A method for increasing the number of early embryo implantations and promoting embryo development in animals

Abstract

The application discloses a method for increasing the number of early embryo implantation and promoting embryo development. The research result of the application shows that after superovulation, the endometrial receptivity is damaged, the embryo implantation site is increased, but the embryo development is slow, the sizes of the embryos are different, and the number of abnormal embryos is increased. The research of the application finds that neomycin can increase the number of embryo implantation after superovulation, and can significantly promote embryo development and reduce the number of abnormal embryos, thereby providing a new method for the production of livestock, poultry and pets.

Description

Technical Field

[0001] This invention belongs to the technical field of artificial animal breeding methods, specifically relating to a method for increasing the number of early embryos implanted in animals and promoting embryo development. Background Technology

[0002] Superovulation (SO) refers to the use of exogenous gonadotropins to induce the development of multiple follicles in a mother, releasing several times or even ten times more fertile eggs than under natural conditions. The purpose is to obtain as many offspring as possible within the effective reproductive lifespan of superior female animals, rapidly expand the superior breed, or obtain a large number of eggs for in vitro fertilization and early embryo culture.

[0003] In production, serum gonadotropin (PMSG) is used to stimulate follicle development, followed by ovulation induction with human chorionic gonadotropin-releasing hormone (hCG) or luteinizing hormone (LH) and gonadotropin-releasing hormone analogs (GnRH-a) to promote maternal ovulation.

[0004] However, hormone-induced estrus synchronization can also impair the establishment of maternal endometrial receptivity, increasing the rate of early embryo implantation failure or post-implantation fetal mortality, reducing the number of surviving fetuses, hindering fetal growth and development, and resulting in lower average birth weight and developmental delays in live births. Studies show that implantation failure due to impaired endometrial receptivity accounts for two-thirds of all implantation failures. Therefore, improving endometrial receptivity after superovulation is of great significance for increasing the number of embryos implanted and promoting embryonic development.

[0005] Given the urgent need to use PMSG to synchronize estrus in female livestock or pets and improve the economic benefits of breeding enterprises, increasing the number of offspring in female livestock or pets is an inevitable trend, and it can also provide technical reference for human in vitro fertilization. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. The technical problem to be solved by the present invention is to increase the number of early embryos implanted and promote embryonic development by regulating the receptivity of the endometrium after superovulation, thereby reducing embryo loss due to impaired endometrial receptivity and thus improving economic efficiency.

[0007] Angiogenin (Ang) is a secreted protein that mainly exerts its effects through endocytosis by binding to the cell surface receptor Plexin-B2, but whether Ang can affect endometrial receptivity has not been reported.

[0008] Neomycin (NM) is a broad-spectrum aminoglycoside antibiotic and an important Ang inhibitor. This invention provides a novel use of neomycin in inhibiting Ang activity to increase the number of early embryos implanted in animals after superovulation and to promote embryonic development.

[0009] The animals mentioned include poultry or pets that are receptive to the uterine lining.

[0010] This invention also provides a method for using neomycin as a drug, starting treatment 7 days before animal mating and continuing until the end of the embryo implantation window.

[0011] Preferably, the neomycin is administered via intraperitoneal injection.

[0012] Preferably, the dose of neomycin administered via intraperitoneal injection is 30-120 mg / kg / day.

[0013] Preferably, the intraperitoneal injection dose of neomycin is 60 mg / kg / day.

[0014] The beneficial effects of this invention are as follows: Research results indicate that superovulation leads to impaired endometrial receptivity, an increase in embryo implantation sites, but also results in delayed embryonic development, inconsistent embryo size, and an increased number of malformed embryos. This invention has found that neomycin can increase the number of embryos implanted after superovulation and significantly promote embryonic development while reducing the number of malformed embryos. This provides a new method for the production of livestock and pets with endometrial receptivity. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0016] Figure 1 The effects of superovulation on uterine angiopoietin (Ang) and endometrial receptivity in mice.

[0017] Figure 2 The effect of Ang on the proliferation-differentiation transition of endometrial epithelial cells.

[0018] Figure 3 Neomycin (NM), an Ang inhibitor, promotes early embryo colonization and development in superovulatory mice. Detailed Implementation

[0019] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0025] 1. Animal husbandry and management

[0026] Both female and male C57 mice (10-12 weeks old) were purchased from Nanjing Annokang Biotechnology Co., Ltd. and housed at the Animal Experiment Center of Nanjing Agricultural University. They were kept in a temperature- and light-controlled environment at 20-25℃ with a 12-hour light-dark interval, with free access to food and water. After 3-5 days of acclimatization, experiments were conducted, and all procedures complied with relevant regulations on laboratory animal welfare.

[0027] 2. Main reagents and instruments

[0028] PMSG, hCG, neomycin, Trizol, reverse transcription kit, Ang antibody, Plexin-B2 antibody, Tubulinα antibody, ANG recombinant protein, EdU kit.

[0029] Example 1:

[0030] Superovulation affects endometrial receptivity and Ang expression in mice:

[0031] Twenty female C57 mice aged 10-12 weeks were randomly divided into two groups: the superovulation group (SO) and the control group (Con). SO mice received an intraperitoneal injection of 10 IU PMSG at 8:00 AM, followed by an injection of 10 IU hCG 48 hours later. Female mice were mated with male mice at a 1:2 ratio at 8:00 PM that evening, and ovulation was detected at 8:00 AM the following morning. The control group (Con) received no hormone treatment, underwent natural estrus, mated, and ovulation was detected at 8:00 AM the following morning. The day of ovulation detection was recorded as 0.5 dpc. At 3.5 dpc, mice were euthanized by cervical dislocation. The peritoneum was opened with sterile scissors and surgical forceps, the uterus was carefully dissected, and the tissue was quickly placed in liquid nitrogen and stored at -80°C. The effects of superovulation on angiopoietin and endometrial receptivity were subsequently detected by RT-PCR and Western blot. Primer sequences are listed in Table 1.

[0032] Table 1 Primer Sequences

[0033]

[0034] The experimental results are shown in Figure 1 , Figure 1 The effects of superovulation on uterine angiopoietin and endometrial receptivity in mice. Figure 1 In the table, A: mRNA expression levels of Ang and Plexin-B2 in the uterus of mice after superovulation; B: protein expression levels of Ang and Plexin-B2 in the uterus of mice after superovulation; C: cell proliferation status in the uterus of mice after superovulation, using Ki67 immunohistochemistry; D: mRNA expression of MUC1, HOXA11, and LIF, which are related to endometrial receptivity. Note: p< 0.05. For example... Figure 1 As shown, uterine angiogenesis increases in mice after superovulation ( Figure 1 A), the mRNA and protein levels of Ang and its receptor Plexin-B2 were significantly elevated ( Figure 1 B), and superovulation significantly affected the mRNA expression of MUC1, HOXA11, and LIF, which are related to endometrial receptivity. Figure 1 C).

[0035] Example 2:

[0036] Effects of recombinant ANG protein on endometrial epithelial cell proliferation-differentiation transition:

[0037] Primary endometrial epithelial cells were isolated from mice. The uterus of 8-10 week old female mice was removed in a clean bench and treated with calcium-free... 2+ Mg 2+Wash the tissue pieces three times with phenol red-free Hanks culture medium and cut them into small pieces. Digest the tissue pieces with Hanks culture medium containing 25 g / L Tryspin and 6 g / L Dispase II at 4°C for 1 h, followed by digestion at room temperature for 30 min. Terminate the digestion with medium containing 10% fetal bovine serum. Repeatedly pipette the tissue pieces, then pass them through a 70 μM cell sieve and centrifuge at 1000 rpm for 5 min. Wash the tissue pieces twice with medium, count the cells, and divide them into groups of 1 x 10⁻⁶ cells. 5 Cells were plated at high density; the medium was changed for the first time after 36-48 hours, and then every 2 days thereafter. Cells were treated with different concentrations of recombinant ANG protein for 24 hours, and cell proliferation was detected by EdU staining. The effect of ANG on the proliferation-differentiation transition of endometrial epithelial cells was analyzed by qPCR.

[0038] Figure 2 The effect of ANG recombinant protein on the proliferation-differentiation transition of endometrial epithelial cells. Figure 2 In the table, A: EdU staining results of mouse endometrial epithelial cells after treatment with different concentrations of recombinant ANG protein; B: Statistical analysis of the number of EdU-positive cells; C: Gene expression levels of MUC1, LIF, and HOXA11 in mouse endometrial epithelial cells after treatment with 10 ng / ml recombinant ANG protein for 24 h. Note: p< 0.05.

[0039] like Figure 2 As shown, 10 ng / ml and 100 ng / ml of recombinant ANG protein significantly promoted the proliferation of mouse endometrial epithelial cells. Figure 2 A, B), and 10 ng / ml ANG recombinant protein affects the gene expression of HOXA11, which is related to endometrial receptivity (A ...). Figure 2 (C) indicates that Ang affects the proliferation-differentiation transition of mouse endometrial epithelial cells.

[0040] Example 3:

[0041] Application of neomycin in early mouse embryo colonization and development:

[0042] Fifty female C57 mice aged 10-12 weeks were randomly divided into three groups: the neomycin + superovulation group (NM), in which 60 mg / kg neomycin (MCE) was injected intraperitoneally at 8:00 AM daily for 12 consecutive days, followed by an injection of 10 IU PMSG (Ningbo Second Hormone Factory) at 8:00 AM on day 6, and then 10 IU hCG (Ningbo Second Hormone Factory) 48 hours later (day 8 after neomycin injection); the female mice were mated with male mice at a 1:2 ratio at 8:00 PM that evening, and ovulation was detected at 8:00 AM the following morning; the superovulation group (SO), in which 10 IU PMSG was injected intraperitoneally at 8:00 AM, followed by 10 IU hCG 48 hours later, and then 1:2 female mice were mated with male mice at a 1:2 ratio at 8:00 PM that evening, and ovulation was detected at 8:00 AM the following morning; and the control group (Con), which received no hormone treatment, came into estrus naturally, mated, and had ovulation detected at 8:00 AM the following morning. The day of thrombus detection was recorded as 0.5 dpc. At 10.5 dpc, the mice were euthanized by cervical dislocation. The abdominal cavity was opened with sterile scissors and surgical forceps, the uterus was carefully dissected, the embryo implantation sites were counted, and the uterine tissue was photographed. The mouse embryos were then separated under a stereomicroscope to observe their development.

[0043] Figure 3 The angiopoietin inhibitor NM promotes early embryo colonization and development in superovulatory mice. Figure 3 In the image, A: Appearance of embryo implantation in the mouse uterus at 10.5 dpc; B: Number of embryos implanted in the mouse uterus at 10.5 dpc; C: Average weight of a single embryo; D: Litter weight; E: Developmental status of a single embryo. Note: p< 0.05, p< 0.01.

[0044] like Figure 3 As shown, the number of embryos implanted in mice after superovulation treatment (SO) was significantly higher than that in the control group (Con). However, the embryos in the SO group were uneven in size and developmental delay, with a higher number of malformed embryos. After NM treatment, the number of embryos implanted at 10.5 dpc was significantly increased, and the embryos were more uniform in size and developmental earlier than in the SO group, with a reduced number of malformed embryos. Figure 3 A, B, E). Furthermore, the weight of a single embryo after superovulation treatment was significantly lower than that in the control group; the average weight of a single embryo in the NM treatment increased by 24.14% compared to the SO group. Figure 3 C), and the weight of a single clutch increased significantly ( Figure 3 D).

[0045] In summary, this invention has discovered that neomycin regulates the proliferation-differentiation transition of endometrial epithelial cells, significantly increasing the number of embryos implanted after superovulation and promoting embryo development, providing a new method for livestock and pet production.

[0046] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for increasing the number of early embryos implanted in animals and promoting embryonic development, characterized in that: Neomycin was administered to mice before and after inducing superovulation. The dosage of neomycin administered is 30-120 mg / kg / day.

2. The method for increasing the number of early embryos implanted and promoting embryonic development in animals according to claim 1, characterized in that: The methods of administering neomycin include intraperitoneal injection, intravenous injection, or oral administration.

3. The method for increasing the number of early embryos implanted and promoting embryonic development according to claim 1, characterized in that: The recommended dose of neomycin is 60 mg / kg / day.

4. The method for increasing the number of early embryos implanted and promoting embryonic development in animals according to claim 1 or 2, characterized in that: Neomycin was administered to female animals 7-10 days before mating and continued until the end of the embryo implantation window; and serum gonadotropins were used to induce superovulation in female animals 2-3 days before mating.

Images