A method for improving embryo implantation or reproductive performance

Abstract

The present invention relates to the field of sexual reproduction technology, and specifically provides a method for improving embryo implantation or reproductive performance. The present invention improves the inflammatory state of the pregnant uterus by applying lactic acid to improve embryo implantation or reproductive performance; in particular, it improves the inflammatory state of the pregnant uterus after exogenous gonadotropin treatment, and finally improves the problems of reduced embryo implantation rate and poor reproductive performance after exogenous gonadotropin treatment. The method described in this application has a wide range of application prospects. For example, it can be used in animal husbandry to achieve reproductive regulation and management of livestock such as pigs, horses, cattle, and sheep, and has high application value.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority of the patent application with the application number "CN 2023118396816" and the invention title "A method for improving embryo implantation or reproductive performance" filed on December 28, 2023, the content of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the field of sexual reproduction technology, and specifically provides a method for improving embryo implantation or reproductive 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 the livestock industry, due to considerations such as cost and half-life, 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 usually selected to induce synchronous follicular development and ovulation in livestock, so as to achieve intensive reproductive management.

[0005] The application of exogenous gonadotropin treatment in livestock production is relatively extensive. For example, embryo transfer reduces the risk of disease transmission, estrus synchronization treatment, accelerates livestock genetic improvement, and protects the genetic resources of endangered livestock. Although exogenous gonadotropin treatment has such a broad application space, there are also many problems at the same time. For example: the proportion of degenerated embryos increases, the fertility of livestock decreases, resulting in embryo implantation failure, the prenatal abortion ratio of livestock increases, and the development quality of offspring decreases (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 view of the above problems, the prior art discloses that, in addition, N-acetylcysteine can specifically improve the uterine environment induced by exogenous gonadotropin and enhance the early embryo development ability, increase the number of implanted embryos, and has the ability to increase the litter size of female livestock (CN107333704A), or improve and enhance the reproductive performance of female animals in precision batch farrowing of sows, effectively increasing the breeding pregnancy rate of sows, etc. The prior art also discloses that the result can be improved or partially rescued by exogenous supplementation of progesterone or the application of some traditional Chinese herbal medicine formulas. In order to provide higher economic benefits for livestock farming, the improvement of the results of exogenous gonadotropin treatment has always been an issue that the livestock industry needs to continuously improve and solve.

[0007] Although studies have shown that an appropriate inflammatory environment may be created during embryo implantation. For example, implantation is a pro-inflammatory state, and local injury of the endometrium helps embryo implantation (N. Dekel, Y. Gnainsky, I. Granot, K. Racicot, G. Mor, The role of inflammation for a successful implantation, Am J Reprod Immunol N Y N 72(2)(2014)141 - 147; Y. Gnainsky, I. Granot, P. Aldo, et al., Biopsy-induced inflammatory conditions improve endometrial receptivity: the mechanism of action, Reprod Camb Engl 149(2015)75 - 85), an excessive inflammatory environment will lead to embryo implantation failure and premature birth (Cha J, Sun X, Dey SK. Mechanisms of implantation: strategies for successful pregnancy. Nat Med. 2012 Dec;18(12):1754 - 67.), indicating that appropriate inflammation is beneficial to embryo implantation, while an excessive inflammatory state will have an adverse effect on pregnancy. How to adjust the uterine inflammatory state to improve embryo implantation or reproductive performance is also an issue of general concern in the industry.

[0008] A large number of previous studies of the present invention have found that changes in lactic acid levels and the subsequent excessive activation of the uterine inflammatory signal NLRP3 inflammasome are the key signals for the decline in uterine receptivity caused by exogenous gonadotropins. The present invention shows that exogenous supplementation of lactic acid can effectively alleviate the abnormal activation of the lactic acid-NLRP3 inflammasome, and thus improve the endometrial receptivity and pregnancy outcomes of female mammals after treatment with exogenous gonadotropins, such as embryo implantation rate, pregnancy rate, and litter size. Previous studies and the prior art cannot indicate the application of exogenous lactic acid in improving endometrial receptivity and pregnancy outcomes after treatment with exogenous gonadotropins. Summary of the Invention

[0009] The purpose of the present application is to provide a method for improving embryo implantation or reproductive performance, by administering lactic acid to improve the inflammatory state of the pregnant uterus, and thus improve embryo implantation or reproductive performance.

[0010] The present application provides a method for improving embryo implantation or reproductive performance for non-disease treatment purposes, which is characterized by including the following steps: administering lactic acid to a pregnant female. The pregnant female is preferably a pregnant female treated with exogenous gonadotropins. The dosage of lactic acid administered is preferably 0.1 - 2.0 mg / Kg.BW (which means 0.1 - 2.0 mg is administered per Kg of the body weight of the pregnant female), preferably 0.2 - 1.5 mg / Kg.BW, more preferably 0.3 - 1.0, 0.3 - 0.5 mg / Kg.BW.

[0011] A lactic acid solution can be administered, such as a lactic acid-PBS solution; preferably a 0.5 - 2 mM lactic acid solution, more preferably a 1 mM lactic acid solution. The administration method of lactic acid can be oral or injection (such as intraperitoneal injection), etc. Lactic acid can also be added to the diet, such as in feed and drinking water.

[0012] In one embodiment, lactic acid is administered during the perimplantation period of the embryo. For livestock, the perimplantation period of embryos in pigs, horses, cattle, and sheep is generally from the 7th - 9th day to the 21st - 23rd day after insemination, and the perimplantation period of embryos in rabbits is generally from the 4th - 6th day to the 10th - 12th day after insemination. For experimental animals, the perimplantation period of embryos in mice is generally from the 3rd day of pregnancy to 1 - 3 days after embryo implantation, and the perimplantation period of embryos in monkeys is generally from the 5th - 7th day to the 12th - 14th day after insemination. The perimplantation period of embryos in rabbits is generally from the 4th - 6th day to the 10th - 12th day after insemination. Preferably, lactic acid is administered daily during the perimplantation period of the embryo.

[0013] As used herein, "insemination" refers to natural mating or artificial insemination.

[0014] In one embodiment, the method comprises the steps of administering lactic acid to a pregnant female during the period from the 3rd day of pregnancy to the time of embryo implantation or administering lactic acid to a pregnant female during the period from the 3rd day of pregnancy to 1 - 3 days after embryo implantation; preferably, lactic acid is administered daily.

[0015] In one embodiment, the method comprises the steps of administering lactic acid to a pregnant female during the perimplantation period of the embryo to a pregnant female who has been treated with exogenous gonadotropin and is pregnant after conception. For livestock, the perimplantation period of embryos in pigs, horses, cattle, and sheep is generally during the period from the 7th - 9th day to the 21st - 23rd day after insemination, and the perimplantation period of embryos in rabbits is generally during the period from the 4th - 6th day to the 10th - 12th day after insemination. For experimental animals, the perimplantation period of embryos in mice is generally from the 3rd day of pregnancy to 1 - 3 days after embryo implantation, the perimplantation period of embryos in monkeys is generally during the period from the 5th - 7th day to the 12th - 14th day after insemination, and the perimplantation period of embryos in rabbits is generally during the period from the 4th - 6th day to the 10th - 12th day after insemination. Preferably, lactic acid is administered daily during the perimplantation period of the embryo. Preferably, lactic acid is administered to a pregnant female during the period from the 3rd day of pregnancy to the time of embryo implantation or administering lactic acid to a pregnant female during the period from the 3rd day of pregnancy to 1 - 3 days after embryo implantation; preferably, lactic acid is administered daily. More specifically, for a pregnant female who has been treated with exogenous gonadotropin and is pregnant after conception, lactic acid is administered continuously for 2 - 5 (2, 3, 4, or 5) days on the 3rd day of pregnancy, such as administering lactic acid continuously on the 3rd and 4th days of pregnancy, or administering lactic acid continuously on the 3rd, 4th, 5th, and 6th days of pregnancy.

[0016] The present application also provides the use of the aforementioned method for improving embryo implantation or reproductive performance in the reproduction of livestock and experimental animals, especially in the reproduction of livestock. For example, it can be used for cattle, sheep, pigs, horses, rabbits, monkeys for experiments, mice for experiments, rabbits for experiments, etc.

[0017] The inventors of the present application have found through research that after treatment with exogenous gonadotropin and conception, there is an obvious state of excessive inflammation and oxidative stress in the endometrium of mice on the fourth day of pregnancy, including the over - activation of the NLRP3 / Caspase - 1 signaling pathway; the over - expression of the oxidative stress molecule NRF2, etc. The present application can solve the problem of reduced embryo implantation rate caused by treatment with exogenous gonadotropin by administering lactic acid, and can also improve breeding performance.

[0018] In this application, female mice treated with exogenous gonadotropin before conception were continuously administered lactic acid (such as lactic acid - PBS solution, preferably 100 μL of 1 mM lactic acid - PBS solution) on the 3rd and 4th days of pregnancy. Through the observation of the ovarian ovulation points and the embryo implantation situation on the 5th day (when embryo implantation occurs), it was found that the embryo implantation situation after exogenous gonadotropin treatment was significantly improved. Specifically, exogenous gonadotropin treatment can be carried out on the 3rd and 1st days before conception to induce follicle development and ovulation.

[0019] In this application, female mice treated with exogenous gonadotropin before conception were continuously intraperitoneally injected with lactic acid (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 parturition showed that the body weight of the newborns after exogenous gonadotropin treatment increased significantly, and the proportion of abnormal fetuses also decreased significantly, and the reproductive performance was significantly improved. Specifically, exogenous gonadotropin treatment can be carried out on the 3rd and 1st days before conception to induce follicle development and ovulation.

[0020] This application also provides the use of lactic acid in the preparation of drugs for improving embryo implantation or reproductive performance; in particular, the use of lactic acid in the preparation of drugs for improving embryo implantation or reproductive performance after exogenous gonadotropin treatment. The drugs are for livestock animals and laboratory animals, especially for domestic animals.

[0021] This application also provides the non - therapeutic use of lactic acid in improving embryo implantation or reproductive performance; in particular, the non - therapeutic use of lactic acid in improving embryo implantation or reproductive performance after exogenous gonadotropin treatment. The application is in livestock animals and laboratory animals, especially in domestic animals.

[0022] The lactic acid described in this application can be L - lactic acid. The exogenous gonadotropin described in this application 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, chorionic gonadotropin, gonadotropin - releasing hormone, and gonadotropin - releasing hormone agonist. The exogenous gonadotropin is used to treat the mother before conception, such as treating the mother on the 3rd, 2nd, and 1st days before conception.

[0023] This application also provides the application of the aforementioned method for improving embryo implantation or reproductive performance in sow production, especially in batch production of sows. Preferably, lactic acid is administered to sows during the period from the 7th - 9th day to the 21st - 23rd day after natural mating or artificial insemination. Preferably, lactic acid is orally administered to sows, such as adding lactic acid to the diet (such as feed and drinking water).

[0024] The present application also provides a method for batch production of sows, characterized by comprising the following steps: 1) performing sexual cycle synchronization on sows; 2) administering exogenous gonadotropin to sows to induce follicular development synchronization; 3) administering ovulation induction drugs to sows; 4) performing artificial insemination on sows; 5) administering lactic acid to sows during the peri-implantation period of embryos.

[0025] In one embodiment, in the step 5), the dosage of lactic acid administered 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.

[0026] In one embodiment, in the step 5), lactic acid is administered every day. Preferably, lactic acid is administered to sows during the period from the 7th - 9th day to the 21st - 23rd day after artificial insemination. Preferably, lactic acid is orally administered to sows, such as by adding lactic acid to the diet such as feed and drinking water. Liquid lactic acid or lactic acid solution, such as lactic acid PBS solution, can be added.

[0027] In one embodiment, in the step 1), allyl trenbolone is administered to sows for sexual cycle synchronization. Preferably, allyl trenbolone is orally administered to sows for sexual cycle synchronization; preferably, sows are continuously administered for 15 - 20 days, such as continuously administered allyl trenbolone for 18 days.

[0028] In one embodiment, in the step 2), after sexual cycle synchronization, exogenous gonadotropin is administered at an interval of 36 - 48 h, such as 42 h, to induce follicular development synchronization. Preferably, exogenous gonadotropin is injected. The exogenous gonadotropin is preferably PMSG.

[0029] In one embodiment, in the step 3), after administering exogenous gonadotropin, an ovulation drug is administered at an interval of 70 - 90 h, such as 80 h. Preferably, the ovulation drug is injected. The ovulation drug is preferably gonadorelin (GnRH).

[0030] In one embodiment, in the step 4), after administering the ovulation drug, artificial insemination is performed at an interval of 20 - 28 h, such as 24 h; preferably, a second artificial insemination is performed after an interval of 14 - 18 h, such as 16 h.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] By administering lactic acid to pregnant females, the embryo implantation and reproductive performance have been significantly improved. Especially for pregnant females treated with exogenous gonadotropins, administering lactic acid can effectively alleviate the abnormal activation of the lactate-NLRP3 inflammasome, improve the uterine inflammatory state, and thus improve the endometrial receptivity and pregnancy outcomes of female mammals after exogenous gonadotropin treatment, such as embryo implantation rate, pregnancy rate, and litter size. Eventually, it corrects the problems of reduced embryo implantation rate and poor reproductive performance after exogenous gonadotropin treatment.

[0033] The present invention provides an important solution to problems such as mid-pregnancy embryo loss and decreased litter size induced by exogenous gonadotropins in the popularization of precision batch production of sows. The present invention effectively improves the pregnancy rate, farrowing rate, number of live piglets per litter, and litter index of batch sows in precision batch production of sows, providing an effective method for cost reduction and efficiency improvement in the entire pig industry. Therefore, the present invention generates great economic value and social value for promoting batch production of live pigs in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is an analysis diagram of the activation of the NLRP3 inflammasome signal in the endometrium of mice during the peri-implantation period induced by exogenous gonadotropins.

[0035] (A) Expression of mRNAs of molecules related to the NLRP3 signaling pathway in the uteri of mice with natural estrus and treated with exogenous gonadotropins on the 4th day of pregnancy; (B) Results of western blot of protein levels of NLRP3 signaling-related proteins in the uteri of mice with natural estrus and treated with exogenous gonadotropins on the 4th day of pregnancy; (C) Contents of IL-1β and IL-18 in the endometrium of mice with natural estrus and treated with exogenous gonadotropins on the 4th day of pregnancy measured by ELISA. The results are expressed as mean ± SEM, *p < 0.05, **p < 0.01.

[0036] Figure 2 It is an analysis diagram of the change in lactic acid content in the endometrium of mice during the peri-implantation period after treatment with exogenous gonadotropins. *p < 0.05.

[0037] Figure 3 It is an analysis diagram of lactic acid inhibiting the over-activation of the NLRP3 inflammasome signal in the endometrium of mice during the peri-implantation period induced by gonadotropins.

[0038] (A) Effect of exogenous supplementation of lactic acid on the protein expression of molecules in the NLRP3 inflammasome signaling pathway in the endometrium of mice in the gonadotropin-treated group during the peri-implantation period; (B) Effect of exogenous supplementation of lactic acid on the contents of key target molecules IL-1β and IL-18 of the NLRP3 inflammasome in the endometrium of mice in the gonadotropin-treated group during the peri-implantation period.

[0039] Figure 4Graph showing the mouse embryo implantation rate statistics.

[0040] CON is the control group, PMSG is experimental group one treated with exogenous gonadotropin, and Lactate is experimental groups two to five treated with different concentrations of lactate-PBS solution after injection following exogenous gonadotropin treatment. **p < 0.01, ***p < 0.001.

[0041] Figure 5 Graph showing the mouse embryo implantation situation.

[0042] Con is the control group, PMSG is experimental group one treated with exogenous gonadotropin, and Lactate is experimental group four treated with 1 mM lactate-PBS solution after injection following exogenous gonadotropin treatment.

[0043] Figure 6 Graph showing the average litter size of mice.

[0044] CON is the control group, PMSG is experimental group one treated with exogenous gonadotropin, and PMSG + Lactate is experimental group four treated with 1 mM lactate-PBS solution after injection following exogenous gonadotropin treatment.

[0045] Figure 7 Graph showing the average birth weight of neonatal mice.

[0046] CON is the control group, PMSG is experimental group one treated with exogenous gonadotropin, and PMSG + Lactate is experimental group four treated with 1 mM lactate-PBS solution after injection following exogenous gonadotropin treatment.

[0047] Figure 8 Graph showing the proportion of abnormal fetuses in neonatal mice.

[0048] CON is the control group, PMSG is experimental group one treated with exogenous gonadotropin, and PMSG + Lactate is experimental group four treated with 1 mM lactate-PBS solution after injection following exogenous gonadotropin treatment. Detailed implementation manners

[0049] The following details the specific implementation manners of the present application. It should be understood that the specific implementation manners described herein are only for the purpose of illustrating and explaining the present application, and are not used to limit the present application.

[0050] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0051] As used in this application, the terms "comprising" and "including" and any variations thereof are intended to cover non-exclusive inclusion.

[0052] 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.

[0053] In the following examples using mice as a model, lactic acid was purchased from Sigma Corporation, product number L6661-100ml. Lactic acid used in the batch production of sows was purchased from Ningxia Shuangta Chemical Co., Ltd., with a purity of 98%.

[0054] Example 1

[0055] This example uses a mouse model to study the molecular mechanism of lactic acid in alleviating uterine inflammation in mice after gonadotropin treatment.

[0056] 1. Experimental animals

[0057] All experimental mice in this experiment were purchased from Beijing Speywood Co., Ltd. Female mice were 8 weeks old and male mice were 10 weeks old. After purchase, the experimental mice were kept in a light-controlled environment (12 hours of light: 12 hours of darkness). Without special experimental requirements, all mice had free access to food and water, and the breeding environment was controlled at 20-24°C.

[0058] 2. Experimental design

[0059] 2.1 Molecular pathway changes in gonadotropin treatment affecting uterine receptive inflammation signals

[0060] For the animal experiment, ICR female mice (SPF grade, 8 weeks old, 28-30 g) were randomly grouped, including a normal control group (CON) and a gonadotropin treatment group (PMSG), with 10-18 mice in each group. The breeding environment temperature was 22°C, with a 12-hour day-night cycle, and free access to food and water. After the animals were purchased, they first went through a 5-7-day adaptation period.

[0061] Control group (CON): Using 8-week-old ICR female mice as the research object, they mated naturally with male mice. After detecting a vaginal plug, they were used as the control group. Then, on the 4th day after detecting the plug, when uterine receptivity was established, the uteri of the mice were isolated for the detection of multiple signaling molecules.

[0062] Gonadotropin treatment group (PMSG): Using 8-week-old ICR female mice as the research object, at 18:00 in the afternoon, 10 IU of PMSG was injected, and 10 IU of hCG was injected 48 hours later to induce superovulation. Immediately afterwards, they were caged with male mice. The next morning, the vaginal plugs were checked. After detecting a vaginal plug, they were used as the gonadotropin treatment group. Then, on the 4th day after detecting the plug, when uterine receptivity was established, the uteri of the mice were isolated for the detection of multiple signaling molecules.

[0063] Endometrial samples of mice in the CON group and the PMSG group were collected on the 4th day of pregnancy for molecular detection. The detection indexes included the changes in the expression of molecules related to NLRP3 inflammasome assembly and activation, as well as the change in lactate content, to explore the molecular mechanism by which exogenous gonadotropins affect endometrial inflammation and then lead to the decline of uterine receptivity.

[0064] 2.2 Molecular regulatory role of lactate in alleviating uterine over-inflammation induced by gonadotropin treatment

[0065] 1) Grouping of experimental animals

[0066] Female ICR mice (SPF grade, 8 weeks old, 28 - 30 g) were randomly grouped for animal experiments, including a normal control group (CON), a gonadotropin treatment group (PMSG), and a gonadotropin treatment and lactate administration group (PMSG + Lactate), with 10 - 12 mice in each group. The breeding environment temperature was 22 °C, with a 12-hour day-night cycle, and free access to food and water. After the animals were purchased, they were first given a 5 - 7-day adaptation period.

[0067] Control group (CON): Female 8-week-old ICR mice were used as the research subjects, mated with male mice during natural estrus, and used as the control group. Subsequently, on the 4th day after detecting the vaginal plug, that is, when uterine receptivity was established, the uteri of the mice were separated for the detection of multiple signaling molecules.

[0068] Gonadotropin treatment group (PMSG): Female 8-week-old ICR mice were used as the research subjects. At 18:00 in the afternoon, 10 IU of PMSG was injected, and 10 IU of hCG was injected after 48 h to induce superovulation. Then, the mice were caged with male mice, and the vaginal plug was checked the next morning. After detecting the vaginal plug, it was used as the gonadotropin treatment group. Subsequently, on the 4th day after detecting the vaginal plug, that is, when uterine receptivity was established, the uteri of the mice were separated for the detection of multiple signaling molecules.

[0069] Gonadotropin treatment and lactate administration group (PMSG + Lactate): Female 8-week-old ICR mice were used as the research subjects. At 18:00 in the afternoon, 10 IU of PMSG was injected, and 10 IU of hCG was injected after 48 h to induce superovulation. Then, the mice were caged with male mice, and the vaginal plug was checked the next morning. After detecting the vaginal plug, it was used as the gonadotropin treatment group. Subsequently, in the afternoon of the 3rd day after detecting the vaginal plug, a lactate-PBS solution (0.3 mg / kg.BW) was supplemented. On the 4th day after detecting the vaginal plug, that is, when uterine receptivity was established, the uteri of the mice were separated for the detection of multiple signaling molecules.

[0070] 2) Detection of molecular indexes

[0071] Endometrial samples of mice in the CON group, PMSG group, and PMSG+Lactate group were collected on the 4th day of pregnancy for molecular detection to detect the changes in the expression of molecules related to the assembly and activation of the NLRP3 inflammasome, and to explore the molecular mechanism by which lactate alleviates the endometrial inflammation affected by exogenous gonadotropins.

[0072] 3. Experimental results

[0073] The molecular detection experiment found that PMSG 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 takes "inhibiting the over-activation of the NLRP3 inflammasome signaling pathway" as a new target for improving the excessive endometrial inflammation caused by exogenous gonadotropin treatment. Further research found that the lactate content decreased ( Figure 2 ), so the present invention takes lactate as a candidate drug. Further signal molecule detection found that lactate can significantly inhibit the assembly and activation of the "NLRP3 inflammasome" ( Figure 3 ).

[0074] Example 2

[0075] This example uses a mouse model to study the effect of lactate on alleviating embryo implantation in mice after gonadotropin treatment.

[0076] 1. Preparation of lactate-PBS solution

[0077] Take the L-lactate solution stored at room temperature and dilute it in cell-grade PBS solution to prepare a 100 mM lactate-PBS solution. Then, gradient dilute the 100 mM lactate-PBS solution to 0.04 mM, 0.2 mM, 1 mM, and 5 mM lactate-PBS solutions.

[0078] 2. Drug treatment

[0079] Female ICR mice were randomly divided into groups for animal experiments, including a normal control group (CON), a gonadotropin treatment group (PMSG), and a gonadotropin treatment and different-dose lactate administration group (PMSG+(different doses)Lactate), with 10-12 mice in each group. The breeding environment temperature was 22 °C, with a 12-hour day-night cycle, and free access to food and water. After the animals were purchased, they first went through a 5-7-day adaptation period.

[0080] Control group: SPF (specific pathogen free)-level sexually mature ICR mice at 6 - 8 weeks of age were caged with adult ICR male mice from 17:00 to 18:00 on the first day. Female mice with vaginal plugs detected in the morning of the next day were collected in one cage, which was recorded as the first day of pregnancy. Then, in the morning of the fifth day of pregnancy, a 1% Direct Blue - saline solution (MACKLIN, D823399) was injected into the tail vein. After 3 minutes, the mice were decapitated and bled from the abdominal artery. Then, the embryo implantation was observed, and the number of ovulation points on both ovaries was counted.

[0081] Experimental group 1: SPF-level sexually mature ICR mice at 6 - 8 weeks of age were treated with exogenous gonadotropin. Specifically, 10 units of PMSG (pregnant mare serum gonadotropin) were injected from 17:00 to 18:00 on the first day, and then 10 units of hCG (human chorionic gonadotropin) were injected from 17:00 to 18:00 on the third day. After the injection, the female mice were caged with adult ICR male mice, and the female mice with vaginal plugs detected in the morning of the next day were collected in one cage, which was recorded as the first day of pregnancy. Then, in the morning of the fifth day of pregnancy, a 1% Direct Blue - saline solution was injected into the tail vein. After 3 minutes, the mice were decapitated and bled from the abdominal artery. Then, the embryo implantation was observed, and the number of ovulation points on both ovaries was counted.

[0082] Experimental groups 2, 3, 4, and 5: SPF-level sexually mature ICR mice at 6 - 8 weeks of age were treated with exogenous gonadotropin. Specifically, 10 units of PMSG were injected from 17:00 to 18:00 on the first day, and then 10 units of hCG were injected from 17:00 to 18:00 on the third day. After the injection, the female mice were caged with adult ICR male mice, and the female mice with vaginal plugs detected in the morning of the next day were collected in one cage, which was recorded as the first day of pregnancy. After that, 100 μL of pre-prepared 0.04 mM, 0.2 mM, 1 mM, 5 mM lactic acid - PBS solution was intraperitoneally injected (corresponding to a mouse weight of 30 g, and the doses were 0.012, 0.06, 0.3, 1.5 mg / Kg respectively) into female mice (weight 30 g ± 3 g) from 17:00 to 18:00 on the third and fourth days of pregnancy. Then, in the morning of the fifth day of pregnancy, a 1% Direct Blue - saline solution was injected into the tail vein. After 3 minutes, the mice were decapitated and bled from the abdominal artery. Then, the embryo implantation was observed, and the number of ovulation points on both ovaries was counted.

[0083] 3. Experimental results

[0084] The embryo implantation on the uterus was observed. By counting the number of ovulation points on the ovary and the number of embryo implantations on the uterus for each mouse, the embryo implantation rate of each group was calculated (the number of embryo implantations on the uterus / the number of ovulation points on the ovary). The method was unpaired T-test, and the results were as followsFigure 4 As shown in the figure. It can be seen that, compared with the control group, the embryo implantation rate of experimental group 1 treated with exogenous gonadotropin was extremely significantly reduced (P<0.0001), that is, the treatment with exogenous gonadotropin would extremely significantly reduce the embryo implantation rate; compared with experimental group 1, experimental group 4 significantly increased the embryo implantation rate (P<0.01), that is, the 1 mM lactate-PBS solution treatment group could significantly increase the embryo implantation rate. From Figure 5 It can also be seen that for the mice in the 1 mM lactate-PBS solution treatment group, the embryo implantation situation on the uterus was significantly improved.

[0085] Example 3

[0086] This example uses a mouse model to study the effect of lactate on alleviating the reproductive performance of mice after gonadotropin treatment

[0087] 1. Preparation of lactate-PBS solution

[0088] Take the L-lactate solution stored at room temperature, dilute it in cell-grade PBS solution to prepare a 100 mM lactate-PBS solution, and then gradient-dilute the 100 mM lactate-PBS solution to a 1 mM lactate-PBS solution.

[0089] 2. Drug treatment

[0090] For the animal experiment, ICR female mice (28 - 30 g) were randomly grouped, including a normal control group (CON), a gonadotropin treatment group (PMSG), and a gonadotropin treatment and lactate application group (PMSG+Lactate), with 10 - 12 mice in each group. The breeding environment temperature was 22 °C, with a 12-hour day-night cycle, and free access to food and water. After the animals were purchased, they first went through a 5 - 7-day adaptation period.

[0091] Control group: SPF-grade sexually mature ICR mice at 6 - 8 weeks of age were caged with adult ICR male mice from 17:00 to 18:00 on the first day afternoon. The female mice with vaginal plugs detected in the morning of the next day were collected in one cage, which was recorded as the first day of pregnancy. Then, on the day of parturition, the newborn pups were counted and weighed.

[0092] Experimental group 1: SPF-grade sexually mature ICR mice at 6 - 8 weeks of age were treated with exogenous gonadotropin. Specifically, 5 units of PMSG were injected from 17:00 to 18:00 on the first day afternoon, and then 5 units of hCG were injected from 17:00 to 18:00 on the third day afternoon. After the injection, the female mice were caged with adult ICR male mice, and the female mice with vaginal plugs detected in the morning of the next day were collected in one cage, which was recorded as the first day of pregnancy. Then, on the day of parturition, the newborn pups were counted and weighed.

[0093] Experimental Group 2: SPF-grade sexually mature ICR mice at 6 - 8 weeks of age were treated with exogenous gonadotropins. Specifically, on the first day at 17:00 - 18:00 in the afternoon, 5 units of PMSG were injected, and then on the third day at 17:00 - 18:00 in the afternoon, 5 units of hCG were injected. After the injection, the female mice were caged with adult ICR male mice, and the female mice with vaginal plugs detected the next morning were collected in one cage, which was recorded as the first day of pregnancy. Then, on the third, fourth, fifth, and sixth days of pregnancy at 17:00 - 18:00 in the afternoon, 100 μL of the prepared 1 mM lactic acid - PBS solution was intraperitoneally injected into the female mice. Then, on the day of parturition, the newborn pups were counted and weighed.

[0094] 3. Experimental Results

[0095] By counting and weighing the newborn pups of each mouse, the statistical data are as Figure 6 、 Figure 7 shown. There was no significant difference in the average litter size among 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 supplementation of lactic acid, the birth weight of the newborn pups in experimental group 2 was significantly higher than that in experimental group 1 (P < 0.05).

[0096] It was also found in the statistics that some pregnant mice after exogenous gonadotropin treatment had abnormal fetal production, such as mummified fetuses, dead fetuses, or mixtures of fetal blood, etc. The pregnant mice with abnormal conditions were counted and statistically analyzed, and the results are as Figure 8 shown. Compared with experimental group 1, the abnormal litter production in experimental group 2 was significantly improved.

[0097] Example 4

[0098] This example studies the application of lactic acid in alleviating the litter production situation of mice after gonadotropin treatment

[0099] In this example, a larger sample size was used for the study to further confirm the effect of lactic acid on the reproductive performance of mice after gonadotropin treatment.

[0100] 1. Preparation of lactic acid - PBS solution

[0101] Take the L-lactic acid solution stored at room temperature and dilute it in cell-grade PBS solution to prepare a 100 mM lactic acid - PBS solution, and then gradient-dilute the 100 mM lactic acid - PBS solution to a 1 mM lactic acid - PBS solution.

[0102] 2. Drug treatment

[0103] 1) Grouping of experimental animals

[0104] ICR female mice (SPF grade, 8 weeks old, 28 - 30 g) were randomly divided into groups for animal experiments, including a normal control group (CON), a gonadotropin treatment group (PMSG), and a gonadotropin treatment and lactate administration group (PMSG + Lactate), with 18 - 20 mice in each group. The breeding environment temperature was 22 °C, with a 12 - hour day - night cycle, and free access to food and water. After the animals were purchased, they were allowed a 5 - 7 - day adaptation period.

[0105] Control group (CON): Female 8 - week - old ICR mice were used as the research subjects, mated with male mice during natural estrus, served as the control group, and then placed in the animal house for normal breeding. On the day of parturition, the number of live offspring per litter was counted as an index.

[0106] Gonadotropin treatment group (PMSG): Female 8 - week - old ICR mice were used as the research subjects. At 18:00 in the afternoon, 10 IU of PMSG was injected, and 10 IU of hCG was injected 48 h later to induce superovulation. Immediately after that, they were caged with male mice. The next morning, the vaginal plug was checked. After the plug was seen, they were regarded as the gonadotropin treatment group and then placed in the animal house for normal breeding. On the day of parturition, the number of live offspring per litter was counted as an index.

[0107] Gonadotropin treatment and lactate administration group (PMSG + Lactate): Female 8 - week - old ICR mice were used as the research subjects. At 18:00 in the afternoon, 10 IU of PMSG was injected, and 10 IU of hCG was injected 48 h later to induce superovulation. Immediately after that, they were caged with male mice. The next morning, the vaginal plug was checked. After the plug was seen, they were regarded as the gonadotropin treatment group. Then, on the 3rd, 4th, 5th, and 6th afternoons after the plug was seen, different doses of lactate (1 mM, that is, 0.3 mg / kg.BW) were supplemented respectively. Then they were placed in the animal house for normal breeding. On the day of parturition, the number of live offspring per litter was counted as an index.

[0108] 2) Counting the number of offspring

[0109] Mice in the CON group, PMSG group, and PMSG + Lactate group were respectively placed in the animal house for normal breeding. On the day of parturition, the delivery rate, average birth weight of offspring, and number of live offspring per litter were counted as indexes.

[0110] 3. Experimental results

[0111] It was found that supplementing lactate in mice could significantly increase the delivery rate, number of live offspring per litter, and average birth weight of offspring (Table 1). The above results indicate that lactate has a significant effect in alleviating the excessive uterine inflammation induced by exogenous gonadotropin and improving pregnancy outcomes.

[0112] Table 1: Effects of lactate on the litter size of mice after gonadotropin treatment

[0113]

[0114] Note: Different lowercase letters ( abc ) indicate that there are significant differences between different groups in pairwise comparisons, and P < 0.05.

[0115] Example 5

[0116] This example is about the application of lactic acid in the precise batch production of sows

[0117] The sow batch production technology (batch management system) divides the population of breeding sows in the pig farm into several batches according to the reproductive cycle and batch interval of sows, and regulates the reproductive synchronization of the participating sows in each batch to achieve an efficient reproductive production technology system of estrus synchronization, ovulation synchronization, insemination synchronization, and farrowing synchronization. Therefore, the essence of sow batch production is batch reproduction.

[0118] 1. Experimental animals

[0119] For all experimental sows in this experiment, replacement sows aged 220 - 250 days and weighing 120 - 140 kg were selected for the experiment.

[0120] 2. Experimental design for sow batch production

[0121] Replacement sows aged 220 - 250 days and weighing 120 - 140 kg were selected for the experiment, and they were divided into two groups: the precise sow batch production (control) group and the precise sow batch production with lactate supplementation group, with 50 - 60 heads in each group.

[0122] The technical process of batch production for replacement sows in the control group (i.e., the precise sow batch production plan) is as follows: 1) Synchronization of the sexual cycle: Sows were orally administered allyl trenbolone and continuously fed for 18 days; 2) 42 hours after the synchronization of the sexual cycle, exogenous gonadotropin PMSG was injected to induce follicular development synchronization; 3) 80 hours after the injection of PMSG, the ovulation induction drug gonadorelin (GnRH) was injected, and the first artificial insemination was performed 24 hours after the injection, followed by the second artificial insemination 16 hours later.

[0123] In the lactate group, based on the existing precise sow batch production plan process, from the 9th day to the 22nd day after the first artificial insemination, lactate (at a dose of 1 mg / Kg·BW) was added to the feed and continuously fed for 14 days.

[0124] 3. Detection of pregnancy rate and litter size

[0125] At 30 - 32 days of pregnancy, B - ultrasound was used to detect whether the sows were pregnant, and the pregnancy rate of sows was statistically analyzed. Then, they were continuously raised, and subsequently, the farrowing rate and litter size of sows were statistically analyzed. The pregnancy rate and farrowing rate of sows were calculated in the following manner:

[0126] Sow pregnancy rate = number of pregnant sows / number of inseminated sows × 100%

[0127] Sow farrowing rate = number of farrowed sows / number of inseminated sows × 100%

[0128] Litter index = number of live piglets per litter × farrowing rate × 100

[0129] Among them, the litter index (Piglet Index, PI) is a comprehensive indicator to measure the overall reproductive performance of the sow population in current production, referring to the number of live piglets produced by every 100 multiparous or replacement sows that have natural mating or artificial insemination.

[0130] 4. Experimental results

[0131] Compared with the pregnancy rate and farrowing rate of sows in precision batch production of sows, the pregnancy rate and farrowing rate of sows in the lactic acid application group were both significantly increased (Table 2).

[0132] Table 2: Effects of lactic acid on the pregnancy rate and farrowing rate of sows in precision batch production of sows

[0133]

[0134] Note: Different lowercase letters (ab) indicate that compared with the control group, the pregnancy rate and farrowing rate of the lactic acid supplementation group are both significantly different, and P < 0.05.

[0135] As can be seen from Table 3, compared with the litter index of sows in precision batch production of sows, the litter index of sows in the precision batch production group combined with lactic acid was significantly improved. Among them, the number of healthy piglets and the number of live piglets per litter in the lactic acid application group were both significantly higher than those of sows in precision batch production of sows, and at the same time, the number of weak piglets in the lactic acid application group was significantly reduced (Table 3).

[0136] Table 3: Effects of lactic acid on the litter production of sows in precision batch production of sows

[0137]

[0138] Note: Different lowercase letters (ab) indicate that compared with the control group, the number of healthy piglets, weak piglets and live piglets in the lactic acid supplementation group are all significantly different, P < 0.05.

[0139] The traditional Chinese continuous production mode has become difficult to adapt to the rapid development of the pig-raising industry. Coupled with the fact that the entire live pig market is facing severe challenges, the technology of batch production of sows has become an important measure for transformation and upgrading. The era of small-scale pig farming will become the past. The emergence of large-scale and batch production methods will surely bring earth-shaking changes to the pig-raising industry in China. This invention provides an important solution path for problems such as mid-pregnancy embryo loss and decreased litter size induced by exogenous gonadotropins in the promotion of precise batch production of sows. This invention effectively improves the pregnancy rate, farrowing rate, number of live piglets per litter, and farrowing index of batch sows in precise batch production of sows. It can achieve an increase of 248 live piglets (weaned piglets) for every 100 replacement sows. Calculated according to the current price of 300 yuan - 400 yuan / weaned piglet, every 100 replacement sows will generate an additional economic value of 74,000 - 99,000 yuan. This provides an effective method for cost reduction and efficiency improvement in the entire pig-raising industry. Therefore, this invention generates huge economic value and social value for promoting batch production of live pigs in China.

[0140] Finally, although the general description and operation process of the present invention have been described in detail above, the actual application situations of different pregnant mothers, such as livestock or laboratory animals, are different. Based on the present invention, some modifications or improvements can be made to it, which are obvious to those skilled in the art. Therefore, these modifications and improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

[0141] In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this application does not separately describe various possible combination methods. In addition, any combination can be made between various different embodiments of this application, as long as it does not violate the idea of this application, it should also be regarded as the content disclosed in this application.

Claims

1. A method for improving embryo implantation or reproductive performance for non-therapeutic purposes, characterized in that, Comprising the following steps: administering lactic acid to a pregnant female; the pregnant female being a pregnant female treated with exogenous gonadotropin, the pregnant female being a livestock animal or a laboratory animal; the dosage of lactic acid administered being 0.1 - 2.0 mg / Kg.

2. The method according to claim 1, characterized in that, The dosage of lactic acid administered is 0.2 - 1.5 mg / Kg.

3. The method according to claim 2, wherein The dosage of lactic acid administered is 0.3 - 1.0 mg / Kg.

4. The method according to any one of claims 1 to 3, characterized in that, Comprising the following steps: for a pregnant female treated with exogenous gonadotropin and after conception, administering lactic acid to the pregnant female during the peri-implantation period of the embryo.

5. The method according to claim 4, characterized in that Administering lactic acid to the pregnant female during the period from the 3rd day of pregnancy to the time of embryo implantation or during the period from the 3rd day of pregnancy to 1 - 3 days after embryo implantation.

6. The method according to claim 5, wherein Administering lactic acid daily.

7. Use of the method according to claims 1 - 6 in the reproduction of livestock animals and laboratory animals.

8. A method for improving embryo implantation or reproductive performance for non-therapeutic purposes, characterized in that, Comprising the following steps: administering lactic acid to a pregnant female; the pregnant female being a pregnant female treated with exogenous gonadotropin, the pregnant female being a domestic animal; the dosage of lactic acid administered being 0.1 - 2.0 mg / Kg.

9. The method according to claim 8, wherein The dosage of lactic acid administered is 0.2 - 1.5 mg / Kg.

10. The method according to claim 9, wherein The dosage of lactic acid administered is 0.3 - 1.0 mg / Kg.

11. According to the method described in any one of claims 8-10, characterized in that, Comprising the following steps: for a pregnant female treated with exogenous gonadotropin and after conception, administering lactic acid to the pregnant female during the peri-implantation period of the embryo.

12. The method according to claim 11, wherein Administering lactic acid to the pregnant female during the period from the 3rd day of pregnancy to the time of embryo implantation or during the period from the 3rd day of pregnancy to 1 - 3 days after embryo implantation.

13. The method according to claim 12, characterized in that, Administering lactic acid daily.

14. Use of the method according to claims 8 - 13 in the reproduction of domestic animals.

15. Non-therapeutic use of lactic acid in improving embryo implantation or reproductive performance after treatment with exogenous gonadotropin, said use being in livestock animals and laboratory animals, the dosage of lactic acid administered being 0.1 - 2.0 mg / Kg.

16. Non-therapeutic use of lactic acid in improving embryo implantation or reproductive performance after treatment with exogenous gonadotropin, said use being in domestic animals, the dosage of lactic acid administered being 0.1 - 2.0 mg / Kg.

17. Use of the method according to claims 1 - 6, 8 - 13 in sow production.

18. The application according to claim 17, characterized in that, Said use being in batch production of sows.

19. The application according to any one of claims 17-18, characterized in that, Administering lactic acid to sows during the period from the 7th - 9th day to the 21st - 23rd day after natural mating or artificial insemination.

20. A method for batch production of sows, characterized in that, Comprising the following steps: 1) Synchronizing the sexual cycle of sows; 2) Administering exogenous gonadotropin to sows to induce synchronization of follicle development; 3) Administering ovulation-inducing drugs to sows; 4) Performing artificial insemination on sows; 5) Administering lactic acid to sows during the peri-implantation period of the embryo, the dosage of lactic acid administered being 0.1 - 2.0 mg / Kg.

21. The method according to claim 20, wherein The dosage of lactic acid administered is 0.2 - 1.5 mg / Kg.

22. The method according to claim 21, characterized in that, The dosage of lactic acid administered is 0.3 - 1.0 mg / Kg.

23. The method according to any one of claims 20-22, characterized in that, In step 5) above, administering lactic acid daily.

24. The method according to claim 23, wherein Administering lactic acid to sows during the period from the 7th - 9th day to the 21st - 23rd day after artificial insemination.

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