Laser-assisted in vitro fertilization

Abstract

The present invention provides a method for improving the efficiency of in vitro fertilization in animals. The method comprises: (a) obtaining oocytes from the animal, wherein the oocytes include the oocyte body and the zona pellucida; (b) obtaining sperm from the animal; (c) maturing the oocytes in a maturation medium; (d) removing cumulus cells from the oocytes; (e) creating holes in the zona pellucida of the oocytes with a laser; (f) contacting the oocytes with the sperm; and (g) incubating the oocytes with the sperm, wherein the incubation enables in vitro fertilization of the oocytes and sperm, and an animal embryo is produced; and the efficiency of in vitro fertilization for producing an animal embryo is improved by creating holes in the zona pellucida of the oocytes with a laser. In certain embodiments, the cumulus cells are removed from the oocytes by treatment with hyaluronidase, pipetting, and / or vortexing. In one embodiment, the oocytes are placed on a microscope stage prior to step (e).

Description

Cross - reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 507,634, filed on June 12, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

Technical Field

[0002] This disclosure generally relates to the field of in vitro fertilization (IVF).

Background Art

[0003] In vitro fertilization (IVF) in horses has not been as successful as in other livestock species. The fertilization rate in horses is extremely low, and the development of embryos after IVF is also poor (Non - Patent Document 1, Non - Patent Document 2, Non - Patent Document 3, Non - Patent Document 4). Although sporadic successful cases of IVF in horses have been reported (Non - Patent Document 5, Non - Patent Document 6, Non - Patent Document 7, Non - Patent Document 8), there is no report that its reproducibility has been confirmed.

[0004] The factors for the failure of IVF in horses are not currently clear. Therefore, there is an unmet need to improve the efficiency of in vitro fertilization (IVF) in horses in order to obtain more embryos and even higher - quality embryos.

Prior Art Documents

Non - Patent Documents

[0005]

Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Non - Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

[0006] This disclosure provides a method for improving the efficiency of in vitro fertilization in animals. The method comprises: (a) obtaining oocytes from the animal, wherein the oocytes include the oocyte body and the zona pellucida; (b) obtaining sperm from the animal; (c) maturing the oocytes in a maturation medium; (d) removing cumulus cells from the oocytes; (e) puncturing the zona pellucida of the oocytes with a laser; (f) contacting the oocytes with the sperm; and (g) incubating the oocytes with the sperm, wherein the incubation enables in vitro fertilization of the oocytes and sperm, and an animal embryo is produced; and the puncture of the zona pellucida of the oocytes with a laser improves the efficiency of the in vitro fertilization that produces an animal embryo. In certain embodiments, the cumulus cells are removed from the oocytes by treatment with hyaluronidase, pipetting, and / or vortexing. In one embodiment, the oocytes are placed on a microscope stage prior to step (e).

[0007] In certain embodiments, the oocyte includes polar bodies. In certain embodiments, the laser is directed to a region of the zona pellucida where a circumferential zona pellucida space exists between the oocyte body and the zona pellucida. In certain embodiments, the laser is directed to a location approximately 2 μm to 5 μm away from the polar bodies of the oocyte.

[0008] In certain embodiments, the sperm are cultured (incubated) in a capacitation medium before being brought into contact with the oocyte. The capacitation medium may contain, for example, a calcium ionophore and caffeine. In certain embodiments, the concentration of the calcium ionophore is about 0.01 μM to about 100 μM. In certain embodiments, the concentration of the caffeine is about 0.01 mM to about 100 mM.

[0009] In certain embodiments, the sperm are not cultured (incubated) in a capacitation medium before being brought into contact with the oocyte.

[0010] In certain embodiments, the oocytes are incubated with the sperm for about 30 minutes to about 24 hours. For example, the oocytes may be incubated with the sperm for about 1 hour to about 3 hours. For example, the oocytes may be incubated with the sperm for about 2 hours.

[0011] In a particular embodiment, the method further includes observing the incubation of the oocyte and sperm under a microscope and terminating the incubation when it is observed that the first sperm cells have entered the zona pellucida space between the zona pellucida and the oocyte body.

[0012] In certain embodiments, the animal is selected from dogs, cats, foxes, tigers, lions, cheetahs, leopards, jaguars, wolves, goats, sheep, elephants, rabbits, opossums, porcupines, lemurs, otters, sloths, kangaroos, wolverines, cows, buffaloes, horses, caribou, deer, camels, elk, llamas, oxen, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinoceroses, and humans. For example, the animal could be a horse.

[0013] Furthermore, this disclosure provides animal embryos produced by the methods disclosed herein. For example, the animal embryos may be horse embryos. [Brief explanation of the drawing]

[0014] The above summary and the following detailed description of the embodiments described herein can be better understood by reading them in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the exact embodiments shown in the drawings.

[0015] [Figure 1]Figure 1 shows an image of a horse embryo three days after the laser perforation treatment of the zona pellucida and the performance of in vitro fertilization.

[0016] [Figure 2] Figure 2 shows an image of a horse embryo on the third day after parthenogenetic activation.

[0017] [Figure 3] Figure 3 shows an image of a horse embryo four days after the laser perforation treatment of the zona pellucida and the performance of in vitro fertilization.

[0018] [Figure 4] Figure 4 shows an image of a horse embryo five days after the laser perforation treatment of the zona pellucida and the performance of in vitro fertilization.

[0019] [Figure 5] Figure 5 shows an image of a horse embryo twelve days after the laser perforation treatment of the zona pellucida and the performance of in vitro fertilization.

Mode for Carrying Out the Invention

[0020] In the background art and throughout this specification, various publications, papers, and patents are cited or described, and each of these references is hereby incorporated by reference in its entirety into this specification. The discussions regarding the documents, acts, materials, devices, articles, etc. included in this specification are for the purpose of explaining the background of the present invention. Such discussions do not admit that some or all of these matters constitute a part of the prior art related to the disclosed or claimed invention.

[0021] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, the specific terms used in this specification have the meaning described in the specification.

[0022] When used herein and in the appended claims, the singular forms “a,” “an,” and “the” should be noted to include plural references unless the context clearly indicates otherwise.

[0023] Unless otherwise specified, numerical values ​​such as concentrations or concentration ranges described herein should be understood in all cases to be modified by the term “approximately.” Therefore, numerical values ​​typically include ±10% of the listed value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Where used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range (including integers and decimal values ​​within such ranges), unless the context clearly indicates otherwise.

[0024] Unless otherwise indicated, the term “at least” preceding a series of elements is understood to refer to all elements within the series. Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of the invention described herein using only ordinary experiments. Such equivalents are intended to be encompassed by the invention.

[0025] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or other variations thereof, are understood to mean the inclusion of a given integer or group of integers, but not the exclusion of other integers or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus containing a list of elements is not necessarily limited to those elements alone and may include other elements not expressly enumerated or specific to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, “or” refers to an inclusive “or” and not an exclusive “or.” For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0026] As used herein, the connecting term “and / or” between multiple enumerated elements is understood to encompass both individual and combined options. For example, when two elements are joined by “and / or,” the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability when the first and second elements are applied together. Any one of these options is understood to be included in its meaning and therefore satisfy the requirements of the term “and / or” as used herein. The simultaneous applicability of two or more of the options is also understood to be included in its meaning and therefore satisfy the requirements of the term “and / or.”

[0027] As used herein, the terms "consists of," or variations such as "consist of" or "consisting of," when used throughout the specification and claims, indicate the inclusion of any enumerated integer or group of integers, but that no further integers or groups of integers may be added to the specified method, structure, or composition.

[0028] As used herein, the terms “consists essentially of,” or variations such as “consist essentially of” or “consisting essentially of,” when used throughout the specification and claims, indicate the inclusion of any enumerated integer or group of integers, and the inclusion of any selection of any enumerated integer or group of integers that does not substantially alter the basic or novel properties of the identified method, structure or composition. See MPEP §2111.03.

[0029] The terms "right," "left," "down," and "up" indicate direction in the referenced drawing.

[0030] Furthermore, the terms “about,” “approximately,” “generally,” “substantially,” and similar terms used herein when referring to the dimensions or characteristics of preferred components of an invention are intended to indicate to those skilled in the art that the described dimensions / characteristics are not strict boundaries or parameters and do not exclude slight variations from those that are functionally the same or similar. At the very least, such references involving numerical parameters include variations that do not change to the lowest digit when using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0031] As used herein, the term “oocyte” refers to the female germ cell involved in reproduction.

[0032] As used herein, the term "sperm" refers to male germ cells involved in reproduction.

[0033] As used herein, the term “zona pellucida” refers to the specialized extracellular matrix surrounding the plasma membrane of an oocyte.

[0034] As used herein, the term “zona pellucida space” refers to the space between the zona pellucida and the cell membrane of an oocyte or fertilized egg.

[0035] As used herein, the term “polar body” refers to a small, haploid cell that forms simultaneously with the egg cell during meiosis but is generally incapable of fertilization.

[0036] As used herein, the term “embryo” refers to the early stages of development of a multicellular organism. “Embryo” or “embryonic development” is part of the life cycle of an organism that begins immediately after fertilization of an egg cell and a sperm cell. The fusion of the egg cell and sperm cell produces a single cell called a zygote, which undergoes numerous cell divisions to eventually become a multicellular organism that implants in the uterine lining, thereby enabling gastrulation, neurogenesis, and organogenesis of the developing individual. “Embryo” may also refer to an unborn or unhatched offspring in the process of development.

[0037] As used herein, the term “cumulus cells” refers to a group of closely related granulosa cells that surround the oocyte and are involved in the processes of oocyte maturation and fertilization.

[0038] As used herein, the term “capacitation” refers to the activation process that prepares sperm for fertilization of oocytes. Capacitation may refer to the activation process that occurs within the female reproductive tract in which sperm are prepared to fertilize oocytes. Alternatively, capacitation may refer to an artificial activation process in which sperm are incubated in a specific culture medium to activate them in preparation for fertilization of oocytes.

[0039] Methods to improve the efficiency of in vitro fertilization (IVF)

[0040] This specification provides methods for improving the efficiency of in vitro fertilization in animals. Improving the efficiency of in vitro fertilization in animals may include, for example, increasing the number of fertilized oocytes after in vitro fertilization, increasing the number of fertilized oocytes that initiate cell division after in vitro fertilization, increasing the number of viable embryos produced by the method, improving the quality of embryos obtained from the in vitro fertilization process, and / or improving the live birth potential of the produced embryos.

[0041] The method for improving the efficiency of in vitro fertilization may include, for example, the following: (a) a step of obtaining oocytes from the animal, wherein the oocytes include the oocyte body and the zona pellucida; (b) a step of obtaining sperm from the animal; (c) a step of maturing the oocytes in a maturation medium; (d) a step of removing cumulus cells from the oocytes; (e) a step of creating a hole in the zona pellucida of the oocytes with a laser; (f) a step of bringing the oocytes into contact with the sperm; (g) a step of incubating the oocytes with the sperm, wherein the incubation enables in vitro fertilization of the oocytes and the sperm, and an animal embryo is produced; and the efficiency of in vitro fertilization for producing an animal embryo is improved by creating a hole in the zona pellucida of the oocytes with a laser.

[0042] While this invention is not intended to be limited by theory, by creating holes in the zona pellucida with a laser, for example, sperm can directly contact the oocyte itself, making it easier for sperm to reach the oocyte for fertilization. This reduces the energy required for sperm to penetrate the zona pellucida, allowing more energy to be used for fertilizing the oocyte. Furthermore, the time required for sperm to reach the oocyte before their effective lifespan expires may be shortened. In addition, this may lead to a reduction in incubation time in the in vitro fertilization process. By creating holes in the zona pellucida with a laser, sperm can come into contact with the oocyte and then fuse to complete the in vitro fertilization process, eliminating the need to inject sperm into the oocyte for fertilization to occur. Such holes allow sperm to directly access the oocyte during the fertilization process.

[0043] Furthermore, by using a laser to create holes in the zona pellucida instead of chemical means, precise holes can be formed with minimal or no damage to the oocyte, potentially leading to the production of more high-quality embryos from the in vitro fertilization process. In addition, lasers are easy to operate (i.e., aiming, intensity adjustment, etc.) and are far faster than chemical means. Lasers can create holes in the zona pellucida without the help of a micromanipulation set, whereas chemical means require a micromanipulation set for aiming.

[0044] The oocytes may be obtained, for example, from female animals, and the sperm may be obtained, for example, from male animals of the same species. The sperm may be obtained, for example, from cryopreserved spermatids.

[0045] By maturing the oocytes in a maturation medium, for example, oocytes having polar bodies that indicate the oocytes are capable of fertilization can be obtained. The maturation medium may contain hormones and / or other chemicals known to induce maturation, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (hCG), estradiol, bovine serum albumin (BSA), epidermal growth factor (EGF), and fetal bovine serum (FBS).

[0046] In certain embodiments, the mature oocyte includes cumulus cells surrounding the oocyte. The cumulus cells can be removed from the oocyte, for example. The cumulus cells may be removed partially or completely, as long as a portion of the zona pellucida is exposed and laser targeting is possible. The cumulus cells can be removed by chemical and / or mechanical means, for example. As an example, the cumulus cells can be removed by treatment with hyaluronidase, pipetting, and / or vortexing.

[0047] In certain embodiments, the oocyte is placed on a microscope stage prior to step (e). The oocyte can be observed for the presence or absence of polar bodies. In certain embodiments, the oocyte includes polar bodies. In certain embodiments, using a microscope, the laser is directed to a region of the zona pellucida where a circumferential zona pellucida space exists between the oocyte body and the zona pellucida. In certain embodiments, the laser is directed to a location about 2 μm to about 5 μm away from the polar body of the oocyte.

[0048] In certain embodiments, the sperm are incubated in a capacitation medium before being brought into contact with the oocyte. The capacitation medium can activate the sperm, for example, toward the fertilization process. The capacitation medium may contain, for example, calcium ionophores, caffeine, heparin, lysophosphatidylcholine (LC), and catecholamines.

[0049] In certain embodiments, the concentration of the calcium ionophore is approximately 0.01 μM to approximately 100 μM. The concentration of the calcium ionophore may be, for example, approximately 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, or values ​​in between these. The concentration of the calcium ionophore is, for example, approximately 0.05 μM to approximately 100 μM, approximately 1 μM to approximately 100 μM, approximately 10 μM to approximately 100 μM, approximately 20 μM to approximately 100 μM, approximately 30 μM to approximately 100 μM, approximately 40 μM to approximately 100 μM, approximately 50 μM to approximately 100 μM, approximately 60 μM to approximately 100 μM, approximately 70 μM to approximately 100 μM, approximately 80 μM to approximately 100 μM, approximately 90 μM to approximately It may be 100 μM, approximately 0.01 μM to 90 μM, approximately 0.01 μM to 80 μM, approximately 0.01 μM to 70 μM, approximately 0.01 μM to 60 μM, approximately 0.01 μM to 50 μM, approximately 0.01 μM to 40 μM, approximately 0.01 μM to 30 μM, approximately 0.01 μM to 20 μM, approximately 0.01 μM to 10 μM, approximately 0.01 μM to 1 μM, or values ​​in between these.

[0050] In certain embodiments, the caffeine concentration is approximately 0.01 mM to approximately 100 mM. The caffeine concentration may be, for example, approximately 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, or values ​​in between. The caffeine concentrations mentioned above are, for example, approximately 0.05 mM to approximately 100 mM, approximately 1 mM to approximately 100 mM, approximately 10 mM to approximately 100 mM, approximately 20 mM to approximately 100 mM, approximately 30 mM to approximately 100 mM, approximately 40 mM to approximately 100 mM, approximately 50 mM to approximately 100 mM, approximately 60 mM to approximately 100 mM, approximately 70 mM to approximately 100 mM, approximately 80 mM to approximately 100 mM, approximately 90 mM to approximately 100 mM. The concentration can be mM, approximately 0.01 mM to 90 mM, approximately 0.01 mM to 80 mM, approximately 0.01 mM to 70 mM, approximately 0.01 mM to 60 mM, approximately 0.01 mM to 50 mM, approximately 0.01 mM to 40 mM, approximately 0.01 mM to 30 mM, approximately 0.01 mM to 20 mM, approximately 0.01 mM to 10 mM, approximately 0.01 mM to 1 mM, or values ​​in between.

[0051] In certain embodiments, the sperm are not cultured (incubated) in a capacitation medium before being brought into contact with the oocyte.

[0052] In certain embodiments, the oocytes may be cultured (incubated) with the sperm for about 30 minutes to about 24 hours. The oocytes can be cultured for, for example, approximately 30 minutes to 20 hours, approximately 30 minutes to 15 hours, approximately 30 minutes to 10 hours, approximately 30 minutes to 5 hours, approximately 30 minutes to 4 hours, approximately 30 minutes to 3 hours, approximately 30 minutes to 2 hours, approximately 30 minutes to 1 hour, approximately 1 hour to 24 hours, approximately 1 hour to 20 hours, approximately 1 hour to 15 hours, approximately 1 hour to 10 hours, approximately 1 hour to 5 hours, approximately 1 hour to 4 hours, approximately 1 hour to 3 hours, approximately 1 hour to 2 hours, approximately 2 hours to 24 hours, approximately 4 hours to 24 hours, approximately 6 hours to 24 hours, approximately 8 hours to 24 hours, approximately 10 hours to 24 hours, approximately 12 hours to 24 hours, approximately 18 hours to 24 hours, approximately 20 hours to 24 hours, or values ​​in between these. The oocytes may, for example, be cultured with the sperm for about 1 to 3 hours. The oocytes may, for example, be cultured with the sperm for about 2 hours.

[0053] In certain embodiments, the method further includes observing the incubation of the oocyte and sperm under a microscope and terminating the incubation when it is visually observed that the first spermatid has entered the zona pellucida space between the zona pellucida and the oocyte body. Visualizing the entry of the first spermatid into the zona pellucida space between the zona pellucida and the oocyte body can, for example, shorten the time required to culture the oocyte with the sperm, thereby reducing the occurrence of polyspermy in the in vitro fertilization process. Visualizing the entry of the first sperm into the zona pellucida space can shorten the incubation time from about 6 hours to about 1-2 hours.

[0054] In certain embodiments, the animal is selected from dogs, cats, foxes, tigers, lions, cheetahs, leopards, jaguars, wolves, goats, sheep, elephants, rabbits, opossums, porcupines, lemurs, otters, sloths, kangaroos, wolverines, cows, buffaloes, horses, caribou, deer, camels, elk, llamas, oxen, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinoceroses, and humans. For example, the animal could be a horse.

[0055] Furthermore, this disclosure provides animal embryos produced by the methods disclosed herein. For example, the animal embryo may be a horse embryo. Embodiments

[0056] The present invention also provides the following non-limiting embodiments.

[0057] Embodiment 1 is a method for improving the efficiency of in vitro fertilization in animals, the method comprising: (a) A step of obtaining oocytes from the animal, wherein the oocytes include the oocyte body and the zona pellucida; (b) A step of obtaining sperm from the animal; (c) A step of maturing the oocytes in a maturation medium; (d) A step of removing cumulus cells from the oocyte; (e) A step of creating a hole in the zona pellucida of the oocyte using a laser; (f) The step of bringing the oocyte into contact with the sperm; (g) A step of incubating the oocyte with the sperm, characterized in that the incubation enables in vitro fertilization of the oocyte and sperm, generates an animal embryo, and improves the efficiency of the in vitro fertilization that generates the animal embryo by creating a hole in the zona pellucida of the oocyte with the laser.

[0058] Embodiment 2 is the method according to Embodiment 1, wherein the cumulus cells are removed from the oocyte by treatment with hyaluronidase, aspiration with a pipette, and / or vortex treatment.

[0059] Embodiment 3 is the method according to Embodiment 1 or 2, wherein the oocytes are placed on a microscope stage prior to step (e).

[0060] Embodiment 4 is the method according to any one of Embodiments 1 to 3, wherein the oocyte includes a polar body.

[0061] Embodiment 5 is the method according to any one of Embodiments 1 to 4, wherein the laser is directed to a region of the zona pellucida where a circumferential zona pellucida space exists between the oocyte body and the zona pellucida.

[0062] Embodiment 6 is the method according to Embodiment 5, wherein the laser is directed to a position approximately 2 μm to 5 μm away from the polar body of the oocyte.

[0063] Embodiment 7 is a method according to any one of Embodiments 1 to 6, wherein the sperm are cultured (incubated) in a capacitation medium before being brought into contact with the oocyte.

[0064] Embodiment 8 is the method according to Embodiment 7, wherein the culture medium for acquiring fertilization ability comprises a calcium ionophore, caffeine, heparin, lysophosphatidylcholine (LC), and / or catecholamines.

[0065] Embodiment 9 is the method according to Embodiment 8, wherein the concentration of the calcium ionophore is approximately 0.01 μM to approximately 100 μM.

[0066] Embodiment 10 is the method according to Embodiment 8, wherein the caffeine concentration is approximately 0.01 mM to approximately 100 mM.

[0067] Embodiment 11 is a method according to any one of Embodiments 1 to 6, wherein the sperm are not cultured (incubated) in a capacitation medium before being brought into contact with the oocyte.

[0068] Embodiment 12 is a method according to any one of Embodiments 1 to 11, wherein the oocytes are cultured (incubated) with the sperm for about 30 minutes to about 24 hours.

[0069] Embodiment 13 is the method according to Embodiment 12, wherein the oocytes are cultured (incubated) with the sperm for about 1 to 3 hours.

[0070] Embodiment 14 is the method according to Embodiment 13, wherein the oocytes are cultured (incubated) with the sperm for about 2 hours.

[0071] Embodiment 15 is the method of Embodiment 13 or 14, further comprising the step of observing the culture (incubation) of the oocyte and sperm with a microscope and terminating the culture (incubation) when it is visually confirmed that the first sperm cell has entered the periphery of the zona pellucida space between the zona pellucida and the oocyte body.

[0072] Embodiment 16 is the method according to any one of Embodiments 1 to 15, wherein the animal is selected from dogs, cats, foxes, tigers, lions, cheetahs, leopards, jaguars, wolves, goats, sheep, elephants, rabbits, opossums, porcupines, lemurs, otters, sloths, kangaroos, wolverines, cows, buffaloes, horses, caribou, deer, camels, elk, llamas, oxen, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinos, and humans.

[0073] Embodiment 17 is the method according to Embodiment 16, wherein the animal is a horse.

[0074] Embodiment 18 is an animal embryo produced by the method described in any one of Embodiments 1 to 17.

[0075] Embodiment 19 is the animal embryo described in Embodiment 18, wherein the animal embryo is a horse embryo. [Examples]

[0076] method

[0077] Maturation of horse oocytes

[0078] Equine oocytes were collected from local equine breeding centers by ultrasound-guided oocyte retrieval and matured in maturation medium in a 4-well plate at 38.5°C and 5% CO2 for 30-35 hours. At the end of maturation, cumulus cells were removed from the equine oocytes by hyaluronidase treatment and repeated aspiration with a thin glass pipette. After washing the oocytes, they were subjected to laser porosity treatment of the zona pellucida (ZP).

[0079] Laser drilling of the transparent zone (ZP)

[0080] The oocytes were transferred into droplets of a mineral oil-coated retention medium placed on a microscope stage to create pores in the zone of projection (ZP) using a laser. The oocytes were positioned using a micromanipulator so that the polar body was at the 12 o'clock position. Laser pulses (e.g., Xyrcos, Hamilton Thorne) were directed towards the ZP at the 3 o'clock position to create pores in the ZP. Prior to in vitro fertilization (IVF), the oocytes were washed and placed in a retention medium in a 4-well plate at 38.5°C and 5% CO2.

[0081] Sperm preparation

[0082] 1 mL of horse semen was placed in a 15 mL centrifuge tube and centrifuged at 328 × g for 5 minutes. 1 mL of pre-warmed semen preparation medium (e.g., EQ-SemenPrep, IVF Scientific) was added on top of the semen layer. The tube containing the semen was incubated at 37°C for 20 minutes. 500 μL of supernatant was taken from the top of the upper phase and transferred to a new empty 1.5 mL centrifuge tube, and 1 mL of pre-warmed semen preparation medium was added. After careful mixing, the mixture was centrifuged at 328 × g for 5 minutes. The supernatant was removed, and 1 mL of pre-warmed semen preparation medium was added to resuspend the sperm pellet. The mixture was centrifuged again at 328 × g for 5 minutes. The supernatant was removed until approximately 200 μL remained. The sperm mixture was carefully mixed and kept at 37°C until IVF.

[0083] Sperm acquire fertilizing ability

[0084] 100 μL of the prepared sperm mixture was added to a new 1.5 mL tube and centrifuged again at 328 × g for 5 minutes. The supernatant was removed and the mixture was added to a sperm fertilization medium (e.g., Hal-L + 1 μM Ca). 2+ 50 μL of ionophore (10 mM caffeine) was added. The sperm mixture was incubated at 38.5°C for 10 minutes.

[0085] in vitro fertilization

[0086] Equine oocytes, whose zona pellucida had been perforated with a laser, were transferred to 100 μL droplets of IVF medium (e.g., BO-IVF medium, IVF Scientific) in a 4-well plate coated with mineral oil. Next, sperm that had acquired fertilizing ability were added to the droplets of IVF medium. The oocytes and spermatids were co-cultured (co-incubated) at 38.5°C, 5% CO2, and air for 30 minutes to 2 hours. The co-culture time was determined by visually observing the first spermatids in the zona pellucida surrounding the oocytes. After IVF, the oocytes were washed and transferred to embryo culture medium (e.g., EQ-IVC, IVF Scientific) and cultured (incubated) at 38.5°C, 5% CO2 and 6% O2, and air.

[0087] result

[0088] Table 1 below shows the number of horse oocytes at the end of maturation. Twenty-four horse oocytes, from which the cumulus cells had been removed, including nine oocytes with polar bodies (PBs) and fifteen oocytes without PBs, were subjected to laser porosalping and IVF. As a control, eleven horse oocytes with intact cumulus cells were parthenogenetically activated.

[0089] [Table 1]

[0090] Table 2 shows the developmental status of horse oocytes 3 days after laser-induced porostomy of the zoned pith (ZP) and IVF. Of the 24 oocytes in the group that underwent laser-induced ZP porostomy and IVF, 13 (54%) cleaved and formed 2-cell or 4-cell embryos. The cleavage rate in the group that underwent laser-induced ZP porostomy and IVF was very similar to the cleavage rate of 55% in the parthenogenesis control group. Figures 1 and 2 show horse embryos formed from both IVF and parthenogenesis activation.

[0091] [Table 2]

[0092] conclusion

[0093] Laser perforation of the zona pellucida prior to IVF has improved the efficiency of IVF in horses. This technique is readily applicable to humans and other species.

[0094] Improving the efficiency of in vitro fertilization (IVF) in horses using laser-induced perforation of the zona pellucida.

[0095] The efficiency of in vitro fertilization (IVF) in horses was significantly improved by using laser-induced zona pellucida treatment prior to in vitro fertilization (IVF). Horse oocytes were collected from local sources and subjected to the laser-assisted IVF process using the laser-induced zona pellucida treatment disclosed above. As described above, of the 139 horse oocytes collected and subjected to laser-assisted IVF, 61.2% were fertilized and cleaved to form two-cell embryos, whereas 0% of the 34 oocytes in the control group that did not undergo laser-induced zona pellucida treatment developed into two-cell embryos. In the group that underwent laser-induced zona pellucida treatment, 52.5% developed to the 8-16 cell stage, 15.8% developed to the morula stage, and 8.6% developed to the blastocyst stage, compared to 0% in the control group that did not undergo laser-induced zona pellucida treatment. Examples of horse embryos produced by laser-assisted IVF are shown in Figures 3 to 5, and the results are shown in Table 3 below.

[0096] [Table 3]

[0097] Laser-assisted in vitro fertilization without the use of a micromanipulator A method for creating holes in the zona pellucida using a laser for in vitro fertilization (IVF) was investigated without the use of a micromanipulator. The objective of this experiment was to simplify the laser-based zona pellucida porosity process and improve the processing speed. This experiment was carried out without the use of a micromanipulator, according to the method described above. The oocytes were transferred to droplets of retention medium coated with mineral oil, placed on a microscope stage, for laser zona pellucida porosity. The oocytes were positioned by moving the microscope stage so that the laser beam was directed towards a portion of the zona pellucida of the oocyte. Laser pulses (e.g., Xyrcos, Hamilton Thorne) were irradiated towards the zona pellucida to create holes in it. This process was repeated for the next oocyte. The oocytes were washed and then placed in retention medium in a 4-well plate at 38.5°C and 5% CO2 prior to in vitro fertilization (IVF). IVF and subsequent culture of the oocytes were carried out as described above. Table 3 shows the development rate of oocytes after the laser-induced puncture treatment of the ZP.

[0098] Determination of incubation time in the IVF process using laser drilling of ZP

[0099] This study investigates the relationship between oocyte and sperm incubation time and polyspermy rates. The objective of this experiment is to determine the optimal incubation time that yields the highest cleavage rate while minimizing polyspermy rates. The experiment will be conducted as described above. After laser puncture of the ZP and IVF, the oocytes will be removed from incubation with the sperm at multiple time points: 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, and 2 hours. The oocytes will be washed and cultured as described above. The optimal incubation time will be determined by comparing the polyspermy rates and cleavage rates of the oocytes at each time point.

[0100] Those skilled in the art will understand that modifications can be made to the embodiments described above without departing from the broad concept of the present invention. Therefore, it will be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to encompass modifications within the spirit and scope of the invention as defined herein.

Claims

1. A method for improving the efficiency of in vitro fertilization in animals, the method comprising: (a) A step of obtaining oocytes from the animal, wherein the oocytes include the oocyte body and the zona pellucida; (b) A step of obtaining sperm from the animal; (c) A step of maturing the oocytes in a maturation medium; (d) The step of removing cumulus cells from the oocyte; (e) A step of creating a hole in the zona pellucida of the oocyte using a laser; (f) The step of bringing the oocyte into contact with the sperm; (g) A step of incubating the oocyte with the sperm, wherein the incubation enables in vitro fertilization of the oocyte and the sperm, and an animal embryo is generated; By creating holes in the zona pellucida of the oocyte with the laser, the efficiency of in vitro fertilization, which generates animal embryos, is improved. A method characterized by the following:

2. The cumulus cells are removed from the oocyte by treatment with hyaluronidase, pipetting, and / or vortexing. The method according to claim 1.

3. The oocytes are placed on a microscope stage prior to step (e). The method according to claim 1.

4. The method according to claim 1, wherein the oocyte includes a polar body.

5. The method according to claim 1, wherein the laser is directed to a region of the zona pellucida where a circumferential zona pellucida space exists between the oocyte body and the zona pellucida.

6. The method according to claim 5, wherein the laser is directed to a position approximately 2 μm to approximately 5 μm away from the polar body of the oocyte.

7. The method according to claim 1, wherein the sperm are cultured (incubated) in a capacitation medium before being brought into contact with the oocyte.

8. The method according to claim 7, wherein the culture medium for acquiring fertilization ability comprises a calcium ionophore, caffeine, heparin, lysophosphatidylcholine (LC), and / or a catecholamine.

9. The method according to claim 8, wherein the concentration of the calcium ionophore is about 0.01 μM to about 100 μM.

10. The method according to claim 8, wherein the concentration of caffeine is approximately 0.01 mM to approximately 100 mM.

11. The method according to claim 1, wherein the sperm are not cultured (incubated) in a capacitation medium before being brought into contact with the oocyte.

12. The method according to claim 1, wherein the oocytes are cultured (incubated) with the sperm for about 30 minutes to about 24 hours.

13. The method according to claim 12, wherein the oocytes are cultured (incubated) with the sperm for about 1 to 3 hours.

14. The method according to claim 13, wherein the oocytes are cultured (incubated) with the sperm for about 2 hours.

15. The method according to claim 13, further comprising the step of observing the culture (incubation) of the oocyte and the sperm with a microscope, and terminating the culture (incubation) when it is visually confirmed that the first sperm cell has entered the periphery of the zona pellucida space between the zona pellucida and the oocyte body.

16. The method according to claim 1, wherein the animal is selected from dogs, cats, foxes, tigers, lions, cheetahs, leopards, jaguars, wolves, goats, sheep, elephants, rabbits, opossums, porcupines, lemurs, otters, sloths, kangaroos, wolverines, cows, buffaloes, horses, caribou, deer, camels, elk, llamas, oxen, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinos, and humans.

17. The method according to claim 16, wherein the animal is a horse.

18. An animal embryo produced by the method described in claim 1.

19. The animal embryo according to claim 18, wherein the animal embryo is a horse embryo.

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