How to grow embryos

Abstract

The present disclosure generally relates to a method for producing a plurality of embryos from one or more donor embryos that contain embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compaction morula. More specifically, the method of the present disclosure includes unzipping the donor embryo and expanding one or more clumps of blastomeres obtained from the donor embryo to produce a plurality of blastocysts from the donor embryo. The present disclosure also relates to the use of such methods in animal breeding.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to Australian Provisional Patent Application No. 2022 / 900164, filed on 31 January 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] (Technical field) The present disclosure generally relates to a method for producing a plurality of embryos from one or more donor embryos that contain embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compaction morula. More specifically, the method of the present disclosure includes unzipping the donor embryo and expanding one or more clumps of blastomeres obtained from the donor embryo to produce a plurality of blastocysts from the donor embryo. The present disclosure also relates to the use of such methods in animal breeding. [Background technology]

[0003] Assisted reproductive technologies (ART) have made tremendous advances, especially during the past few decades. Artificial insemination (AI) remains the most (cost) effective method to achieve genetic gain in cattle populations and is widely used in the dairy industry. In this context, the global market for frozen semen and embryos remains strong, with millions of cattle bred by AI and over one million embryos transferred annually worldwide. Most of the top sires providing semen for AI in the dairy industry are obtained by embryo transfer (ET), and improvements in methods to control estrous cycles and ovulation have led to more effective programs for AI, superovulation of donor cows, and management of ET recipients. Despite these advances in ART, producer use of reproductive technologies such as superovulated embryo transfer (MOET) remains limited due to the costs associated with the creation of each embryo. Thus, unlike conventional AI, MOET is rarely used by producers as a conventional reproductive method.

[0004] More recently, approaches have been reported for generating genetically identical monozygotic twins by embryo bisection and from blastomeres isolated from cleavage-stage embryos. This new approach is an exciting addition to the ART "toolbox," allowing producers to more efficiently capture and select the genetics of females (in addition to the genetics of sires). However, as with other ET- and IVF-based approaches, widespread adoption of embryo twinning at a commercial level is likely to be hindered by the prohibitive costs for producers, as well as challenges associated with scaling this technology.

[0005] Thus, a need exists for improved approaches for embryo propagation to address one or more of these limitations and to aid in industrial applications. Summary of the Invention

[0006] The present disclosure is broadly directed to methods for producing multiple embryos from a single donor embryo, and in particular for improving the efficiency of maturing conceptuses to the blastocyst stage after expansion from a donor. In this regard, the inventors have shown that the efficiency of blastocyst development and maturation is significantly higher for isolated bovine blastomeres expanded in cell aggregates (e.g., duplicates or quadruplets) compared to blastomeres expanded individually, particularly when expanding donor embryos that contain one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compaction morula. The inventors have also shown that implantation of mature blastocysts from embryos produced using the methods of the present disclosure into recipient female cattle can produce healthy calves.

[0007] In one embodiment, the present disclosure provides a method of expanding one or more donor embryos, comprising: (i) obtaining a donor embryo that comprises one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula; (ii) isolating a plurality of embryonic cells from the donor embryo; and (iii) expanding the embryonic cells in vitro under conditions suitable for producing a plurality of conceptuses from the donor embryos, wherein at least a portion of the embryonic cells are expanded as one or more cell clumps, each clump comprising two or more embryonic cells; (iv) culturing the plurality of conceptuses under conditions suitable to produce a plurality of blastocysts.

[0008] In one embodiment, separation of embryonic cells from the or each donor embryo is accomplished by disrupting the zona pellucida (ZP) and isolating the embryonic cells from the donor embryo. This is referred to herein as "unzip" or the "unzip method." According to this embodiment, the ZP can be disrupted and a plurality of embryonic cells can be isolated from each of one or more donor embryos. In one embodiment, the ZP is disrupted enzymatically or mechanically. For example, the ZP can be disrupted enzymatically or mechanically and a micropipette can be used to aspirate embryonic cells from one or more donor embryos, thereby isolating the embryonic cells.

[0009] In one embodiment, substantially all of the embryonic cells isolated from the donor embryo are expanded as a cell clump. For example, all of the embryonic cells isolated from the donor embryo can be expanded within a cell clump.

[0010] As described herein, the donor embryo comprises one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula. In one embodiment, the donor embryo may comprise nine or more embryonic cells, at least one of which is developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula. For example, the donor embryo may comprise 9-64 embryonic cells (e.g., 9-60 embryonic cells, etc.), provided that the embryo has not yet developed into a blastocyst. For example, the donor embryo may comprise 16-64 embryonic cells (e.g., 16-60 embryonic cells, etc.), provided that the embryo has not yet developed into a blastocyst.

[0011] In some examples, the donor embryo may include one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 32-cell embryo. For example, the donor embryo may be an embryo that includes 16-32 embryonic cells (e.g., a pre-morula). Alternatively, the donor embryo may be a pre-compaction morula that includes about 32-64 embryonic cells (e.g., 32-60 embryonic cells, etc.), provided that the embryo has not yet developed into a blastocyst.

[0012] A cell aggregate (i.e., an aggregate of blastomeres isolated from a donor embryo) may comprise about 2-8 embryonic cells prior to expansion. For example, one or more, or each, cell aggregate may comprise 2-4 embryonic cells prior to expansion. For example, one or more, or each, cell aggregate may comprise 2 embryonic cells prior to expansion. For example, one or more, or each, cell aggregate may comprise 4 embryonic cells prior to expansion. For example, one or more, or each, cell aggregate may comprise 8 embryonic cells prior to expansion.

[0013] In one example, the donor embryo comprises 9 or more embryonic cells (e.g., 10, or 11, or 12, or 13, or 14, 15, or 16 or more embryonic cells), at least one of which is developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula, and the embryonic cells isolated from the donor embryo are expanded in a cell clump of 2-4 embryonic cells (e.g., 2 or 4 embryonic cells).

[0014] In one embodiment, the donor embryo comprises 16 or more embryonic cells (e.g., 16-64 embryonic cells), at least one of which is developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula, and the embryonic cells isolated from the donor embryo are expanded in a cell clump of 2-4 embryonic cells (e.g., 2 or 4 embryonic cells). For example, the donor embryo may comprise at least about 16 embryonic cells. For example, the donor embryo may comprise at least about 32 embryonic cells and / or may be classified as a 32-cell embryo. For example, the donor embryo may comprise at least about 64 embryonic cells or may be classified as a pre-compaction morula that has not yet developed into a blastocyst.

[0015] In one embodiment, at least about three conceptuses are generated for each donor embryo in step (iv). In one embodiment, at least about four (e.g., five or six or seven or eight or nine or ten or more) conceptuses are generated for each donor embryo in step (iv). For example, each donor embryo can generate at least four conceptuses. For example, each donor embryo can generate at least five conceptuses. For example, each donor embryo can generate at least six conceptuses. For example, each donor embryo can generate at least seven conceptuses. For example, each donor embryo can generate at least eight conceptuses. For example, each donor embryo can generate at least nine conceptuses. For example, each donor embryo can generate at least ten conceptuses.

[0016] In some embodiments, a portion of the plurality of embryonic cells isolated from the donor embryo is separated as a pre-existing clump (e.g., as a duplicate or quadruplet). Alternatively or additionally, a portion of the plurality of embryonic cells isolated from the donor embryo is aggregated after being separated from the donor embryo. In some embodiments, the aggregated embryonic cells are derived from the same donor embryo. In some embodiments, the aggregated embryonic cells are derived from two or more donor embryos (e.g., two or more embryos from the same animal or from different animals).

[0017] In other embodiments, substantially all of the plurality of embryonic cells isolated from the donor embryo are separated within a pre-existing clump (eg, as duplicates or quadruplets).

[0018] In one embodiment, the embryonic cells or conceptus may be cultured in the presence of one or more factors capable of promoting embryo development, for example, the embryonic cells may be cultured in the presence of one or more factors capable of promoting embryo development to form and expand an embryo.

[0019] In another embodiment, the embryonic cell or an embryo comprising the same may be cultured in the presence of one or more factors capable of maintaining totipotency and / or inhibiting or preventing embryonic development. For example, the embryonic cell or an embryo comprising the same may be cultured in the presence of one or more factors capable of maintaining the elimination of maternal mRNA.

[0020] In each of the foregoing embodiments, the donor embryo may be obtained from a vertebrate animal.

[0021] In one embodiment, the vertebrate may be a mammalian species.

[0022] In one embodiment, the mammalian species may be a domesticated species.

[0023] In one embodiment, the livestock species may be a ruminant species. For example, the livestock species may be a bovine species. For example, the livestock species may be an ovine species (i.e., sheep). For example, the livestock species may be a caprine species (i.e., goats). For example, the livestock species may be a cervid species (i.e., deer). For example, the livestock species may be a camelid species (e.g., camel or alpaca).

[0024] In one embodiment, the livestock species may be a porcine species (i.e., pig).

[0025] In one embodiment, the livestock species may be an equine species (ie, a horse).

[0026] In some embodiments, the one or more donor embryos obtained in step (i) are produced by in vivo fertilization. In other embodiments, the one or more donor embryos obtained in step (i) are produced by in vitro fertilization (IVF).

[0027] In some embodiments, the donor embryo obtained in step (i) is freshly harvested. In other embodiments, the donor embryo obtained in step (i) is cryopreserved. For example, the donor embryo may be thawed. In other embodiments, the donor embryo obtained in step (i) may be stored in an embryo storage medium (e.g., at 4°C).

[0028] In each of the foregoing embodiments, the method may further include selecting one or more of the donor embryos obtained in step (i) based on one or more genetic screening criteria, genetic diagnosis, and / or one or more morphological criteria. For example, the selection step may be performed prior to step (i).

[0029] In one example, the genetic screening criteria may be determined by screening one or more donor embryos for the presence or absence of one or more genetic markers (e.g., SNP alleles or haplotypes) associated with a trait of interest. In one example, the trait of interest is selected from phenotypic production traits, drug resistance, susceptibility to pests and / or parasites, and sex (i.e., determining whether the embryo is male or female).

[0030] In one example, one or more of the donor embryos may be selected based on a genetic diagnosis for one or more conditions, diseases or predispositions thereto.

[0031] In one example, one or more of the donor embryos may be selected based on one or more morphological characteristics indicative of the health of the embryo.

[0032] In each of the above embodiments, one or more of the donor embryos may be genetically modified or edited. For example, one or more of the donor embryos may be genetically modified by introducing foreign nucleic acid into the genome of the embryonic cells contained therein. For example, one or more of the donor embryos may be genetically edited by editing the genome of the embryonic cells contained therein.

[0033] In one example, one or more of the donor embryos includes a unique genetic tag or nucleic acid identifier for traceability of the embryos and / or animals produced therefrom. For example, the unique genetic tag or nucleic acid identifier may be introduced using genetic modification.

[0034] In each of the foregoing embodiments, the disclosed methods include expanding a plurality of embryos in vitro to form blastocysts. For example, the methods may include expanding embryos in vitro to form mature blastocysts ready for implantation.

[0035] The methods of the present disclosure may further include recovering a plurality of embryos produced by the methods of the present disclosure. For example, the methods may include recovering the embryos once they have matured to the blastocyst stage.

[0036] In some embodiments, one or more of the recovered embryos are stored in an embryo storage medium. For example, one or more of the recovered embryos may be stored at about 4° C.

[0037] In some embodiments, one or more of the recovered embryos are cryopreserved. The cryopreserved embryos may be stored at about −180° C. to about −196° C. For example, the cryopreserved embryos may be stored in liquid nitrogen at about −196° C.

[0038] In some embodiments, the methods of the present disclosure further include transferring one or more of the embryos produced by the methods into the oviduct or uterus of one or more recipient females.

[0039] The present disclosure also provides one or more embryos produced by the methods of the present disclosure. In one embodiment, the one or more embryos may be provided in an embryo storage medium or transfer medium at about 4° C. In another embodiment, the one or more embryos may be cryopreserved.

[0040] In one example, the embryo is from a mammalian species (e.g., a non-human mammalian species). In one example, the non-human mammalian species is a livestock species (e.g., a ruminant species). For example, the livestock species may be a bovine species. For example, the livestock species may be an ovine species (i.e., sheep). For example, the livestock species may be a porcine species (i.e., pigs). For example, the livestock species may be an equine species (i.e., horses). For example, the livestock species may be a caprine species (i.e., goats). For example, the livestock species may be a cervid species (i.e., deer). For example, the livestock species may be a camelid species (e.g., a camel or an alpaca).

[0041] The present disclosure also provides a method of breeding an animal, comprising: (i) transferring one or more of the embryos produced by the methods of the present disclosure into the oviduct or uterus of one or more recipient females to establish a pregnancy; (ii) producing an animal by parturition from the pregnant recipient female.

[0042] In one embodiment, the animal is a vertebrate. For example, the vertebrate may be a mammal, an amphibian, a reptile, a fish, or a bird.

[0043] In one particular embodiment, the animal is a mammal (e.g., a non-human mammal). Exemplary non-human mammals that may be produced using the present method include livestock species (e.g., cows, buffalo, pigs, sheep, goats, camels, deer, horses, etc.), companion animals (e.g., dogs, cats, etc.), laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.), non-human primates (e.g., macaques and marmosets, etc.), and wild animal species (e.g., marsupials, cats, rhinos, giant pandas, etc.). In one particular embodiment, the presently disclosed method may be used to breed ruminant livestock species. For example, the presently disclosed method may be used to breed cows. For example, the presently disclosed method may be used to breed sheep. For example, the presently disclosed method may be used to breed goats. For example, the presently disclosed method may be used to breed deer. For example, the presently disclosed method may be used to breed camels. In another embodiment, the methods of the present disclosure may be used to breed pigs. In yet another embodiment, the methods of the present disclosure may be used to breed horses. [Brief description of the drawings]

[0044] [Figure 1] Schematic diagram of normal development of preimplantation conceptus development from the zygote stage to the blastocyst stage. [Diagram 2] Schematic diagram of the sorting of blastomeres in one unzipping (sequential number N = 1) from 8-cell, 16-cell and 32-cell conceptuses. [Diagram 3] Outgrowth of conceptuses derived from 1:8 blastomere duplicates obtained by unzipping bovine conceptuses at approximately the 8-cell stage. [Figure 4] Outgrowth of conceptuses derived from 1:16 blastomere duplicates obtained by unzipping bovine conceptuses at the approximately 16-cell stage. [Diagram 5] Outgrowth of conceptuses derived from 1:32 blastomere tetrads obtained by unzipping bovine conceptuses at approximately the 32-cell stage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in animal nutrition, feed formulation, microbiology, livestock management).

[0046] As used herein, the singular forms "a," "and," and "the" include the plurals of these words unless the context clearly indicates otherwise.

[0047] The term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is intended to explicitly indicate both or either meanings.

[0048] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0049] The term "about" is used herein to mean approximately. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the numerical values ​​recited. In general, the term "about" is used herein to add a numerical value that is 10% above or below (higher or lower) the recited value.

[0050] Those skilled in the art will understand that the present disclosure is capable of variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds referred to or shown herein, individually or collectively, and any and all combinations of any two or more of the above steps or features. Thus, each feature of any particular aspect or embodiment of the present disclosure can be applied mutatis mutandis to any other aspect or embodiment of the present disclosure.

[0051] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0052] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps or group of composition of matter shall be interpreted as encompassing one and more than one (i.e., one or more) of that step, composition of matter, group of steps or group of composition of matter.

[0053] Specific Definitions The term "concept" typically refers to an embryo from fertilization (i.e., a zygote formed when two haploid gamete cells (e.g., an unfertilized oocyte and a sperm cell) combine to form a diploid totipotent cell (e.g., a fertilized egg)) until the emergence of the primitive streak, at which point the entity is called an "embryo". In some cases, however, the terms "embryo" and "concept" are used interchangeably during the period until the emergence of the primitive streak. For example, the term "embryo" can be used to refer not only to a zygote formed at fertilization, but also to an embryo resulting from subsequent cell divisions (i.e., cleavage of the embryo), such as the morula stage (i.e., the embryo is compacting or has compacted) and the blastocyst stage, which has a differentiated trophectoderm and inner cell mass.

[0054] As used herein, the term "morula" refers to a stage of embryonic development. A morula is an early embryo consisting of a ball of cells (called blastomeres) contained within a glycoprotein membrane called the zona pellucida. Morulas are generated from a single-cell fertilized egg through a series of cleavage events (this is illustrated in FIG. 1 for bovine). A key event prior to morula formation is "compaction", where the cell morphology and cell-cell adhesion of the embryo, which contains about 32-64 cells (varies by species), change and initiate the formation of this tightly packed ball of cells. A "morula" usually contains about 32-64 cells (varies by species) and resembles a mulberry, hence the name morula (from the Latin morus: mulberry). For bovine species, the process of compaction usually occurs after the 16-cell stage, and the developing embryo reaches the early morula stage, where cell-cell adhesion between the embryonic cells (or blastomeres) progresses at the 32-cell stage.

[0055] Through a process involving cell differentiation and cavity formation, the morula gives rise to a blastocyst. As used herein, the term "blastocyst" shall be understood to refer to an embryo having an inner cell mass (ICM) or germinal node containing pluripotent embryonic stem cells and an outer layer of cells that will later form the placenta, i.e., the trophectoderm, which contains trophoblast cells. The trophectoderm surrounds the inner cell mass and a fluid-filled blastocyst cavity known as the blastocoel. A blastocyst typically contains between 70 and 300 embryonic cells (this may vary depending on the species and maturity of the embryo). In some embodiments, a blastocyst may contain between about 64 and about 128 cells. In some embodiments, a blastocyst may contain between about 128 and about 256 cells. In some embodiments, a blastocyst may contain between 150 and 256 cells. In some embodiments, a blastocyst may contain between about 256 and 300 cells.

[0056] As used herein, the term "embryonic cells" is intended to encompass all totipotent or pluripotent cells within a developing embryo from the fertilized egg to the blastocyst stage. For example, embryonic cells (also called "blastomeres") obtained from within a developing embryo from the fertilized egg to the morula stage are totipotent or pluripotent embryonic cells (depending on the stage of embryonic development). Similarly, embryonic cells obtained from the inner cell mass of a blastocyst can be pluripotent.

[0057] As used herein, the term "totipotent" is used to describe a cell that can give rise to any cell type. For example, in the case of embryonic cells, a "totipotent" cell is one that can give rise to all cell types in the embryo and eventually differentiate into any one of the specialized cells required for various tissues in the body (e.g., skin, bone, bone marrow, muscle, etc.). The term "totipotent" should be distinguished from the term "pluripotent", which refers to a cell that differentiates into a specific subpopulation of cells in the developing cell mass but cannot give rise to any and all cell types.

[0058] A "pluripotent" cell can differentiate to form the primitive ectoderm, which can then, during gastrulation, differentiate into cells of all three germ layers: the ectoderm (which gives rise to the skin and nervous system), the endoderm (which forms the digestive and respiratory tract, endocrine glands, liver, and pancreas), and the mesoderm, which forms bone, cartilage, most of the circulatory system, muscle, connective tissue, etc. A pluripotent cell is also one that can self-renew, thereby making new copies of itself.

[0059] As used herein, the term "monozygotic embryos" shall be understood to mean two or more embryos formed or derived from a single fertilized egg.

[0060] As used herein, the term "split embryo" shall be understood to mean a portion of an embryo after it is separated from a donor embryo. For example, an embryo split into two parts of embryonic cells can generate two split embryos, each of which contains an embryonic cell. Similarly, an embryo split into three parts, each of which contains an embryonic cell, can produce three split embryos. Those skilled in the art will understand that embryonic cells can be separated from a donor embryo by any means (e.g., "cutting", "unzipping", etc.) to generate a split embryo.

[0061] As used herein, the term "animal" shall be understood to include all vertebrates, such as mammals (i.e., non-human mammals), amphibians, reptiles, fish, and birds. In one embodiment, the animal is a mammal. Exemplary mammals for which the methods of the present disclosure may be useful include livestock animals (e.g., cows, buffalo, pigs, sheep, goats, camels, deer, horses, etc.), companion animals (e.g., dogs, cats, horses, etc.), laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.), non-human primates (e.g., macaques and marmosets), and wild animal species (e.g., marsupials, big cats, rhinos, giant pandas, etc.). In one embodiment, the methods of the present disclosure may be useful in ruminant livestock species (e.g., cows, buffalo, sheep, goats, camels, deer, etc.). In one particular embodiment, the methods of the present disclosure may be useful in bovine species.

[0062] twinning method The present disclosure is directed generally to methods of embryo propagation, also referred to herein as "twinning," and in particular to methods by which multiple embryos can be produced from one or more initial donor embryos.

[0063] In one embodiment, the method of the present disclosure comprises: (i) obtaining a donor embryo that comprises one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula; (ii) isolating a plurality of embryonic cells from the donor embryo; and (iii) expanding the embryonic cells in vitro under conditions suitable for producing a plurality of conceptuses from the donor embryos, wherein at least a portion of the embryonic cells are expanded as one or more cell clumps, each clump comprising two or more embryonic cells; (iv) culturing the plurality of conceptuses under conditions suitable to produce a plurality of blastocysts.

[0064] In this regard, Applicant has unexpectedly discovered that the efficiency with which monozygotic conceptuses can be cultured through to the blastocyst stage is significantly increased when embryonic cells isolated from a donor embryo are expanded as cell clumps, as opposed to when embryonic cells are expanded individually.

[0065] In some embodiments, all or substantially all of the embryonic cells isolated from the donor embryo are expanded as a cell clump. However, in other embodiments, a portion of the embryonic cells isolated from the donor embryo may be expanded as a clump, while other cells may be expanded individually. In this regard, one skilled in the art may modify the process as desired.

[0066] Several techniques for producing multiple embryos from a single donor embryo ("twinning techniques") are known in the art, and each of these techniques is contemplated herein. Exemplary techniques include the "unzip method" and the "cut method," both of which are described in further detail below.

[0067] In a preferred embodiment, the "twinning technique" used in the disclosed method is referred to as the "unzip method." According to this embodiment, a plurality of embryonic cells are isolated from a donor embryo by disrupting or "unzipping" the zona pellucida (ZP) and isolating the embryonic cells from within the donor embryo. According to this embodiment, the zona pellucida of the donor embryo is disrupted to release the embryonic cells contained therein, and then the embryonic cells are isolated (individually or as clumps or clusters of cells) and expanded in vitro according to steps (i)-(iv) above to generate a plurality of embryos. In this manner, each isolated embryonic cell, or each clump of embryonic cells (i.e., two or more cells isolated together), is expanded to become an embryo (e.g., a ZP-removed embryo). As described herein, at least a portion of the embryonic cells isolated from each donor embryo are expanded as one or more cell clumps, each clump comprising two or more embryonic cells. This helps to improve the efficiency of embryo expansion.

[0068] The destruction of the zona pellucida in the unzip method, also known as "assisted hatching", may be carried out using any suitable method known in the art. In this regard, it is known that various techniques are used to assist embryo hatching in the field of assisted reproduction, including partial mechanical zona dissection, zona pellucida opening and zona pellucida thinning using acidic Tyrode's solution, proteolytic enzymes, piezo vibrator manipulator and laser, as described in Hammadeh et al., (2011) J. Assist. Reprod. Genet., 28(2): 119-128. It is also contemplated that the zona pellucida can be destroyed by nanodissection (e.g., using femtosecond laser pulses or atomic force microscope (AFM) with nanoscalpel).

[0069] Any one or more of the techniques described above may be used in the disclosed unzip method for disrupting the zona pellucida.

[0070] After disrupting the zona pellucida, the embryonic cells (e.g., blastomeres) may be isolated and, if appropriate, transferred to fresh medium for expansion. Several methods for isolating individual cells, including embryonic cells, are known in the art and are contemplated herein (e.g., as described in Zhu and Murthy (2013) Curr. Opin. Chem. Eng., 2(1):3-7;). Techniques for isolating cells include, but are not limited to, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), dielectrophoretic digital sorting, immunomagnetic cell separation, immunosurgery, fluid trapping, laser capture microdissection, mechanical dissection, manual picking, microfluidics, micromanipulation, nanodissection, serial dilution, Raman tweezers, and combinations thereof. Any one of these techniques, or combinations thereof, are contemplated for use in the disclosed method for isolating single embryonic cells or clumps of embryonic cells from the disrupted zona pellucida. In one particular embodiment, microfluidics is used to isolate individual embryonic cells.

[0071] In another embodiment, the "twinning technique" used in the method of the present disclosure is called the "cleavage technique." According to this embodiment, one or more early donor embryos of step (ii) are cut (or split) into two or more parts (e.g., three, four, or five or more parts), each of which contains one or more embryonic cells, and at least one of which contains two or more embryonic cells. In some embodiments, all of these parts contain two or more embryonic cells of the donor embryo. The split embryos are then expanded in vitro to generate multiple embryos (e.g., monozygotic embryos), as described in steps (i)-(iv) above.

[0072] According to embodiments in which a "cutting method" is used, the process of cutting (or splitting) the embryo may be carried out using any means known in the art for splitting an embryo. For example, the donor embryo may be split or cut by mechanical dissection using a microsurgical instrument that relies on pressure, such as a blade (e.g., scalpel blade or part thereof) or a fine glass needle. Alternatively or additionally, the donor embryo may be split or cut using a laser, i.e., laser-assisted biopsy. In other examples, the donor embryo may be split or cut using a nanodissection-based tool (e.g., using a femtosecond laser pulse or an atomic force microscope (AFM) with a nanoscalpel). However, it is contemplated that any means known in the art may be used.

[0073] In some embodiments, steps (i)-(iv) of the embryo expansion process can be repeated continuously using newly generated embryos as donor embryos. The process can be repeated "N" times (each referred to as a cycle), with the embryos generated in the previous cycle being used as the donor embryos for the next cycle. The number of cycles performed using the "unzip" method depends on various factors, including the number of embryos to be generated, the number of starting donor embryos, whether any embryos are recovered by the method during intermediate cycles, and the number of embryonic cells in the donor embryos, the last of which determines the upper limit on the number of embryos that can be generated from any one donor embryo.

[0074] Donor embryos for expansion using the methods of the present disclosure include embryos that include one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula. In one embodiment, the donor embryo may include nine or more embryonic cells, at least one of which is developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula. For example, the donor embryo may include 9-64 embryonic cells (e.g., 9-60 embryonic cells), provided that the embryo has not yet developed into a blastocyst. For example, the donor embryo may include 16-64 embryonic cells (e.g., 16-60 embryonic cells, etc.), provided that the embryo has not yet developed into a blastocyst. In some embodiments, it may be advantageous to select a donor embryo for expansion that has more embryonic cells that are totipotent, such as a pre-compaction morula that includes about 32-64 embryonic cells (e.g., 32-60 embryonic cells, etc.), provided that the donor embryo has not yet developed into a blastocyst. For example, the donor embryo may contain one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 32-cell embryo. In other examples, the donor embryo may be a pre-morula embryo that contains between 16 and 32 embryonic cells. In any case, one of skill in the art will understand that the number of embryonic cells within a developing embryo at each stage may vary between species.

[0075] As described herein, applicants have unexpectedly discovered that the efficiency with which monozygotic conceptuses can be cultured through the blastocyst stage after "twinning" is significantly increased when embryonic cells isolated from a donor embryo are expanded as cell clumps (or cell populations), as opposed to the embryonic cells being expanded individually. It is contemplated that prior to expansion, the cell clumps may contain any number of embryonic cells from the donor embryo, e.g., 2, or 3, or 4, or 5, or 6, or 7, or 8 or more cells per cell clump. In some embodiments, the method includes expanding clumps of about 2-4 cells. In other embodiments, the method includes expanding clumps of about 4-6 cells. In other embodiments, the method includes expanding clumps of about 4 cells. However, one of skill in the art will appreciate that the number of cells in each clump may vary depending on the desired expansion outcome and efficiency.

[0076] In one example, the or each donor embryo comprises nine or more embryonic cells, at least one of which is developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula, and at least a portion of the embryonic cells isolated from the donor embryo are expanded in cell clumps comprising two or more embryonic cells (e.g., about 2-4 cells per clump).

[0077] In another example, the or each donor embryo comprises 16-64 embryonic cells (e.g., about 16-60 embryonic cells), at least one of which is developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula, and at least a portion of the embryonic cells isolated from the donor embryo are expanded in cell clumps comprising two or more embryonic cells (e.g., about 2-8 cells per clump, etc.). For example, the method may include isolating a plurality of embryonic cells from a donor embryo comprising about 16 cells and expanding the embryonic cells in vitro under conditions suitable for producing a plurality of conceptuses from the donor embryo, and at least a portion (or all) of the embryonic cells are expanded as part of one or more cell clumps comprising about two embryonic cells per clump prior to expansion. For example, the method may include isolating a plurality of embryonic cells from a donor embryo comprising about 32 cells and expanding the embryonic cells in vitro under conditions suitable for producing a plurality of conceptuses from the donor embryo, where at least a portion (or all) of the embryonic cells are expanded as part of one or more cell clumps comprising about four embryonic cells per clump prior to expansion.

[0078] One of skill in the art will appreciate that the number of conceptuses cultured for each donor embryo in step (iv) to produce multiple blastocysts will depend on the developmental stage (i.e., number of cells) of the donor embryo and the number of cell clumps isolated from the donor. However, it is contemplated that at least about four (e.g., five, six, seven, eight, nine, or ten or more) conceptuses are cultured for each donor embryo in step (iv) to produce multiple blastocysts.

[0079] In some embodiments, a portion of the plurality of embryonic cells separated from the donor embryo is separated with a pre-existing clump (or population). Alternatively or additionally, a portion of the plurality of embryonic cells separated from the donor embryo is aggregated after being separated from the donor embryo. In many cases, the embryonic cells that are aggregated together after being separated from the donor embryo are derived from the same donor embryo. However, in other situations, it may be desirable to form a cell clump using embryonic cells from multiple donor embryos (e.g., multiple donor embryos from the same animal, or donor embryos obtained from different animals of the same species). In each of the above embodiments that describe the formation of a cell clump after separating cells from a donor embryo, the embryonic cells aggregated together may be at the same or similar developmental stage (e.g., cells that are developmentally equivalent to embryonic cells from a 16-cell embryo may be aggregated together, or cells that are developmentally equivalent to embryonic cells from a 32-cell embryo may be aggregated together). However, in other embodiments, the embryonic cells aggregated together may be at different developmental stages (e.g., a cell that is developmentally equivalent to an embryonic cell from an 8-cell embryo may be aggregated with a cell that is developmentally equivalent to an embryonic cell from a 16-cell embryo).

[0080] In some embodiments, the disclosed embryo propagation methods may be used in conjunction with a sequential twinning approach previously developed by the applicant. For example, prior to culturing the plurality of conceptuses in (iv) to generate a plurality of blastocysts, the method may include: (v) isolating one or more of the plurality of conceptuses produced for use as donor embryos in subsequent propagation, wherein each donor embryo isolated for subsequent propagation comprises at least two embryonic cells; (vi) isolating one or more of the embryonic cells from the one or more donor embryos; (vii) expanding the embryonic cells in vitro under conditions suitable to generate a plurality of conceptuses, each of which comprises at least two embryonic cells; (iv) optionally, repeating steps (v) through (vii) "N" times.

[0081] After a desired number of serial expansions, the multiple conceptuses may then be cultured under conditions suitable to generate multiple blastocysts, according to step (iv) of the method of the present disclosure.

[0082] As described herein, steps (v)-(vii) of the method may be repeated "N" times to generate a desired number of embryos from the original donor embryo. Depending on (1) the number of embryonic cells in the initial donor embryo, (2) the technique used to separate the embryonic cells from the initial donor embryo, and (3) unless otherwise specified, the number of repetitions of steps (v)-(vii) performed, i.e., "N", may vary. In this regard, unless otherwise specified, "N" may be ≧1, e.g., 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 or more.

[0083] In each embodiment of the methods described herein, it may be desirable to fix the donor embryo in order to cut or divide it, or to allow for the destruction of the zona pellucida, i.e., "unzipping" the zona pellucida. Methods of fixing the embryo are known in the art, and any one or more of these methods or techniques may be used in the methods of the present disclosure. Exemplary methods contemplated for use in the methods of the present disclosure include aspirating the zona pellucida, creating a depression or dead end in the container, constructing a device to capture the embryo, or attaching the embryo to a surface, for example, by roughening the surface of the container containing the embryo, by using a protein-free medium, or by coating the culture container with a material that adheres to the outer membrane of the embryo.

[0084] As described herein, embryonic cells isolated from a donor embryo using the methods of the present disclosure, or cleaved embryos containing embryonic cells, are cultured and expanded in vitro to generate multiple embryos (e.g., monozygotic embryos).

[0085] Methods for culturing embryos in vitro at various developmental stages are known in the art and contemplated herein. Exemplary methods are described in Examples 1-5 herein. One of skill in the art will appreciate that culture conditions are important for growing developing embryos to the blastocyst stage of development and may be varied / adjusted according to the stage of embryo development as well as to control the rate of embryo development (e.g., cleavage) to provide a sufficient time frame to perform the propagation steps of the disclosed methods. For example, in embryo culture, variables such as temperature and CO2 levels can be controlled to optimize the growth of developing embryos. For example, the optimal temperature for embryo development is about 32°C to about 40°C, preferably about 35°C to 39°C, with a temperature of about 37°C to about 39°C (e.g., 38.5°C) being particularly preferred. The optimal CO2 level in the culture environment for embryo development is about 1% CO2 to about 10% CO2, preferably about 3% CO2 to about 8% CO2, and even more preferably about 5% CO2.

[0086] Suitable media for culturing and expanding embryonic cells and embryos are known in the art. For example, media that mature embryos into blastocysts at rates comparable to those occurring in vivo are described in Summers and Biggers (2003) Human Reprod Update, 9:557-582. Many of these media are loosely based on the concentrations of ions, amino acids, and sugars found in the female reproductive tract during egg release, fertilization, and development (Gardner and Lane (1998) Hum Reprod 13:148-160). Media containing phosphate buffers or HEPES organic buffers are typically used for procedures involving handling gametes outside of an incubator, washing follicles, and micromanipulation. Most media utilize a bicarbonate / CO2 buffer system to keep the pH in the appropriate range, e.g., pH 7.2-7.4. The osmolality of the media is typically in the range of 275-290 mosmol / kg. Embryos may also be cultured under paraffin oil (or an alternative oil that is not toxic to the embryos) to prevent evaporation of the medium, which maintains a constant osmolarity. This oil also minimizes fluctuations in pH and temperature when the embryos are removed from the incubator for microscopic evaluation.

[0087] Suitable media also contain a protein source such as albumin or synthetic serum, which is usually added at a concentration of about 5-20% (w / v or v / v, respectively). Salts such as NaCl, KCl, KH2PO4, CaCl22H2O, MgSO47H2O, or NaHCO3 may also be added to the media. Media also usually contain a carbohydrate source (e.g., glucose) and monocarboxylates (e.g., pyruvate and lactate) due to the presence of carbohydrates and monocarboxylates in the female reproductive tract. Collectively, these are the main energy source for the developing embryo. Media that support the development of fertilized eggs up to 8 cells contain pyruvate and lactate. Some commercially available media are glucose-free, while others contain very low concentrations of glucose to meet the demands of sperm during normal fertilization. Media that support the development of 8-cell embryos up to the blastocyst stage contain low concentrations of pyruvate and lactate, as well as high concentrations of glucose. Supplementing the media with amino acids may also be desirable for embryo development. Media supporting the development of fertilized eggs up to 8 cells are typically supplemented with non-essential amino acids such as proline, serine, alanine, asparagine, aspartic acid, glycine, and glutamic acid. Media supporting the development of 8-cell embryos up to the blastocyst stage are also typically supplemented with essential amino acids such as cysteine, histidine, isoleucine, leucine, lysine, methionine, valine, argentine, glutamine, phenylalanine, threonine, and tryptophan. Media may also contain vitamins.

[0088] The medium may also contain antibiotics. Most ART laboratories use media containing antibiotics to minimize the risk of microbial growth. The most commonly used antibiotics are penicillin (a beta-lactam drug against gram-positive bacteria, inhibiting the integrity of the cell wall) and streptomycin (an aminoglycoside drug against gram-negative bacteria, inhibiting protein synthesis).

[0089] Three examples of sequential media for embryo development that may be useful for culturing embryos in the methods of the present disclosure are G1 / G2 (Gardner et al, (1998) Hum. Reprod. 13:3434), Universal IVF Medium / MS (Bertheussen et al., (1997), and PI / Blastocyst Medium (Behr et al., (1998) Am. Soc. Rep. Med. 0-262). Media for culturing embryos at different developmental stages are commercially available from a variety of sources.

[0090] Other exemplary media for embryo development are described herein in Examples 1-5 and are contemplated for use in the methods of the present disclosure.

[0091] In some embodiments, the embryonic cells and / or developing embryos are cultured in the presence of one or more factors capable of maintaining the totipotency of the embryonic cells and / or inhibiting or preventing embryonic development. Such factors may be added to the culture medium to prevent or delay embryonic development, thereby providing an additional opportunity to perform additional cycles of steps (i)-(iv) before cell differentiation begins to occur. Factors that maintain the totipotency of embryonic cells and / or inhibit or prevent embryonic development are known in the art and are contemplated for use herein. For example, factors that maintain the totipotency of embryonic cells and / or inhibit or prevent embryonic development include anti-miRs and / or ribozymes that inhibit the stability or activity of miRNAs produced by early embryos. Exemplary anti-miRs may target embryo-expressed miRNAs that promote the removal of maternal mRNAs (e.g., anti-miRs targeting the miR-30 family).

[0092] Those skilled in the art will understand that culture conditions can also contribute to maintaining the totipotent state of embryonic cells. Thus, in the culture of embryonic cells, embryos or split embryos, variables such as cell or embryo density, temperature, and CO2 and O2 levels can be controlled to reduce / control the developmental rate of cultured embryos.

[0093] As described herein, the methods of the present disclosure also include culturing and expanding the embryo in vitro to form a blastocyst, which can then be harvested (e.g., for storage and / or transfer into a recipient female). Thus, at some stages of the method, the embryonic cells and / or developing embryo may be cultured in the presence of one or more factors capable of promoting embryonic development. For example, factors capable of promoting embryonic development (i.e., embryonic development factors) may be added to the medium used to culture the embryo through the blastocyst stage for harvesting. Factors that promote embryonic development are known in the art and are contemplated herein.

[0094] One of skill in the art will also appreciate that culture conditions, such as embryo density, temperature and CO2 levels, may be altered and / or optimized to promote embryo development.

[0095] As described herein, the animal species from which the donor embryo is obtained may be any vertebrate, including mammalian species, amphibian species, reptile species, fish species, and avian species (e.g., poultry).

[0096] In one example, the animal is a mammal (e.g., a non-human mammal). Exemplary non-human mammals for which the methods of the present disclosure may be useful include livestock species (e.g., cows, buffalo, pigs, sheep, goats, camels, deer, horses, etc.), companion animals (e.g., dogs, cats, horses, etc.), laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.), non-human primates (e.g., macaques and marmosets), and wild animal species (e.g., marsupials, cats, rhinos, giant pandas, etc.).

[0097] In one particular example, the methods of the present disclosure can be used to generate multiple embryos (e.g., monozygotic embryos) in a ruminant livestock species. For example, the livestock species can be a bovine species. For example, the livestock species can be a sheep species. For example, the livestock species can be a caprine species. For example, the livestock species can be a deer species. For example, the livestock species can be a camel species.

[0098] In another example, the methods of the present disclosure can be used to generate multiple embryos (e.g., monozygotic embryos) from pigs. In another example, the methods of the present disclosure can be used to generate multiple embryos (e.g., monozygotic embryos) from goats. In another example, the methods of the present disclosure can be used to generate multiple embryos (e.g., monozygotic embryos) from horses.

[0099] The donor embryos used in the first cycle of the methods of the present disclosure may be prepared in vivo (e.g., by conventionally rinsing an embryo from a pregnant animal) or may be prepared by in vitro fertilization (IVF) techniques.

[0100] In one embodiment, the donor embryos used in the first cycle of the method are prepared by in vivo methods. For example, oocytes may be fertilized in vivo (e.g., after mating or by artificial insemination) and the embryos subsequently collected from pregnant females by conventional embryo washing. In one embodiment, the donor embryos are prepared by superovulated embryo transfer (MOET). In this method, donor females are administered hormones, primarily follicle stimulating hormone (FSH), prior to fertilization to stimulate the ovaries of cycling female animals to induce superovulation.

[0101] In another embodiment, the donor embryo used in the first cycle of the method is prepared by an in vitro method (i.e., IVF). Methods for preparing embryos using IVF are well known in the art. IVF generally involves producing oocytes from a donor animal by follicular aspiration, followed by in vitro maturation, fertilization, and culturing the resulting embryo until it reaches a desired developmental stage. Advantageously, this approach allows repeated production of embryos from high-value live animals under controlled conditions. Methods for producing embryos by IVF are described in Berlinguer F. "Embryo Production", Animals Production in Livestock, Encyclopedia of Life Support Systems (EOLSS), the contents of which are incorporated herein in their entirety.

[0102] It is also contemplated that the donor embryo used in the first cycle of the method, whether produced in vivo or in vitro, may be freshly collected, stored, or thawed (i.e., a thawed cryopreserved embryo). In one example, the donor embryo is freshly collected. In one example, the donor embryo is stored in embryo storage medium (e.g., at about 4° C.). In another example, the donor embryo is a thawed cryopreserved embryo.

[0103] The donor embryo useful in the disclosed method may also be genetically modified. For example, the embryonic cells in the donor embryo may be genetically modified prior to carrying out the method, such that all embryos produced from the donor embryo have the genetic modification. In one embodiment, the donor embryo is genetically modified by introducing an exogenous nucleic acid into the genome of the embryonic cells contained therein. The exogenous nucleic acid may be an alternative allele of a gene or locus associated with a trait of interest. Alternatively, the exogenous nucleic acid may be a transgene. In another embodiment, the donor embryo may be genetically modified by editing the genome of the embryonic cells contained therein (i.e., genome editing). The genome editing may be selected from the group consisting of an insertion, deletion, substitution, inversion, or translocation. For example, the genome editing may be an insertion, deletion, and / or substitution of a nucleic acid sequence or one or more nucleotide positions therein to replace an existing allele of a gene or locus associated with a trait of interest with an alternative allele.

[0104] Genome editing may also be used to introduce one or more genetic modifications (e.g., nucleotide substitutions) that, when considered alone or in combination, provide a unique genetic profile or fingerprint to the developing embryo. This unique genetic profile or fingerprint can then be used to identify and / or track the embryo (and animal produced from the embryo) produced from the donor embryo. For example, one or more conservative nucleotide substitutions may be made to the embryonic cells in the donor embryo within safe harbor regions of the genome to generate a unique genetic profile or fingerprint.

[0105] Preferably, the genetic modification or editing is performed at the single cell stage, such that all subsequent cells in the developing embryo derived from the modified cell (and the animal resulting therefrom) contain the modification. However, if the genetic modification event is performed after one or more cell divisions, and not all embryonic cells in the donor embryo are modified, the donor embryo may be mosaic for the modification / editing event, having some cells derived from the modified / edited cell and some cells derived from the non-modified / edited cell.

[0106] Several methods have been described in the art for genetically modifying the genome of cells using targeted nucleases.These include, but are not limited to, (1) clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) or other Cas systems, (2) transcription activator-like effector nucleases (TALENs), (3) zinc finger nucleases (ZFNs), and (4) homing endonucleases or meganucleases.It is contemplated that other methods of genetically modifying cells can be used in the methods disclosed herein to genetically modify donor embryos.

[0107] The methods of the present disclosure may further include one or more steps to aid in the selection of a donor embryo to be propagated using the methods. For example, the methods may include selecting a donor embryo prior to step (i) based on one or more genetic screening criteria, genetic diagnosis, and / or one or more morphological criteria.

[0108] In one example, the genetic screening criteria may be determined by screening for the presence or absence of one or more genetic markers (e.g., SNP alleles or haplotypes) associated with (preferred variants of) a phenotypic trait of interest, such as a commercially important production trait as in the case of livestock species. Exemplary phenotypic traits of interest include, but are not limited to, production traits (e.g., growth rate, fecundity, feed conversion efficiency, etc.), drug resistance, susceptibility to pests and / or parasites, and sex (i.e., male or female). In this manner, donor embryos for propagation using the methods of the present disclosure can be obtained from the elite animals.

[0109] Alternatively or additionally, donor embryos may be selected based on genetic diagnosis for one or more conditions, diseases or predispositions thereto. In this regard, preimplantation genetic diagnosis (PGD) or preimplantation genetic testing (PGT) of embryos has become more common in the field of IVF. PGD testing focuses mainly on two methods, namely, fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). However, many techniques for PGD / PGT are known in the art, and one or more of these techniques may be used in the method of the present disclosure to select donor embryos. These include, but are not limited to, polymerase PCR, FISH, single-strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP), primed in situ labeling (PRINS), comparative genomic hybridization (CGH), COMET analysis (single cell gel electrophoresis), heteroduplex analysis, Southern analysis, and denaturing gradient gel electrophoresis (DGGE) analysis.

[0110] Alternatively, or in addition, the donor embryo may be selected based on one or more morphological characteristics, such as morphological characteristics indicative of the health of the embryo.

[0111] After the desired number of embryos (e.g., monozygotic embryos) are produced using the methods of the present disclosure as described herein, the embryos may be matured in vitro to a desired stage of embryonic development (e.g., pre-implantation blastocysts) and retrieved from the medium. The retrieved embryos may then be stored in an appropriate embryo holding or transfer medium until they are transferred to a recipient female and / or until it is time to cryopreserve the embryos. Any commercially available embryo holding and transfer medium is contemplated for use herein. In accordance with embodiments in which embryos are stored for a short period of time prior to transfer into a recipient female (e.g., during transport), the retrieved embryos may be stored at about 2°C to about 8°C, depending on the specifications of the particular holding or transfer medium. In some preferred embodiments, the retrieved embryos are stored at about 4°C.

[0112] The retrieved embryos may also be cryopreserved for storage. The primary techniques used in the art for cryopreservation of embryos are vitrification and programmable slow freezing (SPF), both of which are contemplated herein. According to this example, the retrieved embryos may be transferred to an appropriate cryopreservation medium (e.g., including ethylene glycol freezing medium or similar medium), cryopreserved, and maintained at about -180°C to about -196°C until thawed for use and / or shipped. For example, the cryopreserved embryos may be stored in liquid nitrogen at about -196°C.

[0113] In addition to the application of the method of the present disclosure in commercial livestock breeding, it is also contemplated that the method of the present disclosure can be applied in the field of animal protection and management.For example, embryos generated from donor embryos obtained from endangered or threatened species (including wildlife and livestock species) using the method of the present disclosure can be deposited in a biobank and / or made available to the public for breeding programs.This may be useful for managing breeding programs and populations of endangered or threatened species.Thus, in some embodiments, the method can further include depositing one or more cryopreserved embryos generated by the method of the present disclosure in a biobank.

[0114] According to embodiments in which the retrieved embryos are transferred fresh into a recipient female, the method of the present disclosure may further include transferring one or more of the embryos into the oviduct or uterus of one or more recipient females. Whether the embryo is transferred into the uterus or oviduct depends on the developmental stage of the embryo. Methods for embryo transfer are known in the art. For example, the embryo may be transferred manually using a catheter or other means.

[0115] Animal breeding The present disclosure also provides a method of breeding an animal, comprising: (i) transferring one or more of the plurality of embryos produced by the methods described herein into the oviduct or uterus of one or more recipient females to establish a pregnancy; (ii) producing an animal by parturition from the pregnant recipient female.

[0116] As described herein, the animal may be any vertebrate, including mammalian species, amphibian species, reptile species, fish species, and avian species (e.g., poultry). In one particular embodiment, the animal is a mammal (e.g., a non-human mammal). Exemplary non-human mammals for which the methods of the present disclosure may be useful include livestock species (e.g., cows, buffalo, pigs, sheep, goats, camels, deer, horses, etc.), companion animals (e.g., dogs, cats, etc.), laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.), non-human primates (e.g., macaques and marmosets), and wild animal species (e.g., marsupials, cats, rhinos, giant pandas, etc.). In one particular embodiment, the methods of the present disclosure may be used to breed cattle. In another embodiment, the methods of the present disclosure may be used to breed sheep. In another embodiment, the methods of the present disclosure may be used to breed pigs. In another embodiment, the methods of the present disclosure may be used to breed goats. In another embodiment, the methods of the present disclosure may be used to breed horses. In another embodiment, the methods of the present disclosure may be used to breed camels (e.g., alpacas).

[0117] (Example) Example 1: Growth of bovine conceptuses 1.1 General methods and materials 1.1.1 Preparation of conceptus medium 1.1.1.1 Chemicals and stock solutions Stock solutions for Unzip medium were prepared according to Table 1. Media reagents used in this study were obtained from Sigma unless otherwise stated. Stock solutions were prepared using Milli-Q® water. The osmolarity of NaCl, KCl and NaHCO3 was adjusted to 2000 mOsm, 200 mOsm and 2000 mOsm, respectively, with Milli-Q® water using a vapor pressure osmometer (Wescor). [Table 1]

[0118] 1.1.1.2 Preparation of medium for unzipping For the conceptus unzipping procedure, NbryoIVC-2 Ca 2+ A free medium was used. The medium was prepared by adding the stock solutions (from Table 1) in the order shown in Table 2. The pH of the medium was adjusted to 7.4 by adding 2 M NaOH. The osmolality of the medium was checked using an osmometer (Wescor). The osmolality was adjusted to 270 mOsm by adding Milli-Q® water. Finally, fatty acid-free bovine serum albumin (FAF-BSA) was added to the medium at a concentration of 4 mg / mL and the medium was filter sterilized through a 0.22 μm syringe filter (Millipore). The medium was stored at 4° C. for up to 2 weeks. [Table 2]

[0119] 1.1.1.3 Pronase preparation Pronase is a proteolytic enzyme used to remove the zona pellucida (ZP) in the unzip procedure. Pronase was prepared in HEPES-buffered SOF (synthetic oviductal fluid) medium at a final concentration of 0.3 mg / mL. It was then filter-sterilized through a 0.22 μm syringe filter, aliquoted, and stored at -20°C.

[0120] 1.1.2. In vitro development of bovine conceptuses 1.1.2.1 Production of bovine fertilized eggs Bovine fertilized eggs were produced by IVM and IVF using commercial protocols from Art Lab Solutions. Bovine oocytes were matured in vitro (IVM) for 24 hours from ovaries collected from Nindooinbah cattle farm according to standard procedures. Matured oocytes were then fertilized in vitro for 24 hours using thawed semen from one or more fertile bulls from Nindooinbah cattle farm. After IVF, putative fertilized eggs were transferred to VitroCleave (Art Lab Solutions) in vitro culture (IVC) medium. Fertilized eggs were cultured in VitroCleave (Art Lab Solutions) IVC medium at 38.5°C under 5% O2 and 5% CO2 unless otherwise stated. Fertilized eggs were cultured to approximately 8-cell, 16-cell, or 32-cell stages 96-100 hours after IVF.

[0121] 1.1.3. Preparation of plates and dishes for unzipping 1.1.3.1 0.1% PVA pre-coated plates To avoid ZP-removed conceptuses from adhering to the plate, wells of a 96-well round-bottom plate (Corning) were coated with PVA by adding 50 μL sterile 0.1% PVA (w / v) to each well and incubated overnight at 38.5° C. Each well was then washed three times with sterile water to remove unbound PVA. Plates were then dried, sealed, and stored at 4° C. until use.

[0122] 1.1.3.2 Unzipping dish Prior to the unzip procedure, 55 mm Petri dishes (Corning) were filled with Pronase, NbryoIVC-2 Ca 2+ Separate 20 μL drops of free medium and VitroBlast (Art Lab Solutions) medium were placed and overlaid with mineral oil (Coopers Scientific) and equilibrated at 38.5°C under 5% O2, 5% CO2 for at least 60 min.

[0123] 1.1.3.3 Culture system after unzipping To culture the unzipped blastomeres, 96-well plates precoated with 0.1% PVA were utilized. Each well contained 20 μL of VitroBlast medium (Art Lab Solutions) overlaid with mineral oil to avoid medium evaporation. Before transferring the blastomeres to each well, the plates containing the medium were equilibrated at 38.5°C under 5% O2, 5% CO2 for at least 60 min.

[0124] 1.1.4. Unzipping Procedure All bovine conceptuses underwent a single unzip procedure (N=1 consecutive times). This was performed under a dissecting microscope with a plate heated to 37°C. Conceptuses at approximately 8-, 16- or 32-cell stages were treated with pronase to remove the surrounding ZP for 2 min at 38.5°C in a humidified incubator with 5% O2 and 5% CO2 atmosphere. After the ZP was dissolved, the conceptuses were washed with three drops of 20 μL VitroBlast medium (Art Lab Solution) to wash off residual pronase and incubated at 38.5°C under 5% O2 and 5% CO2 for 10 min. The ZP-removed conceptuses were then incubated in NbryoIVC-2 Ca at 38.5°C under 5% O2 and 5% CO2 for 3 min. 2+ The NbryoIVC-2 cells were then transferred to Ca-free medium to reduce cell-cell contact. 2+ Blastomeres from each conceptus were isolated by aspiration using a micropipette (diameter approx. 120 μm) in free medium. Blastomeres were transferred individually, in duplicates or in quadruplets to PVA pre-coated wells containing VitroBlast medium (Art Lab Solutions) and cultured at 38.5°C under 5% O2 and 5% CO2. Additionally, individual blastomeres were placed together to form duplicate or quadruplet clumps within the wells. In some cases, clumps of blastomeres from different donor conceptuses were formed. Blastomeres were observed every 12-24 hours until the stage equivalent to the blastocyst stage and scored for their developmental status (cleavage, compaction, cavity formation and blastocyst equivalent). Conceptuses were classified as blastocyst equivalents if the blastocyst cavity exceeded half the volume of the conceptus and a clump of ICM cells was present.

[0125] 1.1.5.Naming Individual blastomeres isolated from about 8-cell, 16-cell, and 32-cell conceptuses are designated 1:8, 1:16, and 1:32, respectively. The number before the colon indicates the number of blastomeres, and the number after the colon indicates the stage of the conceptus to which the blastomeres correspond when unzipped. Thus, duplicate and tetrad blastomeres are designated 2 and 4, respectively, before the colon (e.g., 2:8, 2:16, 2:32, and 4:32). In addition, individual blastomeres from about 8-cell, 16-cell, and 32-cell conceptuses that are arranged together to form duplicates are designated 2×1:8, 2×1:16, and 2×1:32, respectively. Blastomeres from about 32-cell conceptuses that are arranged to form tetrads by recombination of four individual blastomeres or two sets of duplicates are designated 4×1:32 or 2×2:32. FIG. 2 illustrates this naming system for unzipping conceptuses with N=1 consecutive unzips. As shown in FIG. 2, in the unzipping procedure, the intact conceptuses after removal of the zona pellucida (ZP) are separated into individual, duplicated or quadruplet blastomeres within the conceptuses. The cleavage of cells in the conceptuses is not simultaneous, and there is a non-uniform developmental stage of the blastomeres in these conceptuses, which may include 1:8, 1:16 and 1:32 blastomeres. The unzipped duplicated and quadruplet blastomeres are indicated as 2 and 4, respectively, before the colon (e.g., 2:8, 2:16, 2:32 and 4:32). The formation of clumps can also be achieved by recombining individual blastomeres (e.g., 2×1:8, 2×1:16, 2×1:32 and 4×1:32) or by combining duplicates (2×2:32).

[0126] 1.2 Results A single unzip procedure was performed on bovine conceptuses at approximately 8-cell, 16-cell and 32-cell stages. Since the cleavage of cells in the conceptuses is not simultaneous, there are heterogeneous developmental stages of the blastomeres in these conceptuses, including 1:8, 1:16 and 1:32 blastomeres. Therefore, each type of blastomere was analyzed separately.

[0127] 1.2.1 Formation of blastocyst equivalents from single and duplicate 1:8 blastomeres From six replicates, 186 1:8 blastomeres were obtained by unzipping, of which 37 (19.2%) formed blastocyst equivalents (Table 3). Additionally, unzipped conceptuses were separated into duplicate blastomeres (2:8). From six replicates, 32 pairs of 2:8 blastomeres were obtained, of which 9 (28.1.5%) formed blastocyst equivalents. Furthermore, individual 1:8 blastomeres were cultured together to form duplicate clumps (2 × 1:8). From five replicates, 81 recombined duplicates were obtained, of which 21 (25.9%) formed blastocyst equivalents. Individual 1:8 blastomeres from different conceptuses were also recombined to form duplicate clumps. From three replicates, 6 pairs of 2 × 1:8 blastomeres were obtained, of which 1 (16.7%) formed blastocyst equivalents.

[0128] As reported in standard commercial IVF procedures, intact conceptuses can usually be cultured to the blastocyst stage with an efficiency of about 30%. The left side of Figure 3 shows the formation of blastocyst equivalents from unzipped dyads (2:8 and 2x1:8). 113 dyads (32 2:8 + 81 2x1:8) were obtained from donor conceptuses, of which 30 (27%) formed blastocyst equivalents. The right side of the figure shows the expected commercial outcome for intact conceptuses versus 113 dyads from unzipped conceptuses. The 113 dyads from unzipped conceptuses are equivalent to 28 intact 8-cell conceptuses. From the 28 intact conceptuses, 8.5 intact blastocysts are expected to form (30%, as reported in commercial procedures). As a result, the unzip procedure allows for a 3.5-fold increase in blastocyst development rates compared to standard commercial procedures. [Table 3]

[0129] 1.2.2 Formation of blastocyst equivalents from single and duplicate 1:16 blastomeres From six replicates, 352 1:16 blastomeres were obtained by unzipping, of which 39 (11.1%) formed blastocyst equivalents (Table 4). Additionally, unzipped conceptuses were separated into duplicate blastomeres (2:16). From seven replicates, 538 pairs of 2:16 blastomeres were obtained, of which 277 (51.5%) formed blastocyst equivalents. Furthermore, individual 1:16 blastomeres were cultured together to form duplicates (2 × 1:16). From five replicates, 553 recombined duplicates were obtained, of which 238 (43.0%) formed blastocyst equivalents. Individual 1:16 blastomeres from different conceptuses were also recombined to form duplicate clumps. From three replicates, 15 pairs of 2 × 1:16 blastomeres were obtained, of which 4 (26.7%) formed blastocyst equivalents. As can be seen from the data in Table 4, expansion of duplicate (2 × 1:16) blastomeres from 16-cell conceptuses yielded significantly more blastocyst equivalents compared with individually expanded 16-cell blastomeres.

[0130] The left side of FIG. 4 shows the formation of blastocyst equivalents from unzipped duplicated blastomeres (2:16 and 2×1:16). 1091 duplicated blastomeres (538 2:16+553 2×1:16) were obtained, of which 515 (47%) formed blastocyst equivalents. The right side of the figure shows the expected commercial outcome for 1091 duplicated unzipped conceptuses versus intact conceptuses. 1091 unzipped duplicated blastomeres are equivalent to 136 intact 16-cell conceptuses. From the 136 intact conceptuses, 40.9 intact blastocysts are expected to form (30%, as reported in the commercial procedure). As a result, the unzipped procedure allows a 12.6-fold increase in blastocyst development rate compared to the standard commercial procedure. [Table 4]

[0131] 1.2.3 Formation of blastocyst equivalents from single, duplicate and quadruplet 1:32 blastomeres From three replicates, 27 1:32 blastomeres were obtained by unzipping, none of which formed blastocyst equivalents (Table 5). Unzipped conceptuses were also separated into duplicate blastomeres (2:32). Seven replicates yielded 71 pairs of 2:32 blastomeres, of which 28 (39.4%) formed blastocyst equivalents. Furthermore, individual 1:32 blastomeres were cultured together to form duplicates (2 × 1:32). Five replicates yielded 46 recombined duplicates, of which 6 (13.0%) formed blastocyst equivalents. Individual 1:32 blastomeres from different conceptuses were also recombined to form duplicate clumps. One replicate yielded one pair of 2 × 1:32 blastomeres, of which 1 (100.0%) formed blastocyst equivalents.

[0132] Four blastomere groups (quadruplets) were also evaluated. Five repeats were performed for each of the 4:32, 2x2:32, and 4x1:32 blastomere groups. The 4:32 group yielded 34 blastomere pairs, of which 24 (70.6%) formed blastocyst equivalents. The 2x2:32 group yielded 46 blastomere pairs, of which 25 (54.3%) formed blastocyst equivalents. The 4x1:32 group yielded 60 blastomere pairs, of which 37 (61.7%) formed blastocyst equivalents. Individual 1:32 blastomeres from different conceptuses were also recombined to form quadruplet clumps. One repeat yielded one 4-cell clump consisting of 1:32+3:32, of which one (100.0%) formed a blastocyst equivalent.

[0133] The left side of Figure 5 shows the formation of blastocyst equivalents from unzipped quadruplets (4:32, 2x2:32, 4x1:32). 140 quadruplets (34 4:32 + 46 2x2:32 + 60 1x4:32) were obtained, of which 86 (61%) formed blastocyst equivalents. The right side of the figure shows the expected commercial outcome for the intact conceptus versus the 140 quadruplets of the unzipped conceptus. The 140 unzipped dyads are equivalent to 19 intact 32-cell conceptuses. From the 18 intact conceptuses, 5.4 intact blastocysts are expected to form (30%, as reported in the commercial procedure). As a result, the unzipped procedure allows a 15.9-fold increase in the blastocyst development rate compared to the standard commercial procedure. [Table 5]

[0134] Example 2: Transfer of blastocyst equivalents derived from 2:16 and 4:32 aggregates 1.1 General methods and materials 1.1.1 Synchronization of recipient cows For conceptus implantation, estrous cycles were synchronized using standard hormonal treatment protocols on day 7 or 8. Recipients were examined by rectal palpation for the presence of corpora lutea, and conceptuses were implanted into the uterine horn ipsilateral to the ovary where the corpus luteum was present.

[0135] 1.1.2 Transfer of blastocyst equivalents into recipient cows Blastocyst equivalents were derived from either 2:16 or 4:32 clumps as described in Example 1 (bovine conceptus expansion). Day 7 and day 8 blastocyst equivalents were scored for the presence or absence of an inner cell mass (ICM). Blastocyst equivalents with ICM in holding medium (Transport VitroBlast, Art Lab Solutions) were placed into 0.25 mL straws either individually or together with blastocyst equivalents lacking ICM.

[0136] 1.1.3 Pregnancy diagnosis Pregnancy was confirmed by either a blood test (Idexx) or transrectal ultrasound 2-3 weeks and 1-3 months after implantation.

[0137] 1.2 Results 1.2.1 Pregnancy and birth rates of blastocyst equivalents derived from 2:16 and 4:32 aggregates In two replicate experiments, blastocyst equivalents derived from either the 2:16 or 4:32 aggregates were transferred into a total of 140 recipient cows. Pregnancy rates 2-3 weeks and 1-3 months after transfer are shown in Table 2.1. A total of 12 calves were born to the 60 recipients (Table 2.1). [Table 6]

[0138] It will be appreciated by those skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0139] (Additional Note) (Appendix 1) 1. A method for expanding a donor embryo, comprising: (i) obtaining a donor embryo that comprises one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 16-cell embryo or a pre-compaction morula; (ii) isolating a plurality of said embryonic cells from said donor embryo; (iii) expanding the embryonic cells in vitro under conditions suitable for producing a plurality of conceptuses from the donor embryos, wherein at least a portion of the embryonic cells are expanded as one or more cell clumps, each clump comprising two or more embryonic cells; (iv) culturing said plurality of conceptuses under conditions suitable to produce a plurality of blastocysts.

[0140] (Appendix 2) 2. The method of claim 1, wherein isolating one or more of the embryonic cells from the one or more donor embryos is accomplished by disrupting the zona pellucida (ZP) and isolating the embryonic cells from the one or more donor embryos.

[0141] (Appendix 3) 3. The method of claim 2, wherein the ZP is enzymatically or mechanically disrupted and a micropipette is used to aspirate one or more of the embryonic cells and / or clumps thereof from the one or more donor embryos, thereby isolating the embryonic cells.

[0142] (Appendix 4) 4. The method of any one of claims 1 to 3, wherein all or substantially all of the embryonic cells isolated from the donor embryo are expanded as cell clumps.

[0143] (Appendix 5) 5. The method of any one of claims 1-4, wherein the donor embryo comprises about 9-64 cells, and the embryo has not yet developed into a blastocyst.

[0144] (Appendix 6) 6. The method of any one of claims 1 to 5, wherein the donor embryo comprises about 16-64 cells, and the embryo has not yet developed into a blastocyst.

[0145] (Appendix 7) 7. The method of any one of claims 1-6, wherein the donor embryo comprises about 32-64 cells, and the embryo has not yet developed into a blastocyst.

[0146] (Appendix 8) 5. The method of any one of claims 1 to 4, wherein the donor embryo comprises one or more embryonic cells that are developmentally equivalent to an embryonic cell from a 32-cell embryo.

[0147] (Appendix 9) 9. The method of any one of claims 1 to 8, wherein each cell aggregate comprises 2 to 8 embryonic cells prior to expansion.

[0148] (Appendix 10) 10. The method of claim 9, wherein each cell aggregate contains 2-4 embryonic cells prior to expansion.

[0149] (Appendix 11) 11. The method of claim 10, wherein each cell clump comprises four embryonic cells prior to expansion.

[0150] (Appendix 12) 12. The method of any one of claims 1-11, wherein at least about four conceptuses are cultured for each donor embryo in step (iv) to generate a plurality of blastocysts.

[0151] (Appendix 13) 13. The method of claim 12, wherein at least about 6-8 conceptuses are cultured for each donor embryo in step (iv) to generate a plurality of blastocysts.

[0152] (Appendix 14) (i) at least a portion of the plurality of embryonic cells isolated from the donor embryo is isolated as a pre-existing clump; or (ii) The method of any one of claims 1 to 13, wherein at least a portion of the plurality of embryonic cells isolated from the donor embryo are aggregated after being separated from the donor embryo.

[0153] (Appendix 15) 15. The method of claim 14, wherein the embryonic cells that are aggregated are derived from the same donor embryo or from two or more donor embryos.

[0154] (Appendix 16) 16. The method of claim 15, wherein the embryonic cells to be aggregated are derived from a donor embryo obtained from a different animal.

[0155] (Appendix 17) (i) the embryonic cells are cultured in the presence of one or more factors capable of promoting embryonic development, and / or (ii) The method according to any one of claims 1 to 16, wherein the embryonic cells are cultured in the presence of one or more factors capable of maintaining totipotency.

[0156] (Appendix 18) 18. The method according to any one of claims 1 to 17, wherein the donor embryo is from a mammalian species.

[0157] (Appendix 19) The mammalian species is (i) livestock species; (ii) ruminant species; and / or (iii) The method according to claim 18, wherein the animal is a bovine, ovine or caprine species.

[0158] (Appendix 20) 20. The method of any one of claims 1 to 19, wherein the one or more donor embryos of step (i) are produced by in vivo fertilization (IVF).

[0159] (Appendix 21) The one or more donor embryos of step (i) are freshly harvested; one or more donor embryos of step (i) are stored in embryo storage medium; and / or 21. The method according to any one of claims 1 to 20, wherein one or more donor embryos in step (i) are cryopreserved.

[0160] (Appendix 22) 22. The method of any one of claims 1-21, further comprising selecting the one or more donor embryos prior to step (i) based on one or more genetic screening criteria, genetic diagnosis, and / or one or more morphological criteria.

[0161] (Appendix 23) 23. The method of any one of claims 1-22, wherein the one or more donor embryos are genetically modified.

[0162] (Appendix 24) 24. The method of claim 23, wherein the one or more donor embryos comprise a unique genetic tag or identifier for traceability of the embryos produced therefrom and / or animals produced from the embryos.

[0163] (Appendix 25) 25. The method of any one of claims 1-24, wherein the produced plurality of embryos are expanded in vitro to form blastocysts.

[0164] (Appendix 26) 26. The method of any one of claims 1-25, further comprising recovering the embryo produced by said method.

[0165] (Appendix 27) 27. The method of claim 26, wherein one or more of the recovered embryos are stored in embryo storage medium and / or at 4° C.

[0166] (Appendix 28) 28. The method of claim 27, wherein one or more of the recovered embryos are cryopreserved.

[0167] (Appendix 29) 29. The method of any one of claims 1-28, further comprising transferring one or more of the embryos produced by said method into the uterus or oviduct of one or more recipient females.

[0168] (Appendix 30) Prior to culturing the plurality of conceptuses to produce the plurality of blastocysts in (iv), the method further comprises: (v) isolating one or more of the plurality of conceptuses produced in (iii) for use as donor embryos in subsequent propagation, wherein each donor embryo isolated for subsequent propagation comprises at least two embryonic cells; (vi) isolating one or more of the embryonic cells from the one or more donor embryos; (vii) expanding the embryonic cells in vitro under conditions suitable to generate a plurality of conceptuses, each of which comprises at least two embryonic cells; (viii) optionally, repeating steps (i)-(iii) "N" times, where "N" is 1 to 10.

[0169] (Appendix 31) 10. One or more embryos produced by the method according to any one of claims 1 to 30.

[0170] (Appendix 32) 1. A method for breeding animals, comprising: (i) transferring one or more of the embryos produced by the method according to any one of claims 1 to 30 into the uterus or oviduct of one or more recipient females to establish a pregnancy; (ii) producing said animal by parturition from said pregnant recipient female.

[0171] (Appendix 33) (i) the animal is a mammalian species; (ii) the mammalian species is a livestock species; (iii) the livestock species is a ruminant species; and / or (iv) The method of claim 32, wherein the livestock species is a bovine, ovine or caprine species.

Claims

1. A method for growing donor embryos, (i) Obtaining a donor embryo containing one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a morula before compaction, (ii) Separating multiple embryonic cells from the donor embryo, (iii) Expanding the embryonic cells in vitro under conditions suitable for producing multiple conception products from the donor embryo, wherein at least a portion of the embryonic cells is expanded into one or more cell aggregates, and each aggregate contains two or more embryonic cells. (iv) Culturing the multiple conception products under conditions suitable for producing multiple blastocysts, The donor embryo is derived from a domesticated ruminant species. method.

2. The isolation of one or more embryonic cells from one or more donor embryos is achieved by disrupting the zona pellucida (ZP) and isolating the embryonic cells from the one or more donor embryos, or The isolation of one or more embryonic cells from one or more donor embryos is achieved by enzymatically or mechanically disrupting the zona pellucida and using a micropipette to aspirate one or more of the embryonic cells and / or their aggregates from the one or more donor embryos, thereby isolating the embryonic cells. The method according to claim 1.

3. The method according to claim 1, wherein all or substantially all of the embryonic cells isolated from the donor embryo are expanded as a cell aggregate.

4. The donor embryo contains approximately 9 to 64 cells, and the embryo has not yet developed into a blastocyst. The donor embryo contains approximately 16 to 64 cells, and the embryo has not yet developed into a blastocyst, or The donor embryo contains approximately 32 to 64 cells, and the embryo has not yet developed into a blastocyst. The method according to claim 1.

5. The method according to claim 1, wherein the donor embryo comprises one or more embryonic cells that are developmentally equivalent to embryonic cells from a 32-cell embryo.

6. The method according to claim 1, wherein each cell aggregate contains 2 to 8 embryonic cells before expansion, contains 2 to 4 embryonic cells before expansion, or contains 4 embryonic cells before expansion.

7. The method according to claim 1, wherein at least about four conception products are cultured for each donor embryo in step (iv) to produce multiple blastocysts.

8. The method according to claim 7, wherein at least about 6 to 8 conception products are cultured for each donor embryo in step (iv) to produce multiple blastocysts.

9. (i) At least a portion of the plurality of embryonic cells separated from the donor embryo are separated as existing aggregates, or (ii) The method according to claim 1, wherein at least a portion of the plurality of embryonic cells separated from the donor embryo are aggregated after being separated from the donor embryo.

10. The method according to claim 9, wherein the aggregated embryonic cells are derived from the same donor embryo or from two or more donor embryos.

11. The aggregated embryonic cells are derived from donor embryos obtained from different animals, or In step (i), one or more of the donor embryos are produced by in vivo fertilization (IVF). The method according to claim 10.

12. (i) The embryonic cells are cultured in the presence of one or more factors that can promote embryonic development, (ii) The embryonic cells are cultured in the presence of one or more factors that can maintain totipotency, or Combinations of process (i) and process (ii), The method according to claim 1, including the method described in claim 1.

13. The method according to claim 1, wherein the donor embryo is derived from a cattle, sheep, or goat.

14. The method according to claim 13, wherein the donor embryo is of bovine origin.

15. (i) one or more donor embryos in step (i) are freshly collected, (ii) One or more donor embryos from step (i) are stored in embryo retention medium. (iii) One or more donor embryos from step (i) are cryopreserved, (iv) The multiple embryos created are expanded in vitro to form blastocysts. (v) The one or more embryos prepared by the above method are collected and stored in an embryo-holding medium and / or stored at 4°C. (vi) The one or more embryos produced by the above method are collected and cryopreserved, or (vii) Any combination of process (i) to process (vi), The method according to claim 1, including the method described in claim 1.

16. (i) Selecting one or more donor embryos prior to step (i) based on one or more genetic screening criteria, genetic diagnosis, and / or one or more morphological criteria (ii) The one or more donor embryos have been genetically modified. (iii) The one or more donor embryos are genetically modified to include a unique genetic tag or identifier for traceability of the embryos and / or animals produced from them, or (iv) Any combination of process (i) to process (iii), The method according to claim 1, further comprising:

17. Before the step of culturing the plurality of conception products to produce the plurality of blastocysts in step (iv), the method is performed (v) Isolating one or more of the multiple conception products prepared in step (iii) for use as donor embryos in subsequent proliferation, wherein each donor embryo isolated for subsequent proliferation contains at least two embryonic cells. (vi) Isolating one or more of the embryonic cells from the one or more donor embryos, (vii) Expanding the embryonic cells in vitro under conditions suitable for producing multiple conception products, each containing at least two embryonic cells, (viiii) The method according to claim 1, further comprising (viiii) optionally repeating steps (i) to (iii) N times, wherein N is optionally between 1 and 10.

18. A method of breeding animals, (i) Obtaining one or more donor embryos produced by a method for propagating one or more donor embryos as defined in any one of claims 1 to 17, (ii) Transferring one or more of the above embryos into the uterus or fallopian tube of one or more recipient females to establish pregnancy, (iii) Producing the animal by giving birth from the pregnant recipient female, The aforementioned animal is a domesticated ruminant. method.

19. The method according to claim 18, wherein one or more donor embryos are obtained by carrying out a method for growing one or more donor embryos.

20. The method according to claim 18, wherein the ruminant livestock species is a cattle, sheep, or goat.

21. The method according to claim 18, wherein the ruminant livestock species is a cattle species.

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