Laser-assisted in vitro fertilization

Abstract

Provided herein are methods of increasing the efficiency of in vitro fertilization in an animal. The methods comprise (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 oocyte in a maturation medium; (d) removing cumulus cells from the oocyte; (e) drilling a hole in the zona pellucida of the oocyte with a laser; (f) contacting the oocyte with the sperm; (g) incubating the oocyte with the sperm, whereby the incubation allows for the in vitro fertilization of the oocyte and sperm to produce an animal embryo, and whereby the efficiency of the in vitro fertilization to produce an animal embryo is increased by drilling a hole in the zona pellucida of the oocyte with the laser.

Description

LASER-ASSISTED IN VITRO FERTILIZATIONCROSS REFERENCE TO RELATED APPLICATIONS

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

[0002] The present disclosure generally relates to the field of in vitro fertilization (IVF).BACKGROUND OF THE INVENTION

[0003] In vitro fertilization (IVF) in horse is not as successful as in other domestic species. The fertilization rate in horse is very low, and the development of the embryos after IVF is poor (Blue et al., Equine Vet J. Supp 8: 111-116 (1989); Zhang et al., Mol. Reprod. Dev. 26:361-5 (1990); Roasa et al., Theriogenology 68:560-6 (2007); Palmer et al., J. Reprod. Fertil. Supp. 44:375-384 (1991)). There have been sporadic reports of success in IVF in horse (Bezard et al., Proc. Intern. Scient. Conf. Biotechnics Horse Reprod., Agricultural University of Crakow, Poland p. 12 (1992); Feliz et al., Biol. Reprod. 107(6): 1551-64 (2022); Li et al., Biol. Reprod.Monograph 1 (1):613-622 (1995); Leemans et al., Reprod. 152:R233-R245 (2016)) without reports of confirmed repeatability.

[0004] The barrier to successful IVF in horse is currently unclear. Thus, there currently is an unmet need to increase the in vitro fertilization (IVF) efficiency in horse to produce more embryos and embryos with greater quality.BRIEF SUMMARY OF THE INVENTION

[0005] Provided herein is a method of increasing the efficiency of in vitro fertilization in an animal. The methods comprise (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 oocyte in a maturation medium; (d) removing cumulus cells from the oocyte; (e) drilling a hole in the zona pellucida of the oocyte with a laser; (f) contacting the oocyte with the sperm; (g) incubating the oocyte with the sperm, whereby the incubation allows for the in vitro fertilization of the oocyte and sperm to produce an animal embryo, and whereby the efficiency ofthe in vitro fertilization to produce an animal embryo is increased by drilling a hole in the zona pellucida of the oocyte with the laser. In certain embodiments, the cumulus cells are removed from the oocyte by treating with hyaluronidase, aspirating with a pipet, and / or with vortexing. In certain embodiments, the oocytes are placed on a microscope stage prior to step (e).

[0006] In certain embodiments, the oocyte comprises a polar body. In certain embodiments, the laser is aimed at a portion of the zona pellucida where a perivitelline space exists between the oocyte body and the zona pellucida. In certain embodiments, the laser is aimed about 2 m to about 5 pm away from the polar body of the oocyte.

[0007] In certain embodiments, the sperm is incubated in a capacitation medium prior to being contacted with the oocyte. The capacitation medium can, for example, comprise a calcium ionophore and caffeine. In certain embodiments, the calcium ionophore is at a concentration of about 0.01 pM to about 100 pM. In certain embodiments, the caffeine is at a concentration of about 0.01 mM to about 100 mM.

[0008] In certain embodiments, the sperm is not incubated in a capacitation medium prior to being contacted with the oocyte.

[0009] In certain embodiments, the oocyte is incubated with the sperm for about 30 minutes to about 24 hours. The oocyte can, for example, be incubated with the sperm for about 1 hour to about 3 hours. The oocyte can, for example, be incubated with the sperm for about 2 hours.

[0010] In certain embodiments, the methods further comprise observing the incubation of the oocyte and the sperm with a microscope and ending the incubation upon visualization of the first sperm cell entering the perivitelline space between the zona pellucida and the oocyte body.

[0011] In certain embodiments, the animal is selected from a dog, a cat, a fox, a tiger, a lion, a cheetah, a leopard, a jaguar, a wolf, a goat, a sheep, an elephant, a rabbit, an opossum, a porcupine, a lemur, an otter, a sloth, a kangaroo, a wolverine, a cattle, a buffalo, a horse, a caribou, a deer, a camel, an elk, a llama, an ox, a moose, a bear, a panda, a koala, a chimpanzee, a gorilla, a monkey, a giraffe, a seal, a hippopotamus, a rhinoceros, and a human. The animal can, for example, be a horse.

[0012] Also provided are animal embryos produced by the methods disclosed herein. The animal embryos can, for example, be horse embryos.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing summary, as well as the following detailed description of embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

[0014] FIG. 1 shows an image of a horse embryo 3 days after laser zona pellucida drilling and in vitro fertilization.

[0015] FIG. 2 shows an image of a horse embryo 3 days post parthenogenetic activation.

[0016] FIG. 3 shows an image of a horse embryo 4 days after laser zona pellucida drilling and in vitro fertilization.

[0017] FIG. 4 shows an image of a horse embryo 5 days after laser zona pellucida drilling and in vitro fertilization.

[0018] FIG. 5 shows an image of a horse embryo 12 days after laser zona pellucida drilling and in vitro fertilization.DETAILED DESCRIPTION OF THE INVENTION

[0019] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

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

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

[0022] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of1% to 10% (w / v) includes 0.9% (w / v) to 1 1% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0023] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0024] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0025] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0026] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integeror group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.

[0027] As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. § 2111.03.

[0028] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made.

[0029] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art ( .g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0030] As used herein, the term “oocyte” refers to the female germ cell involved in reproduction.

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

[0032] As used herein, the term “zona pellucida” refers to a specialized extracellular matrix that surrounds the plasma membrane of oocytes.

[0033] As used herein, the term “perivitelline space” refers to the space between the zona pellucida and the cell membrane of an oocyte or a fertilized ovum.

[0034] As used herein, the term “polar body” refers to a small haploid cell that is formed at the same time as an egg cell during meiosis, but generally does not have the ability to be fertilized.

[0035] As used herein, the term “embryo” refers to an initial stage of development of a multicellular organism. “Embryo” or “embryonic development” is the part of the life cycle that begins just after fertilization of the egg cell with the sperm cell. The resulting fusion of the egg cell and the sperm cell results in a single-celled zygote that undergoes many cell divisions, ultimately resulting in a multicellular organism implanted in the lining of a womb, which allowsfor gastrulation, neurulation, and organogenesis of the developing organism. An “embryo” can also refer to an unborn or unhatched offspring in the process of development.

[0036] As used herein, the term “cumulus cells” refers to a group of closely associated granulosa cells that surround the oocyte and participate in the processes of oocyte maturation and fertilization.

[0037] As used herein, the term “capacitation” refers to the activation process that prepares the sperm for fertilizing an oocyte. Capacitation can refer to the activation process that occurs in the female reproductive tract that prepares the sperm to fertilize an oocyte. Alternatively, capacitation can refer to an artificial activation process comprising incubating the sperm in a specific medium for activating the sperm to prepare the sperm for fertilizing the oocyte.

[0038] Methods of increasing the efficiency of in vitro fertilization (IVF)

[0039] Provided herein is a method of increasing the efficiency of in vitro fertilization in an animal. Increasing the efficiency of in vitro fertilization for an animal can, for example, comprise increasing the number of fertilized oocytes after in vitro fertilization, increasing the number of fertilized oocytes undergoing cell divisions after in vitro fertilization, increasing the number of viable embryos produced by the method, increasing the quality of the produced embryo from the in vitro fertilization process, and / or increasing the capability of the produced embryos to generate live birth.

[0040] The methods of increasing the efficiency of in vitro fertilization can, for example, comprise (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 oocyte in a maturation medium; (d) removing cumulus cells from the oocyte; (e) drilling a hole in the zona pellucida of the oocyte with a laser; (f) contacting the oocyte with the sperm; (g) incubating the oocyte with the sperm, whereby the incubation allows for the in vitro fertilization of the oocyte and sperm to produce an animal embryo, and whereby the efficiency of the in vitro fertilization to produce an animal embryo is increased by drilling a hole in the zona pellucida of the oocyte with the laser.

[0041] Without intending to be limited by theory, drilling a hole in the zona pellucida with a laser can, for example, allow for easier access of the sperm to fertilize the oocyte by providing direct access for the sperm to contact the oocyte body. This can result in reduced energy spent by the sperm in penetrating the zona pellucida and more energy in fertilizing the oocytes. This canalso result in reduction in the time spent by the sperm to contact the oocytes before the limited useful lifespan of the sperm expires. This can also result in reduction in reduced incubation times for the in vitro fertilization process. By utilizing a laser to drill the hole in the zona pellucida, the sperm can get in contact with the oocyte and then fuse with the oocyte to complete the in vitro fertilization process, making it unnecessary to inject the sperm into the oocyte for fertilization to occur. The hole will provide the sperm direct access to the oocyte for the fertilization process.

[0042] Further, utilizing a laser for drilling a hole in the zona pellucida, instead of utilizing chemical means to drill a hole, allows for a precise hole to be drilled with minimal to no damage to the oocyte, which can allow for the production of more higher quality embryos from the in vitro fertilization process. Additionally, the laser is easier to manipulate (i.e., aim, adjust strength, etc.) and is much faster than utilizing chemical means. The laser can drill a hole through the zona pellucida without the help of a micromanipulation set while utilizing chemical means results in the need for a micromanipulation set for aiming.

[0043] The oocyte can, for example, be obtained from a female animal, and the sperm can, for example, be obtained from a male animal of the same species. The sperm can, for example, be obtained from cryopreserved sperm cells.

[0044] Maturing the oocyte in a maturation medium can, for example, result in an oocyte with a polar body indicating that the oocyte is ready for fertilization. The maturation medium can, for example, comprise 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).

[0045] In certain embodiments, the mature oocyte comprises cumulus cells surrounding the oocyte. The cumulus cells can, for example, be removed from the oocyte. The cumulus cells can be partially removed or completely removed, as long as a portion of the zona pellucida is exposed and capable of being targeted with the laser. The cumulus cells can, for example, be removed by chemical methods and or mechanical methods. By way of an example, the cumulus cells can be removed by treating with hyaluronidase, aspirating with a pipet, and / or with vortexing.

[0046] In certain embodiments, the oocytes are placed on a microscope stage prior to step (e). The oocyte can be observed for the presence of a polar body. In certain embodiments, the oocytecomprises a polar body. In certain embodiments, using the microscope, the laser is aimed at a portion of the zona pellucida where a perivitelline space exists between the oocyte body and the zona pellucida. In certain embodiments, the laser is aimed about 2 pm to about 5 pm away from the polar body of the oocyte.

[0047] In certain embodiments, the sperm is incubated in a capacitation medium prior to being contacted with the oocyte. The capacitation medium can, for example, activate the sperm for the fertilization process. The capacitation medium can, for example, comprise a calcium ionophore, caffeine, heparin, lysophosphatidylcholine (LC), and catecholamines.

[0048] In certain embodiments, the calcium ionophore is at a concentration of about 0.01 p.M to about 100 pM. The calcium ionophore can, for example, be at a concentration of about 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1 pM, 5 pM, 10 pM, 15 M, 20 pM, 25 ptM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 |1M, 70 pM, 75 pM, 80 pM, 85 pM, 90 |1M, 95 pM, 100 pM, or any value in between. The calcium ionophore can, for example, be about 0.05 pM to about 100 pM, about 1 pM to about 100 pM, about 10 pM to about 100 p.M, about 20 p.M to about 100 pM, about 30 pM to about 100 p.M, about 40 pM to about 100 pM, about 50 pM to about 100 pM, about 60 pM to about 100 pM, about 70 pM to about 100 pM, about 80 pM to about 100 pM, about 90 pM to about 100 p.M, about 0.01 pM to about 90 pM, about 0.01 pM to about 80 pM, about 0.01 pM to about 70 pM, about 0.01 pM to about 60 p.M, about 0.01 p.M to about 50 pM, about 0.01 pM to about 40 pM, about 0.01 pM to about 30 p.M, about 0.01 p.M to about 20 pM, about 0.01 pM to about 10 pM, about 0.01 pM to about 1 pM, or any value in between.

[0049] In certain embodiments, the caffeine is at a concentration of about 0.01 mM to about 100 mM. The caffeine can, for example, be at a concentration of 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 in between. The caffeine can, for example, be about 0.05 mM to about 100 mM, about 1 mM to about 100 mM, about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, about 60 mM to about 100 mM, about 70 mM to about 100 mM, about 80 mM to about 100 mM, about 90 mM to about 100 mM, about 0.01 mM to about 90 mM, about 0.01 mM to about 80 mM, about 0.01 mM to about 70 mM, about 0.01 mM to about 60 mM, about 0.01 mM to about 50mM, about 0.01 mM to about 40 mM, about 0.01 mM to about 30 mM, about 0.01 mM to about 20 mM, about 0.01 mM to about 10 mM, about 0.01 mM to about 1 mM, or any value in between.

[0050] In certain embodiments, the sperm is not incubated in a capacitation medium prior to being contacted with the oocyte.

[0051] In certain embodiments, the oocyte is incubated with the sperm for about 30 minutes to about 24 hours. The oocyte can, for example, be incubated with the 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 in between. The oocyte can, for example, be incubated with the sperm for about 1 hour to about 3 hours. The oocyte can, for example, be incubated with the sperm for about 2 hours.

[0052] In certain embodiments, the methods further comprise observing the incubation of the oocyte and the sperm with a microscope and ending the incubation upon visualization of the first sperm cell entering the peri-vitelline space between the zona pellucida and the oocyte body. Visualization of the first sperm cell entering the perivitelline space between the zona pellucida and oocyte body can, for example, reduce the amount time needed for the oocyte to be incubated with the sperm, which can result in a reduction in the amount of polyspermy in the in vitro fertilization process. The visualization of the first sperm entering the perivitelline space can reduce the incubation time from about 6 hours to about 1-2 hours.

[0053] In certain embodiments, the animal is selected from a dog, a cat, a fox, a tiger, a lion, a cheetah, a leopard, a jaguar, a wolf, a goat, a sheep, an elephant, a rabbit, an opossum, a porcupine, a lemur, an otter, a sloth, a kangaroo, a wolverine, a cattle, a buffalo, a horse, a caribou, a deer, a camel, an elk, a llama, an ox, a moose, a bear, a panda, a koala, a chimpanzee,a gorilla, a monkey, a giraffe, a seal, a hippopotamus, a rhinoceros, and a human. The animal can, for example, be a horse.

[0054] Also provided are animal embryos produced by the methods disclosed herein. The animal embryos can, for example, be horse embryos.EMBODIMENTS

[0055] The invention provides also the following non-limiting embodiments.

[0056] Embodiment l is a method of increasing the efficiency of in vitro fertilization in an animal, the method comprising:(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 oocyte in a maturation medium;(d) removing cumulus cells from the oocyte;(e) drilling a hole in the zona pellucida of the oocyte with a laser;(f) contacting the oocyte with the sperm;(g) incubating the oocyte with the sperm, whereby the incubation allows for the in vitro fertilization of the oocyte and sperm to produce an animal embryo, and whereby the efficiency of the in vitro fertilization to produce an animal embryo is increased by drilling a hole in the zona pellucida of the oocyte with the laser.

[0057] Embodiment 2 is the method of embodiment 1, wherein the cumulus cells are removed from the oocyte by treating with hyaluronidase, aspirating with a pipet, and / or with vortexing.

[0058] Embodiment 3 is the method of embodiment 1 or 2, wherein the oocytes are placed on a microscope stage prior to step (e).

[0059] Embodiment 4 is the method of any one of embodiments 1-3, wherein the oocyte comprises a polar body.

[0060] 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 a perivitelline space exists between the oocyte body and the zona pellucida.

[0061] Embodiment 6 is the method of embodiment 5, wherein the laser is aimed about 2 pm to about 5 pm away from the polar body of the oocyte.

[0062] Embodiment 7 is the method of any one of embodiments 1-6, wherein the sperm is incubated in a capacitation medium prior to being contacted with the oocyte.

[0063] Embodiment 8 is the method of embodiment 7, wherein the capacitation medium comprises a calcium ionophore, caffeine, heparin, lysophosphatidylcholine (LC), and / or catecholamines.

[0064] Embodiment 9 is the method of embodiment 8, wherein the calcium ionophore is at a concentration of about 0.01 pM to about 100 pM.

[0065] Embodiment 10 is the method of embodiment 8, wherein the caffeine is at a concentration of about 0.01 mM to about 100 mM.

[0066] Embodiment 11 is the method of any one of embodiments 1-6, wherein the sperm is not incubated in a capacitation medium prior to being contacted with the oocyte.

[0067] Embodiment 12 is the method of any one of embodiments 1-11, wherein the oocyte is incubated with the sperm for about 30 minutes to about 24 hours.

[0068] Embodiment 13 is the method of embodiment 12, wherein the oocyte is incubated with the sperm for about 1 hour to about 3 hours.

[0069] Embodiment 14 is the method of embodiment 13, wherein the oocyte is incubated with the sperm for about 2 hours.

[0070] Embodiment 15 is the method of embodiment 13 or 14, further comprising observing the incubation of the oocyte and the sperm with a microscope and ending the incubation upon visualization of the first sperm cell entering the perivitelline space between the zona pellucida and the oocyte body.

[0071] Embodiment 16 is the method of any one of embodiments 1-15, wherein the animal is selected from a dog, a cat, a fox, a tiger, a lion, a cheetah, a leopard, a jaguar, a wolf, a goat, a sheep, an elephant, a rabbit, an opossum, a porcupine, a lemur, an otter, a sloth, a kangaroo, a wolverine, a cattle, a buffalo, a horse, a caribou, a deer, a camel, an elk, a llama, an ox, a moose, a bear, a panda, a koala, a chimpanzee, a gorilla, a monkey, a giraffe, a seal, a hippopotamus, a rhinoceros, and a human.

[0072] Embodiment 17 is the method of embodiment 16, wherein the animal is a horse.

[0073] Embodiment 18 is an animal embryo produced by the method of any one of embodiments 1-17.

[0074] Embodiment 19 is the animal embryo of embodiment 18, wherein the animal embryo is a horse embryo.EXAMPLES

[0075] Methods

[0076] Horse oocyte maturation

[0077] Horse oocytes were collected from local equine breeding centers through ultrasound guided ovum pickup and matured in maturation medium in a 4-well plate at 38.5°C in 5% CO2 for 30-35 hours. At the end of the maturation, horse oocytes were removed of cumulus cells by hyaluronidase treatment and repeated aspiration by a fine glass pipet. The oocytes were washed and then used for laser zona pellucida (ZP) drilling.

[0078] Laser zona pellucida (ZP) drilling

[0079] The oocytes were transferred into a droplet of holding medium covered by mineral oil on the microscope stage for laser ZP drilling. The oocytes were positioned by micromanipulators so that the polar body was at the 12 o’clock position. A laser pulse (e.g., Xyrcos, Hamilton Thorne) was fired toward the ZP at the 3 o’clock position to cut a hole through the ZP. The oocytes were washed and placed into holding medium in a 4-well plate at 38.5°C in 5% CO2 before in vitro fertilization (IVF).

[0080] Sperm preparation

[0081] One milliliter (ml) of the horse semen was placed into a 15 ml centrifuge tube and centrifuged for 5 minutes at 328 x g. One ml of pre-heated 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. A volume of 500 pl of the supernatant from the very top of the upper phase was taken into a new empty 1.5 ml centrifuge tube, and 1 ml of preheated semen preparation medium was added. After carefully mixing, the mix was centrifuged at 328 x g for 5 minutes. The supernatant was removed, 1 ml of preheated semen preparation medium was added, and the sperm pellet was re-suspended. The mix was centrifuged again at 328 x g for 5 minutes. The supernatant was removed until approximately 200 pl was left. The sperm mix was mixed carefully, and the sperm mix was kept at 37°C until IVF.

[0082] Sperm capacitation

[0083] A volume of 100 l of the prepared sperm mix was added into a new 1.5 ml tube and centrifuged again at 328 x g for 5 minutes. The supernatant was removed and 50 pl of sperm capacitation medium (e.g., 1 ml Hal-L + luM Ca2 ionophore + 10 mM caffeine) was added. The sperm mix was incubated at 38.5°C for 10 minutes.

[0084] In vitro fertilization

[0085] The laser-drilled horse oocytes were transferred into 100 pl droplets of IVF medium (e.g., BO-IVF medium, IVF Scientific) in a 4-well plate covered with mineral oil. The capacitated sperm was then added to the IVF droplets. The oocytes and sperm cells were co-incubated for 30 minutes to 2 hours at 38.5°C in 5% CO2 in air. The coincubation interval was determined by the visualization of the first sperm cell in the perivitelline space of the oocytes. At the end of IVF, the oocytes were washed and transferred into embryo culture medium (e.g., EQ-IVC, IVF Scientific) and incubated at 38.5°C in 5% CO2 and 6% O2 in air.

[0086] Results

[0087] Table 1 below shows the number of horse oocytes at the end of maturation. Twenty -four denuded horse oocytes, including 9 oocytes with polar body (PB) and 15 without PB were subjected to laser ZP drilling and IVF. Eleven non-denuded horse oocytes were parthenogenetically activated as the control.

[0088] Table 1 : Number of horse oocytes at the end of maturation

[0089] Table 2 shows the development of horse oocytes on Day 3 post laser ZP drilling and IVF. Of the 24 oocytes in the group with laser ZP drilling and IVF, 13 (54%) cleaved to form 2-cell or 4-cell embryos. The cleavage rate of the group of laser ZP drilling and IVF was very similar to that of the group of parthenogenetic control of 55%. FIGs. 1 and 2 show the horse embryos formed from both IVF and parthenogenetic activation.

[0090] Table 2: Development of horse oocytes 3 days post laser zona pellucida drilling and in vitro fertilization

[0091] Conclusion

[0092] Laser zona pellucida drilling before IVF improved the IVF efficiency in horse. This technology could be easily applicable to human and other species.

[0093] Improved Efficiency of In vitro fertilization (IVF) in Horse utilizing laser zona drilling.

[0094] By utilizing laser zona drilling before in vitro fertilization (IVF), the IVF efficiency in horse was dramatically improved. Horse oocytes were collected from a local source and used for the laser-assisted IVF procedure as disclosed above. Out of the 139 horse oocytes collected and subjected to the laser-assisted IVF, as described above, 61.2% were fertilized and divided to form 2-cell embryos, compared to 0% in the control group of 34 oocytes in which the laser- assisted zona drilling was not performed. Of the laser-assisted zona drilling group, 52.5% developed to the 8 to 16 cell stage, 15.8% to morulae, and 8.6% to blastocysts, compared to 0% at those stages in the control group in which laser-assisted zona drilling was not performed. Examples of horse embryos produced by the laser-assisted IVF are shown in the pictures in FIGs. 3-5 and the results are provided below in Table 3.

[0095] Table 3: Improved efficiency of IVF in horse utilizing laser zona drillingLaser-assisted in vitro fertilization without the use of micromanipulators.

[0096] The use of a laser to drill the zona pellucida for in vitro fertilization without the use of micromanipulators was studied. The aim of the experiment was to simplify the laser ZP drilling and to increase the speed of the process. The experiment was conducted as described above without the use of micromanipulators. The oocytes were transferred into a droplet of holding medium covered by mineral oil on the microscope stage for laser ZP drilling. The oocytes were positioned by moving the microscope stage around so that the laser beam was aimed at a portion of the zona pellucida of the oocytes. A laser pulse (e.g., Xyrcos, Hamilton Thome) was fired toward the ZP to cut a hole through the ZP. The process was repeated on the next oocyte, and so on. The oocytes were washed and placed into holding medium in a 4-well plate at 38.5°C in 5% CO2 before in vitro fertilization (IVF). IVF and subsequent culture of the oocytes was conducted as described above. The developmental rate of the oocytes after ZP laser drilling was shown in Table 3.

[0097] Determination of incubation time for IVF process utilizing laser ZP drilling.

[0098] The relationship between the rate of polyspermy and the incubation duration of the oocytes and sperm is to be studied. The aim of the experiment is to determine the optimal incubation duration for the highest cleavage rate with the lowest polyspermy rate. The experiment is to be conducted as described above. The oocytes after laser ZP drilling and IVF are removed from the incubation with the sperm at various time points of 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, and 2 hours. The oocytes are washed and cultured as described above. The polyspermy rate and cleavage rate of the oocytes at various time points are compared to determine the optimal incubation duration.

[0099] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

Claims

CLAIMS1. A method of increasing the efficiency of in vitro fertilization in an animal, the method comprising:(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 oocyte in a maturation medium;(d) removing cumulus cells from the oocyte;(e) drilling a hole in the zona pellucida of the oocyte with a laser;(f) contacting the oocyte with the sperm;(g) incubating the oocyte with the sperm, whereby the incubation allows for the in vitro fertilization of the oocyte and sperm to produce an animal embryo, and whereby the efficiency of the in vitro fertilization to produce an animal embryo is increased by drilling a hole in the zona pellucida of the oocyte with the laser.

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

3. The method of claim 1, wherein the oocytes are 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 about 2 pm to about 5 pm away from the polar body of the oocyte.

7. The method of claim 1, wherein the sperm is incubated in a capacitation medium prior to being contacted with the oocyte.

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

9. The method of claim 8, wherein the calcium ionophore is at a concentration of about 0.01 pM to about 100 pM.

10. The method of claim 8, wherein the caffeine is at a concentration of about 0.01 mM to about 100 mM.11 . The method of claim 1 , wherein the sperm is not incubated in a capacitation medium prior to being contacted with the oocyte.

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

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

14. The method of claim 13, wherein the oocyte is 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 with a microscope and ending the incubation upon visualization of the first sperm cell entering the peri-vitelline space between the zona pellucida and the oocyte body.

16. The method of claim 1, wherein the animal is selected from a dog, a cat, a fox, a tiger, a lion, a cheetah, a leopard, a jaguar, a wolf, a goat, a sheep, an elephant, a rabbit, an opossum, a porcupine, a lemur, an otter, a sloth, a kangaroo, a wolverine, a cattle, a buffalo, a horse, a caribou, a deer, a camel, an elk, a llama, an ox, a moose, a bear, a panda, a koala, a chimpanzee, a gorilla, a monkey, a giraffe, a seal, a hippopotamus, a rhinoceros, and a human.

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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