Method for increasing colonization number of early embryo of animal and promoting embryo development

Abstract

The invention discloses a method for increasing the colonization number of early embryos of animals and promoting embryonic development, and research results show that after superovulation, endometrial receptivity is damaged, embryo colonization sites are increased, but embryos are slow in development and different in size, and the number of malformed embryos is increased; research finds that neomycin can increase the embryo colonization number after superovulation, can significantly promote embryo development and reduce the number of abnormal embryos, and provides a new method for livestock and poultry and pet production.

Description

Technical Field

[0001] The invention belongs to the technical field of animal artificial propagation methods, and in particular relates to a method for increasing the number of early embryo implantation and promoting embryo development in animals. Background Art

[0002] Superovulation (SO) refers to the application of exogenous gonadotropins to induce the development of multiple follicles in the mother, releasing several times or even dozens of times more fertile eggs than under natural conditions. The purpose is to obtain as many offspring as possible from excellent female livestock within their effective reproductive years, to quickly expand the excellent population, or to obtain more eggs for in vitro fertilization and early embryo culture.

[0003] During production, serum gonadotropin (PMSG) is used to stimulate follicular development, and then human chorionic gonadotropin-releasing hormone (hCG) or luteinizing hormone (LH) and gonadotropin-releasing hormone analogue (GnRH-a) are used to induce ovulation to promote maternal ovulation.

[0004] However, hormone-induced estrus synchronization can also lead to impaired maternal endometrial receptivity, increased early embryo implantation failure rate or increased fetal mortality after implantation, reduced number of surviving fetuses, stunted fetal growth and development, reduced average weight of live births, and developmental delay. Studies have shown 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 embryo development.

[0005] Under the urgent need to use PMSG to synchronize estrus of female livestock or pets and improve the economic benefits of breeding enterprises, increasing the litter size of 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 summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. The technical problem to be solved by the present invention is to increase the number of early embryo implantation and promote embryo development by regulating the receptivity of the endometrium after superovulation, and reduce embryo loss due to impaired endometrial receptivity, thereby improving economic benefits.

[0007] Angiogenin (Ang) is a secretory protein that exerts its effects mainly by binding to the cell surface receptor Ple×in-B2 and undergoing internalization. However, there are no reports on whether Ang can affect endometrial receptivity.

[0008] Neomycin (NM) is an aminoglycoside broad-spectrum antibiotic and an important Ang inhibitor. The present invention provides a new use of neomycin in inhibiting Ang activity, increasing the number of early embryos implanted in animals after superovulation and promoting embryonic development.

[0009] Wherein, the animals include livestock or pets.

[0010] The present invention also provides a method for using neomycin as a drug, which starts from 7 days before animal mating and ends at the embryo implantation window period.

[0011] Preferably, the administration of neomycin includes intraperitoneal injection.

[0012] Preferably, the dosage of neomycin for intraperitoneal injection is 30 to 120 mg / kg / day.

[0013] Preferably, the dosage of neomycin injected intraperitoneally is 60 mg / kg / day.

[0014] Beneficial effects of the invention: The research results of the invention show that after superovulation, the endometrial receptivity is impaired, the embryo implantation sites increase, but the embryos develop slowly, the sizes vary, and the number of deformed embryos increases. The research of the invention finds that neomycin can increase the number of embryo implantation after superovulation, and can significantly promote embryo development and reduce the number of deformed embryos. It provides a new method for livestock and pet production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0016] Figure 1 This study is about the effect of superovulation on uterine angiogenin (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 The Ang inhibitor neomycin (NM) promotes early embryonic colonization and development in superovulated mice. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0020] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present 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 term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor is it a separate 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, the materials and reagents used in the following examples are all commercially available.

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

[0025] 1. Animal breeding and management

[0026] C57 female mice (10-12 weeks old) and male mice (10-12 weeks old) were purchased from Nanjing Annuokang Biotechnology Co., Ltd. and housed in the Animal Experimental Center of Nanjing Agricultural University in a temperature-controlled light environment of 20-25°C and 12-h light-dark intervals. They were allowed to eat and drink freely. Experiments were conducted after 3-5 days of adaptation. All operations were in compliance with relevant regulations on experimental 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] Embodiment 1:

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

[0031] Twenty C57 female mice aged 10 to 12 weeks were selected and randomly divided into two groups: the superovulation group (SO), first injected with 10 IU PMSG at 8:00 am, and then injected with 10 IU hCG 48 hours later. At 8:00 pm on the same day, the female mice were mated with male mice in a 1:2 ratio in the same cage, and the thrombus was detected at 8:00 am the next day; the control group (Con), without hormone treatment, natural estrus, mating, and thrombus detection at 8:00 am the next day. The day of thrombus detection was recorded as 0.5 dpc. At 3.5 dpc, the mice were killed by cervical dislocation, the abdominal cavity was opened with sterile scissors and surgical forceps, the uterus was carefully removed, and the tissue was quickly placed in liquid nitrogen and stored at -80 ℃. The effects of superovulation on angiogenin and endometrial receptivity were subsequently detected by RT-PCR and western blot. The primer sequences are listed in Table 1.

[0032] Table 1 Primer sequences

[0033]

[0034]

[0035] The experimental results are shown in Figure 1 , Figure 1 The effect of superovulation on uterine angiogenin and endometrial receptivity in mice. Figure 1 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, Ki67 immunohistochemistry; D: mRNA expression of MUC1, HOXA11, and LIF related to endometrial receptivity. Note: *p<0.05. Figure 1 As shown, the angiogenesis of the uterus of mice increased after superovulation ( Figure 1 A), the mRNA and protein levels of Ang and its receptor Plexin-B2 were significantly increased ( Figure 1 B), and superovulation significantly affected the mRNA expressions of MUC1, HOXA11, and LIF, which are related to endometrial receptivity ( Figure 1 C).

[0036] Embodiment 2:

[0037] Effects of ANG recombinant protein on proliferation-differentiation transition of endometrial epithelial cells:

[0038] Primary endometrial epithelial cells were isolated from female mice aged 8-10 weeks. The uterus was removed in a clean bench and the cells were washed with a Ca-free 2+ Mg 2+, washed 3 times with Hanks without phenol red, cut into small pieces; digested with Hanks containing 25g / L Tryspin and 6g / L Dispase II at 4℃ for 1h, then digested at room temperature for 30min; terminated digestion with medium containing 10% fetal bovine serum; repeatedly blown the tissue pieces, then passed through a 70μM cell sieve, centrifuged at 1000rpm for 5min; washed twice with medium, counted the cells, and calculated the number of cells as 1×10 5 Density plating; the first medium change was performed after 36 to 48 hours, and then every 2 days. The cells were treated with different concentrations of recombinant ANG protein for 24 hours, and cell proliferation was detected by EdU staining. qPCR was used to analyze the effect of ANG on the proliferation-differentiation transition of endometrial epithelial cells.

[0039] Figure 2 The effect of ANG recombinant protein on the proliferation-differentiation transition of endometrial epithelial cells. Figure 2 In the figure, A: EdU staining results after treating mouse endometrial epithelial cells with different concentrations of ANG recombinant protein; B: Statistical analysis results of EdU positive cell numbers; C: Gene expression levels of MUC1, LIF, and HOXA11 after treating mouse endometrial epithelial cells with 10 ng / ml ANG recombinant protein for 24 hours. Note: *p<0.05.

[0040] like Figure 2 As shown, 10ng / ml and 100ng / ml of ANG recombinant protein can significantly promote the proliferation of mouse endometrial epithelial cells ( Figure 2 A, B), and 10ng / ml ANG recombinant protein affects the gene expression of HOXA11 related to endometrial receptivity ( Figure 2 C), indicating that Ang affects the proliferation-differentiation transition of mouse endometrial epithelial cells.

[0041] Embodiment 3:

[0042] Application of neomycin in early embryonic colonization and development in mice:

[0043] Fifty C57 female mice aged 10 to 12 weeks were selected and randomly divided into three groups, namely, the neomycin + superovulation group (NM), which was intraperitoneally injected with 60 mg / kg neomycin (MCE Company) at 8:00 every day for 12 consecutive days, and after the injection of neomycin at 8:00 on the 6th day, it was followed by an injection of 10 IU PMSG (Ningbo Second Hormone Factory), and 48 hours later (the 8th day after the injection of neomycin), 10 IU hCG (Ningbo Second Hormone Factory) was injected. At 8:00 pm on the same day, the female mice were mated with male mice in a 1:2 ratio in the same cage, and the thrombus was checked at 8:00 am the next day; the superovulation group (SO), firstly, 10 IU PMSG was intraperitoneally injected at 8:00 in the morning, and 10 IU hCG, at 8:00 pm on the same day, female mice were mated with male mice in a ratio of 1:2 in the same cage, and the plug was detected at 8:00 am the next day; the control group (Con) was not treated with hormones, naturally estrus, mated, and the plug was detected at 8:00 am the next day. The day of plug detection was recorded as 0.5 dpc. At 10.5 dpc, the mice were killed by cervical dislocation, and the abdominal cavity was opened with sterile scissors and surgical forceps. The uterus was carefully removed, the embryo implantation sites were counted, and the uterine tissue was photographed. After that, the mouse embryos were separated under a stereo microscope to observe their development.

[0044] Figure 3 The angiopoietin inhibitor NM promotes early embryonic colonization and development in superovulated mice. Figure 3 In the figure, A: the appearance of embryo implantation in the mouse uterus at 10.5 dpc; B: the number of embryo implantation in the mouse uterus at 10.5 dpc; C: the average weight of a single embryo; D: the weight of a single litter; E: the development of a single embryo. Note: *p<0.05, **p<0.01.

[0045] like Figure 3 As shown in the figure, the number of embryos implanted in mice after superovulation treatment (SO) was significantly higher than that in the control group (Con), but the embryos in the SO group had uneven development and delayed development, and there were more malformed embryos. After NM treatment, the number of embryos implanted at 10.5dpc increased significantly, and the size of embryos tended to be uniform, and the development was earlier than that in the SO group, and the number of malformed fetuses decreased ( Figure 3 A, B, E). The weight of a single embryo after superovulation was significantly lower than that of the control group. The average weight of a single embryo in the NM treatment increased by 24.14% compared with the SO group ( Figure 3 C), and the weight of each litter increased significantly ( Figure 3 D).

[0046] In summary, the present invention found that neomycin regulates the proliferation-differentiation conversion of endometrial epithelial cells, significantly increases the number of embryo implantation after superovulation, and can promote embryo development, providing a new method for livestock, poultry and pet production.

[0047] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for increasing the number of early embryo implantation and promoting embryo development in animals, characterized in that: Neomycin was administered to animals before and after superovulation was induced.

2. The method for increasing the number of early embryo implantation and promoting embryo development in animals according to claim 1, characterized in that: The animals include livestock or pets.

3. The method for increasing the number of early embryo implantation and promoting embryo development in animals according to claim 1 or 2, characterized in that: The administration of neomycin includes intraperitoneal injection, intravenous injection or oral administration.

4. The method for increasing the number of early embryo implantation and promoting embryo development in animals according to claim 1 or 2, characterized in that: The dosage of neomycin administered is 30 to 120 mg / kg / day.

5. The method for increasing the number of early embryo implantation and promoting embryo development in animals according to claim 4, characterized in that: The dose of neomycin administered was 60 mg / kg / day.

6. The method for increasing the number of early embryo implantation and promoting embryo development in animals according to claim 1 or 2, characterized in that: Neomycin is administered to female animals starting from 7 to 10 days before mating until the end of the embryo implantation window period; and serum gonadotropin is used to induce superovulation in female animals 2 to 3 days before mating.

7. The method for increasing the number of early embryo implantation and promoting embryo development in animals according to claim 1 or 2, characterized in that: The neomycin is used for regulating the proliferation-differentiation conversion of animal endometrial epithelial cells after superovulation.

8. The method for increasing the number of early embryo implantation and promoting embryo development in animals according to claim 1 or 2, characterized in that: The neomycin can improve the endometrial receptivity and reduce the number of abnormal embryos.

Images