Method for obtaining mature egg cells of non-human animals, assisted reproduction method for non-human animals, and use of said methods in endangered animal protection

Abstract

The present invention relates to the technical field of animal reproduction, and relates in particular to a method for obtaining mature egg cells of non-human animals, an assisted reproduction method for non-human animals, and use of said methods in endangered animal protection. The method uniquely uses the spleen of other animals (such as mice) and has an excellent effect. An ideal number of mature egg cells can be rapidly obtained by means of the method.

Description

Methods for obtaining mature oocytes from non-human animals, assisted reproductive methods for non-human animals, and their applications in endangered animal protection Technical Field

[0001] The present invention relates to the field of animal reproductive technology, and in particular to a method for obtaining mature egg cells of non-human animals, an assisted reproductive method for non-human animals, and an application thereof in the protection of endangered animals. Background Art

[0002] Endangered species face numerous challenges to survival, including environmental changes, disease, human activities, and other external factors, which can negatively impact their reproductive capacity and population size. Therefore, developing assisted reproductive technology that can effectively enhance the reproductive capacity of endangered species is crucial for preserving biodiversity and maintaining ecological balance.

[0003] Currently, the protection measures taken for endangered animals mainly include wildlife protection, ecological restoration, gene bank preservation, etc. However, these protection measures are often difficult to effectively solve the reproductive problems of endangered animals.

[0004] As for assisted reproductive technologies, existing technologies primarily include artificial in vitro fertilization (IVF) and embryo transfer. While these technologies can address reproductive issues for some animals, they still have certain limitations and shortcomings for certain endangered species. For example, the small number of endangered species, their short estrus periods, and the limited number of mature eggs that can be collected through artificial egg collection make it difficult to substantially improve the reproductive success rate and population size of endangered species.

[0005] Summary of the Invention

[0006] In view of this, the present invention provides a method for obtaining mature egg cells from non-human animals, an assisted reproductive method for non-human animals, and its application in the protection of endangered animals, so as to solve the problem in the related technology that the number of mature egg cells collected by artificial egg collection is small, making it difficult to substantially improve the reproductive success rate and population size of endangered animals.

[0007] In a first aspect, the present invention provides a method for obtaining mature oocytes from non-human animals, comprising the following steps:

[0008] The donor animal's ovarian tissue pieces are transplanted into the recipient animal's spleen;

[0009] After the oocytes in the ovarian tissue mass mature in the recipient animal, the mature oocytes are extracted.

[0010] In the above method provided by the present invention, the ovarian tissue block of the donor animal is transplanted into the spleen of the recipient animal for preservation, and after the egg cells in the ovarian tissue block mature, the mature egg cells of the donor animal are extracted. Among them, transplanting the ovarian tissue block of the donor animal into the spleen of the recipient animal for preservation can provide the ovarian tissue block with an external environment similar to its natural growth environment, which is conducive to the survival and growth of the ovarian tissue; at the same time, by performing transplantation and egg retrieval on the recipient animal, the harm of these processing operations to the donor animal can be avoided to a great extent, ensuring its health and safety. Therefore, the method of the present invention can safely and quickly obtain a large number of mature egg cells from donor animals in a short period of time, can effectively protect the genetic diversity of donor animals, expand their animal population, and enhance their ability to adapt to environmental changes, thereby better protecting biodiversity and ecological balance.

[0011] In an optional embodiment, after the ovarian tissue piece is transplanted into the spleen of the recipient animal, the transplanted recipient animal is raised;

[0012] Preferably, gonadotropin is administered to the recipient animal after the transplantation to promote the maturation of oocytes in the ovarian tissue mass.

[0013] In an optional embodiment, after the ovarian tissue piece is transplanted, the transplanted recipient animal is raised for a first effective period of time, and then the gonadotropin is administered thereto to obtain a target recipient animal;

[0014] feeding the target recipient animal for a second effective period of time, and then obtaining mature oocytes from the spleen of the target recipient animal;

[0015] Optionally, the first effective period is 1 to 20 weeks;

[0016] Optionally, the second effective duration is 18 to 96 hours.

[0017] In an alternative embodiment, the gonadotropin comprises pregnant mare serum gonadotropin and / or human chorionic gonadotropin.

[0018] In an optional embodiment, when the gonadotropin is administered to the recipient animal after the transplantation, the administration amount of the gonadotropin is 2 to 7 IU per kilogram of body weight of the recipient animal.

[0019] In an optional embodiment, the diameter of the ovarian tissue block is 0.1-1 mm.

[0020] In an optional embodiment, the extracting of mature oocytes comprises:

[0021] obtaining a cumulus-oocyte complex tightly wrapped by cumulus cells and granulosa cells and having a glossy appearance from the ovarian tissue block;

[0022] A naked egg with obvious and uniform germinal vesicles and a small perivitelline space is obtained from the cumulus-oocyte complex.

[0023] In an optional embodiment, the method further includes:

[0024] The obtained naked eggs are cleaned and then cultured to obtain mature egg cells;

[0025] Optionally, the washing comprises washing with an oocyte in vitro maturation culture medium;

[0026] Optionally, the culturing includes culturing using an oocyte in vitro maturation culture medium, and the culturing time is 15 to 16 hours.

[0027] In an optional embodiment, the donor animals include endangered animals, rare and protected animals, or animals with high reproduction or population demand, such as sika deer, Siberian tigers, giant pandas and other rare and protected animals or endangered animals.

[0028] Optionally, the recipient animal is an easily accessible animal, a low-cost animal, an easy-to-raise animal, or an experimental animal, such as mice, rats, guinea pigs, rabbits, cats, dogs, pigs, monkeys, and other animals used in experiments.

[0029] Preferably, the donor animal and the recipient animal are both viviparous mammals; further, the donor animal and the recipient animal are both sexually mature female viviparous mammals.

[0030] In an optional embodiment, the ovarian tissue piece is from a sexually mature living donor animal or a deceased donor animal that died no more than 3 hours ago.

[0031] That is, any sexually mature donor animal is suitable for the present invention, including donor animals with reduced or no fertility, and elderly donor animals. It should also be noted that donor animals that have been dead for no more than 3 hours are also suitable, such as donor animals that have been dead for 1 hour. Of course, the dead animal should also be a sexually mature animal.

[0032] In a second aspect, the present invention provides a method for assisted reproduction in non-human animals, comprising the following steps:

[0033] Step 1: taking the mature oocytes obtained by the above method and performing in vitro fertilization to obtain in vitro fertilized eggs;

[0034] Step 2a: transplanting the in vitro fertilized egg obtained in step 1 into the uterus or fallopian tube of a mother animal or a surrogate mother animal; or

[0035] Step 2b: culturing the in vitro fertilized eggs obtained in step 1 in vitro to obtain embryonic cells, and then transplanting the embryonic cells into the uterus or fallopian tube of the mother animal or surrogate mother animal.

[0036] It should be noted that the fertilized egg here usually refers to a single-cell fertilized egg, and the embryonic cell usually refers to the fertilized egg that develops and divides into two cells or four cells. These situations are all acceptable. When transplanted, it can be transplanted directly into the uterus or into the fallopian tube. If it is in the fallopian tube, it will also enter the uterus during the later development process.

[0037] A surrogate mother animal generally refers to any mammal, such as a fertile female of the same species as the donor animal.

[0038] In a third aspect, the present invention provides the application of the above-mentioned method for obtaining mature oocytes of non-human animals or the assisted reproductive method of non-human animals in the protection of endangered animals.

[0039] In an optional embodiment, the endangered animal is a viviparous mammal;

[0040] Preferably, the endangered animals include at least one of sika deer, Siberian tigers and giant pandas.

[0041] In an optional embodiment, the application also includes application to rare and protected animals or animals with high reproduction or population requirements.

[0042] The present invention has the following beneficial technical effects:

[0043] (1) The present invention provides a method for rapidly obtaining mature oocytes from animals. This method uniquely utilizes the spleen of other animals (such as mice) and has excellent results. Furthermore, this method can rapidly obtain an ideal number of mature oocytes.

[0044] (2) The present invention also provides an animal assisted reproductive method, which can effectively assist the donor animal in reproduction.

[0045] (3) The present invention has extremely low requirements for donor animals. Any sexually mature donor animal can be used, including elderly animals that have reduced or lost their reproductive capacity. Therefore, this application is of great significance for the effective or relatively rapid reproduction of donor animals.

[0046] (4) The method of the present invention is of great significance and value, particularly for the protection of endangered animals. It can significantly increase the genetic diversity of endangered animals, expand their populations, and enhance their ability to adapt to environmental changes. When the method of the present invention is used to protect endangered animals, ovarian tissue pieces can be obtained not only from fertile donor animals, but also from older donor animals, or even from recently deceased donor animals, thereby ensuring the reproduction of endangered animals to the greatest extent possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0048] FIG1 is a diagram showing the H&E staining results of a pathological section of spleen tissue of a target recipient mouse in step (4) of Example 1 of the present invention;

[0049] FIG2 is a photograph of the first generation of mice produced by one of the surrogate mother mice in Example 1 of the present invention;

[0050] FIG3 is an electron microscope photograph of the fertilized egg obtained in step (7) of Example 2 of the present invention. DETAILED DESCRIPTION

[0051] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0052] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0053] The present invention is further described in detail below with reference to specific examples. These examples should not be construed as limiting the scope of protection claimed in the present invention.

[0054] Example 1

[0055] Assisted reproduction in mice was performed as follows:

[0056] (1) C57BL / 6J female mice carrying fluorescent protein throughout their bodies were used as donor mice. The flank was opened and the ovaries were removed from the opening.

[0057] (2) The ovary obtained in step (1) is trimmed to remove the capsule and fallopian tube, and then divided into ovarian tissue blocks with a diameter of 0.1 to 1 mm, and placed in HBSS (Hank's balanced salt solution) for storage. To avoid major damage to the ovarian tissue, this step can be completed under a microscope;

[0058] (3) Using a 10 ml syringe, puncture and implant the ovarian tissue block obtained in step (2) into the spleen of a recipient mouse that has undergone spleen translocation. After implantation, press the puncture site to prevent the ovarian tissue block from falling off, and obtain a transplanted recipient mouse;

[0059] (4) The transplanted recipient mice obtained in step (3) are conventionally raised, and 8 weeks after the transplantation, the transplanted recipient mice are intraperitoneally injected with pregnant mare serum gonadotropin (PMSG) at an injection volume of 7 IU per kilogram of body weight to obtain target recipient mice; the target recipient mice are conventionally raised until 48 hours after the injection, the spleen tissue of the target recipient mice is taken for pathological sectioning, and then relatively mature oocytes are extracted from the spleen of the target recipient mice by surgery;

[0060] (5) selecting a cumulus oocyte complex with compact and glossy cumulus cells and granulosa cells from the more mature ovum extracted in step (4), and then selecting a naked egg with obvious, uniform germinal vesicles and a smaller perivitelline space from the cumulus oocyte complex for subsequent maturation culture;

[0061] (6) The naked eggs selected in step (5) were washed twice with IVM culture medium (oocyte in vitro maturation culture medium), and then transferred to the balanced IVM culture medium and cultured for 16 hours to allow the oocytes to mature and obtain mature oocytes. All the obtained mature oocytes were washed and transferred to the balanced HTF fertilization dish for use;

[0062] (7) A C57BL / 6J male mouse carrying fluorescent protein was killed by cervical dislocation. Sperm from the tail of the epididymis was placed in a C-TYH drop after equilibration for half an hour. After mixing and capacitation for 0.5 hours, 5 μl of the capacious sperm was dropped into the HTF fertilization dish containing mature oocytes obtained in step (6) for in vitro fertilization. The mouse was then placed in an incubator at 5% CO2 and 37°C for about 24 hours to obtain mouse embryonic cells (2-cell).

[0063] (8) Pseudo-pregnant ICR / Gpt mice were used as surrogate mother mice, and the 2-cells obtained in step (7) were transplanted into the oviducts of the surrogate mother mice. Twenty 2-cells were transplanted into each surrogate mother mouse, and a total of three surrogate mother mice were transplanted. The remaining 2-cells were transferred to the balanced KSOM culture dish for culture, 20 cells / drop, 50 μl KSOM / drop, and placed in a 5% CO2, 37°C incubator for culture.

[0064] (9) Properly feed and manage the surrogate mother mice (including providing appropriate nutrition, environment, and health checks) to ensure that they can conceive normally and give birth to healthy pups; during the normal gestation period of the surrogate mother mice, monitor the growth and development of the fetus to ensure that they can give birth to healthy pups;

[0065] (10) After about 19 days of rearing, the surrogate mother mice gave birth to pups, and the number of pups was counted.

[0066] The H&E staining results of the pathological sections of the spleen tissue of the target recipient mouse in step (4) are shown in Figure 1. As can be seen from Figure 1, the ovarian tissue mass can continue to survive in the mouse spleen and develop into mature oocytes under the stimulation of gonadotropin.

[0067] In this embodiment, steps (1) to (6) took a total of 59 days, and a total of 100 mature oocytes were obtained. It can be seen that the method of the present invention can obtain an ideal number of mature oocytes from donor animals in a short period of time.

[0068] In step (7) of this embodiment, a total of 78 mouse embryonic cells (2-cell) were obtained. After repeated multiple times according to the above method, the fertilization rate was 78.3%. This shows that the method of the present invention does not affect the activity of the egg cells, and the obtained mature egg cells can successfully complete fertilization.

[0069] In step (10) of this embodiment, the number of pups born by the three surrogate mother mice was 8, 9, and 11, respectively. According to the above method, multiple verifications were performed, and the statistical results showed that the ratio of 2-cell survival and development into viable mice was 73%, respectively. This shows that the mature oocytes obtained by the method of the present invention can successfully develop into viable pups after forming fertilized eggs.

[0070] In step (10) of this embodiment, a photo of the first-generation mice produced by one of the surrogate mother mice is shown in Figure 2. As can be seen from Figure 2, the first-generation mice produced by the surrogate mother mouse are all fluorescent protein-positive mice, with the same characteristics as the parent, which shows that the mature egg cells obtained by the method of the present invention and the assisted reproductive method can stably inherit the maternal characteristics.

[0071] Example 2

[0072] Assisted reproduction of sika deer is performed as follows:

[0073] (1) The lateral abdomen of the donor sika deer (source: female deer purchased from a sika deer farm in Northeast China) was opened and the ovaries were removed from the opening;

[0074] (2) The ovary obtained in step (1) is trimmed to remove the capsule and fallopian tube, and then divided into ovarian tissue blocks with a diameter of 0.1 to 1 mm, and placed in HBSS (Hank's balanced salt solution) for storage. To avoid major damage to the ovarian tissue, this step can be completed under a microscope;

[0075] (3) Using a 10 ml syringe, puncture and implant the ovarian tissue block obtained in step (2) into the spleen of a recipient mouse that has undergone spleen translocation. After implantation, press the puncture site to prevent the ovarian tissue block from falling off, and obtain a transplanted recipient mouse;

[0076] (4) conventionally raising the transplanted recipient mice obtained in step (3), and 8 weeks after the transplantation, intraperitoneally injecting pregnant mare serum gonadotropin (PMSG) into the transplanted recipient mice at a dose of 7 IU per kilogram of body weight to obtain target recipient mice; conventionally raising the target recipient mice until 48 hours after the injection, and surgically extracting relatively mature oocytes from the spleen of the target recipient mice;

[0077] (5) selecting a cumulus oocyte complex with compact and glossy cumulus cells and granulosa cells from the more mature ovum extracted in step (4), and then selecting a naked egg with obvious, uniform germinal vesicles and a smaller perivitelline space from the cumulus oocyte complex for subsequent maturation culture;

[0078] (6) The naked eggs selected in step (5) were washed twice with IVM culture medium (oocyte in vitro maturation culture medium), and then transferred to the balanced IVM culture medium and cultured for 16 hours to allow the oocytes to mature and obtain mature oocytes. All the obtained mature oocytes were washed and transferred to the balanced HTF fertilization dish for use;

[0079] (7) Frozen sika deer sperm was thawed and placed in a C-TYH droplet that had been equilibrated for half an hour. After mixing and capacitation for 0.5 h, 5 μl of the capacious sperm was dropped into the HTF fertilization dish containing mature oocytes obtained in step (6) for in vitro fertilization. The dish was then placed in a 5% CO2, 37°C incubator and cultured for about 24 h to obtain sika deer embryonic cells.

[0080] (8) Using pseudo-pregnant sika deer (source: female deer purchased from a sika deer farm in Northeast China) as surrogate mother sika deer, and transplanting the sika deer embryonic cells obtained in step (7) into the oviduct of the surrogate mother sika deer, with 5 embryonic cells transplanted into each surrogate mother sika deer, and a total of 3 surrogate mother sika deer were transplanted;

[0081] (9) Properly feed and manage the surrogate mother sika deer (including providing appropriate nutrition, environment, and health checks) to ensure that the surrogate mother sika deer has a normal pregnancy and gives birth to healthy cubs; during the normal pregnancy of the surrogate mother sika deer, monitor the growth and development of the fetus to ensure that the surrogate mother gives birth to healthy cubs;

[0082] (10) After about 240 days of rearing, the surrogate mother sika deer gave birth and the number of cubs was counted.

[0083] An electron microscope photograph of the fertilized egg obtained in step (7) of this embodiment is shown in FIG3 . As can be seen from FIG3 , the rare animal sika deer can be cultivated to form a fertilized egg by the method provided by the present invention, and meets the conditions for embryo transplantation.

[0084] In this embodiment, steps (1) to (6) took a total of 59 days, and a total of 14 mature oocytes were obtained. It can be seen that the method of the present invention can obtain an ideal number of mature oocytes from donor animals in a short period of time.

[0085] In step (7) of this embodiment, a total of 9 sika deer embryonic cells were obtained. After repeated use of the above method, the fertilization rate was 63.2%. This shows that the method of the present invention does not affect the activity of the egg cells, and the obtained mature egg cells can successfully complete fertilization.

[0086] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for obtaining mature oocytes of non-human animals, characterized in that, It includes the following steps: Transplant ovarian tissue blocks of a donor animal into the spleen of a recipient animal; When the egg cells in the ovarian tissue blocks mature in the recipient animal, extract the mature egg cells.

2. The method according to claim 1, characterized in that, After transplanting the ovarian tissue blocks into the spleen of the recipient animal, raise the transplanted recipient animal; Preferably, administer gonadotropin to the transplanted recipient animal to promote the maturation of the egg cells in the ovarian tissue blocks.

3. The method according to claim 2, characterized in that, After transplanting the ovarian tissue blocks, raise the transplanted recipient animal for a first effective duration, and then administer the gonadotropin to obtain a target recipient animal; Raise the target recipient animal for a second effective duration, and then obtain mature egg cells from the spleen of the target recipient animal; Optionally, the first effective duration is 1 to 20 weeks; Optionally, the second effective duration is 18 to 96 h.

4. The method according to claim 2 or 3, characterized in that, The gonadotropin includes pregnant mare serum gonadotropin and / or human chorionic gonadotropin.

5. The method according to claim 4, wherein When administering the gonadotropin to the transplanted recipient animal, the dosage of the gonadotropin for each kilogram of body weight of the recipient animal is 2 to 7 IU.

6. The method according to claim 1, wherein The diameter of the ovarian tissue block is 0.1 to 1 mm.

7. The method according to claim 1, wherein The extraction of the mature egg cells includes: Obtain a cumulus-oocyte complex in which the cumulus cells and granulosa cells are tightly wrapped and shiny from the ovarian tissue block; Obtain a denuded oocyte with an obvious, uniform germinal vesicle and a small perivitelline space from the cumulus-oocyte complex.

8. The method according to claim 7, wherein The method further includes: Wash and culture the obtained denuded oocytes to obtain mature egg cells; Optionally, the washing includes washing with an in vitro maturation medium for oocytes; Optionally, the culture includes culturing with an in vitro maturation medium for oocytes, and the culture time is 15 to 16 h.

9. The method according to claim 1, characterized in that, The donor animal includes endangered animals, rare and protected animals, or animals with high requirements for reproduction or quantity; Optionally, the recipient animal is an animal that is easy to obtain, has low cost, is easy to raise, or is a laboratory animal; Preferably, both the donor animal and the recipient animal are viviparous mammals.

10. The method according to claim 1, characterized in that, The ovarian tissue block is from a sexually mature surviving donor animal or a dead donor animal whose death time does not exceed 3 hours.

11. An assisted reproduction method for non-human animals, characterized in that, It includes the following steps: Step 1: Take the mature egg cells obtained by the method according to any one of claims 1 to 10, perform in vitro fertilization to obtain in vitro fertilized eggs; Step 2a: Transplant the in vitro fertilized eggs obtained in Step 1 into the uterus or fallopian tube of a maternal animal or a surrogate maternal animal; or, Step 2b: Perform in vitro culture on the in vitro fertilized eggs obtained in Step 1 to obtain embryonic cells, and then transplant the embryonic cells into the uterus or fallopian tube of a maternal animal or a surrogate maternal animal.

12. The application of the method for obtaining mature egg cells of non-human animals according to any one of claims 1 to 10 or the assisted reproductive method of non-human animals according to claim 11 in the protection of endangered animals.

13. The application according to claim 12, characterized in that, The endangered animal is a viviparous mammal; Preferably, the endangered animal includes at least one of sika deer, Amur tiger, and giant panda.

14. The application according to claim 12, wherein The application also includes the application in animals under protection or those with high requirements for reproduction or quantity.

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