: A method to improve the availability of recipient cows in timed embryo transfer involves first performing superovulation and artificial insemination on the donor cow, and then performing live embryo flushing on the donor cow on the 7th day after insemination to obtain grade A and grade B embryos for later use.
[0028] The recipient cows were then selected: First, the body condition of the cattle was observed, and Angus cattle with a body condition score of 3.0-3.5, smooth coat, appropriate fat coverage on the back, ribs and rump, no prominent bones such as ribs, hip bones, and ischium, no excessive fat deposition in the reproductive organs due to obesity, and no disease characteristics were selected as recipient cows. The recipient cows were then divided into two groups, one as the experimental group and the other as the control group.
[0029] Recipient cow restraint: The recipient cows were herded into restraint frames and their heads were fixed to prevent disturbance. Subsequently, ultrasound examination was used to select individuals with normal uterine morphology, no fluid accumulation, pus, adhesions, or endometritis, moderate uterine wall thickness, normal ovarian size and shape, no cysts, and normal corpus luteum function. The vagina and cervix were free of inflammation, adhesions, or malformations, and mucus secretion was normal. The selected recipient cows were then transferred and separated from other cattle for rearing.
[0030] Estrus synchronization treatment was performed on the recipient cows: On day D0, from 8:00 to 10:00 AM, progesterone suppositories were implanted in recipient cows, and 150 mg of progesterone, 4 mg of estradiol, and 10 mL of vitamins A, D, and E were injected intramuscularly to promote follicle development and inhibit follicle maturation and ovulation, thereby synchronizing the estrous cycles of the recipient cow population. In contrast, recipient cows in the control group were only injected with 4 mg of estradiol.
[0031] At 8:00 AM on Day 9, recipient cows were injected intramuscularly with 0.5 mg of cloprostenol. The experimental group was simultaneously injected intramuscularly with 10 mL of vitamins A, D, and E, while the control group recipient cows were not injected with vitamins A, D, and E. At 16:00 in the afternoon, 0.5 mg of cloprostenol was injected intramuscularly to dissolve the corpus luteum in the recipient cow.
[0032] On day 12, the progesterone suppository was removed to relieve the inhibition on the hypothalamus and pituitary gland, inducing estrus in the recipient cows. 200 mL of physiological saline was injected into the recipient cow's vagina using a vaginal irrigation tube and a large-volume syringe to maintain normal vaginal physiological function.
[0033] At 8:00 AM on Day 13, the recipient cow was injected with 100 μg of GnRH to achieve the transition from "synchronized estrus" to "synchronized ovulation".
[0034] Observe the estrus status of recipient cows from 5:00 to 20:00 on days 13-15. Use the stable acceptance of mounting behavior by the cows as the standard for estrus. Accurately record the first and last estrus dates of each cow.
[0035] D20 assesses the corpus luteum quality of recipient cows using ultrasound examination, marking grade A, grade B, grade C, and no obvious corpus luteum, and recording the side on which the corpus luteum is located.
[0036] Based on the last estrus date of the recipient cow, embryo transfer was performed on day 20 for cows that came into estrus on day 13; on day 21 for cows that came into estrus on day 14; and on day 22 for cows that came into estrus on day 15. The embryo tubes were removed from the liquid nitrogen using tweezers, placed at room temperature (25°C) for 10 seconds, and then immersed in a 37°C constant temperature water bath for 40-60 seconds, before being wiped clean. The embryo tubes were then inserted into the transfer gun and transferred into the uterine horn on the same side as the corpus luteum. After transfer, the recipient cows received an intramuscular injection of 500 mg of progesterone, while the control group recipient cows did not receive such an injection.
[0037] After embryo transfer, ensure the recipient cows receive adequate nutrition to guarantee successful pregnancy and calving. Regularly check the pens, observe for signs of estrus, and promptly perform pregnancy checks. On day 60 post-transfer, perform an ultrasound pregnancy check to confirm the pregnancy rate of the recipient cows.
[0038] Subsequently, the pregnancy outcomes of recipient cows in the experimental group and the control group (who did not receive progesterone and vitamin injections) were statistically compared, and the following table shows the comparison of experimental results: control group 132 71.2%（94 / 132） 36.4%（48 / 132） 28.8%（38 / 132） 65.1%（86 / 132） 41.9%（36 / 86） experimental group 176 89.7%（158 / 176） 72.7%（128 / 176） 11.9%（21 / 176） 84.7%（149 / 176） 67.8%（101 / 149）
[0039] Among them, the recipient availability rate refers to the proportion of cows that meet the conditions for embryo transfer out of the total number of treated cows. The criteria for judgment are that the cows show obvious and normal estrus symptoms after treatment (such as accepting mounting, normal reproductive tract mucus, etc.), and the corpus luteum of the ovary is well developed and has A or B grade corpus luteum that meet the physiological conditions for embryo transfer.
[0040] The pregnancy rate after transplantation is the result of a pregnancy test 60 days after estrus. The pregnancy rate refers to the proportion of pregnant cows out of the total number of available recipient cows.
[0041] The above comparison results show that, based on the implantation of progesterone suppositories and injection of cloprostenol in recipient cows, the injection of progesterone and vitamins A, D, and E increased the rate of grade A corpora lutea in recipient cows from 36.4% to 72.7%, and the recipient availability rate from 65.1% to 84.7%. The pregnancy rate after embryo transfer increased from 41.9% to 67.8%.
[0042] Application Example 2: Referring to the treatment method given in Example 1, the effect of shortening the progesterone suppository treatment time on the availability rate of recipient cows and the pregnancy rate of embryo transfer was studied. The availability rate of recipient cows and the pregnancy rate of embryo transfer were compared between 9 days and 12 days of progesterone suppository treatment time. The procedure for administering progesterone suppositories to recipient cows for 9 days is as follows: On day D0, from 8:00 to 10:00 AM, progesterone suppositories are implanted in recipient cows, and at the same time, 150 mg of progesterone, 4 mg of estradiol, and 10 mL of vitamins A, D, and E are injected intramuscularly.
[0043] On day 7, at 8:00 AM, the recipient cows were given an intramuscular injection of 0.5 mg of cloprostenol and 10 mL of vitamins A, D, and E. At 4:00 PM, they were given another intramuscular injection of 0.5 mg of cloprostenol.
[0044] On day 9, the progesterone suppository was removed, and 200 mL of physiological saline was injected into the recipient cow's vagina using a vaginal irrigation tube and a large-volume syringe for irrigation.
[0045] At 8:00 AM on day 10, the recipient cow was injected with 100 μg of GnRH.
[0046] From 5:00 to 20:00 on days 10-12, observe the estrus status of the recipient cows. Use the stable acceptance of mounting behavior by the cows as the standard for estrus. Accurately record the first and last estrus dates for each cow.
[0047] D17 uses ultrasound to assess the quality of the corpus luteum in recipient cows, marking grade A, grade B, grade C, and no obvious corpus luteum, and recording the side on which the corpus luteum is located.
[0048] Embryo transfer was performed on day 17. After transfer, recipient cows received an intramuscular injection of 500 mg progesterone. The effects of different progesterone suppository administration times on the availability and pregnancy rate of recipient cows were statistically analyzed. The results are shown in the table below:
[0049] The data comparison above shows that shortening the duration of progesterone suppository administration to 9 days reduced the rate of grade A corpus luteum in recipient cows from 74.1% to 45.1%, and the availability rate of recipient cows from 85.2% to 68.6%. The pregnancy rate after embryo transfer decreased from 69.6% to 54.3%. Therefore, a 12-day duration of progesterone suppository administration is more effective.
[0050] The data comparison results of the two application examples above show that this treatment method effectively overcomes the core pain points of low effective utilization rate of embryo transfer recipient cows and declining pregnancy rate after embryo transfer, and significantly improves the overall breeding efficiency and application stability of bovine embryo transfer, which will help to further promote the industrialization of bovine embryo transfer technology in my country.
[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for improving the availability of recipient cattle in timed embryo transfer, characterized in that, Includes the following steps: S1: Select healthy cows with a body condition score of 3.0-3.5, and then select individuals with normal uterine morphology and normal ovarian size and morphology as recipient cows through ultrasound examination; S2: On the morning of day D0, progesterone plugs were implanted into the recipient cows, and progesterone, estradiol, and vitamins A, D, and E were injected intramuscularly. S3: On the morning of day D9, administer intramuscular injections of cloprostenol, vitamins A, D, and E to the recipient cows, and continue intramuscular injections of cloprostenol in the afternoon. S4: Remove the progesterone plug from the recipient cow on day 12 and flush the cow's vagina with saline. S5: Inject GnRH intramuscularly into recipient cows on day 13, and then observe and record estrus in recipient cows from day 13 to day 15. S6: On day D20, assess the quality of the corpus luteum in recipient cows by rectal examination, labeling grade A, grade B, grade C, and no corpus luteum, and recording whether the corpus luteum is located on the left or right side; S7: On day D20, anesthetize the recipient cow by injecting 5-7 mL of 2% procaine hydrochloride injection solution between the first and second caudal vertebrae. Then, transfer the embryo into the uterine horn on the side of the recipient cow with well-developed corpus luteum. After the transfer, administer progesterone intramuscularly. S8: After transplantation, strengthen the nutrition and feeding management of the recipient cow, conduct regular checks on the pen, pay attention to the return to estrus status of the recipient cow, and perform ultrasound pregnancy checks on the 30th and 60th days after transplantation to confirm the pregnancy status of the recipient cow.
2. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 1, characterized in that, In step S1, after the recipient cow is restrained with a restraint frame, an ultrasound examination is performed on the recipient cow's uterus.
3. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 1, characterized in that, In S2, the injection volume of progesterone is 100-200 mg, the injection volume of estradiol is 2-6 mg, and the injection volume of vitamins A, D, and E is 10-15 mL.
4. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 1, characterized in that, In S3, cloprostol is injected on the 9th day after thrombectomy, the injection volume of cloprostol is 0.3-0.7 mg, the injection volume of vitamins A, D, and E is 10-15 mL, and the interval between the two injections of cloprostol is 12 hours.
5. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 1, characterized in that, In step S4, a vaginal irrigation tube and a large-volume syringe are used to inject 200-250 mL of physiological saline into the vagina of the recipient cow to flush it and maintain the normal physiological state of the recipient cow's vagina.
6. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 1, characterized in that, In S5, the injection dose of GnRH is 100-120 μg, which realizes the transformation of the recipient cow from "synchronized estrus" to "synchronized ovulation".
7. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 6, characterized in that, In S5, the estrus status of the recipient cows is observed from 5:00 to 20:00 on days D13-15. The stable acceptance of mounting behavior by the cows is used as the standard for estrus. The first and last estrus dates of each cow are accurately recorded.
8. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 1, characterized in that, In S6, the corpus luteum quality of the recipient cow is assessed by rectal examination combined with ultrasound examination.
9. The method for improving the availability of recipient cattle in timed embryo transfer according to claim 1, characterized in that, In S7, based on the last estrus date of the recipient cow, the cows that came into estrus on D13 will undergo embryo transfer on D20; the cows that came into estrus on D14 will undergo embryo transfer on D21; and the cows that came into estrus on D15 will undergo embryo transfer on D22. After the transfer, the recipient cows will receive an intramuscular injection of 450-550 mg of progesterone.