Method for improving embryo implantation status or reproductive performance

Abstract

A method for improving embryo implantation status or reproductive performance, for use in applying lactic acid or salt thereof to improve the embryo implantation status or reproductive performance, and finally mitigating the problems that the embryo implantation rate decreases and the reproductive performance is poor after exogenous gonadotropin treatment. The method has wide application prospects, for example, the method can be used in animal husbandry to achieve the regulation and management of the reproduction of livestock such as pigs, horses, cattle and sheep, and can also be used in female assisted reproduction, and thus the method has high application value.

Description

A method for improving embryo implantation or reproductive performance

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to patent applications with application number “CN 2023118396816” filed on December 28, 2023, and invention name “A method for improving embryo implantation or reproductive performance”, and with application number “CN 2024106532941” filed on May 24, 2024, and invention name “A method for improving embryo implantation or reproductive performance”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The invention relates to the technical field of sexual reproduction, and specifically provides a method for improving embryo implantation or reproduction performance. Background Art

[0004] Gonadotropins are glycoprotein hormones that regulate the development of mammalian gonads and gametes and promote the production and secretion of sex hormones. In human assisted reproduction, exogenous follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG, used to replace luteinizing hormone (LH) to stimulate ovulation) are often used to induce superovulation in patients to obtain more eggs. In the livestock industry, due to cost and half-life considerations, equine chorionic gonadotropin (eCG, also known as pregnant mare serum gonadotropin, abbreviated as PMSG) and gonadotropin-releasing hormone analogs (GnRH analogs, used to induce endogenous LH release) are often used to induce synchronous follicular development and ovulation in livestock, achieving intensive reproductive management.

[0005] Exogenous gonadotropin treatment is widely used in assisted reproductive technology and is also widely used in livestock production, such as embryo transplantation to reduce the risk of disease transmission, estrus synchronization, accelerated livestock genetic improvement and protection of endangered livestock genetic resources. Although exogenous gonadotropin treatment has such a broad application space, it also has many problems. For example, ovarian hyperstimulation often leads to an increase in the proportion of degenerated embryos, an increase in the rate of prenatal miscarriage, and a decrease in the developmental quality of offspring (Ziecik AJ, Biallowicz M, Kaczmarek M, Demianowicz W, Rioperez J, Wasielak M, Bogacki M. Influence of estrus synchronization of prepubertal gilts on embryo quality. J Reprod Dev. 2005 Jun; 51(3): 379-84; Laster DB. Ovulation, fertility and prenatal mortality in heifers treated with PMSG or porcine FSH. J Reprod Fertil. 1973 May; 33(2): 275-82.).

[0006] In response to the above problems, the prior art discloses that N-acetylcysteine ​​can improve the pregnancy rate of in vitro fertilization-embryo transfer by relieving uterine contractions (EP2916914B1, CN104768614B); in addition, N-acetylcysteine ​​can specifically improve the uterine environment induced by exogenous gonadotropins and improve the ability of early embryonic development, increase the number of implanted embryos, and has the ability to increase the litter size of female livestock (CN107333704A), or improve and improve the reproductive performance of female animals using precision sow batch production, effectively improving the breeding pregnancy rate of sows, etc. The prior art also discloses that the results can be improved or partially rescued by the application of exogenous progesterone supplementation or some traditional Chinese herbal medicine formulas. In order to provide higher economic benefits to livestock breeding, the improvement of the results of exogenous gonadotropin treatment has always been a problem that the animal husbandry industry needs to continuously improve and solve.

[0007] In addition, in the field of assisted reproduction, frozen embryo transfer is often used to reduce the impact of exogenous gonadotropins on uterine receptivity and pregnancy outcomes. That is, after a woman receives exogenous gonadotropin treatment and undergoes live oocyte retrieval, the IVF embryos (fresh embryos) obtained are first frozen and preserved. When the woman's uterus gradually returns to a state not treated with exogenous gonadotropins, the frozen embryos are transferred at an appropriate time to improve the pregnancy rate. However, embryo freezing has a certain impact on embryonic development. However, the causes and specific molecular regulatory mechanisms of gonadotropin-induced embryo loss, post-implantation fetal growth restriction, and decreased pregnancy rates have not been clear. Therefore, there has been a lack of measures that can effectively improve endometrial receptivity and pregnancy outcomes after exogenous gonadotropin treatment. Summary of the Invention

[0008] The purpose of the present application is to provide a method for improving embryo implantation or reproductive performance by administering lactic acid or its salts to improve embryo implantation or reproductive performance.

[0009] The present application provides a method for improving embryo implantation or reproductive performance for non-disease treatment purposes, characterized by comprising the steps of administering lactic acid or a salt thereof to a pregnant mother. The pregnant mother is preferably a pregnant mother treated with exogenous gonadotropins. The dosage of lactic acid or a salt thereof is preferably 0.1-2.0 mg / kg.BW (meaning 0.1-2.0 mg per kg of the pregnant mother's body weight), preferably 0.2-1.5 mg / kg.BW, and more preferably 0.3-1.0 or 0.3-0.5 mg / kg.BW.

[0010] Lactic acid or its salt solution, such as a lactic acid-PBS solution, can be administered; preferably a 0.5-2 mM lactic acid solution, more preferably a 1 mM lactic acid solution. Lactic acid or its salt can be administered orally or by injection (e.g., intraperitoneal injection). Lactic acid or its salt can also be added to the diet, such as feed and drinking water.

[0011] In one embodiment, lactic acid or its salt is applied during the embryo implantation period. For livestock, the embryo implantation period of pigs, horses, cattle, and sheep is generally between the 7th to 9th day after insemination and the 21st to 23rd day, and the embryo implantation period of rabbits is generally between the 4th to 6th day after insemination and the 10th to 12th day. For experimental animals, the embryo implantation period of mice is generally between the 3rd day of gestation and the 1st to 3rd day after embryogenesis, the embryo implantation period of monkeys is generally between the 5th to 7th day after insemination and the 12th to 14th day, and the embryo implantation period of rabbits is generally between the 4th to 6th day after insemination and the 10th to 12th day. Preferably, lactic acid or its salt is applied every day during the embryo implantation period.

[0012] As used herein, "insemination" refers to natural mating or artificial insemination.

[0013] In one embodiment, the method comprises the steps of administering lactic acid or a salt thereof to the pregnant mother from day 3 of pregnancy to embryo implantation or administering lactic acid or a salt thereof to the pregnant mother from day 3 of pregnancy to day 1-3 after embryo implantation; preferably, administering lactic acid or a salt thereof every day.

[0014] In one embodiment, the method comprises the following steps, for a pregnant mother treated with exogenous gonadotropins and conceived, administering lactic acid or a salt thereof to the pregnant mother during the embryo peri-implantation period. For livestock, the embryo peri-implantation period of pigs, horses, cattle, and sheep is generally between the 7th to 9th day and the 21st to 23rd day after insemination, and the embryo peri-implantation period of rabbits is generally between the 4th to 6th day and the 10th to 12th day after insemination. For experimental animals, the embryo peri-implantation period of mice is generally between the 3rd day of gestation and the 1st to 3rd day after embryo implantation, the embryo peri-implantation period of monkeys is generally between the 5th to 7th day and the 12th to 14th day after insemination, and the embryo peri-implantation period of rabbits is generally between the 4th to 6th day and the 10th to 12th day after insemination. Preferably, lactic acid or a salt thereof is administered every day during the embryo peri-implantation period. Preferably, lactic acid or a salt thereof is administered to the pregnant mother from day 3 of pregnancy to the period of embryo implantation, or from day 3 of pregnancy to day 1-3 after embryo implantation; preferably, lactic acid or a salt thereof is administered daily. More specifically, lactic acid or a salt thereof is administered to the pregnant mother after conception, starting from day 3 of pregnancy, for 2-5 consecutive days (2, 3, 4, or 5), such as continuously administered on day 3 and day 4 of pregnancy, or continuously administered on day 3, day 4, day 5, and day 6 of pregnancy.

[0015] This application also provides the use of the aforementioned method for improving embryo implantation or reproductive performance in the breeding of livestock and experimental animals, especially livestock breeding, such as cattle, sheep, pigs, horses, rabbits, experimental monkeys, experimental mice, and experimental rabbits.

[0016] The present application can solve the problem of reduced embryo implantation rate caused by exogenous gonadotropin treatment by administering lactic acid or its salts, and can also improve breeding performance.

[0017] This application studies mice treated with exogenous gonadotropins before conception and after conception, by continuously administering lactic acid or its salt (such as a lactic acid-PBS solution, preferably 100 μL of a 1 mM lactic acid-PBS solution) on days 3 and 4 of gestation. Observation of the ovarian ovulation site and embryo implantation on day 5 (embryogenesis and implantation) revealed that embryo implantation was significantly improved after exogenous gonadotropin treatment. Specifically, exogenous gonadotropin treatment can be performed on days 3 and 1 before conception to induce follicular development and ovulation.

[0018] The present application treats mice treated with exogenous gonadotropins and after conception, continuously intraperitoneally injects lactic acid or its salt (such as lactic acid-PBS solution, preferably 100 μL of 1 mM lactic acid-PBS solution) on the 3rd, 4th, 5th, and 6th days of pregnancy; the results after delivery show that the weight of newborn pups treated with exogenous gonadotropins is significantly increased, the proportion of abnormal fetuses is also significantly reduced, and reproductive performance is significantly improved. Specifically, exogenous gonadotropins can be treated on the 3rd and 1st days before conception to induce follicular development and ovulation.

[0019] The present application also provides the use of lactic acid or its salts in the preparation of a medicament for improving embryo implantation or reproductive performance; in particular, the use of lactic acid or its salts in the preparation of a medicament for improving embryo implantation or reproductive performance after treatment with exogenous gonadotropins. The medicament is for use in humans, livestock, and experimental animals, and in particular, for livestock.

[0020] The present application also provides non-therapeutic uses of lactic acid or its salts for improving embryo implantation or reproductive performance, particularly for improving embryo implantation or reproductive performance after exogenous gonadotropin treatment. Such uses are in livestock and experimental animals, particularly livestock.

[0021] The lactic acid or its salt described herein can be L-lactic acid or its salt. The exogenous gonadotropin described herein can be at least one of PMSG (pregnant mare serum gonadotropin), hCG (human chorionic gonadotropin), follicle stimulating hormone (FSH), luteinizing hormone (LH), gonadorelin, buserelin, recombinant porcine luteinizing hormone, clomiphene, chorionic gonadotropin, postmenopausal gonadotropin, gonadotropin-releasing hormone and gonadotropin-releasing hormone agonist. The exogenous gonadotropin described herein is treated with the mother before conception, such as on the 3rd day, 2nd day, or 1st day before conception.

[0022] The present application also provides the aforementioned method for improving embryo implantation or reproductive performance, and its use in sow production, particularly in batch production. Preferably, lactic acid or a salt thereof is administered to the sow between days 7-9 and days 21-23 after natural mating or artificial insemination. Preferably, lactic acid or a salt thereof is administered orally to the sow, such as by adding lactic acid or a salt thereof to the sow's diet, such as feed or drinking water.

[0023] The present application also provides a sow batch production method, which is characterized in that it includes the following steps: 1) synchronizing the sexual cycle of the sow; 2) administering exogenous gonadotropin to the sow to induce synchronization of follicle development; 3) administering ovulation-inducing drugs to the sow; 4) performing artificial insemination on the sow; 5) administering lactic acid or its salt to the sow during the peri-implantation period of the embryo.

[0024] In one embodiment, in step 5), the dosage of lactic acid or its salt is 0.1-2.0 mg / Kg.BW, preferably 0.2-1.5 mg / Kg.BW, more preferably 0.3-1.0, 0.3-0.5 mg / Kg.BW.

[0025] In one embodiment, in step 5), lactic acid or a salt thereof is administered daily, preferably between days 7-9 and 21-23 after artificial insemination. Preferably, lactic acid or a salt thereof is administered orally to the sow, such as by adding lactic acid or a salt thereof to the diet, such as feed or drinking water. Liquid lactic acid or a salt thereof or a solution thereof, such as a lactic acid PBS solution, may be added.

[0026] In one embodiment, in step 1), the sow is administered alprogesterone for sexual cycle synchronization. Preferably, the sow is orally administered alprogesterone for sexual cycle synchronization; preferably, the sow is administered alprogesterone continuously for 15-20 days, such as for 18 consecutive days.

[0027] In one embodiment, in step 2), exogenous gonadotropin is administered 36-48 hours, such as 42 hours, after the sexual cycle synchronization treatment to induce synchronization of follicular development. Preferably, the exogenous gonadotropin is injected. The exogenous gonadotropin is preferably PMSG.

[0028] In one embodiment, in step 3), an ovulation drug is administered 70-90 hours, such as 80 hours, after the administration of exogenous gonadotropin. Preferably, the ovulation drug is injected. The ovulation drug is preferably gonadorelin (GnRH).

[0029] In one embodiment, in step 4), artificial insemination is performed after 20-28 hours, such as 24 hours, after the administration of the ovulation drug; preferably, the second artificial insemination is performed after 14-18 hours, such as 16 hours.

[0030] Compared with the prior art, this application has the following beneficial effects:

[0031] The present application significantly improves embryo implantation and reproductive performance by administering lactic acid or its salts to pregnant mothers. In particular, administration of lactic acid or its salts to pregnant mothers treated with exogenous gonadotropins can effectively improve endometrial receptivity and pregnancy outcomes, such as embryo implantation rate, pregnancy rate, and litter size, in female mammals treated with exogenous gonadotropins, ultimately correcting the reduced embryo implantation rate and poor reproductive performance associated with exogenous gonadotropin treatment.

[0032] This invention provides an important solution to the problems of mid-gestation embryo loss and reduced litter size caused by exogenous gonadotropins during the promotion of precision sow batch production. This invention effectively improves the pregnancy rate, farrowing rate, number of live pigs born per litter, and litter index of sows in precision sow batch production, providing an effective method for reducing costs and increasing efficiency for the entire pig farming industry. Therefore, this invention has significant economic and social value in promoting batch production of live pigs in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is an analysis of NLRP3 inflammasome signaling activation in the peri-implantation endometrium of mice induced by exogenous gonadotropin.

[0034] (a) Expression of NLRP3 signaling pathway-related molecules in the uterus of mice in natural estrus and treated with exogenous gonadotropins on day 4 of gestation; (b and c) Expression levels of NLRP3 signaling-related proteins in the uterus of mice in natural estrus and treated with exogenous gonadotropins on day 4 of gestation; (df) Western blotting results of NLRP3 signaling-related proteins in the uterus of mice in natural estrus and treated with exogenous gonadotropins on day 4 of gestation; (g and h) ELISA assay for IL-1β and IL-18 levels in the endometrium of mice in natural estrus and treated with exogenous gonadotropins on day 4 of gestation. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01.

[0035] Figure 2 shows the changes in lactic acid content in the mouse endometrium during the peri-implantation period after treatment with exogenous gonadotropin. *p<0.05.

[0036] Figure 3 is an analysis of lactic acid or its salts inhibiting gonadotropin-induced overactivation of NLRP3 inflammasome signaling in the peri-implantation endometrium of mice.

[0037] (a) Statistical graph of embryo implantation rates in mice supplemented with exogenous lactate or its salts; (b and c) Effects of exogenous lactate or its salts on immunofluorescence expression of NLRP3 inflammasome signaling pathway molecules in the endometrium of gonadotropin-treated mice during the periimplantation period; (df) Effects of exogenous lactate or its salts on expression of NLRP3 inflammasome signaling pathway molecules in the endometrium of gonadotropin-treated mice during the periimplantation period; (g and h) Effects of exogenous lactate or its salts on the levels of IL-1β and IL-18, key targets of the NLRP3 inflammasome, in the endometrium of gonadotropin-treated mice during the periimplantation period. NC represents the control group, OS represents the exogenous gonadotropin-treated experimental group 1, and OS+L represents the exogenous gonadotropin-treated group followed by injection of lactate-PBS solution. **p<0.01, ***p<0.001.

[0038] Figure 4 shows the implantation status of mouse embryos.

[0039] NC was the control group, OS was the exogenous gonadotropin-treated group, and OS+L was the exogenous gonadotropin-treated group followed by injection of 1 mM lactic acid-PBS solution.

[0040] FIG5 is a graph showing the average litter size of mice.

[0041] NC was the control group, OS was the exogenous gonadotropin-treated group, and OS+L was the exogenous gonadotropin-treated group followed by injection of 1 mM lactic acid-PBS solution.

[0042] FIG6 is a graph showing the average birth weight of newborn mice.

[0043] NC is the control group, OS is the exogenous gonadotropin treatment group, and OS+L is the exogenous gonadotropin treatment group followed by injection of 1 mM lactic acid-PBS solution.

[0044] FIG7 is a diagram showing the proportion of abnormal fetuses in newborn mice.

[0045] NC was the control group, OS was the exogenous gonadotropin-treated group, and OS+L was the exogenous gonadotropin-treated group followed by injection of 1 mM lactic acid-PBS solution. DETAILED DESCRIPTION

[0046] The following is a detailed description of the specific embodiments of the present application. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application and are not intended to limit the present application.

[0047] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0048]

[0026] Throughout this application, the terms "include" and "comprises" and any variations thereof, are intended to cover a non-exclusive inclusion.

[0049] 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.

[0050] In the following examples using mice as a model, lactic acid was purchased from Sigma, product number L6661-100ml. Lactic acid for sow batch production was purchased from Ningxia Shuangta Chemical Co., Ltd. with a purity of 98%.

[0051] Example 1

[0052] This example uses a mouse model to study the molecular mechanism of action of lactic acid or its salts

[0053] 1. Experimental Animals

[0054] All experimental mice in this experiment were purchased from Beijing Sibeifu Company. The female mice were 8 weeks old and the male mice were 10 weeks old. Since purchase, the experimental mice have been kept in a light-controlled environment (12 hours of light: 12 hours of darkness). There are no special experimental requirements. All mice have free access to food and water, and the breeding environment is controlled at 20-24°C.

[0055] 2. Experimental Design

[0056] 2.1 Gonadotropin treatment affects changes in molecular pathways of uterine inflammatory signals

[0057] For animal experiments, ICR female mice (SPF, 8 weeks old, 28-30g) were randomly divided into two groups: a normal control group (NC) and a gonadotropin-treated group (OS), with 10-18 mice per group. The animals were housed at 22°C, on a 12-hour day / night cycle, with free access to food and water. Following purchase, the animals underwent a 5-7-day acclimatization period.

[0058] Control group (NC): Female 8-week-old ICR mice were used as research subjects. They were naturally estrus and mated with male mice. After the plug was seen, they served as the control group. Then, on the 4th day after the plug was seen, when uterine receptivity was established, the mouse uterus was isolated and multiple signal molecules were detected.

[0059] Gonadotropin-treated group (OS): Female 8-week-old ICR mice were used as research subjects. They were injected with 10 IU PMSG at 18:00 in the afternoon, and 10 IU hCG was injected 48 hours later to induce superovulation. Then they were caged with male mice. The vaginal plug was checked the next morning. After the plug was found, they were designated as the gonadotropin-treated group. Subsequently, on the 4th day after the plug was found, when uterine receptivity was established, the mouse uterus was isolated and multiple signal molecules were detected.

[0060] Endometrial samples from mice in the NC group and OS group on the fourth day of pregnancy were collected for molecular testing. The detection indicators were changes in the expression of molecules related to NLRP3 inflammasome assembly and activation, as well as changes in the content of lactic acid or its salts, to explore the molecular mechanism by which exogenous gonadotropins affect endometrial inflammation and thus lead to decreased uterine receptivity.

[0061] 2.2 Molecular regulatory effects of lactic acid or its salts

[0062] 1) Experimental animal grouping

[0063] For animal experiments, ICR female mice (SPF, 8 weeks old, 28-30g) were randomly divided into three groups: a normal control group (NC), a gonadotropin-treated group (OS), and a gonadotropin-treated and lactate-administered group (OS+L), with 10-12 mice per group. The animals were housed at 22°C, on a 12-hour day / night cycle, with free access to food and water. Following purchase, the animals underwent a 5-7-day acclimatization period.

[0064] Control group (NC): Female 8-week-old ICR mice were used as research subjects and mated with male mice in natural estrus as the control group. Then, on the 4th day after uterine plugging, when uterine receptivity was established, the mouse uterus was isolated and multiple signal molecules were detected.

[0065] Gonadotropin-treated group (NC): Female 8-week-old ICR mice were used as research subjects. They were injected with 10 IU PMSG at 18:00 in the afternoon, and 10 IU hCG was injected 48 hours later to induce superovulation. Then they were caged with male mice. The vaginal plug was checked the next morning. The mice were designated as the gonadotropin-treated group after the plug was found. Subsequently, on the fourth day after the plug was found, when uterine receptivity was established, the mouse uterus was isolated and multiple signal molecules were detected.

[0066] Gonadotropin treatment and lactic acid and its salts administration group (OS+L): Female 8-week-old ICR mice were used as research subjects, and 10 IU PMSG was injected at 18:00 in the afternoon, and 10 IU hCG was injected 48 hours later to induce superovulation. Then they were caged with male mice, and the vaginal plug was checked the next morning. After the plug was found, they were used as the gonadotropin treatment group. Subsequently, on the afternoon of the third day after the plug was found, lactic acid-PBS solution (0.3 mg / kg.BW) was supplemented. On the fourth day after the plug was found, when uterine receptivity was established, the mouse uterus was isolated and multiple signal molecules were detected.

[0067] 2) Molecular marker detection

[0068] Endometrial samples from mice in the NC, OS, and OS+L groups on the fourth day of pregnancy were collected for molecular testing to detect changes in the expression of molecules related to NLRP3 inflammasome assembly and activation, and to explore the molecular mechanism by which lactate or its salts alleviate endometrial inflammation induced by exogenous gonadotropins.

[0069] 3. Experimental Results

[0070] Molecular detection experiments found that exogenous gonadotropin treatment led to the assembly and activation of the "NLRP3 inflammasome" in the endometrium (Figure 1), which in turn caused abnormal endometrial receptivity. Therefore, the present invention uses "inhibiting the excessive activation of the NLRP3 inflammasome signaling pathway" as a new target for improving excessive endometrial inflammation caused by exogenous gonadotropin treatment. Further studies found that the lactic acid content decreased (Figure 2), for which the present invention uses lactic acid or its salts as candidate drugs. Further signal molecule detection found that lactic acid or its salts can significantly inhibit the assembly and activation of the "NLRP3 inflammasome" (Figure 3).

[0071] Example 2

[0072] This example uses a mouse model to study the effect of lactic acid or its salts on alleviating embryo implantation in mice after gonadotropin treatment.

[0073] 1. Preparation of Lactic Acid-PBS Solution

[0074] L-lactic acid or its salt solution stored at room temperature was diluted in cell-grade PBS solution to prepare a 100 mM lactic acid-PBS solution. The 100 mM lactic acid-PBS solution was then gradiently diluted to prepare 0.04 mM, 0.2 mM, 1 mM, and 5 mM lactic acid-PBS solutions.

[0075] 2. Drug treatment

[0076] For animal experiments, ICR female mice were randomly divided into three groups: a normal control group (NC), a gonadotropin-treated group (OS), and a gonadotropin-treated group plus different doses of lactate (OS+L), with 10-12 mice per group. The animals were housed at 22°C, with a 12-hour day and night cycle, and had free access to food and water. Following purchase, the animals underwent a 5-7-day acclimatization period.

[0077] Control group: 6-8 week old, SPF (specific pathogen free) sexually mature ICR mice were co-bred with adult ICR male mice between 5:00 PM and 6:00 PM on the first day. Female mice with vaginal plugs detected the following morning were collected and placed in a single cage, marking the first day of pregnancy. On the morning of the fifth day of pregnancy, 1% direct blue-saline solution (MACKLIN, D823399) was injected into the tail vein. Three minutes later, the mice were sacrificed by cervical dislocation and exsanguination performed via the abdominal artery. Embryonic implantation was observed, and the number of ovulation sites on both ovaries was counted.

[0078] Experimental Group 1: 6-8 week old, sexually mature, SPF-qualified ICR mice were treated with exogenous gonadotropins. Specifically, 10 units of PMSG (pregnant mare serum gonadotropin) were injected between 5:00 PM and 6:00 PM on the first day, followed by 10 units of hCG (human chorionic gonadotropin) between 5:00 PM and 6:00 PM on the third day. Following these injections, the female mice were co-housed with adult male ICR mice. Female mice with vaginal plugs detected the following morning were collected together and marked as pregnant on the first day of pregnancy. On the morning of the fifth day of pregnancy, 1% direct blue-saline solution was injected through the tail vein. Three minutes later, the mice were sacrificed by cervical dislocation and exsanguination performed via abdominal artery exsanguination. Embryonic implantation was observed, and the number of ovarian follicles was counted.

[0079] Experimental Groups 2, 3, 4, and 5: 6-8 week-old, sexually mature SPF ICR mice were treated with exogenous gonadotropins. Specifically, 10 units of PMSG were injected between 5:00 PM and 6:00 PM on the first day, followed by 10 units of hCG between 5:00 PM and 6:00 PM on the third day. Following these injections, female mice were housed with adult male ICR mice. Female mice with vaginal plugs detected the following morning were collected from the same cage and designated as the first day of pregnancy. Subsequently, between 5:00 PM and 6:00 PM on the third and fourth days of pregnancy, 100 μL of 0.04 mM, 0.2 mM, 1 mM, or 5 mM lactate-PBS solutions (doses of 0.012, 0.06, 0.3, and 1.5 mg / kg, respectively, were injected intraperitoneally into female mice (weighing 30 g ± 3 g). Then, on the morning of the fifth day of pregnancy, 1% direct blue-normal saline solution was injected into the tail vein. Three minutes later, the mice were killed by cervical dislocation and bleeding was performed on the abdominal artery. The embryo implantation was then observed and the ovulation points on both ovaries were counted.

[0080] 3. Experimental Results

[0081] Uterine embryo implantation was observed. The number of ovulation sites and embryo implantations in each mouse's ovaries was counted, and the embryo implantation rate (number of uterine embryo implantations / number of ovulation sites) was calculated for each group using an unpaired T-test. The results are shown in Figure 3. As can be seen, compared with the control group, the embryo implantation rate in experimental group 1, treated with exogenous gonadotropins, was significantly reduced (P < 0.0001), indicating that exogenous gonadotropin treatment significantly reduced the embryo implantation rate. Compared with experimental group 1, the embryo implantation rate in experimental group 4 was significantly increased (P < 0.01), indicating that the 1mM lactic acid-PBS solution treatment group significantly increased the embryo implantation rate. Figure 4 also shows that the 1mM lactic acid-PBS solution-treated group showed a significant improvement in uterine embryo implantation.

[0082] Example 3

[0083] This example uses a mouse model to study the effect of lactic acid or its salts on the reproductive performance of mice after gonadotropin treatment.

[0084] 1. Preparation of Lactic Acid-PBS Solution

[0085] Take the L-lactic acid solution stored at room temperature and dilute it in a cell-grade PBS solution to prepare a 100 mM lactic acid-PBS solution. Then, the 100 mM lactic acid-PBS solution is gradiently diluted to prepare a 1 mM lactic acid-PBS solution.

[0086] 2. Drug treatment

[0087] For animal experiments, ICR female mice (28-30 g) were randomly divided into three groups: a normal control group (NC), a gonadotropin-treated group (OS), and a gonadotropin-treated and lactate-administered group (OS+L), with 10-12 mice per group. The animals were housed at 22°C, on a 12-hour day / night cycle, with free access to food and water. Following purchase, the animals underwent a 5-7-day acclimatization period.

[0088] Control group: 6-8 week old, sexually mature SPF ICR mice were co-bred with adult ICR male mice between 5:00 PM and 6:00 PM on the first day. Female mice with vaginal plugs detected the following morning were collected in a single cage and marked as having the first day of pregnancy. On the day of delivery, newborns were counted and weighed.

[0089] Experimental Group 1: 6-8 week-old, sexually mature, SPF-qualified ICR mice were treated with exogenous gonadotropins. Specifically, 5 units of PMSG were injected between 5:00 PM and 6:00 PM on the first day, followed by 5 units of hCG between 5:00 PM and 6:00 PM on the third day. Following injection, female mice were co-housing with adult male ICR mice. Female mice with vaginal plugs detected the following morning were collected from the same cage and marked as having reached day one of pregnancy. On the day of delivery, newborns were counted and weighed.

[0090] Experimental Group 2: 6-8 week-old, sexually mature, SPF-qualified ICR mice were treated with exogenous gonadotropins. Specifically, 5 units of PMSG were injected between 5:00 PM and 6:00 PM on the first day, followed by 5 units of hCG between 5:00 PM and 6:00 PM on the third day. Following these injections, the female mice were co-housing with adult male ICR mice. Female mice with vaginal plugs detected the following morning were collected from the same cage, marking the first day of pregnancy. Subsequently, 100 μL of a 1 mM lactate-PBS solution was intraperitoneally injected into the female mice between 5:00 PM and 6:00 PM on the third, fourth, fifth, and sixth days of pregnancy. Newborns were counted and weighed on the day of delivery.

[0091] 3. Experimental Results

[0092] The number of newborn pups was counted and weighed, and the statistical data are shown in Figures 5 and 6. There was no significant difference in the average litter size between the control group, experimental group 1, and experimental group 2. However, the average birth weight of experimental group 1 was significantly lower than that of the control group (P < 0.001). After exogenous lactate supplementation, the birth weight of the pups in experimental group 2 was significantly higher than that in experimental group 1 (P < 0.05).

[0093] Statistics also revealed that some pregnant mice treated with exogenous gonadotropins gave birth to abnormal fetuses, such as mummified fetuses, stillborn fetuses, or fetal blood mixtures. Counting and statistical analysis of these abnormal pregnant mice are shown in Figure 7. Compared with Experimental Group 1, the abnormal litter size in Experimental Group 2 was significantly improved.

[0094] Example 4

[0095] This example studies the application of lactic acid or its salts in alleviating the birthing of mice after gonadotropin treatment

[0096] This example uses a larger sample size to conduct research and further confirm the effect of lactic acid or its salts on the reproductive performance of mice after gonadotropin treatment.

[0097] 1. Preparation of Lactic Acid-PBS Solution

[0098] Take the L-lactic acid solution stored at room temperature and dilute it in a cell-grade PBS solution to prepare a 100 mM lactic acid-PBS solution. Then, the 100 mM lactic acid-PBS solution is gradiently diluted to prepare a 1 mM lactic acid-PBS solution.

[0099] 2. Drug treatment

[0100] 1) Experimental animal grouping

[0101] For animal experiments, ICR female mice (SPF, 8 weeks old, 28-30g) were randomly divided into three groups: a normal control group (CON), a gonadotropin-treated group (PMSG), and a gonadotropin-treated and lactate-treated group (PMSG+Lactate), with 18-20 mice per group. The animals were housed at 22°C, on a 12-hour day / night cycle, with free access to food and water. Following purchase, the animals underwent a 5-7-day acclimatization period.

[0102] Control group (CON): Female 8-week-old ICR mice were used as research subjects. They were naturally estrus and mated with male mice as the control group. They were then placed in the animal room and raised normally. On the day of delivery, the number of live pups born was counted.

[0103] Gonadotropin-treated group (PMSG): Female 8-week-old ICR mice were used as research subjects. They were injected with 10 IU PMSG at 18:00 in the afternoon, and 10 IU hCG was injected 48 hours later to induce superovulation. Then they were caged with male mice. The vaginal plug was checked the next morning. The mice with a plug were designated as the gonadotropin-treated group and then placed in the animal room for normal feeding. On the day of delivery, the number of live pups born was counted.

[0104] Gonadotropin treatment and lactate administration group (PMSG+Lactate): Female 8-week-old ICR mice were used as research subjects. They were injected with 10 IU PMSG at 18:00 in the afternoon and 10 IU hCG was injected 48 hours later to induce superovulation. Then they were caged with male mice. The vaginal plug was checked the next morning. After the plug was found, they were designated as the gonadotropin treatment group. Subsequently, different doses of lactate (1 mM, i.e. 0.3 mg / kg.BW) were supplemented on the afternoon of the 3rd, 4th, 5th, and 6th days after the plug was found. They were then placed in the animal room for normal feeding. On the day of delivery, the number of live pups born was counted.

[0105] 2) Litter size statistics

[0106] The mice in the CON group, PMSG group, and PMSG+Lactate group were placed in the animal room and raised normally. On the day of delivery, the delivery rate, average birth weight of pups, and number of live pups born per litter were counted.

[0107] 3. Experimental Results

[0108] The results showed that lactate supplementation significantly increased the birth rate, number of live pups born, and average birth weight of pups in mice (Table 1). These results indicate that lactate has a significant effect in improving pregnancy outcomes.

[0109] Table 1: Effects of lactate on litter size in mice treated with gonadotropin Note: Different lowercase letters (abc) indicate significant differences between the two groups, with P < 0.05.

[0110] Example 5

[0111] This example is the application of lactic acid or its salts in precision sow batch production

[0112] Batch management technology divides the breeding sow herd into batches based on the sow's reproductive cycle and batch intervals. The system synchronizes the reproduction of each batch of sows, achieving a highly efficient reproductive production system with synchronized estrus, ovulation, breeding, and farrowing. Therefore, the essence of batch sow production is batch breeding.

[0113] 1. Experimental Animals

[0114] All experimental sows in this experiment were gilts aged 220-250 days and weighing 120-140 kg.

[0115] 2. Sow batch experimental design

[0116] Gilts aged 220-250 days and weighing 120-140 kg were selected for the experiment. They were divided into two groups: precision sow batch production (control) group and precision sow batch production and lactate supplementation group, with 50-60 sows in each group.

[0117] The technical process for batch production of gilts in the control group (i.e., precision sow batch production program) is as follows: 1) Sexual cycle synchronization treatment: sows are orally administered allerglucose for 18 consecutive days; 2) After sexual cycle synchronization treatment, exogenous gonadotropin PMSG is injected at an interval of 42 hours to induce synchronization of follicular development; 3) After the injection of PMSG, the ovulation-inducing drug gonadorelin (GnRH) is injected at an interval of 80 hours. The first artificial insemination is performed 24 hours after the injection, and the second artificial insemination is performed 16 hours after the injection.

[0118] For the lactic acid group, based on the existing precision sow batch production process, lactic acid (1 mg / kg·BW) was added to the feed from day 9 to day 22 after the first artificial insemination injection and fed continuously for 14 days.

[0119] 3. Detection of pregnancy rate and birth status

[0120] On the 30th to 32nd day of gestation, the sows are checked for pregnancy by B-ultrasound, and the pregnancy rate of the sows is calculated. The sows are then kept and the farrowing rate and litter size are calculated. The sow pregnancy rate and farrowing rate are calculated as follows: Sow pregnancy rate = number of pregnant sows / number of bred sows × 100%, Sow farrowing rate = number of farrowing sows / number of bred sows × 100%, Farrowing index = number of live piglets born per litter × farrowing rate × 100,

[0121] Among them, the Piglet Index (PI) is a comprehensive indicator used in current production to measure the overall reproductive performance of sow groups. It refers to the number of live piglets born per 100 sows or gilts that undergo natural mating or artificial insemination.

[0122] 4. Experimental Results

[0123] Compared with the pregnancy rate and farrowing rate of sows in precision sow batch production, the pregnancy rate and farrowing rate of sows in the lactic acid group were significantly increased (Table 2).

[0124] Table 2: Effects of lactic acid on pregnancy and farrowing rates in precision sow batch production Note: Different lowercase letters (ab) indicate that the pregnancy rate and delivery rate of the lactate supplementation group were significantly different from those of the control group, with P < 0.05.

[0125] As shown in Table 3, the farrowing index of sows in the precision sow batch production group combined with lactic acid was significantly improved compared to the sow farrowing index in precision sow batch production. The number of healthy piglets and the number of piglets born alive in the lactic acid group were significantly higher than those in the precision sow batch production group, while the number of weak piglets in the lactic acid group was significantly reduced (Table 3).

[0126] Table 3: Effect of lactic acid on farrowing in precision sow batch production Note: Different lowercase letters (ab) indicate that compared with the control group, the number of healthy piglets, the number of weak piglets and the number of live piglets in the lactic acid supplementation group were significantly different (P < 0.05).

[0127] Since the first outbreak of African swine fever in my country in August 2018, China's traditional continuous production model has become difficult to adapt to the rapid development of the pig farming industry. In addition, the entire pig market faces severe challenges. Sow batch production technology has become a key measure for transformation and upgrading. In the future, the era of small-scale pig farming will become a thing of the past. The emergence of large-scale, batch production methods will inevitably bring earth-shaking changes to China's pig farming industry. This invention provides an important solution to the problems of mid-gestation embryo loss and reduced litter size caused by exogenous gonadotropins in the promotion of precision sow batch production. The present invention effectively improves the pregnancy rate, farrowing rate, number of live piglets born per litter and farrowing index of batch sows in precision sow batch production, and can increase the number of live piglets (weaned piglets) by 248 for every 100 reserve sows. According to the current price of 300-400 yuan / weaned piglet, every 100 reserve sows will generate an additional economic value of 74,000-99,000 yuan, which provides an effective method for reducing costs and increasing efficiency for the entire pig farming industry. Therefore, the present invention creates huge economic and social value for promoting batch production of live pigs in my country.

[0128] Finally, while the general description and operational procedures of the present invention have been thoroughly described above, it will be readily apparent to those skilled in the art that modifications or improvements may be made based on the present invention, as the actual application conditions vary with different pregnant mothers, such as livestock or experimental animals. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0129] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations. In addition, the various different embodiments of this application can also be arbitrarily combined, as long as they do not violate the concept of this application, they should also be regarded as the content disclosed in this application.

Claims

1. A method for improving embryo implantation or reproductive performance, characterized in that, It includes the following steps: administering lactic acid or its salt to a pregnant female.

2. The method according to claim 1, characterized in that, The pregnant female is a pregnant female treated with exogenous gonadotropin.

3. The method according to claim 1 or 2, characterized in that, The dosage of lactic acid or its salt administered is 0.1 - 2.0 mg / Kg.BW, preferably 0.2 - 1.5 mg / Kg.BW, more preferably 0.3 - 1.0 mg / Kg.BW.

4. The method according to claim 3, characterized in that, It includes the following steps: administering lactic acid or its salt to a pregnant female during the peri-implantation period of the embryo; Preferably, lactic acid or its salt is administered to the pregnant female during the 3rd day of pregnancy to the period of embryo implantation or during the 1 - 3 days after the embryo implantation from the 3rd day of pregnancy; More preferably, lactic acid or its salt is administered daily.

5. The method according to claim 4, wherein It includes the following steps: for a pregnant female treated with exogenous gonadotropin and after conception, administering lactic acid or its salt to the pregnant female during the peri-implantation period of the embryo; Preferably, lactic acid or its salt is administered to the pregnant female during the 3rd day of pregnancy to the period of embryo implantation or during the 1 - 3 days after the embryo implantation from the 3rd day of pregnancy; More preferably, lactic acid or its salt is administered daily.

6. Use of the method according to any one of claims 1 - 5 in the reproduction of livestock and experimental animals, especially in the reproduction of domestic animals.

7. Use of lactic acid or its salt in the preparation of a drug for improving embryo implantation or reproductive performance.

8. Use of lactic acid or its salt in the preparation of a drug for improving embryo implantation or reproductive performance after treatment with exogenous gonadotropin.

9. The application according to claim 7 or 8, characterized in that, The drug is a drug for humans, livestock and experimental animals, especially for domestic animals.

10. Use of lactic acid or its salt in improving embryo implantation or reproductive performance.

11. Use of lactic acid or its salt in improving embryo implantation or reproductive performance after treatment with exogenous gonadotropin.

12. The application according to claim 10 or 11, characterized in that, The application is in livestock and experimental animals, especially in domestic animals.

13. Use of the method according to any one of claims 1 - 5 in sow production, especially in batch production of sows.

14. The application according to claim 13, wherein Lactic acid or its salt is administered to sows during the period from the 7th - 9th day to the 21st - 23rd day after natural mating or artificial insemination.

15. A method for batch production of sows, characterized in that, It includes the following steps: 1) Synchronize the sexual cycle of sows; 2) Administer exogenous gonadotropin to sows to induce synchronous follicular development; 3) Administer ovulation-inducing drugs to sows; 4) Perform artificial insemination on sows; 5) Administer lactic acid or its salt to sows during the peri-implantation period of the embryo.

16. The method according to claim 15, wherein In step 5), the dosage of lactic acid or its salt administered is 0.1 - 2.0 mg / Kg.BW, preferably 0.2 - 1.5 mg / Kg.BW, more preferably 0.3 - 1.0 mg / Kg.BW.

17. The method according to claim 15 or 16, characterized in that In step 5), lactic acid or its salt is administered daily. Preferably, lactic acid or its salt is administered to sows during the period from the 7th - 9th day to the 21st - 23rd day after artificial insemination.

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