Preparation for improving bovine embryo implantation survival rate as well as preparation method and application thereof

Abstract

The invention relates to the technical field of animal husbandry breeding and biology, and particularly discloses a preparation for improving the bovine embryo implantation survival rate and a preparation method and application thereof. The invention provides a preparation for improving the survival rate of bovine embryo implantation. The preparation comprises a preparation A and a preparation B, the preparation A is prepared from the following components in concentration: 5 to 10g / L of cane sugar and 0.2 to 0.8 g / L of immunosuppressant; the solvent is water; the immunosuppressive agent is prepared by mixing inositol and 6-mercaptopurine in a weight ratio of (0.1-1): (0.1-1); the preparation B is prepared from the following components in concentration: 1 to 5g / L of water-soluble chitosan, 1 to 5g / L of rhizoma anemarrhenae extracting solution, 0.5 to 1.5 g / L of water-soluble maca powder and 0.1 to 0.5 g / L of magnesium salicylate; the solvent is normal saline. When the preparation provided by the invention is applied to a bovine embryo transplantation test, the breeding performance of the conception rate, the calving rate and the calf survival rate of a receptor cow can be obviously improved.

Description

Technical Field

[0001] The present application relates to the technical field of animal husbandry and biology, and specifically to a preparation for improving the survival rate of bovine embryo implantation, and a preparation method and application thereof. Background Art

[0002] At present, with the development of animal husbandry and the application of biotechnology, embryo transplantation technology has become an important means to improve the reproductive efficiency of cattle. However, in actual operation, the survival rate of embryos after implantation is affected by many factors, among which rejection is one of the key issues. Existing solutions include improving surgical techniques, but the effect is limited and may bring side effects.

[0003] Therefore, developing a safe and efficient method to reduce rejection reactions and improve the survival rate of bovine embryo implantation is a technical problem that needs to be urgently solved in this field. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a preparation for improving the survival rate of bovine embryo implantation and a preparation method and application thereof.

[0005] The present application provides a preparation for improving the implantation survival rate of bovine embryos, specifically comprising preparation A and preparation B; The preparation A is composed of the following components: sucrose 5-10 g / L, immunosuppressant 0.2-0.8 g / L; the solvent is water; the immunosuppressant is a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.1-1:0.1-1; The preparation B is composed of the following components in the following concentrations: 1-5 g / L water-soluble chitosan, 1-5 g / L Anemarrhena asphodeloides extract, 0.5-1.5 g / L water-soluble maca powder, and 0.1-0.5 g / L magnesium salicylate; the solvent is physiological saline.

[0006] In the technical scheme provided by the present application, preparation A utilizes sucrose as a protective agent, which can help maintain the osmotic pressure balance in the subsequent embryo implantation body, and is conducive to adapting to the intrauterine tissue fluid environment faster after embryo transplantation. Low-dose inositol and 6-mercaptopurine are used to act together as immunosuppressants before embryo transplantation, which has the effect of suppressing the activity of immune cells, can reduce the generation of inflammatory factors and proteins, reduce the in vivo immune response of cows in transplantation experiments and after the test, and reduce the prevention of maternal immune response to adversely affect the embryo, thereby creating an immune environment that is more conducive to embryo implantation and development, and then the conception rate and survival rate of the embryo can be significantly improved. And inositol plays an important role in signal transduction and metabolic regulation in cells, can affect the signaling pathway of endometrial cells, improve endometriosis, enhance endometrial receptivity, and enhance the attachment of embryos to the surface of the uterus to make them more suitable for embryo implantation.

[0007] In addition, after the embryo transplantation experiment, the present application uses water-soluble chitosan, rhizoma anemarrhenae extract, water-soluble maca powder, and magnesium salicylate as preparation B to inject the donor cow. Water-soluble chitosan: has good biocompatibility and biodegradability, can form a protective film, covers the surface of the endometrium, and provides a stable microenvironment for embryo implantation. Rhizoma anemarrhenae extract contains a variety of active ingredients, has anti-inflammatory, antioxidant and immunomodulatory effects, can improve the inflammatory response of the endometrium, and improve the success rate of implantation; water-soluble maca powder is rich in macaamide, macaene, protein, fatty acids, dietary fiber, amino acids, vitamins, minerals and other nutrients and alkaloids, has the effects of regulating endocrine and enhancing immunity, helps to regulate maternal hormone levels, and provides favorable conditions for embryo implantation; magnesium salicylate has sedative, antispasmodic effects, can relieve uterine contraction, and reduce mechanical pressure during implantation. The combined use of water-soluble chitosan, Anemarrhena asphodeloides extract, water-soluble maca powder, and magnesium salicylate can stimulate the production and secretion of progesterone, effectively promote early embryonic development and subsequent pregnancy, and enhance the attachment of the embryo to the surface of the uterus, thereby achieving the effect of improving the conception rate, calving rate, and calf survival rate.

[0008] Preferably, the preparation A is composed of the following components in concentrations: sucrose 6-9 g / L, immunosuppressant 0.4-0.6 g / L; and the solvent is water.

[0009] Preferably, the preparation B is composed of components with the following concentrations: 2-4 g / L water-soluble chitosan, 2-4 g / L Anemarrhena asphodeloides extract, 0.8-1.2 g / L water-soluble maca powder, and 0.2-0.4 g / L magnesium salicylate; and the solvent is normal saline.

[0010] Preferably, in the immunosuppressant, the amount of the inositol is greater than the amount of the 6-mercaptopurine.

[0011] Preferably, the immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.8-1:0.1-0.3.

[0012] Preferably, the preparation method of the Anemarrhena extract is: drying and crushing the Anemarrhena, adding 5-8 times the mass of water of the Anemarrhena dry powder, extracting at 50-80°C for 4-8h, filtering, centrifuging the filtrate, taking the supernatant, and concentrating under reduced pressure to 5-10% of the mass of the supernatant.

[0013] Preferably, the preparation method of the Anemarrhena extract is: drying and crushing the Anemarrhena, adding 5-8 times the mass of water of the Anemarrhena dry powder, extracting at 60-70°C for 5-7h, filtering, centrifuging the filtrate, taking the supernatant, and concentrating under reduced pressure to 5-10% of the mass of the supernatant.

[0014] In a second aspect, the present application provides a method for preparing a preparation for improving the implantation survival rate of bovine embryos, which specifically comprises the following steps in sequence: Preparation of Preparation A: According to the 1L specification, weigh the corresponding weight of each raw material component, then fully dissolve it in 950mL of deionized water, stir and mix evenly, add deionized water to make the volume to 1L, filter and sterilize with a 0.22μm microporous filter membrane to obtain Preparation A; Preparation of Preparation B: According to the 1L specification, weigh the corresponding weight of each raw material component respectively, and then fully dissolve them in physiological saline, stir and mix evenly, add physiological saline to make the volume to 1L, filter and sterilize with a 0.22μm microporous filter membrane to obtain Preparation B.

[0015] In a third aspect, the present application provides a method for improving the implantation survival rate of bovine embryos, characterized in that the preparation for improving the implantation survival rate of bovine embryos according to any one of claims 1 to 8 is used; specifically comprising the following steps in sequence: Preparation A was perfused into the uterus of donor cows in estrus and ovulation, with the perfusion dose of Preparation A being 2-10 g / 100 kg body weight. Embryo transplantation was performed on the donor cows 24-48 hours later. Preparation B was perfused into the donor cows 6-12 hours after embryo transplantation, with the perfusion dose of Preparation B being 0.5-1.5 g / 100 kg body weight. During the experiment, the donor cows were raised normally until they gave birth.

[0016] Preferably, the method for improving the implantation survival rate of bovine embryos specifically comprises the following steps in sequence: Preparation A was perfused into the uterus of donor cows in estrus and ovulation, with the perfusion dose of Preparation A being 4-8 g / 100 kg body weight. Embryo transplantation was performed on the donor cows 32-40 hours later. Preparation B was perfused into the donor cows 8-10 hours after embryo transplantation, with the perfusion dose of Preparation B being 0.8-1.2 g / 100 kg body weight. During the experiment, the donor cows were raised normally until they gave birth.

[0017] In summary, the technical solution of this application has the following effects: The present application utilizes a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.1-1:0.1-1 to form an immunosuppressant, and sucrose as the raw material of preparation A component, and water-soluble chitosan, Anemarrhena extract, water-soluble maca powder, and magnesium salicylate of specific concentrations as the raw materials of preparation B component. Preparation A and preparation B are used in combination in a bovine embryo transplantation experiment, which can significantly improve the breeding performance of the recipient cows in terms of conception rate, calving rate, and calf survival rate. DETAILED DESCRIPTION

[0018] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application.

[0019] Water-soluble chitosan (article number KJT01) and water-soluble maca powder (specification 30:1) were purchased from Xi'an Shizeyuan Biotechnology Co., Ltd.; the remaining raw materials can be obtained commercially. Example

[0020] Examples 1-5 Examples 1-5 respectively provide a preparation for improving the implantation survival rate of bovine embryos and a method for improving the implantation survival rate of bovine embryos.

[0021] The difference between the above embodiments is that the concentrations of the components in preparation A or the concentrations of the components in preparation B are different, as shown in Table 1.

[0022] The preparation method of the preparation for improving the implantation survival rate of bovine embryos in the above embodiment is as follows: Preparation of Preparation A: According to Table 1, based on the 1L specification, weigh the corresponding weight of each raw material component, then fully dissolve it in 950mL deionized water, stir and mix, add deionized water to make the volume to 1L, filter and sterilize with a 0.22μm microporous filter membrane to obtain Preparation A; the immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.9:0.2.

[0023] Preparation of Preparation B: According to Table 1, according to the 1L specification, weigh the corresponding weight of each raw material component respectively, then fully dissolve them in physiological saline, stir and mix to dissolve, add physiological saline to make the volume to 1L, filter and sterilize with a 0.22μm microporous filter membrane to obtain Preparation B; wherein, the preparation method of Anemarrhena extract is: dry and crush 100g of Anemarrhena, add 600g of Anemarrhena dry powder and deionized water, extract at 65°C for 6h, filter, centrifuge the filtrate at 5000rpm for 10min, take the supernatant, and concentrate under reduced pressure to 8% of the mass of the supernatant.

[0024] The method for improving the implantation survival rate of bovine embryos in the above embodiment is: The recipient cows were more than 2 months postpartum. The recipient cows were empty-stomach, non-estrus multiparous cows, and the rest of their shapes were the same as the donor cows. The recipient cows were synchronized. The donor cows on the 5th day of estrus were perfused with preparation A in their uterus. The perfusion dose of preparation A was 6g / 100kg body weight. After 36 hours, the donor cows were transplanted with embryos. 9 hours after the embryo transplantation, the donor cows were perfused with preparation B. The perfusion dose of preparation B was 1g / 100kg body weight. During the experiment, the donor cows were raised normally until they gave birth.

[0025] Table 1 Amount of each component in formulation A and formulation B in Examples 1-5 Embodiment 6-9 Examples 6-9 respectively provide a preparation for improving the implantation survival rate of bovine embryos and a method for improving the implantation survival rate of bovine embryos.

[0026] The difference between the above embodiment and embodiment 1 is that the dosage ratio of inositol and 6-mercaptopurine in the immunosuppressant in preparation A is as follows.

[0027] In Example 6: the immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.1:1.0.

[0028] In Example 7: the immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 1:1.

[0029] In Example 8: the immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.8:0.3.

[0030] In Example 9: the immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 1:0.1.

[0031] The other process parameters in the above embodiment are the same as those in embodiment 1.

[0032] Examples 10-13 Examples 10-13 respectively provide a preparation for improving the implantation survival rate of bovine embryos and a method for improving the implantation survival rate of bovine embryos.

[0033] The difference between the above embodiment and embodiment 1 is that in preparation B, the preparation method of the Anemarrhena asphodeloides extract is specifically as follows.

[0034] In Example 10: The preparation method of the Anemarrhena extract is as follows: dry and crush 100g of Anemarrhena, add 600g of Anemarrhena dry powder and deionized water, extract at 50°C for 8h, filter, centrifuge the filtrate at 5000rpm for 10min, take the supernatant, and concentrate under reduced pressure to 8% of the supernatant mass.

[0035] In Example 11: The preparation method of the Anemarrhena extract is as follows: dry and crush 100g of Anemarrhena, add 600g of Anemarrhena dry powder and deionized water, extract at 80°C for 4h, filter, centrifuge the filtrate at 5000rpm for 10min, take the supernatant, and concentrate under reduced pressure to 8% of the supernatant mass.

[0036] In Example 12: The preparation method of the Anemarrhena extract is as follows: dry and crush 100g of Anemarrhena, add 600g of Anemarrhena dry powder and deionized water, extract at 60°C for 7h, filter, centrifuge the filtrate at 5000rpm for 10min, take the supernatant, and concentrate under reduced pressure to 8% of the supernatant mass.

[0037] In Example 13: The preparation method of the Anemarrhena extract is as follows: dry and crush 100g of Anemarrhena, add 600g of Anemarrhena dry powder and deionized water, extract at 70°C for 5h, filter, centrifuge the filtrate at 5000rpm for 10min, take the supernatant, and concentrate under reduced pressure to 8% of the supernatant mass.

[0038] The other process parameters in the above embodiment are the same as those in embodiment 1.

[0039] Examples 14-15 Examples 14-15 respectively provide a preparation for improving the survival rate of bovine embryo implantation and a method for improving the survival rate of bovine embryo implantation.

[0040] The difference between the above embodiment and embodiment 1 is that the method for improving the implantation survival rate of bovine embryos is specifically as follows.

[0041] In Example 14, cows over 2 months postpartum were used as recipient cows. The recipient cows were empty-stomach, non-estrus multiparous cows, and the rest of their shapes were the same as the donor cows. The recipient cows were synchronized, and the donor cows on the 5th day of estrus were perfused with preparation A in their uterus, with the perfusion dose of preparation A being 6g / 100kg body weight. After 24 hours, the donor cows were transplanted with embryos; 6 hours after the embryo transplantation, the donor cows were perfused with preparation B, with the perfusion dose of preparation B being 1g / 100kg body weight; during the experiment, the donor cows were raised normally until they gave birth.

[0042] In Example 15, cows over 2 months postpartum were used as recipient cows. The recipient cows were empty-stomach, non-estrus multiparous cows, and the rest of their shapes were the same as the donor cows. The recipient cows were synchronized, and the donor cows on the 5th day of estrus were perfused with preparation A in their uterus, with the perfusion dose of preparation A being 6g / 100kg body weight. After 48 hours, the donor cows were transplanted with embryos; 12 hours after the embryo transplantation, the donor cows were perfused with preparation B, with the perfusion dose of preparation B being 1g / 100kg body weight; during the experiment, the donor cows were raised normally until they gave birth.

[0043] The other process parameters in the above embodiment are the same as those in embodiment 1.

[0044] Comparative Example Comparative Examples 1-3 Comparative Examples 1-3 respectively provide a preparation for improving the implantation survival rate of bovine embryos and a method for improving the implantation survival rate of bovine embryos.

[0045] The difference between the comparative example and Example 1 is that the concentrations of the components in Preparation A or the concentrations of the components in Preparation B are different, as shown in Table 1.

[0046] The other process parameters in the above comparative example are the same as those in Example 1.

[0047] Comparative Examples 4-5 Comparative Examples 4-5 respectively provide a preparation for improving the implantation survival rate of bovine embryos and a method for improving the implantation survival rate of bovine embryos.

[0048] The differences between the above comparative example and Example 1 are specifically as follows.

[0049] In Comparative Example 4, the immunosuppressant is composed of a mixture of inositol and azathioprine in a weight ratio of 0.9:0.2.

[0050] In Comparative Example 5: an equal amount of Leonurus japonicus extract was used to replace the Anemarrhena asphodeloides extract; the preparation method of the Anemarrhena asphodeloides extract was as follows: 100 g of Leonurus japonicus was dried and crushed, 600 g of Leonurus japonicus dry powder and deionized water were added, extracted at 65°C for 6 h, filtered, the filtrate was centrifuged at 5000 rpm for 10 min, the supernatant was taken, and reduced pressure was concentrated to 8% of the mass of the supernatant.

[0051] The other process parameters in the above comparative example are the same as those in Example 1.

[0052] Performance testing Evaluation of embryo transplantation performance of bovine embryos: There were 50 recipient cows in each experimental group, and 50 thawed surviving bovine embryos were taken for embryo transplantation.

[0053] Pregnancy judgment and conception rate calculation: blood was collected from the anterior vena cava on the 50th day after embryo transfer. The blood was collected in a centrifuge tube and centrifuged at 3500r / min and 4°C for 15min. The supernatant was taken to test the serum progesterone level, the number of cows that successfully conceived was recorded, and the conception rate was calculated; where conception rate = number of conceptions / 50×100%.

[0054] Group management: pregnant cows are raised separately in groups until they give birth, and their calving rate and calf survival rate are calculated; among which, calving rate = number of calves born / 50×100%, calf survival rate = number of calves alive / number of calves born×100%.

[0055] Test results: as shown in Table 2.

[0056] Table 2 Performance test results of embryo transplantation performance in Examples and Comparative Examples Combined with Table 2, by comparing the test results of the embodiments and the comparative examples, in comparative example 1, preparation A did not add immunosuppressants, and the breeding performance of the conception rate and calving rate of the recipient cows was poor. In comparative example 2, preparation B did not add Anemarrhena extract, water-soluble maca powder, and magnesium salicylate, and the breeding performance of the calving rate and calf survival rate of the recipient cows was poor. In comparative example 3, the concentrations of the components in preparation A or the concentrations of the components in preparation B did not match, and the comprehensive breeding performance of the conception rate, calving rate, and calf survival rate of the recipient cows was poor. In comparative example 4, the immunosuppressant in preparation A was composed of a mixture of inositol and azathioprine in a weight ratio of 0.9:0.2, and the breeding performance of the conception rate and calving rate of the recipient cows was poor. In comparative example 5, preparation B replaced the Anemarrhena extract with an equal amount of Leonurus extract, and the breeding performance of the calving rate and calf survival rate of the recipient cows was poor.

[0057] In contrast, embryo transplantation experiments were conducted on cattle using the preparations provided in the examples of the present application, and the recipient cows had excellent breeding performance in terms of conception rate, calving rate, and calf survival rate.

[0058] 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 may be made thereto 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 preparation for improving the survival rate of bovine embryo implantation, characterized in that: Specifically including preparation A and preparation B; The preparation A is composed of the following components: sucrose 5-10 g / L, immunosuppressant 0.2-0.8 g / L; the solvent is water; the immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.1-1:0.1-1; The preparation B is composed of the following components in the following concentrations: 1-5 g / L water-soluble chitosan, 1-5 g / L Anemarrhena asphodeloides extract, 0.5-1.5 g / L water-soluble maca powder, and 0.1-0.5 g / L magnesium salicylate; the solvent is physiological saline.

2. The preparation for improving the implantation survival rate of bovine embryos according to claim 1, characterized in that: The preparation A is composed of the following components in the following concentrations: sucrose 6-9 g / L, immunosuppressant 0.4-0.6 g / L; the solvent is water.

3. The preparation for improving the implantation survival rate of bovine embryos according to claim 1, characterized in that: The preparation B is composed of the following components in the following concentrations: 2-4 g / L water-soluble chitosan, 2-4 g / L Anemarrhena asphodeloides extract, 0.8-1.2 g / L water-soluble maca powder, and 0.2-0.4 g / L magnesium salicylate; the solvent is physiological saline.

4. The preparation for improving the implantation survival rate of bovine embryos according to claim 1, characterized in that: In the immunosuppressant, the amount of the inositol is greater than the amount of the 6-mercaptopurine.

5. The preparation for improving the implantation survival rate of bovine embryos according to claim 4, characterized in that: The immunosuppressant is composed of a mixture of inositol and 6-mercaptopurine in a weight ratio of 0.8-1:0.1-0.

3.

6. The preparation for improving the implantation survival rate of bovine embryos according to claim 1, characterized in that: The preparation method of the Anemarrhena extract is as follows: drying and crushing the Anemarrhena, adding 5-8 times the mass of water of the Anemarrhena dry powder, extracting at 50-80° C. for 4-8 hours, filtering, centrifuging the filtrate, taking the supernatant, and concentrating under reduced pressure to 5-10% of the mass of the supernatant.

7. The preparation for improving the implantation survival rate of bovine embryos according to claim 6, characterized in that: The preparation method of the Anemarrhena extract is as follows: drying and crushing the Anemarrhena, adding water 5-8 times the mass of the Anemarrhena dry powder, extracting at 60-70° C. for 5-7 hours, filtering, centrifuging the filtrate, taking the supernatant, and concentrating under reduced pressure to 5-10% of the mass of the supernatant.

8. The method for preparing the preparation for improving the implantation survival rate of bovine embryos according to any one of claims 1 to 7, characterized in that: Specifically, the following steps are performed in sequence: Preparation of Preparation A: According to the 1L specification, weigh the corresponding weight of each raw material component, then fully dissolve it in 950mL of deionized water, stir and mix evenly, add deionized water to make the volume to 1L, filter and sterilize with a 0.22μm microporous filter membrane to obtain Preparation A; Preparation of Preparation B: According to the 1L specification, weigh the corresponding weight of each raw material component respectively, and then fully dissolve them in physiological saline, stir and mix evenly, add physiological saline to make the volume to 1L, filter and sterilize with a 0.22μm microporous filter membrane to obtain Preparation B.

9. A method for improving the implantation survival rate of bovine embryos, characterized in that: Using the preparation for improving the implantation survival rate of bovine embryos according to any one of claims 1 to 7; specifically comprising the following steps in sequence: Preparation A was perfused into the uterus of donor cows in estrus and ovulation, with the perfusion dose of Preparation A being 2-10 g / 100 kg body weight. Embryo transplantation was performed on the donor cows 24-48 hours later. Preparation B was perfused into the donor cows 6-12 hours after embryo transplantation, with the perfusion dose of Preparation B being 0.5-1.5 g / 100 kg body weight. During the experiment, the donor cows were raised normally until they gave birth.

10. The method for improving the implantation survival rate of bovine embryos according to claim 9, characterized in that: Specifically, the following steps are performed in sequence: Preparation A was perfused into the uterus of donor cows in estrus and ovulation, with the perfusion dose of Preparation A being 4-8 g / 100 kg body weight. Embryo transplantation was performed on the donor cows 32-40 hours later. Preparation B was perfused into the donor cows 8-10 hours after embryo transplantation, with the perfusion dose of Preparation B being 0.8-1.2 g / 100 kg body weight. During the experiment, the donor cows were raised normally until they gave birth.

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