Laser assisted in vitro fertilization

Abstract

Provided herein are methods for improving the efficiency of in vitro fertilization of animals. The method comprises (a) obtaining an oocyte from the animal, wherein the oocyte comprises an oocyte body and a zona pellucida; (b) obtaining sperm from the animal; (c) maturing the oocytes in a maturing medium; (d) removing cumulus cells from the oocytes; (e) drilling in the zona pellucida of the oocyte with a laser; (f) contacting the oocyte with the sperm; (g) co-incubating the oocytes with sperms, whereby the incubation enables in vitro fertilization of the oocytes and sperms to produce animal embryos, and whereby drilling is performed in the zona pellucida of the oocytes by laser to improve the efficiency of in vitro fertilization of the production of animal embryos.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application 63 / 507,634, filed June 12, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

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

[0003] In vitro fertilization (IVF) in horses is less successful than in other livestock species. Fertilization rates in horses are very low, and embryos develop poorly after IVF (Blue et al., Equine Vet J. Supp [Equine Veterinary Journal Supplement] 8:111-116 (1989); Zhang et al., Mol. Reprod.Dev. [Molecular Reproduction and Development] 26:361-5 (1990); Roasa et al., Theriogenology [Veterinary Obstetrics] 68:560-6 (2007); Palmer et al., J. Reprod. Fertil.Supp. [Journal of Reproduction and Fertility Supplement] 44:375-384 (1991)). There have been sporadic reports of successful equine IVF (Bezard et al., Proc. Intern. Scient. Conf. Biotechnics Horse Reprod. [Proceedings of the International Scientific Conference on Equine Reproductive Biotechnology], Agricultural University of Crakow, Poland, p. 12 (1992); Feliz et al., Biol. Reprod. [Reproductive Biology] 107(6):1551-64 (2022); Li et al., Biol. Reprod. Monograph [Reproductive Biology Monograph] 1(1):613-622 (1995); Leemans et al., Reprod. [Reproduction] 152:R233-R245 (2016)), but no reports have confirmed reproducibility.

[0004] The obstacles to successful equine IVF are currently unclear. Therefore, the need to improve the efficiency of equine in-vitro fertilization (IVF) to produce more and higher-quality embryos remains unmet. Summary of the Invention

[0005] This article provides a method for improving the efficiency of in vitro fertilization (IVF) in animals. The method includes (a) obtaining oocytes from an animal, wherein the oocytes comprise an oocyte body and a zona pellucida; (b) obtaining sperm from the animal; (c) maturing the oocytes in a maturation medium; (d) removing cumulus cells from the oocytes; (e) drilling a hole in the zona pellucida of the oocytes using a laser; (f) contacting the oocytes with sperm; and (g) co-incubating the oocytes with sperm, thereby enabling in vitro fertilization of the oocytes and sperm to produce animal embryos, and thereby improving the efficiency of in vitro fertilization to produce animal embryos by drilling a hole in the zona pellucida of the oocytes using a laser. In some embodiments, cumulus cells are removed from the oocytes by treatment with hyaluronidase, aspiration with a pipette, and / or vortexing. In some embodiments, the oocytes are placed on a microscope stage prior to step (e).

[0006] In some embodiments, the oocyte includes a polar body. In some embodiments, the laser is aimed at a portion of the zona pellucida, which is located between the oocyte body and the zona pellucida. In some embodiments, the laser is aimed at a distance of approximately 2 μm to approximately 5 μm from the polar body of the oocyte.

[0007] In some embodiments, the sperm is incubated in a capacitating medium before contacting the oocyte. The capacitating medium may contain, for example, a calcium ionophore and caffeine. In some embodiments, the concentration of the calcium ionophore is from about 0.01 μM to about 100 μM. In some embodiments, the concentration of caffeine is from about 0.01 mM to about 100 mM.

[0008] In some embodiments, the sperm is not incubated in a capacitating medium before contacting the oocyte.

[0009] In some embodiments, the oocyte is co-incubated with sperm for about 30 minutes to about 24 hours. For example, the oocyte may be co-incubated with sperm for about 1 hour to about 3 hours. For example, the oocyte may be co-incubated with sperm for about 2 hours.

[0010] In some embodiments, the method further includes observing the incubation of oocytes and sperm under a microscope and ending the incubation when the first sperm cell is observed to enter the perioval space between the zona pellucida and the oocyte body.

[0011] In some implementations, 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, cattle, buffalo, horses, caribou, deer, camels, elk, llamas, bulls, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinoceroses, and humans. For example, the animal could be a horse.

[0012] Animal embryos produced by the methods disclosed herein are also provided. For example, the animal embryo could be a horse embryo. Attached Figure Description

[0013] The foregoing overview and the following detailed description of embodiments of this application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the precise embodiments shown in the drawings.

[0014] Figure 1 Images of equine embryos 3 days after laser-drilled transparent tubes and in vitro fertilization are shown.

[0015] Figure 2 Images of equine embryos 3 days after parthenogenesis activation are shown.

[0016] Figure 3 Images of equine embryos 4 days after laser-drilled transparent tubes and in vitro fertilization are shown.

[0017] Figure 4 Images of equine embryos 5 days after in vitro fertilization are shown, obtained by laser-drilled transparent tubes.

[0018] Figure 5 Images of equine embryos 12 days after laser-drilled zona pellucida and in vitro fertilization are shown. Detailed Implementation

[0019] Background and references throughout this specification include or describe various publications, articles, and patents; each of these references is incorporated herein by reference in its entirety. Discussions of documents, actions, materials, devices, articles, or other content contained herein are intended to provide background for the invention. Such discussions are not an admission that any or all of them constitute prior art with respect to any disclosed or claimed invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Otherwise, certain terms used herein have the meanings set forth in the specification.

[0021] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references.

[0022] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges, mentioned herein should be understood in all cases to be modified by the term “about.” Thus, numerical values ​​typically include ±10% of the listed values. 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). Unless the context clearly indicates otherwise, as used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions of values ​​within such ranges.

[0023] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Such equivalents are intended to be covered by the invention.

[0024] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, shall be understood to include the single or plurality of integers said therein, but not exclude any other single or plurality of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, “or” means inclusive or, not exclusive or. For example, conditions A or B satisfy any 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).

[0025] As used herein, the connecting term “and / or” should be understood to encompass both individual and combined options when connecting multiple enumerated elements. For example, when two elements are connected by “and / or”, the first option refers to the applicability of the first element in the absence of the second element. The second option refers to the applicability of the second element in the absence of the first element. The third option refers to the applicability of the first and second elements together. Any of these options should be understood to fall within this meaning and therefore satisfy the requirement of the term “and / or” as used herein. The simultaneous applicability of more than one option should also be understood to fall within this meaning and therefore satisfy the requirement of the term “and / or”.

[0026] As used herein, the term “consists of” or such as “consist of” or “consisting of” as used throughout the specification and claims includes any of the listed integers or groups of integers, but no additional integers or groups of integers may be added to the method, structure, or composition.

[0027] As used herein, the terms “consistsessentially of” or such as “consist essentially of” or “consisting essentially of” as used throughout the specification and claims mean that any of the listed integers or groups of integers is included, and may optionally include any of the listed integers or groups of integers that do not substantially alter the essential or novel characteristics of the method, structure, or composition. See MPEP § 2111.03.

[0028] The words “right,” “left,” “down,” and “up” indicate directions in the attached diagram.

[0029] It should also be understood that the terms “about,” “approximately,” “usually,” “substantially,” and similar terms used herein, when referring to the size or feature of a component of a preferred embodiment of the invention, indicate that the stated size / characteristic is not a strict limit or parameter, and does not exclude minor variations that are functionally identical or similar as understood by one of ordinary skill in the art. At least, such references including numerical parameters include variations that do not change the least significant digits when using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0030] As used in this article, the term "oocyte" refers to the female reproductive cell involved in reproduction.

[0031] As used in this article, the term "sperm" refers to the male reproductive cells involved in reproduction.

[0032] As used in this article, the term "zona pellucida" refers to the specialized extracellular matrix surrounding the oocyte plasma membrane.

[0033] As used in this article, the term "periovolumic space" refers to the space between the zona pellucida and the cell membrane of the oocyte or fertilized egg.

[0034] As used in this article, the term "polar body" refers to a small haploid cell that forms simultaneously with the egg cell during meiosis, but which is usually not capable of fertilization.

[0035] As used herein, the term "embryo" refers to the initial stage of development in a multicellular organism. "Embryonic development" is the part of the life cycle that begins after the fertilization of an egg and sperm cell. The resulting egg and sperm cell fuse to form a single-celled zygote, which, through multiple cell divisions, eventually develops into a multicellular organism implanted in the uterine lining. This process allows the developing organism to undergo gastrulation, neurogenesis, and organogenesis. "Embryonic development" can also refer to an unborn or unhatched offspring during its development.

[0036] As used in this article, the term "cumulus cell" refers to a closely associated group of granulosa cells surrounding the oocyte and involved in the oocyte maturation and fertilization process.

[0037] As used herein, the term “capacitation” refers to the activation process that prepares sperm for fertilization of an oocyte. Capacitation can refer to the activation process that occurs in the female reproductive tract to prepare sperm for fertilization of an oocyte. Alternatively, capacitation can refer to an artificial activation process that includes incubating sperm in a specific culture medium to activate the sperm and prepare them for fertilization of an oocyte.

[0038] Methods to improve the efficiency of in vitro fertilization (IVF) This article provides a method for improving the efficiency of in vitro fertilization (IVF) in animals. Improving the efficiency of IVF in animals includes, for example, increasing the number of fertilized oocytes after IVF, increasing the number of fertilized oocytes undergoing cell division after IVF, increasing the number of live embryos produced by this method, improving the quality of embryos produced during IVF, and / or improving the ability of the produced embryos to produce live births.

[0039] Methods for improving in vitro fertilization efficiency, for example, include (a) obtaining oocytes from an animal, wherein the oocytes comprise an oocyte body and a zona pellucida; (b) obtaining sperm from the animal; (c) maturing the oocytes in a maturation medium; (d) removing cumulus cells from the oocytes; (e) drilling a hole in the zona pellucida of the oocytes using a laser; (f) contacting the oocytes with sperm; and (g) co-incubating the oocytes with sperm, thereby enabling in vitro fertilization of the oocytes and sperm to produce animal embryos, and thereby improving the in vitro fertilization efficiency for producing animal embryos by drilling a hole in the zona pellucida of the oocytes using a laser.

[0040] Not limited to theoretical limitations, for example, drilling a hole in the zona pellucida with a laser can provide sperm with a direct channel to contact the oocyte body, making it easier for sperm to access and fertilize the oocyte. This can lead to a reduction in the energy consumed by sperm to penetrate the zona pellucida and the use of more energy to fertilize the oocyte. It can also reduce the time sperm spends contacting the oocyte before the end of their limited sperm viable lifespan. This can also lead to a reduction in the incubation time during in vitro fertilization. By using a laser to drill a hole in the zona pellucida, sperm can contact and fuse with the oocyte to complete the in vitro fertilization process, thus eliminating the need for sperm injection into the oocyte for fertilization. The hole will provide sperm with a direct route to contact the oocyte for the fertilization process.

[0041] Furthermore, using lasers to drill holes in the zona pellucida, rather than chemical methods, allows for the creation of precise pores with minimal or no damage to the oocytes, resulting in more and higher-quality embryos during in-vitro fertilization. Additionally, lasers are easier to manipulate (i.e., aiming, intensity adjustment, etc.) and much faster than chemical methods. Lasers can drill holes in the zona pellucida without the aid of micromanipulation equipment, whereas chemical methods require aiming with micromanipulation equipment.

[0042] For example, the oocytes can be obtained from female animals, and the sperm can be obtained from male animals of the same species. For example, the sperm can be obtained from cryopreserved sperm cells.

[0043] Maturation of oocytes in a maturation medium produces oocytes with polar bodies, indicating that the oocytes are ready for fertilization. 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).

[0044] In some embodiments, the mature oocyte comprises cumulus cells surrounding the oocyte. For example, cumulus cells can be removed from the oocyte. Cumulus cells can be removed partially or completely, provided that a portion of the zona pellucida is exposed and can be targeted by a laser. For example, cumulus cells can be removed by chemical and / or mechanical methods. For instance, cumulus cells can be removed by treatment with hyaluronidase, aspiration with a pipette, and / or vortexing.

[0045] In some embodiments, prior to step (e), the oocyte is placed on a microscope stage. The presence of a polar body in the oocyte can be observed. In some embodiments, the oocyte contains a polar body. In some embodiments, a laser is aimed at a portion of the zona pellucida, the perivitelline space between the oocyte body and the zona pellucida, using a microscope. In some embodiments, the laser is aimed at a distance of approximately 2 μm to approximately 5 μm from the oocyte's polar body.

[0046] In some embodiments, sperm are incubated in a capacitation medium before contacting the oocyte. For example, the capacitation medium can activate the sperm to carry out the fertilization process. For example, the capacitation medium may contain calcium ionophores, caffeine, heparin, lysophosphatidylcholine (LC), and catecholamines.

[0047] In some embodiments, the concentration of the calcium ion carrier is from about 0.01 μM to about 100 μM. For example, the concentration of the calcium ion carrier can be about 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 any value between the two. For example, calcium ion carriers can be 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 100 μM, approximately 0.01 μM to approximately 90 μM, approximately 0.01 μM to approximately 80 μM, approximately 0.01 μM to approximately 70 μM, approximately 0.01 μM to approximately 60 μM, approximately 0.01 μM to approximately 50 μM, approximately 0.01 μM to approximately 40 μM, approximately 0.01 μM to approximately 30 μM, approximately 0.01 μM to approximately 20 μM, etc. μM, about 0.01 μM to about 10 μM, about 0.01 μM to about 1 μM, or any value in between.

[0048] In some embodiments, the concentration of caffeine is from about 0.01 mM to about 100 mM. For example, the concentration of caffeine can be about 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 any value between the two. For example, caffeine can be 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, approximately 0.01 mM to approximately 90 mM, approximately 0.01 mM to approximately 80 mM, approximately 0.01 mM to approximately 70 mM, approximately 0.01 mM to approximately 60 mM, approximately 0.01 mM to approximately 50 mM, approximately 0.01 mM to approximately 40 mM, approximately 0.01 mM to approximately 30 mM, approximately 0.01 mM to approximately 20 mM. mM, about 0.01 mM to about 10 mM, about 0.01 mM to about 1 mM, or any value in between.

[0049] In some implementations, the sperm are not incubated in a capacitating medium before they come into contact with the oocyte.

[0050] In some embodiments, the oocyte is co-incubated with sperm for about 30 minutes to about 24 hours. For example, the oocyte may be co-incubated with sperm for about 30 minutes to about 20 hours, about 30 minutes to about 15 hours, about 30 minutes to about 10 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1 hour, about 1 hour to about 24 hours, about 1 hour to about 20 hours, about 1 hour to about 15 hours, about 1 hour to about 10 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 24 hours, about 4 hours to about 24 hours, about 6 hours to about 24 hours, about 8 hours to about 24 hours, about 10 hours to about 24 hours, about 12 hours to about 24 hours, about 18 hours to about 24 hours, about 20 hours to about 24 hours, or any value between the two. For example, oocytes can be co-incubated with sperm for approximately 1 to 3 hours. For example, oocytes can be co-incubated with sperm for approximately 2 hours.

[0051] In some embodiments, the method further includes microscopic observation of the incubation of the oocyte and sperm, and termination of incubation when the first sperm cell is observed to enter the perivitelline space between the zona pellucida and the oocyte body. For example, observing the first sperm cell entering the perivitelline space between the zona pellucida and the oocyte body can reduce the time required for the oocyte and sperm to incubate together, which can lead to a reduction in the amount of polysperm entering the egg during in vitro fertilization. Observing the first sperm cell entering the perivitelline space can reduce the incubation time from about 6 hours to about 1-2 hours.

[0052] In some 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, cattle, buffalo, horses, caribou, deer, camels, elk, llamas, bulls, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinoceroses, and humans. For example, the animal could be a horse.

[0053] Animal embryos produced by the methods disclosed herein are also provided. For example, the animal embryo may be a horse embryo.

[0054] Implementation The present invention also provides the following non-limiting embodiments.

[0055] Implementation method 1 is a method for improving the efficiency of in vitro fertilization in animals, the method comprising: (a) Obtaining oocytes from the animal, wherein the oocytes comprise an oocyte body and a zona pellucida; (b) Obtaining sperm from the animal; (c) Mature the oocytes in a maturation culture medium; (d) Remove cumulus cells from the oocyte; (e) Drilling a hole in the zona pellucida of the oocyte using a laser; (f) Bring the oocyte into contact with the sperm; (g) The oocyte is co-incubated with the sperm, thereby enabling in vitro fertilization of the oocyte and sperm to produce an animal embryo, and Therefore, the efficiency of in vitro fertilization for producing animal embryos is improved by drilling holes in the zona pellucida of the oocytes using the laser.

[0056] Implementation 2 is the method described in Implementation 1, wherein the cumulus cells are removed from the oocyte by treatment with hyaluronidase, aspiration with a pipette, and / or vortexing.

[0057] Implementation 3 is the method described in Implementation 1 or 2, wherein the oocyte is placed on a microscope stage before step (e).

[0058] Embodiment 4 is the method of any one of Embodiments 1-3, wherein the oocyte comprises a polar body.

[0059] Embodiment 5 is the method of any one of Embodiments 1-4, wherein the laser is aimed at a portion of the zona pellucida where there is a perivitelline space between the oocyte body and the zona pellucida.

[0060] Implementation 6 is the method described in Implementation 5, wherein the laser is aimed at a distance of about 2 μm to about 5 μm from the polar body of the oocyte.

[0061] Embodiment 7 is the method of any one of Embodiments 1-6, wherein the sperm is incubated in a capacitating medium before contacting the oocyte.

[0062] Implementation method 8 is the method described in implementation method 7, wherein the capacitating medium contains a calcium ion carrier, caffeine, heparin, lysophosphatidylcholine (LC) and / or catecholamines.

[0063] Embodiment 9 is the method described in Embodiment 8, wherein the concentration of the calcium ion carrier is from about 0.01 μM to about 100 μM.

[0064] Embodiment 10 is the method described in Embodiment 8, wherein the concentration of caffeine is from about 0.01 mM to about 100 mM.

[0065] Embodiment 11 is the method of any one of Embodiments 1-6, wherein the sperm is not incubated in a capacitating culture medium before contacting the oocyte.

[0066] Embodiment 12 is the method of any one of Embodiments 1-11, wherein the oocyte and sperm are co-incubated for about 30 minutes to about 24 hours.

[0067] Implementation 13 is the method of implementation 12, wherein the oocyte is co-incubated with the sperm for about 1 hour to about 3 hours.

[0068] Implementation 14 is the method described in Implementation 13, wherein the oocyte and the sperm are co-incubated for about 2 hours.

[0069] Implementation 15 is the method of Implementation 13 or 14, further comprising observing the incubation of the oocyte and the sperm under a microscope, and ending the incubation when the first sperm cell is observed to enter the perioval space between the zona pellucida and the oocyte body.

[0070] Implementation method 16 is the method of any one of implementation methods 1-15, 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, cattle, buffalo, horses, caribou, deer, camels, elk, llamas, bulls, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinoceroses, and humans.

[0071] Implementation 17 is the method described in Implementation 16, wherein the animal is a horse.

[0072] Embodiment 18 is an animal embryo produced by any one of Embodiments 1-17.

[0073] Embodiment 19 is the animal embryo described in Embodiment 18, wherein the animal embryo is a horse embryo.

[0074] Example method Horse oocyte maturation Horse oocytes were collected from a local horse breeding center using ultrasound-guided oocyte retrieval technology and placed in maturation medium in 4-well plates for 30-35 hours at 38.5°C and 5% CO2. At the end of maturation, cumulus cells were removed from the oocytes by hyaluronidase treatment and repeated aspiration with a fine glass pipette. After washing, the oocytes were used for laser zona pellucida (ZP) drilling.

[0075] Laser-guided transparent (ZP) drilling Oocytes were transferred to a droplet of maintenance medium coated with mineral oil on a microscope stage for laser ZP drilling. The oocytes were positioned using a micromanipulator so that the polar bodies were at the 12 o'clock position. A laser pulse (e.g., Xyrcos, Hamilton-Thorne) was fired at the ZP at the 3 o'clock position to cut a hole penetrating the ZP. Prior to in vitro fertilization (IVF), the oocytes were washed and placed in maintenance medium in a 4-well plate (38.5°C, 5% CO2).

[0076] Sperm preparation Place 1 ml of horse semen in a 15 ml centrifuge tube and centrifuge at 328 xg for 5 minutes. Add 1 ml of preheated semen preparation medium (e.g., EQ-SemenPrep, IVF Scientific) to the semen layer. Incubate the tube containing the semen at 37°C for 20 minutes. Transfer the top 500 μl of supernatant from the upper phase to a new 1.5 ml empty centrifuge tube and add 1 ml of preheated semen preparation medium. After careful mixing, centrifuge the mixture at 328 xg for 5 minutes. Remove the supernatant, add 1 ml of preheated semen preparation medium, and resuspend the sperm pellet. Centrifuge the mixture again at 328 xg for 5 minutes. Remove the supernatant until approximately 200 μl remains. Carefully mix the sperm mixture and maintain it at 37°C until IVF.

[0077] sperm capacitation Add 100 μl of the prepared sperm mixture to a new 1.5 ml tube and centrifuge again at 328 xg for 5 minutes. Remove the supernatant and add 50 μl of sperm capacitation medium (e.g., 1 ml Hal-L + 1 uM Ca2+ ionophore + 10 mM caffeine). Incubate the sperm mixture at 38.5°C for 10 minutes.

[0078] In vitro fertilization Laser-drilled equine oocytes were transferred to 100 μl droplets of IVF medium (e.g., BO-IVF medium, IVF Scientific) in 4-well plates coated with mineral oil. Capacitated sperm were then added to the IVF droplets. Oocytes and sperm were co-incubated at 38.5°C in air with 5% CO2 for 30 minutes to 2 hours. The co-incubation interval was determined by visualization of the first sperm cell in the perivitelline space of the oocyte. At the end of IVF, the oocytes were washed and transferred to embryo culture medium (e.g., EQ-IVC, IVF Scientific) and incubated at 38.5°C in air containing 5% CO2 and 6% O2.

[0079] result Table 1 below shows the number of horse oocytes at the end of maturation. Twenty-four naked horse oocytes (including 9 oocytes with polar bodies (PB) and 15 oocytes without PB) were subjected to laser ZP drilling and IVF. Eleven non-naked horse oocytes were activated by parthenogenesis as a control.

[0080] Table 1: Number of horse oocytes at the end of maturity

[0081] Table 2 shows the development of tegument oocytes on day 3 after laser ZP drilling and IVF. Of the 24 oocytes in the laser ZP drilling and IVF group, 13 (54%) cleaved to form 2-cell or 4-cell embryos. The cleavage rate in the laser ZP drilling and IVF group was very similar to that in the parthenogenetic control group (55%). Figure 1 and Figure 2 The image shows a equine embryo formed by both IVF and parthenogenesis activation.

[0082] Table 2: Development of equine oocytes 3 days after laser zona pellucida drilling and in vitro fertilization

[0083] in conclusion Laser-guided perforation before IVF improves IVF efficiency in horses. This technique can be easily applied to humans and other species.

[0084] Improved equine in vitro fertilization (IVF) efficiency using laser-guided transparent tube drilling.

[0085] Equine IVF efficiency was significantly improved by using laser zona pellucida drilling prior to in vitro fertilization (IVF). Equine oocytes were collected from local sources and used in the laser-assisted IVF procedure disclosed above. As described above, of the 139 equine oocytes collected and subjected to laser-assisted IVF, 61.2% fertilized and divided to form 2-cell embryos, compared to 0% in the control group of 34 oocytes that did not undergo laser-assisted zona pellucida drilling. In the laser-assisted zona pellucida drilling group, 52.5% developed to the 8- to 16-cell stage, 15.8% to the morula stage, and 8.6% to the blastocyst stage, compared to 0% for all corresponding cell stages in the control group that did not undergo laser-assisted zona pellucida drilling. Examples of equine embryos produced by laser-assisted IVF include... Figures 3 to 5 The images are shown in the table below, and the results are provided in Table 3.

[0086] Table 3: Improved IVF efficiency using laser-guided transparent strip drilling

[0087] Laser-assisted in vitro fertilization without the use of micromanipulators.

[0088] This study investigated the use of laser-drilled zona pellucida for in vitro fertilization (IVF) without the use of a micromanipulator. The aim of this experiment was to simplify laser-drilled zona pellucida and increase the speed of the process. The experiment was performed as described above, without the use of a micromanipulator. Oocytes were transferred to a droplet of mineral oil-coated maintenance medium on a microscope stage for laser-drilled zona pellucida. The oocytes were positioned by moving the microscope stage so that the laser beam was aimed at a portion of the zona pellucida. A laser pulse (e.g., Xyrcos, Hamilton-Thorne) was fired at the zona pellucida to cut a hole penetrating it. The process was repeated on the next oocyte, and so on. Prior to IVF, the oocytes were washed and placed in maintenance medium in a 4-well plate (38.5°C, 5% CO2). IVF and subsequent culture of the oocytes were performed as described above. The oocyte development rate after laser-drilled zona pellucida is shown in Table 3.

[0089] Determination of incubation time for IVF process using laser ZP drilling.

[0090] This study investigated the relationship between polyspermyation rate and the incubation duration of oocytes and sperm. The aim of this experiment was to determine the optimal incubation duration for achieving the highest cleavage rate and the lowest polyspermyation rate. The experiment was conducted as described above. Oocytes following laser ZP drilling and IVF were removed from co-incubation with sperm at different time points: 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, and 2 hours. The oocytes were washed and cultured as described above. The polyspermyation rate and cleavage rate of the oocytes at different time points were compared to determine the optimal incubation duration.

[0091] Those skilled in the art will understand that changes can be made to the above embodiments without departing from their broad inventive concept. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined in this specification.

Claims

1. A method for improving the efficiency of in vitro fertilization in animals, the method comprising: (a) Obtaining oocytes from the animal, wherein the oocytes comprise an oocyte body and a zona pellucida; (b) Obtaining sperm from the animal; (c) Mature the oocytes in a maturation culture medium; (d) Remove cumulus cells from the oocyte; (e) Drilling a hole in the zona pellucida of the oocyte using a laser; (f) Bring the oocyte into contact with the sperm; (g) The oocyte is co-incubated with the sperm, thereby enabling in vitro fertilization of the oocyte and sperm to produce an animal embryo, and Therefore, the efficiency of in vitro fertilization for producing animal embryos is improved by drilling holes in the zona pellucida of the oocytes using the laser.

2. The method of claim 1, wherein the cumulus cells are removed from the oocyte by treatment with hyaluronidase, aspiration with a pipette, and / or vortexing.

3. The method of claim 1, wherein the oocyte is placed on a microscope stage prior to step (e).

4. The method of claim 1, wherein the oocyte comprises a polar body.

5. The method of claim 1, wherein the laser is aimed at a portion of the zona pellucida where a perivitelline space exists between the oocyte body and the zona pellucida.

6. The method of claim 5, wherein the laser is aimed at a distance of about 2 μm to about 5 μm from the polar body of the oocyte.

7. The method of claim 1, wherein the sperm is incubated in a capacitating medium before contacting the oocyte.

8. The method of claim 7, wherein the capacitation medium comprises a calcium ion carrier, caffeine, heparin, lysophosphatidylcholine (LC) and / or catecholamines.

9. The method of claim 8, wherein the concentration of the calcium ion carrier is from about 0.01 μM to about 100 μM.

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

11. The method of claim 1, wherein the sperm is not incubated in a capacitating medium before contacting the oocyte.

12. The method of claim 1, wherein the oocyte is co-incubated with the sperm for about 30 minutes to about 24 hours.

13. The method of claim 12, wherein the oocyte is co-incubated with the sperm for about 1 hour to about 3 hours.

14. The method of claim 13, wherein the oocyte is co-incubated with the sperm for about 2 hours.

15. The method of claim 13, further comprising observing the incubation of the oocyte and the sperm under a microscope, and ending the incubation when the first sperm cell is observed to enter the perivitelline space between the zona pellucida and the oocyte body.

16. The method of 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, cattle, buffalo, horses, caribou, deer, camels, elk, llamas, bulls, moose, bears, pandas, koalas, chimpanzees, gorillas, monkeys, giraffes, seals, hippos, rhinoceroses, and humans.

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

18. An animal embryo produced by the method of claim 1.

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

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