An embryonic life element and its application in improving animal reproductive performance

Abstract

The present invention relates to the field of animal breeding technology, and in particular to an embryonic vital element and its application in improving animal reproductive performance. The embryonic vital element comprises: butyric acid or its derivatives, methionine, NCG and resveratrol; the mass ratio of butyric acid or its derivatives, methionine and NCG is (5-15): (2.5-60): (1-15); the mass percentage of resveratrol is 0.01-1%. The present invention promotes steroid hormone synthesis, adhesion factor secretion and embryonic trophoblast development in the process of animal embryonic development through the synergistic effect between multiple key substances, thereby improving the reproductive performance of animals. The embryonic vital element provided by the present invention can effectively improve the reproductive performance of mammals such as embryo implantation rate, litter size, litter live piglets or litter weight, which is of great significance for improving the production efficiency of the pig farming industry.

Description

Technical Field

[0001] The invention relates to the technical field of animal reproduction, in particular to an embryo vital element and application thereof in improving animal reproduction performance. Background Art

[0002] The reproductive performance of sows is very important to the production efficiency of the pig farming industry, and the unsatisfactory development of early embryos will significantly affect the embryo survival rate and pregnancy outcomes, which in turn has a negative impact on the reproductive performance of sows. Poor development of early embryos may lead to an increase in early embryo mortality, limiting the reproductive performance of sows.

[0003] In order to ensure the healthy development of the sow's early embryo, it is very important to provide a good maternal nutritional environment. Ensuring adequate and balanced nutrition supply before conception and early pregnancy is essential to support optimal embryo development. Sows need to obtain sufficient nutrients such as protein, energy, vitamins and minerals to support the normal growth and development of the embryo. In addition, factors such as sow feeding management, environmental conditions and disease management also affect embryonic development and reproductive performance.

[0004] At present, some studies have proposed a variety of nutrients related to animal embryonic development, such as protein, amino acids, NCG or other nutrients, which have certain potential to improve animal reproductive performance, but the degree of improvement is limited. It is still necessary to explore strategies or intervention methods to more significantly improve animal reproductive performance. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an embryo life element and its application in improving the reproductive performance of animals. Through the synergistic effect of multiple key substances, the reproductive performance of animals is significantly improved.

[0006] In a first aspect, the present invention provides a composition comprising: butyric acid or a derivative thereof, methionine, NCG and resveratrol;

[0007] The mass ratio of butyric acid or its derivative, methionine and NCG is (5-15):(2.5-60):(1-15);

[0008] The mass percentage of the resveratrol is 0.01-1%.

[0009] Sodium butyrate, as a short-chain fatty acid, can be absorbed and utilized by cells, and has a regulatory effect on gene expression related to the embryonic development process; methionine is an amino acid related to embryonic development, which is important for the synthesis of proteins related to the embryonic development process, and is also involved in maintaining the normal cell metabolism; N-carbamylglutamate (hereinafter referred to as NCG) is also involved in the expression of key genes in the physiological metabolism process of the embryo implantation period. The present invention combines the three, significantly promoting the synthesis of key proteins in the embryonic development stage, significantly promoting the synthesis of steroid hormones, the secretion of adhesion factors and the development of embryonic trophoblasts during the animal embryonic development process, and thus improving multiple reproductive performances such as embryo implantation rate.

[0010] Furthermore, the mass ratio of butyric acid or its derivatives, methionine, and NCG is (8-12): (4-60): (2-15).

[0011] Within a further defined preferred range, the three can work better synergistically to improve the reproductive performance of sows.

[0012] Furthermore, the mass percentage of the resveratrol is 0.02-0.5%.

[0013] Resveratrol has antioxidant and anti-inflammatory functions. It can improve ovarian function through specific signal pathways and improve embryonic development potential. The present invention further compounds resveratrol on the basis of butyric acid or its derivatives, methionine and NCG to further improve the reproductive performance of animals. However, the anti-inflammatory properties of resveratrol may inhibit the decidual process related to inflammation, resulting in reduced endometrial receptivity, so the dosage should not be too high.

[0014] In a second aspect, the present invention provides an embryonic vital element, which comprises the composition.

[0015] Furthermore, in terms of mass percentage, the embryonic vital elements include 0.08-0.12% sodium butyrate, 0.04%-0.6% methionine, 0.02-0.15% NCG and 0.02-0.5% resveratrol.

[0016] In a third aspect, the present invention provides an animal feed, comprising the composition or the embryo vital element.

[0017] In a fourth aspect, the present invention provides the use of the composition, or the embryo vital element, or the animal feed in improving the reproductive performance of animals.

[0018] Furthermore, the reproductive performance includes: one or more of embryo implantation rate, reproduction rate, litter size, litter live number or litter weight.

[0019] The present invention further provides the use of the composition, the embryo vital element, or the animal feed in promoting steroid hormone synthesis, adhesion factor secretion or embryo trophoblast development during animal embryo development.

[0020] Furthermore, the animal is a mammal, preferably a rat or a pig.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a composition that can synergistically improve the survival rate of early embryos and the number of embryo implantation. The multiple key components in the composition synergistically enhance the effect by regulating the physiological metabolic process of the endometrium, promoting progesterone synthesis, and improving the embryo environment, thereby significantly improving the reproductive performance of animals, such as increasing the litter size, the litter live number and the litter weight, and the degree of improvement is significant, exceeding the improvement effect of general nutrients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a schematic diagram of the changes in genes related to ovarian steroid hormone synthesis after embryonic vitalin treatment provided in Example 1 of the present invention.

[0025] Figure 2 This is a schematic diagram of the changes in genes related to endometrial adhesion factor secretion after being treated with embryonic vitalin provided in Example 1 of the present invention.

[0026] Figure 3 This is a schematic diagram of changes in genes related to early embryo development after treatment with embryo vitalin provided in Example 1 of the present invention.

[0027] Figure 4 It is a schematic diagram of the change of embryo adhesion rate after the embryo adhesion test is carried out using the culture medium of the experimental group and the control group provided in Example 1 of the present invention.

[0028] Figure 5 This is a schematic diagram of changes in early embryo survival rate and embryo implantation number after feeding a diet containing embryo vital elements provided by Example 2 of the present invention.

[0029] Figure 6 This is a schematic diagram of the changes in the levels of estrogen and progesterone in the serum of rats during the embryo implantation period after being fed a diet containing embryo vital elements provided by Example 2 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] This example provides a method for verifying the effect of embryonic vital elements on sow reproductive related cells. The specific process is as follows:

[0033] 1. Test materials

[0034] The porcine trophectoderm cells (pTc), porcine ovarian granulosa cells (pGCs), and porcine endometrial epithelial cells (pEECs) used in this experiment were all from the laboratory of Professor Zeng Xiangfang, College of Animal Science and Technology, China Agricultural University.

[0035] 2. Test methods

[0036] 2.1 Preparation of embryo vital essence culture medium

[0037] Add 5 mL of FBS and 0.5 mL of penicillin-streptomycin mixture to a 50 mL centrifuge tube, add DMEM high-glucose medium to 50 mL, mix thoroughly, and obtain complete cell culture medium containing 10% FBS, 100 U / mL penicillin and 100 U / mL streptomycin. Then add 5.5 mg of sodium butyrate, 1.86 mg of methionine, 0.95 mg of NCG, and 50 μl of 10 mM resveratrol.

[0038] The final concentrations of embryo vital element medium are: 1 mM sodium butyrate, 0.25 mM methionine, 0.1 mM NCG, 10 μM resveratrol.

[0039] 2.2 Gene expression assay

[0040] pTc cells, pGCs cells and pEECs cells were inoculated into 6-well plates respectively, and when the cell confluence reached 80%, they were cultured for 12 hours with ordinary culture medium and embryonic vital element culture medium (ordinary culture medium is the embryonic vital element culture medium in 2.1 above without embryonic vital element component), cells were collected with trizol, RNA was extracted, and RNA was reverse transcribed with Polymer reverse transcription kit. Subsequently, the expression of genes related to reproduction in the three cells was detected respectively.

[0041] 2.3 Adhesion test

[0042] The pEECs cells were inoculated into a 6-well plate and cultured with a culture medium for 12 hours. When the cell confluence reached 80%, the pTc cells cultured in the culture medium for 12 hours were counted (A1) and inoculated onto the pEECs cells to simulate the embryo adhesion process for 1 hour. Subsequently, the non-adherent pTc cells were gently washed off with PBS and counted with a cell counter (A2). The embryo adhesion rate was calculated as (A1-A2) / A1*100%.

[0043] 3. Experimental results

[0044] 3.1 Gene expression

[0045] In order to explore the molecular mechanism by which embryonic vitalin affects the reproductive performance of sows, the present invention determined the expression of embryonic vitalin on reproductive-related cell functional genes from the aspects of ovarian steroid hormone synthesis, endometrial adhesion factor secretion, early embryonic development and embryo adhesion.

[0046] The results of the effect of embryonic vital factors on ovarian steroid hormone synthesis are as follows Figure 1 As shown, the results showed that embryonic vitalin promoted the STAR gene and CYP19A1 gene of pig ovarian granulosa cells, but had no promoting effect on CYP11A1, CYP17A1, FSHR and LHR. STAR gene and CYP19A1 are both key genes for the synthesis of estrogen and progesterone.

[0047] The results of the effect of embryo vital factors on the secretion of endometrial adhesion factors are as follows Figure 2 As shown, the results showed that embryonic vitalin promoted the Hoxa10 gene and LIF gene of pig endometrial epithelial cells, among which the expression of LIF gene was significantly promoted. This gene encodes leukemia inhibitor factor and is an important signal factor in the embryonic adhesion window period.

[0048] The effects of embryo vital factors on early embryonic development are as follows Figure 3As shown, the results showed that embryonic vitalin promoted the GATA3 gene of pig embryonic trophoblast cells, which is a key gene for trophoblast development. At the same time, embryonic vitalin promoted the expression of amino acid transporter and glucose transporter LAT1, ASCT2 and GLUT4 genes.

[0049] 3.2 Adhesion test

[0050] In order to further verify the effect of embryonic vital essence on embryo implantation, the present invention adopts the adhesion test shown in 2.3 for detection. The experimental group and the control group use different culture media. The experimental group uses embryonic vital essence culture medium (the same as step 2.1), sodium butyrate culture medium (all embryonic vital essence components in embryonic vital essence culture medium are replaced with equal mass of sodium butyrate), methionine culture medium (all embryonic vital essence components in embryonic vital essence culture medium are replaced with equal mass of methionine), NCG culture medium (all embryonic vital essence components in embryonic vital essence culture medium are replaced with equal mass of NGC), resveratrol culture medium (all embryonic vital essence components in embryonic vital essence culture medium are replaced with equal mass of resveratrol) and control group (all embryonic vital essence components are removed from embryonic vital essence culture medium).

[0051] The results are as follows Figure 4 As shown in Table 2, Figure 4 It can be seen that the embryo adhesion rate of the control group is 59.8%, and the embryo adhesion rate of the embryo life essence group is 71.1%, and the improvement rate is significantly improved to 18.9%. The improvement rate results of each experimental group are shown in Table 1, from which it can be seen that embryo life essence can significantly improve the embryo adhesion rate, and the effect is better than the sum of different components.

[0052] Table 1 Results of embryo adhesion rate in the control group and different experimental groups

[0053] Group Embryo adhesion rate improvement Sodium butyrate medium 1.20％ Methionine medium 12.29％ NCG Medium 6.83％ Resveratrol culture medium 15.54％ Embryo Life Medium 18.90％

[0054] The results showed that Embryo Vitality can significantly improve embryo adhesion rate, and the effect is better than the sum of different components.

[0055] The above results indicate that embryo vitalin may improve the reproductive performance of sows by promoting steroid hormone synthesis, adhesion factor secretion and embryo trophoblast development, thereby increasing embryo adhesion rate.

[0056] Example 2

[0057] This example provides the effect of embryo vital factors on the reproductive performance of maternal mice. The specific process is as follows:

[0058] 1. Test methods

[0059] (1) This experiment selected healthy 8-week-old female primiparous SD rats weighing 180-220 g, purchased from Beijing Huafukang Biotechnology Co., Ltd. After acclimation for 3 days, the female SD rats were caged with male SD rats of the same species with normal fertility at a ratio of 3:1 at 8:00 pm. Vaginal smears were performed on the female rats at 8:00 am the next day, and the presence of sperm was observed under a microscope. If there was sperm, it was recorded as the first day of pregnancy.

[0060] Forty pregnant rats were randomly divided into two groups, a control group and an embryo vital element group, with 20 replicates in each group.

[0061] The rats in the embryonic life element group were fed with pelleted feed containing embryonic life element (including 0.1% sodium butyrate, 0.6% methionine, 0.1% NCG, and 0.05% resveratrol by mass percentage) from 1 to 7 days of gestation, and the rats in the control group were fed with pelleted feed of standard clean-grade breeding mouse feed from 1 to 7 days of gestation. Feed processing was completed at Beijing Huafukang Biotechnology Co., Ltd.

[0062] (2) Sample collection

[0063] On the 7th day of pregnancy, all rats were anesthetized with sodium pentobarbital, blood was collected from the anterior vena cava, the abdominal cavity was opened, the uterus was exposed, the embryo implantation site tissue and ovarian tissue were collected, and the number of embryo implantations was recorded.

[0064] (3) Serum hormone measurement

[0065] The serum estradiol concentration was measured using a radioimmunoassay kit (HY-10029).The serum estradiol concentration was measured using a radioimmunoassay kit (HY-10029).

[0066] 2. Results

[0067] (1) Production performance

[0068] The results are as follows Figure 5 As shown in the figure, on the seventh day of pregnancy, the number of embryo implantation in the control group of SD rats was 12.63, while that in the embryo life element group was 14.06, an increase of 1.43 compared with the control group, with a significant difference (P<0.01). This indicates that feeding embryo life element diet in early pregnancy can significantly increase the survival rate of early embryos and the number of embryo implantation.

[0069] (2) Serum results

[0070] The addition of embryo vital factors to the diet during early pregnancy affected the levels of estrogen and progesterone in the serum of rats during the embryo implantation period. Figure 6As shown, on the seventh day of pregnancy, the serum estrogen levels in the control group and the embryo life element group were 9.83pg / mL and 24.10pg / mL, respectively, and the statistical test showed a significant difference (P<0.05); the serum progesterone levels in the control group and the embryo life element group were 12.27ng / mL and 26.52ng / mL, respectively, and the statistical test showed an extremely significant difference (P<0.01).

[0071] Example 3

[0072] This example verifies the effect of embryonic vital elements on the reproductive performance of sows.

[0073] 1. Test methods

[0074] The experiment selected 100 (Long × Large) binary hybrid sows and randomly divided them into 5 groups, 20 in each group, according to the sow back fat, parity and reproductive performance of the first three litters. The control group and the experimental group were fed with early pregnancy diets from 1 to 28 days of pregnancy. There were 4 experimental groups, and sodium butyrate, methionine, NCG and embryo life element were added to the control group diet, and the addition amount was so that the final mass percentage was 0.46%. Embryo life element included 0.1% sodium butyrate, 0.16% methionine, 0.15% NCG and 0.05% resveratrol, and the total was also 0.46%. After that, they were fed the same pregnant sow diet until delivery. The nutritional level of the diet meets the nutritional needs of pregnant sows in GB / T 39235-2020 National Standard for Nutrient Requirements of Pigs. The dietary formulas of the control group and the embryo life element group are shown in Table 2.

[0075] Table 2 Diet composition of embryo vital element group

[0076]

[0077]

[0078]

[0079] 2. Feeding and management

[0080] The experiment was conducted at the Fengning Animal Experimental Base of the National Feed Engineering Technology Research Center and the Fengning Experimental Station of China Agricultural University (Chengde Jiuyun Agriculture and Animal Husbandry Co., Ltd.). The experimental pigs were raised in a closed pig house with a cement floor and good ventilation. Disinfection, deworming and immunization were strictly carried out in accordance with the routine procedures of the pig farm. All sows were raised in stalls (2.2×0.65m 2) and fed twice a day at 06:00 in the morning and 16:00 in the afternoon. Feed 2.5kg / d in the early pregnancy and 3.5kg / d in the late pregnancy. The day of mating was recorded as the 0th day of pregnancy. From the 1st day to the 28th day of pregnancy, the corresponding diet was fed according to the grouping. All sows ate the diet every day. Water was free to drink during the experiment.

[0081] 3. Data Collection

[0082] On the day of sow farrowing, litter size, number of live piglets born, stillbirths, mummies, piglet sex, birth weight and litter weight were recorded.

[0083] 4. Results

[0084] The test results are shown in Table 1, litter size improvement rate = (average litter size of experimental group - average litter size of control group) / litter size of control group; litter weight improvement rate = (average litter weight of experimental group - average litter weight of control group) / average litter weight of control group.

[0085] Table 3 Comparison of litter size and litter weight between the control group and each experimental group

[0086] Group Increase in litter size Litter weight increase rate Embryo Life 14.88％ 16.75％ Sodium Butyrate 10.26％ 3.31％ Methionine 8.91％ 5.83％ NCG 10.12％ 13.31％

[0087] The results showed that compared with the control group, the litter size and litter weight of sows fed embryonic vital diet in early pregnancy were significantly increased. This shows that feeding embryonic vital can effectively improve the reproductive performance of sows.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An embryo vital element, characterized in that: The embryonic vital elements include: butyrate, methionine, NCG, and resveratrol; The mass ratio of butyric acid, methionine and NCG is (8-12): (4-60): (2-15); The mass percentage of the resveratrol is 0.05-0.5%.

2. The embryo vital element according to claim 1, characterized in that Calculated by mass percentage, it includes 0.08%-0.12% sodium butyrate, 0.04%-0.6% methionine, 0.02%-0.15% NCG and 0.05%-0.5% resveratrol.

3. An animal feed, characterized in that: The animal feed comprises the embryo vital element according to claim 1.

4. Use of the embryo vital element according to claim 1 or 2, or the animal feed according to claim 3, in the preparation of a medicine for improving the reproductive performance of animals.

5. The use according to claim 4, characterized in that: The reproductive performance includes: one or more of embryo implantation rate, litter size, litter live number or litter weight.

6. The use according to claim 4, characterized in that: The reproductive performance is the reproductive rate.

7. The use according to any one of claims 4 to 6, characterized in that: The animal is a mammal.

8. The use according to claim 7, characterized in that: The animal is a rat or a pig.

Images