embryo digonization

Abstract

To support industrial applications, we provide an improved approach to embryo proliferation. [Solution] This disclosure generally relates to a method for producing multiple embryos from one or more donor embryos by performing continuous proliferation, for example, three or more times, and to the application of this method in animal breeding. This disclosure also relates to a method for producing multiple identical embryos from a donor embryo containing embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compressed morula.

Description

Technical Field

[0001] The present disclosure generally relates to a method of producing multiple embryos from one or more donor embryos by performing a continuous growth cycle, for example, three or more growths, and the application of such a method in animal breeding. The present disclosure also relates to a method of producing multiple monozygotic embryos from a donor embryo containing embryonic cells equivalent in development to embryonic cells from a 16-cell embryo or a pre-compressed morula embryo.

Background Art

[0002] Reproductive assisted technology (ART) has made great progress especially in the past few decades. Artificial insemination (AI) is still the most (cost-effectively) effective method to realize the genetic gain of the cattle population and is widely used in the dairy industry. In this regard, the global market for frozen semen and embryos has continued to be booming. Millions of cattle are bred by AI, and more than one million embryos are transplanted worldwide every year. In the dairy industry, most top-level sires that provide semen for AI are derived from embryo transfer (ET). Improvements in methods for controlling the estrous cycle and ovulation have led to more effective programs for AI, superovulation of donor cows, and management of ET recipients. Despite these advances in ART, due to the cost associated with the production of each embryo, the use of reproductive technologies such as multiple ovulation and embryo transfer (MOET) by producers is still limited. Therefore, unlike conventional AI, the possibility that MOET will be used by producers as a conventional breeding method is low.

[0003] Recently, an approach has been reported to produce genetically identical monozygotic twins from blastomeres isolated from cleavage-stage embryos by embryo bisection. This new addition to the "toolbox" of ART is exciting and allows producers to more effectively capture and select the genes of the female (dam) in addition to the genes of the sire. However, the widespread adoption at the commercial level of embryo twinning, like other ET- and IVF-based approaches, may be hindered by the exorbitant costs for producers and challenges related to scaling up the technology.

[0004] Therefore, improved approaches to embryo proliferation are needed to overcome one or more of these limitations and support industrial application. [Overview of the Initiative] [Means for solving the problem]

[0005] This disclosure broadly relates to a method for producing multiple embryos from one or more donor embryos. In this regard, the inventors have shown for the first time that bovine donor embryos containing at least two embryonic cells can be continuously proliferated three or more times before the resulting embryos expand to the blastocyst stage. In this way, the inventors have shown that the efficiency of blastocyst retrieval from early donor embryos using the continuous proliferation method described herein is significantly higher (for example, up to 6 times) than simply culturing early intact donor embryos directly to the blastocyst stage.

[0006] Furthermore, the inventors have shown for the first time that multiple monozygous embryos can be produced using a bovine embryo as a donor embryo, which contains one or more embryonic cells that are developmentally equivalent to those from a 16-cell embryo or a pre-compressed morula, including cases where the embryo is further grown by employing the continuous growth steps described herein. Starting with a more developed donor embryo, typically containing a heterogeneous mixture of blastomeres that are developmentally equivalent to those from 8-cell, 16-cell, 32-cell, and 64-cell embryos, the inventors have shown that the efficiency of blastocyst retrieval using the growth method described herein is significantly higher (for example, up to 10 times higher when continuous growth is employed) than simply culturing an early, intact donor embryo directly to a blastocyst.

[0007] For example, this disclosure provides a method for growing one or more donor embryos, and this method is (i) The step of obtaining one or more donor embryos containing at least two embryonic cells, (ii) The step of separating one or more embryonic cells from one or more donor embryos, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple embryos, each containing at least two embryonic cells, (iv) a step of isolating one or more of the multiple embryos produced in step (iii) to be used as donor embryos in subsequent proliferation, and (v) A step which includes repeating steps (i) to (iv) above "n" ("n" ≥ 3) times.

[0008] In another example, this disclosure provides a method for growing donor embryos, and this method is: (i) A step of obtaining a donor embryo containing one or more embryonic cells that are developmentally equivalent to the embryonic cells from a 16-cell embryo or a pre-compressed morula, (ii) the step of separating one or more embryonic cells from the donor embryo, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple identical embryos from the donor embryo, and (iv) A step comprising culturing the multiple monozygous embryos under conditions suitable for producing multiple monozygous blastocysts.

[0009] Prior to the step of culturing the plurality of monozygotic embryos to produce the plurality of blastocysts, the method is as follows: (i) a step of isolating one or more of the plurality of monozygotic embryos produced for use as donor embryos in subsequent proliferation, wherein each donor embryo isolated for subsequent proliferation contains at least two embryonic cells, (ii) The step of separating one or more embryonic cells from one or more donor embryos, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple embryos, each containing at least two embryonic cells, (iv) a step of isolating one or more of the multiple embryos produced in step (iii) to be used as donor embryos in subsequent proliferation, and (v) Before culturing the multiple embryos under conditions suitable for producing multiple blastocysts, the step may further include repeating steps (i) to (iv) n times.

[0010] In one example, the separation of one or more embryonic cells from each donor embryo is achieved by dividing the donor embryo into two or more parts, each part (or demi-embryo) containing one or more embryonic cells. In one example, at least one donor embryo may be divided into two parts. In one example, at least one donor embryo may be divided into three parts. In one example, at least one donor embryo may be divided into four parts. In one example, at least one donor embryo may be divided into five or more parts.

[0011] In each of the above examples, the donor embryo may be divided or cut using microsurgical instruments. For example, the donor embryo may be divided or cut by microdissection using a blade (e.g., a scalpel blade or part thereof), a fine glass needle, or a laser (e.g., laser-assisted biopsy). In other examples, the donor embryo may be divided or cut by nanodissection (e.g., an atomic force microscope (AFM) with a femtosecond laser pulse or nanoscalpel).

[0012] In another example, the isolation of one or more embryonic cells from each donor embryo is achieved by disrupting the zona pellucida (ZP) and isolating one or more of the embryonic cells from the donor embryo. This is referred to herein as “thawing” or “thawing method.” According to this example, the ZP can be disrupted, and one or more embryonic cells can be isolated from one or more donor embryos. In one example, the ZP is disrupted enzymatically or mechanically. For example, the embryonic cells can be isolated by enzymatically or mechanically disrupting the ZP and aspirating one or more of the embryonic cells from the one or more donor embryos using a micropipette.

[0013] Unless otherwise stated, "n" is 1 or greater. For example, "n" may be 2 or greater. For example, "n" may be 3 or greater. Following the example where "n" is 3 or greater, "n" may also be 4 or greater. For example, "n" may be 5 or greater. For example, "n" may be 6, 7, 8, 9, or 10 or greater.

[0014] In one example, the method of this disclosure produces 16 or more identical embryos. In another example, the method of this disclosure produces 32 or more identical embryos. In another example, the method of this disclosure produces 64 or more identical embryos. In another example, the method of this disclosure produces 128 or more identical embryos. In another example, the method of this disclosure produces 256 or more identical embryos. In another example, the method of this disclosure produces 512 or more identical embryos.

[0015] For example, the embryo cells or embryos containing them may be cultured in the presence of one or more factors that can promote embryonic development. For instance, embryo cells may be cultured in the presence of one or more factors that can promote embryonic development in order to form and expand monozygous embryos (for example, for harvesting purposes).

[0016] In another example, the embryo cells or embryos containing them may be cultured in the presence of one or more factors capable of promoting totipotency and / or inhibiting or preventing embryonic development. For example, the embryo cells or embryos containing them may be cultured in the presence of one or more factors capable of promoting maternal mRNA clearance.

[0017] Unless otherwise specified, when no compression has occurred yet, the donor embryo or each donor embryo contains from about 2 to about 300 embryonic cells, for example, from about 2 to about 256 embryonic cells or from about 2 to about 64 embryonic cells. For example, when no compression has occurred yet, the donor embryo or each donor embryo may contain from about 100 to about 256 embryonic cells. For example, when no compression has occurred yet, the donor embryo or each donor embryo may contain from about 64 to about 128 embryonic cells. For example, when no compression has occurred yet, the donor embryo or each donor embryo may contain from about 32 to about 64 embryonic cells. For example, when no compression has occurred yet, the donor embryo or each donor embryo may contain from about 16 to about 32 embryonic cells. For example, when no compression has occurred yet, the donor embryo or each donor embryo may contain from about 2 to about 32 embryonic cells. For example, when no compression has occurred yet, the donor embryo or each donor embryo may contain from about 2 to about 16 embryonic cells. For example, the donor embryo or each donor embryo may contain from about 2 to about 8 embryonic cells.

[0018] According to an example where the donor embryo contains one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compressed morula, the donor embryo may contain from about 12 to about 32 uncompressed embryonic cells. According to this example, the donor embryo may contain one or more embryonic cells that are developmentally equivalent to embryonic cells from an 8-cell embryo, one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo, one or more embryonic cells that are developmentally equivalent to embryonic cells from a 32-cell embryo, and / or one or more embryonic cells that are developmentally equivalent to embryonic cells from a 64-cell embryo.

[0019] In each of the above examples, donor embryos can be obtained from vertebrates.

[0020] In one example, the vertebrate may be a mammalian species.

[0021] In one example, the mammalian species may be a livestock species. For example, the livestock species may be a bovine species. For example, the livestock species may be a sheep species (i.e., a sheep). For example, the livestock species may be a swine species (i.e., a pig). For example, the livestock species may be a horse species (i.e., a horse). For example, the livestock species may be a goat species (i.e., a goat). For example, the livestock species may be a deer species (i.e., a deer). For example, the livestock species may be a camelid species (e.g., a camel or an alpaca).

[0022] In some examples, one or more of the donor embryos obtained in step (i) are produced by in vivo fertilization. In other examples, one or more of the donor embryos obtained in step (i) are produced by in vitro fertilization (IVF).

[0023] In some examples, one or more of the donor embryos obtained in step (i) are fresh. In other examples, one or more of the donor embryos obtained in step (i) are cryopreserved. For example, the donor embryos may be thawed.

[0024] In each of the above examples, 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 diagnostic criteria, and / or one or more morphological criteria. For example, the selection step may be performed before step (i).

[0025] In one example, the genetic screening criteria can be determined by screening one or more donor embryos for the presence of one or more gene markers (e.g., SNP alleles or haplotypes) associated with a trait of interest. In one example, a 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).

[0026] In one example, one or more of the donor embryos can be selected based on a genetic diagnosis related to one or more symptoms, diseases, or predispositions thereto.

[0027] In one example, one or more donor embryos may be selected based on one or more morphological features indicating embryonic health.

[0028] In each of the above examples, one or more donor embryos can be genetically modified. For example, one or more donor embryos can be genetically modified by introducing exogenous nucleic acids into the genome of the embryonic cells contained within them. For example, one or more donor embryos can be genetically modified by editing the genome of the embryonic cells contained within them.

[0029] For example, one or more donor embryos contain a unique genetic tag or nucleic acid identifier for traceability of the embryos produced therefrom and / or the animals produced from those embryos. For example, genetic modification may be used to introduce a unique genetic tag or nucleic acid identifier.

[0030] In each of the above examples, the method includes the step of inflating multiple embryos in vitro to form blastocysts. For example, the method may include the step of expanding embryos in vitro to form mature blastocysts ready for implantation.

[0031] The method may further include a step of harvesting multiple embryos produced by this method. For example, the method may include a step of harvesting the embryos when they have matured to the blastocyst stage.

[0032] In some cases, one or more of the harvested embryos are stored in an embryo-retaining medium. For example, one or more of the harvested embryos may be stored at approximately 4°C.

[0033] In some cases, one or more of the harvested embryos are cryopreserved. The cryopreserved embryos may be stored at approximately -180°C to approximately -196°C. For example, the cryopreserved embryos may be stored in liquid nitrogen at approximately -196°C.

[0034] In some examples, the method of the present disclosure further includes the step of implanting one or more of the embryos produced by the method into the fallopian tubes of one or more recipient females.

[0035] Furthermore, this disclosure provides one or more embryos produced by the method of this disclosure. In one example, the one or more embryos may be provided in embryo storage or transfer medium at approximately 4°C. In another example, the one or more embryos may be cryopreserved.

[0036] In one example, the embryo may originate from a mammalian species (e.g., a non-human mammalian species). In one example, the non-human mammalian species may be a domesticated species. For example, the domesticated species may be a cattle species. For example, the domesticated species may be a sheep species (i.e., a sheep). For example, the domesticated species may be a pig species (i.e., a pig). For example, the domesticated species may be a horse species (i.e., a horse). For example, the domesticated species may be a goat species (i.e., a goat). For example, the domesticated species may be a deer species (i.e., a stag). For example, the domesticated species may be a camel species (i.e., a camel or an alpaca).

[0037] Furthermore, this disclosure provides a method for raising animals, and this method is, (i) The step of implanting one or more embryos produced by the method of the present disclosure into the fallopian tube of one or more recipient females to establish pregnancy, and (ii) A step comprising producing an animal from a pregnant recipe female by birth.

[0038] For example, the animal in question is a vertebrate. For instance, a vertebrate may be a mammal, amphibian, reptile, fish, or bird.

[0039] In one particular example, the animal is a mammal, for example, a non-human mammal. Exemplary non-human mammals that can be produced using the above method include domesticated species (e.g., cattle, 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., rhesus monkeys, marmosets, etc.), and wild animal species (e.g., marsupials, cats, rhinoceroses, giant pandas, etc.). In one particular example, the method of the present disclosure can be used to raise cattle. In another example, the method of the present disclosure can be used to raise sheep. In another example, the method of the present disclosure can be used to raise pigs. In another example, the method of the present disclosure can be used to raise goats. In another example, the method of the present disclosure can be used to raise horses. [Brief explanation of the drawing]

[0040] [Figure 1] Figure 1 is a schematic diagram of the classification scheme for blastomeres and developing embryos in four thawings from a two-cell stage embryo. The left side represents the normal development of the preimplantation product from the fertilized egg stage to the blastocyst stage. In the thawing procedure, the zona pellucida (ZP) (the gray band surrounding the embryo) is removed to separate the individual blastomeres within the embryo (number of consecutive divisions n=1). The individual blastomeres isolated from the two-cell embryo are called 1:2. These blastomeres are then developed to form pairs named 2:4. After cleavage, these 2:4 blastomeres can be thawed again (number of consecutive divisions n=2), in which case the blastomeres are separated into 1:4. This process is repeated, resulting in two more consecutive divisions of n=3 and n=4, respectively. As these blastomeres develop, they transition to the corresponding subsequent stages of the preimplantation product, and therefore the denominator changes accordingly. After 1:16 steps, the blastomeres can be compressed and cavitated to form something equivalent to a blastocyst. [Modes for carrying out the invention]

[0041] General technology and definition Unless otherwise defined, all technical and scientific terms used herein should be considered to have the same meaning as those commonly understood by those skilled in the art (e.g., animal nutrition, feed formulation, microbiology, livestock management, etc.).

[0042] As used herein, even in the singular form, these words include their complex forms unless otherwise specified in the context.

[0043] The term "and / or," for example, "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be considered to clearly support both meanings or either one of them.

[0044] Throughout this specification, variations of the word such as “contains,” “contains,” or “includes” shall be understood to indicate that they contain the elements, integers, or steps, or groups of elements, integers, or steps described herein, but not to indicate that they exclude other elements, integers, or steps, or other groups of elements, integers, or steps.

[0045] In this specification, the term "approximately" is used to indicate an approximation. When the term "approximately" is used in relation to a numerical range, it modifies that range by extending the boundary above or below the listed numbers. Generally, in this specification, the term "approximately" is used to modify a number by 10% above or below (higher or lower) the stated value.

[0046] Those skilled in the art will understand that this disclosure is susceptible to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure includes all steps, features, compositions and compounds mentioned or indicated individually or collectively herein, as well as any and all combinations of any two or more of these steps or features. Therefore, each feature of any particular aspect or embodiment of this disclosure may be applied to any other aspect or embodiment of this disclosure with the necessary modifications.

[0047] This disclosure is not limited in scope by the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods as described herein are clearly within the scope of this disclosure.

[0048] Throughout this specification, unless otherwise specified or the context requires otherwise, references to a single step, a single material composition, a group of steps, or a group of material compositions should encompass one or more (i.e., one or more) of these steps, material compositions, groups of steps, or groups of material compositions.

[0049] Define specifics In this specification, the term “embryo” means the fertilized egg, which is formed when two haploid gamete cells (e.g., an unfertilized oocyte and a spermatocyte) combine to form a diploid totipotent cell (e.g., a fertilized egg), and the embryo that arises from subsequent cell division (i.e., embryonic cleavage) (including the morula stage (i.e., while the inner cell mass is compressed) and the blastocyst stage, when the trophectoderm and inner cell mass have differentiated).

[0050] As used herein, the term “morula” refers to a stage in embryonic development. A morula is an early embryo consisting of cell spheres (called blastomeres) enclosed in a glycoprotein membrane called the zona pellucida. A series of cleavage events (as shown in Figure 1 for cattle) produce a morula from a single-cell fertilized egg. Before morula formation, there is a crucial event called “compression,” during which the embryo, containing approximately 8 to 32 cells (depending on the species), undergoes changes in cell morphology and intercellular adhesion, initiating the formation of this solid cell sphere. Morulas typically contain around 16 to 32 cells (depending on the species) and are so named because they resemble mulberries (Latin: morus: mulberry). In cattle, the compression process usually occurs after the 16-cell stage, and the developing embryo reaches the early morula stage at the 32-cell stage, at which point intercellular adhesion between embryonic cells (or blastomeres) progresses, and the embryo contains a compressed inner cell mass (ICM).

[0051] Through the processes of cell differentiation and cavitation, the morula gives rise to a blastocyst. As used herein, the term “blastocyst” should be understood to refer to an embryo having an inner cell mass (ICM) or embryonic embryo containing totipotent embryonic stem cells and an extracellular layer or trophoblast that later forms the placenta. The trophoblast surrounds the inner cell mass and the blastocyst cavity, which is filled with fluid called the blastocoel. A blastocyst typically contains 70 to 300 embryonic cells (this may vary depending on the embryo's species and maturity). In some cases, a blastocyst may contain approximately 64 to 128 cells. In some cases, a blastocyst may contain approximately 128 to 256 cells. In some cases, a blastocyst may contain 150 to 256 cells. In some cases, a blastocyst may contain approximately 256 cells.

[0052] As used herein, the terms “embryonic cells (singular)” or “embryonic cells (plural)” are intended to encompass all totipotent cells in a developing embryo from the oocyte stage to the blastocyst stage. For example, embryonic cells obtained from within a developing embryo (also called a “blastomeres”) from the oocyte stage to the morula stage are totipotent embryonic cells. Similarly, embryonic cells obtained from the inner cell mass of a blastocyst may be totipotent.

[0053] As used herein, the term “totipotent” is used to describe a cell that can produce any type of cell. For example, in the case of embryonic cells, a “totipotent” cell is a cell that can produce any type of cell in the embryo and can ultimately differentiate into any of the specialized cells required for different tissues in the body (e.g., skin, bone, bone marrow, muscle, etc.). The term “totipotent” is distinguished from the term “pluripotent,” which refers to a cell that may differentiate into a specific subpopulation of cells within a developing cell mass, but may not produce any type of cell or any type of cell.

[0054] As used herein, the term “monozygous embryo” should be understood to refer to two or more embryos formed or derived from a single fertilized egg.

[0055] As used herein, the term “hemiembryo” should be understood to mean a part of an embryo that has been divided or cut. For example, an embryo that has been cut in half will produce two hemiembryos, each containing embryonic cells. Similarly, an embryo that has been cut into three parts, each containing embryonic cells, will produce three hemiembryos.

[0056] As used herein, the term “animal” should be understood to include all vertebrates, such as mammals (i.e., non-human mammals), amphibians, reptiles, fish, and birds. In one example, the animal is a mammal. Exemplary mammals for which the methods of this disclosure may be useful include domestic animals (e.g., cattle, 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., rhesus monkeys, marmosets, etc.), and wild animal species (e.g., marsupials, large cats, rhinoceroses, giant pandas, etc.). In one particular example, the methods of this disclosure may be useful for cattle (i.e., bovine).

[0057] twinning method This disclosure relates in general to methods for amplifying embryos, also referred to herein as “twinization,” and more particularly to methods for producing multiple embryos from one or more early donor embryos. Several approaches or “twinization techniques” for producing multiple embryos from one or more early donor embryos are described herein.

[0058] For example, the method of this disclosure is: (i) The step of obtaining one or more donor embryos containing at least two embryonic cells, (ii) The step of separating one or more embryonic cells from one or more donor embryos, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple embryos, each containing at least two embryonic cells, (iv) a step of isolating one or more of the multiple embryos produced in step (iii) to be used as donor embryos in subsequent proliferation, and (v) A general method step which is to repeat steps (i) to (iv) above "n" ("n" ≥ 3) times.

[0059] In another example, the method of this disclosure for growing donor embryos using the “twinization technique” described herein is: (i) A step of obtaining a donor embryo containing one or more embryonic cells that are developmentally equivalent to the embryonic cells from a 16-cell embryo or a pre-compressed morula, (ii) the step of separating one or more embryonic cells from the donor embryo, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple identical embryos from the donor embryo, and (iv) A general method step comprising the step of culturing the multiple monozygous embryos under conditions suitable for producing multiple monozygous blastocysts.

[0060] In the latter method, prior to the step of culturing the plurality of monozygotic embryos to produce the plurality of blastocysts, the method, (i) a step of isolating one or more of the plurality of monozygotic embryos produced for use as donor embryos in subsequent proliferation, wherein each donor embryo isolated for subsequent proliferation contains at least two embryonic cells, (ii) The step of separating one or more embryonic cells from one or more donor embryos, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple embryos, each containing at least two embryonic cells, (iv) a step of isolating one or more of the multiple embryos produced in step (iii) to be used as donor embryos in subsequent proliferation, and (v) Before culturing the multiple embryos under conditions suitable for producing multiple blastocysts, the step may further include repeating steps (i) to (iv) n times.

[0061] As described herein, steps (i) to (iv) of this method can be repeated "n" times to produce multiple embryos from the original donor embryo. The number of repetitions (i.e., "n") in which steps (i) to (iv) are performed may vary depending on (1) the number of embryonic cells in the initial donor embryo, (2) the technique used to separate the embryonic cells therefrom, and (3) unless otherwise stated. In this regard, "n" may be 1 or greater unless otherwise stated. For example, it may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. According to the example of a method that requires "n" to be 3 or greater, "n" may be 3, 4, 5, 6, 7, 8, 9, or 10 or more.

[0062] In one embodiment, the "twinization technique" employed is the "cutting method." According to this embodiment, in step (i), one or more early donor embryos are cut (or divided) into two parts, each containing one or more embryonic cells, for example, in approximately equal proportions. Next, as described in steps (i) to (iii) above, both hemifloras are expanded in vitro to produce two identical embryos. Then, in step (iv), the newly grown embryos are used as donor embryos, and the process in steps (i) to (iii) is repeated. As shown in step (v), the embryos produced in the previous cycle are used as donor embryos for the next cycle, and this process can be repeated "n" times (one cycle is called one repetition). In this way, proliferation using the cutting method can double the number of identical embryos produced in each new cycle compared to the previous cycle. The number of cycles performed using the cutting method can vary, as it is influenced by many factors (e.g., the number of embryos produced, the number of initiating donor embryos, the developmental stage of the donor embryos, and whether embryos are harvested from this method in the intervening cycle).

[0063] In one example, one or more early donor embryos each contain at least two embryonic cells, and the minimum number of cycles performed using the cutting method may be three (i.e., n≧3).

[0064] In another example, one or more early donor embryos each contain one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compressed morula. In this example, the minimum number of cycles performed using the cutting method may be one (i.e., n≧1).

[0065] In yet another example, the initial donor embryo is a 4-cell embryo, and the desired outcome is the production of at least 16 identical embryos. In this example, at least 4 cycles (i.e., n≧4) of the "cutting method" alone are required to produce at least 16 identical embryos from the initial donor embryo. However, the cutting method may also include at least 5 cycles (i.e., n=5), at least 6 cycles (i.e., n=6), or at least 7 cycles (i.e., n=7), and so on. In this regard, those skilled in the art will understand that the number of cycles performed using the cutting method can easily be varied (e.g., increased) based on the number of embryos produced from the initial donor embryo and the efficiency of embryo retrieval after each cycle.

[0066] In another embodiment, the “twinization technique” employed is the “cookie-cutter method.” According to this embodiment, in step (i), one or more early donor embryos are cut (or divided) into three, four, five or more parts, each containing one or more embryonic cells, for example, in roughly equal proportions. Then, according to steps (i) to (iii) above, these are in vitro expanded to produce three or more identical embryos. Next, in step (iv), the newly produced embryos (or “twinized” embryos) are used as donor embryos, and this process is repeated. As shown in step (v), the embryos produced in the previous cycle are used as donor embryos for the next cycle, and this process can be repeated “n” times (one is called one cycle). The number of cycles performed using the “cookie-cutter” method depends on many factors, including the number of embryos produced, the number of parts to which the donor embryo is cut in each cycle (which may be three or more parts and may vary from cycle to cycle), the number of starting donor embryos, the developmental stage of the donor embryos, and whether embryos are harvested from the method in the intervening cycle. Therefore, the number of cycles may vary.

[0067] In one example, one or more early donor embryos each contain at least four embryonic cells, and the minimum number of cycles performed using the cookie-cutter method may be three (i.e., n≧3).

[0068] In another example, one or more early donor embryos each contain one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compressed morula. In this example, the minimum number of cycles performed using the cookie-cutter method may be one (i.e., n≧1).

[0069] In yet another example, the initial donor embryo is a 4-cell embryo, and the desired outcome is the production of at least 16 identical embryos. In this example, assuming the donor embryo is cut into three parts in each cycle, the “cookie cutter” method may involve at least three cycles (i.e., n≧3) to produce at least 16 identical embryos from the initial donor embryo. However, the cookie cutter method may involve at least four cycles (i.e., n=4), at least five cycles (i.e., n=5), or at least six cycles (i.e., n=6), and so on. With this cutting method, a person skilled in the art will understand that the number of cycles performed using the cookie cutter method may vary depending on the number of embryos produced from the initial donor embryo and the efficiency of embryo retrieval after each cycle.

[0070] Furthermore, it is conceivable that the number of divisions in each donor embryo using the "cookie-cutter" method (i.e., the number of half-embryos produced) may vary within and between cycles. In this regard, the number of divisions in a donor embryo may depend on the number of embryos produced, as well as other factors such as embryo health and embryonic stage (e.g., number of embryonic cells).

[0071] Embryos that can be propagated using the cutting and / or cookie-cutter methods of this disclosure include pre-implantation embryos from the two-cell stage to the blastocyst stage. In some cases, it may be advantageous to select a donor embryo for division that has more totipotent embryonic cells, for example, a blastocyst containing about 70–300 embryonic cells. In other cases, the donor embryo may be from the late morula to the early blastocyst stage, for example, consisting of about 30–70 embryonic cells. In yet another case, the donor embryo may be a morula consisting of about 16–32 embryonic cells. In yet another case, the donor embryo may be a pre-morula stage embryo consisting of 2–about 16 embryonic cells. In any case, those skilled in the art will understand that the number of embryonic cells in an embryo developing at each stage may vary by species.

[0072] In each embodiment of cutting or dividing one or more donor embryos into multiple half-embryos, the process of cutting (or dividing) the embryo may be carried out using any embryo division means known in the art. For example, the donor embryo may be divided or cut by mechanical dissection using pressure-dependent microsurgical instruments such as blades (e.g., scalpel blades or parts thereof) or fine glass needles. Alternatively or additionally, the donor embryo may be divided or cut using a laser, i.e., laser-assisted biopsy. In other examples, the donor embryo may be divided or cut by nanodissection-based tools (e.g., femtosecond laser pulses or atomic force microscopes (AFM) with nanoscalpels). However, it is conceivable that any means known in the art may be employed.

[0073] In further embodiments, the “twinization technique” employed is referred to as the “thawing method.” According to this embodiment, one or more embryonic cells are separated from a donor embryo by destroying or “thawing” the zona pellucida (ZP) and isolating one or more embryonic cells from within the donor embryo. According to this embodiment, the zona pellucida of the donor embryo is destroyed to release the embryonic cells contained therein, and then, according to steps (i) to (iii) above, the embryonic cells are isolated (individually or in groups / clusters) and independently inflated in vitro to produce multiple embryos. In this way, each isolated embryonic cell or each embryonic cell cluster (i.e., if two or more cells are isolated together) is inflated to become an embryo (e.g., an embryo without a ZP). In step (iv), the newly produced embryo is used as a donor embryo and this process is repeated. As shown in step (v), the embryo produced in the previous cycle is used as the donor embryo for the next cycle and this process can be repeated “n” times (one is called one cycle). The number of cycles performed using the "thawing" method depends on various factors, including the number of embryos produced, the number of initiating donor embryos, whether embryos are harvested from this method in the intervening cycle, and the number of embryonic cells in the donor embryo, the latter of which determines the upper limit on the number of monozygotic embryos that can be produced from any single donor embryo.

[0074] The thawing method can be performed on any donor embryo containing totipotent embryonic cells surrounded by the zona pellucida before complete compression (i.e., from 2-cell stage embryos to early pre-compressed morula stage embryos). In one example, the thawing method is performed using a 2-cell donor embryo. In another example, the thawing method is performed using a 4-cell donor embryo. In another example, the thawing method is performed using an 8-cell donor embryo. In another example, the thawing method is performed using a 16-cell donor embryo. In specific cases, the method is performed using donor embryos at different developmental stages, including the early morula stage where compression is not complete. In preferred cases, the embryonic cells within the zona pellucida are not compressed.

[0075] In one example, one or more early donor embryos each contain at least two embryonic cells, and the minimum number of cycles performed using the thawing method may be three (i.e., n≧3).

[0076] In another example, one or more early donor embryos each contain one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or a pre-compressed morula. According to this example, the minimum number of cycles performed using the cutting method may be one (i.e., n≧1). In one example where a single donor embryo containing 16 blastomeres is used as the early donor embryo, the thawing method may require only one cycle to produce at least about 16 identical embryos from the early donor embryo. In another example, the thawing method may be performed starting with an early donor embryo containing 8 to 12 blastomeres (i.e., an 8-cell stage embryo). According to this example, the thawing method may require two cycles (i.e., n=2) to produce at least about 16 identical embryos from the early donor embryo. For example, two cycles of the thawing method, in which the donor embryo in each cycle is "thawed" at the 8-cell stage, can produce about 64 to about 100 identical embryos. Therefore, it will be understood that using the "thawing method" may require fewer cycles to achieve the desired number of embryos compared to the "cutting method" or "cookie cutter method" described herein.

[0077] Similar to the "cutting method" and the "cookie cutter method," those skilled in the art will understand that the number of cycles performed using the "thawing method" may vary depending on the number of identical embryos produced from the early donor embryos and the number of embryonic cells within each donor embryo.

[0078] The disruption of the zona pellucida in thawing (also known as "assisted incubation") can be performed using any suitable method known in the art. In this regard, various techniques are known to be employed in the field of assisted reproductive technology to support embryo incubation, such as partial mechanical zona dissection, zona drilling, and zona thinning using acidic Tyrode's solution, proteinase, pieson vibrator manipulator, and laser, as described in Hammadehetal., (2011) J. Assist. Reprod. Genet., 28(2):119-128. It is also conceivable that the zona pellucida may be disrupted by nanodissection (e.g., femtosecond laser pulses or atomic force microscopy (AFM) with nanoscalpel).

[0079] The defrosting method of this disclosure for disrupting the zona pellucida may employ one or more of the techniques described above.

[0080] When the zona pellucida is disrupted, embryonic cells (e.g., blastomeres) can be isolated and, if appropriate, transplanted into fresh culture medium and allowed to expand. Many methods for isolating individual cells, including embryonic cells, are known in the art and are conceivable 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, immunomagnetocellular separation, immunosurgery, hydrodynamic trapping, laser-captured microdissection, mechanical dissection, manual picking, microfluidics, micromanipulation, nanodissection, serial dilution, Raman forceps, and combinations thereof. To isolate a single embryonic cell or embryonic cell aggregate from a disrupted zona pellucida, one or a combination of these techniques may be used in the methods of this disclosure. In one particular example, microfluidics are employed to isolate individual embryonic cells.

[0081] In each embodiment of the methods described herein, it may be desirable to immobilize the donor embryo in order to cut or divide the donor embryo or to disrupt the zona pellucida (i.e., "thaw" the zona pellucida). Methods for immobilizing embryos are known in the art, and one or more of those methods or techniques may be used in the methods of the present disclosure. Exemplary methods that may be used in the methods of the present disclosure include, for example, roughening the surface of the container containing the embryo, using a protein-free culture medium, applying a material to the culture vessel that adheres to the outer membrane of the embryo, applying aspiration to the zona pellucida, forming depressions or dead ends in the container, constructing a device for capturing the embryo, or attaching the embryo to a surface.

[0082] As described herein, embryonic cells or hemiflora containing embryonic cells isolated from a donor embryo are cultured in vitro and expanded using the method of this disclosure to produce multiple embryos (e.g., monozygous embryos).

[0083] Methodologies for culturing embryos in vitro at various developmental stages are known in the art and are considered herein. Exemplary methods are described herein in Examples 1-5. Those skilled in the art will understand that culture conditions are important for growing a developing embryo to the blastocyst stage and can be modified / adjusted according to the developmental stage of the embryo, and that the rate of embryonic development (e.g., cleavage) can be controlled to provide a sufficient time window to perform the proliferation step of the method disclosed herein. For example, in embryo culture, variables such as temperature and CO2 level can be controlled to optimize the growth of the developing embryo. For example, the optimal temperature for embryonic development is about 32°C to about 40°C, preferably about 35°C to 39°C, and particularly preferably 37°C. The optimal CO2 level for embryonic development in the culture environment is about 1% CO2 to about 10% CO2, preferably about 3% CO2 to about 8% CO2, and more preferably about 5% CO2.

[0084] Appropriate culture media for culturing and developing embryonic cells and embryos are well known in the art. For example, culture media that allow embryos to mature to the blastocyst stage at a rate comparable to that of in vivo development are described in Summers and Biggers (2003) Human Reprod Update, 9:557-582. Many of these culture media are roughly 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) HumReprod 13:148-160). Typically, culture media containing phosphate buffer or HEPES organic buffer are used for procedures involving gamete handling outside of incubators, follicle washing, and micromanipulation. Most culture media utilize a bicarbonate / CO2 buffer system to maintain the pH within an appropriate range (e.g., pH 7.2-7.4). The osmotic pressure of culture media is typically in the range of 275-290 mosmol / kg. Additionally, embryos can be cultured in paraffin oil (or an alternative oil harmless to embryos) to prevent evaporation of the culture medium and maintain a constant osmotic pressure. This oil also minimizes fluctuations in pH and temperature when removing embryos from the incubator for microscopic evaluation.

[0085] Appropriate culture media typically include protein sources such as albumin or synthetic serum, usually added at concentrations of approximately 5-20% (w / v or v / v, respectively). Salt sources such as NaCl, KCl, KH2PO4, CaCl22H2O, MgSO47H2O, or NaHCO3 may also be added to the medium. Since carbohydrates are present in the female reproductive tract, culture media usually also include carbohydrate sources. Along with amino acids, these are the primary energy sources for developing embryos. Culture media supporting 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 sperm requirements during normal fertilization. Media supporting the development of 8-cell embryos up to the blastocyst stage contain low concentrations of pyruvate and lactate, and high concentrations of glucose. Supplementing the culture medium with amino acids may also be desirable for embryo development. Culture media that support 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. Culture media that support the development of 8-cell embryos up to the blastocyst stage are also typically supplemented with essential amino acids such as cystine, histazine, isoleucine, leucine, lysine, methionine, valine, argentine, glutamine, phenylalanine, threonine, and tryptophan. The culture medium may also contain vitamins.

[0086] Furthermore, culture media may contain antibiotics. Most ART labs actually use culture media containing antibiotics to minimize the risk of microbial growth. The most commonly used antibiotics are penicillin (a β-lactam antibiotic that inhibits cell wall integrity in gram-positive bacteria) and streptomycin (an aminoglycoside antibiotic that inhibits protein synthesis in gram-negative bacteria).

[0087] Three examples of sequential embryonic development media that may be useful for culturing embryos in the method disclosed herein are G1 / G2 (Gardneretal, (1998) Hum. Reprod 13:3434), Universal IVFMedium / MS (Bertheussenetal, (1997), and PI / BlastocystMedium (Bertheussenetal, (1998) Am.Soc.Rep.Med. 0-262). Media for culturing embryos at different developmental stages can be purchased from various sources.

[0088] Other exemplary culture media for embryo development are described herein in Examples 1-5 and may be used in the methods of this disclosure.

[0089] In some cases, embryonic cells and / or developing embryos are cultured in the presence of one or more factors capable of promoting embryonic cell totipotency and / or inhibiting or preventing embryogenesis. Such factors may be added to the culture medium to prevent or slow embryogenesis, thereby providing further opportunities to perform additional cycles of steps (i) to (iv) before cell differentiation begins. Factors that promote embryonic cell totipotency and / or inhibit or prevent embryogenesis are known in the art and their use is considered herein. For example, factors that promote embryonic cell totipotency and / or inhibit or prevent embryogenesis include anti-miRs and / or ribozymes that inhibit the stability and activity of miRNAs produced by early embryos. Exemplary anti-miRs may target miRNAs expressed by the embryo that promote maternal mRNA clearance (e.g., anti-miRs targeting the miR-30 family).

[0090] Those skilled in the art will understand that culture conditions can also contribute to maintaining the totipotency of embryonic cells. Therefore, during the culture of embryonic cells, embryos, or hemifleurs, the developmental rate of the cultured embryo can be regulated / controlled by controlling variables such as cell or embryo density, temperature, and CO2 levels.

[0091] As described herein, the methods of this disclosure may also include the step of culturing the embryo in vitro to expand it and form a blastocyst. The blastocyst can then be harvested, for example, for storage and / or implantation in a recipient embryo. Thus, in some steps of the above methods, embryonic cells and / or developing embryos can be cultured in the presence of one or more factors that can promote embryogenesis. For example, factors that can promote embryogenesis (i.e., embryogenetic factors) can be added to the culture medium used to culture the embryo to the blastocyst stage for harvest. Factors that promote embryogenesis are known in the art and are conceivable herein.

[0092] Those skilled in the art will understand that culture conditions, such as embryo density, temperature, and CO2 levels, can be modified and / or optimized to promote embryonic development.

[0093] It is conceivable that the various twinning techniques described herein (i.e., “cutting,” “cookie-cutter,” and “thawing”) may be used in combination with each other. For example, the method of the present disclosure may include one or more cycles in which a donor embryo is divided / cut into two or more hemiembryos using the “cutting” or “cookie-cutter” method, followed by one or more cycles of the “thawing” method performed on the expanded hemiembryos produced by the early cycle. By combining these approaches, the number of embryos produced can be optimized while simultaneously minimizing the number of cycles that need to be performed. According to another example of combining these techniques, the method of the present disclosure may include one or more cycles of the “thawing” method for producing multiple blastomeres (e.g., 16 blastomeres), followed by one or more cycles of the “cutting” or “cookie-cutter” method performed on the expanded embryos from the blastomeres to further increase the number of embryos by a factor of two, three, or four. According to this last example, one or more cycles of the “thawing” method can be performed in the laboratory, and the subsequent cycles of the “cutting” or “cookie-cutter” method can be performed in situ before implantation. In this regard, those skilled in the art will understand that the methods disclosed herein can be combined with the various twinning techniques described herein.

[0094] As described herein, the donor embryo obtained in step (i) of this method may be at any embryonic stage containing multiple totipotent embryonic cells, for example, from a pre-implantation 2-cell stage embryo to a blastocyst stage embryo. However, in some embodiments of the method herein, embryos at a specific developmental stage and / or containing a specific number of embryonic cells may be preferred. To illustrate this point, pre-compressed embryos are preferred when a “thawing method” is performed to allow isolation of blastomeres. In some examples, the donor embryo is at the 2-cell stage. In some examples, the donor embryo is at the 4-cell stage. In some examples, the donor embryo is at the 8-cell stage. In some examples, the donor embryo is at the 16-cell stage. In some examples, the donor embryo is a morula. In other examples, the donor embryo is a blastocyst (pre-hatching). Therefore, a donor embryo useful for the above method may contain approximately 2 to 300 embryo cells, such as approximately 4 to 256 embryo cells, or approximately 100 to 256 embryo cells, or approximately 70 to 100 embryo cells, or approximately 30 to 70 embryo cells, or approximately 16 to 30 embryo cells, or approximately 4 to 16 embryo cells, or approximately 4 to 8 embryo cells, or approximately 2 to 4 embryo cells.

[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 bird species (e.g., poultry).

[0096] In one example, the animal is a mammal, such as a non-human mammal. Exemplary non-human mammals for which the methods of this disclosure may be useful include domesticated species (e.g., cattle, 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., rhesus monkeys, marmosets, etc.), and wild animal species (e.g., marsupials, cats, rhinoceroses, giant pandas, etc.).

[0097] In one particular example, the method of the present disclosure can be used to produce multiple embryos (e.g., identical embryos) in a bovine. In another example, the method of the present disclosure can be used to produce multiple embryos (e.g., identical embryos) from a sheep. In yet another example, the method of the present disclosure can be used to produce multiple embryos (e.g., identical embryos) from a pig. In yet another example, the method of the present disclosure can be used to produce multiple embryos (e.g., identical embryos) from a goat. In yet another example, the method of the present disclosure can be used to produce multiple embryos (e.g., identical embryos) from a horse. In yet another example, the method of the present disclosure can be used to produce multiple embryos (e.g., identical embryos) in a camel.

[0098] The donor embryos used in the first cycle of the method of this disclosure may be prepared in vivo (for example, by conventionally washing embryos from a pregnant animal) or by in vitro fertilization (IVF) methods.

[0099] In one example, donor embryos used in the first cycle of this method are prepared by an in vivo method. For example, oocytes may be fertilized in vivo (e.g., post-mating or by artificial insemination), and the embryos may then be retrieved from pregnant females by conventional embryo washing. In another example, donor embryos are produced by multiple ovulatory embryo transfer (MOET), in which hormones, mainly follicle-stimulating hormone (FSH), are administered to the donor female before fertilization to stimulate the ovaries of the circulating female animal and induce multiple ovulation.

[0100] In another example, in vitro methodologies (i.e., IVF) are used to prepare donor embryos for use in the first cycle of this method. Methods of embryo production using IVF are well known in the art. IVF typically involves the production of oocytes from a donor animal by follicular aspiration, followed by in vitro maturation, fertilization, and culture until the produced embryos reach the desired developmental stage. Conveniently, this approach allows for the repeated production of embryos from living animals of specific value under controlled conditions. Methods of embryo production by IVF are described in Berlinguer F. "Embryo Production," In: Animals Production in Livestock, Encyclopedia of Life Support Systems (EOLSS), and their entirety is incorporated herein.

[0101] The donor embryos used in the first cycle of this method may be fresh or thawed (i.e., thawed cryopreserved embryos), regardless of whether they are produced in vitro or in vitro. In one example, the donor embryos are fresh. In another example, the donor embryos are thawed cryopreserved embryos.

[0102] The donor embryos useful in the methods of this disclosure may be genetically modified. For example, the embryonic cells within the donor embryo may be genetically modified before performing the methods so that all embryos produced from the donor have genetic modification. In one example, 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 a substitute allele for a gene or locus associated with the trait of interest. Alternatively, the exogenous nucleic acid may be a transgene. In another example, the donor embryo may be genetically modified by editing the genome of the embryonic cells contained therein (i.e., genome editing). Genome editing may be selected from the group consisting of insertions, deletions, substitutions, inversions, or translocations. For example, genome editing may involve insertions, deletions, and / or substitutions of a nucleic acid sequence or one or more nucleotide positions therein to replace an existing allele for a gene or locus associated with the trait of interest with a substitute allele.

[0103] Genome editing, when employed alone or in combination, can also be used to introduce one or more genetic modifications (e.g., nucleotide substitutions) into a developing embryo, providing a unique genetic profile or fingerprint. This unique genetic profile or fingerprint can then be used to identify and / or track embryos (and animals produced from those embryos) that were originally produced from a donor embryo. For example, to generate a unique genetic profile or fingerprint, one or more conservative nucleotide substitutions within a safe harbor region of the genome may be performed on embryonic cells within a donor embryo.

[0104] Preferably, the genetic recombination or editing occurs at the single-cell stage such that all subsequent cells in the developing embryo derived from the recombinated cell (and the animal produced therefrom) contain the recombination. However, if the genetic recombination event occurs after one or more cell divisions, and not all embryonic cells in the donor embryo are recombinated, the donor embryo may be a mosaic of the recombination / editing event, having some cells derived from the recombinated / edited cell and some cells derived from the non-recombined / edited cell.

[0105] In the art, many methods have been described for genetically modifying the genome of cells using targeted nucleases. These include, but are not limited to, (1) clustered and regularly arranged short palindromic sequence repeats (CRISPR)-CRISPR-related 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. These other methods for genetically modifying cells may be used in the methods of the present disclosure to genetically modify donor embryos.

[0106] The method of the present disclosure may further include one or more steps to assist in the selection of donor embryos to be grown using the method described above. For example, the method may include selecting donor embryos based on one or more genetic screening criteria, genetic diagnostic criteria, and / or one or more morphological criteria prior to step (i).

[0107] For example, genetic screening criteria can be determined by screening for the presence of one or more genetic markers (e.g., SNP alleles or haplotypes) associated with a phenotypic trait of interest (or a favorable variant thereof), such as a commercially important production trait, as in the case of livestock breeds. Examples of phenotypic traits of interest include, but are not limited to, production traits (e.g., growth rate, fertility, feed conversion efficiency), drug resistance, susceptibility to pests and / or parasites, and sex (i.e., male or female). In this way, donor embryos for propagation by the method of this disclosure can be obtained from elite animals.

[0108] Alternatively or additionally, donor embryos may be selected based on genetic diagnosis for one or more symptoms, diseases, or predispositions thereof. In this regard, preimplantation genetic diagnosis (PGD) of embryos is becoming more common in the field of IVF. PGD tests primarily focus on two methodologies: fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). However, many techniques for PGD are publicly known in the art, and one or more of these techniques may be used in the methods of this disclosure to select donor embryos. These techniques include, but are not limited to, methods that rely on PCR, FISH, single-stranded higher-order polymorphism (SSCP), restriction fragment length polymorphism (RFLP), primed in situ labeling (PRINS), comparative genomic hybridization (CGH), COMET analysis (single-cell gel electrophoresis), heterozygous double-strand analysis, Southern spectroscopy, and denaturant concentration gradient gel electrophoresis (DGGE) analysis.

[0109] Alternatively or additionally, donor embryos may be selected based on one or more morphological features, such as morphological features indicating embryonic health.

[0110] As described herein, once a desired number of embryos (e.g., monozygous embryos) are produced using the method of this disclosure, these embryos can be matured in vitro to a desired embryonic stage (e.g., preimplantation blastocyst) and harvested from the culture medium. The harvested embryos can then be stored in a suitable embryo retention or transfer medium until they are ready for transfer to a recipient embryo and / or until they are ready for cryopreservation. This specification may suggest the use of any commercially available embryo retention and transfer medium. In cases where the embryos are stored for a short period before transfer to a recipient embryo (e.g., during transport), the harvested embryos may be stored at approximately 2°C to approximately 8°C, depending on the specifications of the particular retention or transfer medium. In some preferred examples, the harvested embryos are stored at approximately 4°C.

[0111] Furthermore, harvested embryos may be cryopreserved. The main techniques used for cryopreservation of embryos in the art are vitrification and slow programmable freezing (SPF), both of which are conceivable herein. According to this example, harvested embryos can be transferred to a suitable cryopreservation medium (e.g., ethylene glycol cryopreservation medium or similar) and cryopreserved, and maintained at approximately -180°C to approximately -196°C until thawing, use and / or shipment. For example, cryopreserved embryos may be stored in liquid nitrogen at approximately -196°C.

[0112] In addition to its application to commercial livestock breeding, the method of this disclosure may also be applicable to the fields of animal conservation and management. For example, embryos produced from donor embryos obtained using the method of this disclosure from endangered or threatened species (including wild and domesticated species) can be deposited in a biobank and / or distributed for breeding programs. This could support breeding programs and population management of endangered or threatened species. Therefore, in some examples, the method may further include the step of depositing one or more cryopreserved embryos prepared by the method of this disclosure into a biobank.

[0113] In embodiments in which harvested embryos are transferred to recipient embryos while fresh, the method of the present disclosure may further include the step of transferring one or more monozygotic embryos to the fallopian tubes of one or more recipient embryos. Methods of embryo transfer are known in the art. For example, embryos may be transferred manually using a catheter or other means.

[0114] Therefore, this disclosure further provides a method for raising animals, and this method is, (i) The step of implanting one or more embryos produced by the method described herein into the fallopian tube of one or more recipient females to establish pregnancy, and (ii) A step comprising producing an animal from a pregnant recipe female by birth.

[0115] As described herein, the animal may be any vertebrate, including mammalian species, amphibian species, reptile species, fish species, and bird species (e.g., poultry). In one particular example, the animal is a mammal, e.g., a non-human mammal. Exemplary non-human mammals for which the method of the Disclosure may be useful include domesticated species (e.g., cattle, 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., rhesus monkeys, marmosets, etc.), and wild animal species (e.g., marsupials, cats, rhinoceroses, giant pandas, etc.). In one particular example, the method of the Disclosure may be used for raising cattle. In another example, the method of the Disclosure may be used for raising sheep. In yet another example, the method of the Disclosure may be used for raising pigs. In yet another example, the method of the Disclosure may be used for raising goats. In another example, the method of the present disclosure can be used for raising horses. In yet another example, the method of the present disclosure can be used for raising camelid animals (e.g., alpacas).

[0116] Examples Example 1: Continuous twinning of embryos using the "cutting method" This example outlines the experimental steps involved in performing a "cutting method" to produce multiple embryos, such as identical embryos.

[0117] The 36 donor (blastocyst) embryos obtained from Ultrablack dairy cows produced by MOET are sourced from Nindooinbah (Beaudesert, QLD, Australia). The 36 MOET embryos are divided into the following test groups. Control group: Six embryos left uncut. Test group 1: Six embryos that have undergone one bisection. Test group 2: Six embryos undergoing two consecutive bisections (i.e., two consecutive cycles of bisection and expansion). Test group 3: Six embryos undergoing three consecutive bisections (i.e., three consecutive cycles of bisection and expansion). Test group 4: Six embryos undergoing four consecutive bisections (i.e., four consecutive cycles of bisection and expansion). Test group 5: Six embryos undergoing five consecutive bisections (i.e., five consecutive cycles of bisection and expansion).

[0118] In Test Group 1, donor blastocyst embryos are bisected by microdissection. After bisection, half of the embryos are cultured for 2, 4, or 6 days to assess the recovery of the number of inner cell masses (ICMs), the number of trophoblasts, and total embryonic viability. Optimal culture conditions are determined by analyzing fixed embryos using morphological embryo scoring, immunohistochemistry of ICM markers (e.g., Nanog, SOX2, OCT4), trophoblast markers (CDX2), and live / dead cell staining. Results are validated by qPCR on RNA isolated from 3–8 embryos.

[0119] The culture conditions identified in Test Group 1 as optimal for ICM expansion in bisected embryos will be applied to Test Groups 2-5. The success of consecutive bisections will be evaluated using the analysis described for Test Group 1 above.

[0120] For each of the test groups 3, 4, and 5, healthy-looking embryos that had expanded after a sequential bisection process were implanted into recipients, and pregnancy rates and healthy development of offspring were evaluated.

[0121] Example 2: Production of multiple monozygotic embryos using the "cookie cutter method" This example outlines the experimental steps taken when performing the "cookie-cutter method" for producing multiple identical embryos.

[0122] The 24 donor (blastocyst) embryos produced by MOET from Ultrablack dairy cows are sourced from Nindooinbah (Beaudesert, QLD, Australia). The 24 MOET embryos are divided into the following test groups. Control group: Six embryos left uncut. Test group 1: Six embryos that have undergone bisection. Test group 2: 6 embryos that will develop into one-quarter Test group 3: Six embryos cut into eighths.

[0123] All embryo division is performed by veterinarians at Nindooinbah, according to the standards of the test group.

[0124] Six embryos in the control group remain uncultured, but embryo fragments / sections produced by cleavage are cultured for up to 7 days to evaluate the number of inner cell masses, trophoblasts, and the recovery of embryonic viability.

[0125] After culturing, 3 to 8 fixed embryos are analyzed by morphological embryo scoring, immunohistochemistry of ICM markers (e.g., Nanog, SOX2, OCT4, etc.), trophoblast marker (CDX2), and live / dead cell staining. The results are validated by qPCR on RNA isolated from each of the 3 to 8 embryos.

[0126] Example 3: Production of multiple identical embryos using the "thawing method" This example outlines the experimental steps of performing a "thawing method" to produce multiple identical embryos from a single donor embryo.

[0127] Using a mobile laboratory in Nindooinbah (Beaudesert, QLD, Australia), we will provide time-fed oocytes inseminated with semen from one bull. Eight 4-cell donors and four 8-cell embryos will be provided.

[0128] After disrupting the zona pellucida of the embryo using pronase treatment of the embryo according to a published protocol, single blastomeres and diblastomere aggregates are isolated and deposited in separate wells of a multi-well plate containing culture medium for expansion.

[0129] After culturing, fixed embryos are analyzed by morphological embryo scoring, immunohistochemistry of ICM markers (e.g., Nanog, SOX2, OCT4, etc.), trophoblast marker (CDX2), and live / dead cell staining. The results are validated by qPCR on RNA isolated from 3-8 embryos.

[0130] Twelve of the most aesthetically pleasing swollen blastomeres were cultured for seven days, and four of the most aesthetically pleasing blastocysts (whether derived from a single or double blastomeres) were implanted into recipients to evaluate pregnancy rates and healthy development of offspring.

[0131] Example 4: Production of multiple embryos by continuous division This example describes an experiment in which the inventors performed continuous proliferation of donor embryos (also referred to herein as conception products) using the "thawing method" to produce multiple blastocysts. This example demonstrates that the continuous proliferation method of this disclosure using the "thawing" technique can significantly improve the efficiency of blastocyst production compared to standard culture of intact conception products without proliferation.

[0132] method Preparation of culture medium for conception products Drugs and stock solutions Unless otherwise noted, stock solutions for culturing and thawing the culture media were prepared according to Table 1. All culture medium reagents used in this study were obtained from Sigma. Stock solutions were prepared using Milli-Q® water. Using a vapor pressure osmometer (Wescor), the osmotic pressures of NaCl, KCl, and NaHCO3 were adjusted to 2000 mOsm, 200 mOsm, and 2000 mOsm, respectively, with Milli-Q® water.

[0133] Table 1, Stock solutions used to prepare culture and thawing media. [Table 1]

[0134] Preparation of culture media for embryonic product culture and thawing. NbryoIVC-2Ca 2+ The culture medium was used for the conception product thawing procedure, and NbryoIVC-3 medium was used for conception product culture before and after thawing (up to day 8 of development). The culture medium was prepared by adding stock solutions in the order shown in Table 2. The pH of the medium was adjusted to 7.4 by adding 2MNaOH. The osmotic pressure of the medium was tested using an osmometer (Wescor). The osmotic pressure was adjusted to 270 mOsm by adding MilliQ® 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 filtered and sterilized using a 0.22 μm syringe filter (Millipore). The medium was stored at 4°C for up to 2 weeks.

[0135] Table 2, NbryoIVC-2 Ca 2+ Composition of free and NbryoIVC-3 medium [Table 2]

[0136] Pronase preparation Pronase is a proteolytic enzyme used to remove the zona pellucida (ZP) during the thawing procedure. Pronase was prepared at a final concentration of 0.3 mg / mL in HEPES buffer-NbryoIVC-3. It was then filtered and sterilized using a 0.22 μm syringe filter, divided into equal portions, and stored at -20°C.

[0137] In vitro development of bovine conception products Production of bovine fertilized eggs Bovine embryos were produced by IVF using ArtSolutions' commercial protocol. Bovine oocytes were matured in vitro (IVM) from ovaries collected from a cattle farm in Nindoonibah, following standard procedures. After 24 hours of IVM, the matured oocytes were fertilized in vitro with thawed semen from one fertile bull from the Nindoonibah cattle farm. 24 hours after IVF, the assumed embryos were transferred to VitroCleave (ArtSolutions) in vitro culture (IVC) medium. Unless otherwise noted, embryos were cultured in VitroCleave (ArtSolutions) IVC medium at 38.5°C under 7% O2 and 5% CO2. After IVF, embryos were cultured to 2, 4, 8, and 16 cells for 25–32, 32–42, 42–52, and 96–100 hours, respectively.

[0138] Prepare the thawing plate and dish. 0.1% PVA pre-coated plate To prevent ZP-free conception products from adhering to the plate, the wells of a 96-well round-bottom plate (Corning) were coated by adding 50 μL of sterile 0.1% PVA in sterile water to each well, and incubated overnight at 38.5°C. Each well was then washed three times with sterile water to remove unbound PVA. The plate was then dried, sealed, and stored at 4°C until use.

[0139] Defrosted dish Before the thawing procedure, add 20 μL of pronase droplets, NbryoIVC-2 Ca 2+A 55 mm petri dish (Corning) containing NbryoIVC-3 medium layered with free and mineral oil (Coopers Scientific) was equilibrated at 38.5°C under 7% O2 and 5% CO2 for at least 60 minutes.

[0140] Post-thawing culture system After thawing, individual blastomeres were cultured using 96-well plates pre-coated with 0.1% PVA. Each well contained 50 μL of NbryoIVC-3 or 20 μL of VitroBlast (ArtSolutions) IVC medium, topped with mineral oil to prevent evaporation of the medium. Before transplanting the isolated blastomeres into each well, the plates containing the medium were equilibrated at 38.5°C under 7% O2 and 5% CO2 for at least 60 minutes.

[0141] Sequential decompression procedure All bovine embryo thawing procedures were performed under a dissecting microscope with plates heated to 37°C. In the first serial thawing (sequence n=1), one of the 2-cell, 4-cell, 8-cell, or 16-cell embryos was pronase-treated at 38.5°C for 2 minutes in a humidified incubator with a 7% O2, 5% CO2 atmosphere to remove surrounding ZP. Once the ZP was dissolved, the embryo was washed with three 20 μL NbryoIVC-3 droplets of medium to wash away any remaining pronase and incubated at 38.5°C under 7% O2, 5% CO2 for 10 minutes. Subsequently, the ZP-free embryo was treated with NbryoIVC-2Ca 2+ The cells were transplanted into free medium and placed at 38.5°C under 7% O2 and 5% CO2 for 3 minutes to reduce intercellular contact. Then, NbryoIVC-2Ca 2+ Blastomes of each conception product were separated from free medium by aspiration using a micropipette (approximately 120 μm in diameter). Each blastomere was cultured individually in PVA pre-coated wells containing assigned medium at 38.5°C under 7% O2 and 5% CO2 conditions.

[0142] Blastomeres that had undergone several thawing procedures (n=2, n=3, or n=4 consecutive times) were thawed after division. In the second thawing procedure (n=2 consecutive times), blastomeres that had undergone cleavage were thawed at 38.5°C under 7% O2 and 5% CO2 and then treated with NbryoIVC-2Ca 2+ The cells were left in free medium for 1–3 minutes. As previously mentioned, blastomeres were separated by aspiration using a micropipette and cultured individually in PVA pre-coated wells containing assigned medium. This process was repeated in the third thawing procedure (n=3 consecutive) and the fourth thawing procedure (n=4 consecutive). Based on the developmental stage of the blastomeres (cleavage, compression, cavitation, small blastocyst), blastomeres were scored every 12–24 hours until day 8 of preimplantation development. If the cavity was larger than half the volume of the embryo and there were aggregated clusters of ICM cells, the embryo was classified as a blastocyst.

[0143] Naming and Definition of Example 4 As described herein, the term “embryo” refers to the embryo formed when two haploid gamete cells (e.g., an unfertilized oocyte and a spermatocyte) combine to form a diploid totipotent cell (e.g., a fertilized egg), as well as the embryo resulting from subsequent cell division (i.e., embryonic cleavage) (including the morula stage (i.e., the approximately 16-cell stage) and the blastocyst stage, in which the trophectoderm and inner cell mass have differentiated). As described herein, “conception product” refers to the developing embryo from fertilization to the appearance of the primitive streak (corresponding to day 18 of development in cattle). Since the inventors cultured cattle fertilized eggs and individual blastomeres up to 8 days post-fertilization (up to the blastocyst stage), the term “conception product” has been used to describe the developing entity.

[0144] Blastomes isolated from 2-cell, 4-cell, 8-cell, and 16-cell stage embryonic products are referred to herein as "1:2," "1:4," "1:8," and "1:16" blastomeres, respectively, where the numerator represents the number of blastomeres in the cell mass and the denominator represents the corresponding embryonic product stage of the blastomere. Figure 1 illustrates this naming system for thawing 2-cell embryonic products via n=4 consecutive thaws. Figure 1 is a schematic diagram of the classification scheme of blastomeres and developing embryos in four thaws from a 2-cell stage embryo. The left side of Figure 1 represents the normal development of a preimplantation embryonic product from the oocyte stage to the blastocyst stage. In the thawing procedure shown on the right side of Figure 1, the ZP is removed to separate the individual blastomeres within the embryonic product (n=1 consecutive). Individual blastomeres isolated from a 2-cell embryonic product are called 1:2. These blastomeres are then developed to form pairs named 2:4. After cleavage, these 2:4 blastomeres can be thawed again (n=2 consecutively), in which case the blastomeres are separated into a 1:4 ratio. This process can be repeated n times (e.g., n=3 and / or n=4 or more). As these blastomeres develop, they progress to the subsequent corresponding preimplantation product stages, so the denominator changes accordingly. After the 1:16 stage, the blastomeres can be compressed and cavitated to form something equivalent to a blastocyst.

[0145] result Thawing of two-cell bovine conception products via a sequential n=4 thawing procedure A total of 28 two-cell bovine conception products were thawed. After a sequential n=1 thawing procedure, 56 1:2 blastomeres were obtained (see Table 2 and the nomenclature in Figure 1). 55 / 56 of the 1:2 blastomeres were divided into 2:4, of which 48 underwent another thawing (sequential n=2). After sequential n=2 thawing, 91 / 96 of the 1:4 blastomeres were divided into 2:8, of which 81 underwent another thawing (sequential n=3). After a sequential n=3 thawing procedure, 145 / 162 of the 1:8 blastomeres were divided into 2:16, of which 100 underwent another thawing (sequential n=4). All individual blastomeres from sequential n=1, n=2, and n=3 were cultured in 50 μL of NbryoIVC-3 medium. Finally, after a series of n=4 thawing procedures, 199 1:16 blastomeres were obtained and allowed to progress to the blastocyst stage in 20 μL of VitroBlast (ArtSolutions) IVC medium. Of these 199 1:16 blastomeres, 171 (85.9%) underwent cleavage, 156 (78.4%) underwent compression, 127 (63.8%) underwent cavitation, and 53 (26.6%) progressed to form small blastocysts.

[0146] Literature consistently reports that intact bovine embryos can be cultured to the blastocyst stage with an efficiency of approximately 30% (as shown in Table 2), which is the experience of the inventors, who have extensive experience in the culture and transplantation of livestock embryos. Therefore, if 28 two-cell donor embryos were cultured without the thawing procedure described herein, the yield would be expected to be 8 blastocysts, based on an efficiency of approximately 30%. By using the serial thawing procedure described above, the inventors were able to produce 53 blastocysts from the initial 28 donor embryos, thereby improving the efficiency of blastocyst production by approximately six times compared to culturing intact embryos.

[0147] Table 2, Development of blastocyst-like structures derived from two-cell bovine conception products thawed by a serial thawing procedure (n=4 consecutively). [Table 2]

[0148] Example 5: Production of multiple embryos by thawing ≥8 cell bovine embryos This example describes an experiment in which the inventors thawed ≥8 cell donor embryos using a "thawing method" and produced multiple blastocysts.

[0149] method The culture medium, culture conditions, and thawing procedure for the conception product are generally as described in the "Methods" section of Example 4.

[0150] In short, we thawed ≥8-cell stage embryonic products from a total of 19 cows. The thawed embryonic products contained 8 to 43 cells. The isolated blastomeres were cultured individually in 20 μL of VitroBlast (ArtSolutions) IVC medium. Because cell cleavage in the embryonic products is not synchronized, these embryonic products contain heterogeneous stages of blastomere development consisting of 1:8, 1:16, 1:32, and 1:64 blastomeres. As a result, each type of blastomere was analyzed individually.

[0151] result From 19 conception products, 53 1:8 blastomeres were obtained, of which 38 (71.7%) cleaved, 34 (64.1%) compressed, 25 (47.2%) cavitated, and 21 (39.6%) formed blastocysts (Table 2.2). Additionally, 162 1:16 blastomeres were obtained, of which 126 (50%) cleaved, 105 (64.8%) compressed, 99 (61.1%) cavitated, and 36 (22.2%) formed blastocysts. 54 1:32 blastomeres were obtained, of which 47 (87.0%) cleaved, 45 (83.3%) compressed, 44 (81.4%) cavitated, and 3 (5.6%) formed blastocysts. Some 1:32 blastomeres were cultured in pairs (i.e., 2:32). Specifically, two 2:32 blastomeres were obtained from the thawing procedure, of which two (100%) cleaved, one (50.0%) compressed, one (50.0%) cavitated, and zero (0%) formed blastocysts. Finally, 27 1:64 blastomeres were obtained, of which 13 (48.1%) cleaved, 11 (40.7%) compressed, 11 (40.7%) cavitated, and one (3.7%) formed blastocysts. In total, 61 blastocysts were produced from the initial 19 donor embryos. This is summarized in Table 3.

[0152] Starting with ≥8-cell stage embryos containing embryonic products with blastomeres equivalent to 16-cell, 32-cell, and 64-cell embryos in development, the inventors demonstrated that embryo proliferation using the "thawing" method can significantly improve the efficiency of blastocyst production. In this regard, the inventors were able to produce 61 blastocysts from 19 initial donor embryonic products, which represents a tenfold increase in blastocyst production efficiency compared to culturing intact embryonic products (which typically achieves an efficiency of approximately 30%, as mentioned above).

[0153] Table 3, Development of blastocysts equivalent to bovine conception products with ≥8 cells thawed by a single sequential thawing procedure (n=1 consecutively). [Table 3]

[0154] Those skilled in the art will understand that many changes and / or modifications can be made to the embodiments described above without departing from the broad general scope of this disclosure. Therefore, these embodiments are considered illustrative rather than restrictive in all respects.

[0155] (Related applications) This application claims priority to Australian Provisional Application No. 2020902691, filed on 31 July 2020, the entirety of which is incorporated herein by reference.

[0156] (Note) (Note 1) A method for growing one or more donor embryos, (i) The step of obtaining one or more donor embryos containing at least two embryonic cells, (ii) The step of separating one or more embryonic cells from one or more donor embryos, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple embryos, each containing at least two embryonic cells, (iv) a step of isolating one or more of the multiple embryos produced in step (iii) to be used as donor embryos in subsequent proliferation, and (v) A method comprising the step of repeating steps (i) to (iv) above "n" ("n" ≥ 3) times.

[0157] (Note 2) n is 4 or greater, as described in Appendix 1.

[0158] (Note 3) The method according to Appendix 1 or 2, wherein 16 or more identical embryos are produced from the donor embryos obtained in step (i) above.

[0159] (Note 4) The method according to any one of the appendices 1 to 3, wherein each of the one or more donor embryos contains 2 to 64 embryonic cells.

[0160] (Note 5) The method according to any one of the appendices 1 to 4, wherein each of the one or more donor embryos contains 2 to 16 embryonic cells.

[0161] (Note 6) A method for growing donor embryos, (i) A step of obtaining a donor embryo containing one or more embryonic cells that are developmentally equivalent to the embryonic cells from a 16-cell embryo or a pre-compressed morula, (ii) the step of separating one or more embryonic cells from the donor embryo, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple identical embryos from the donor embryo, and (iv) A method comprising the step of culturing the multiple monozygous embryos under conditions suitable for producing multiple monozygous blastocysts.

[0162] (Note 7) Prior to the step of culturing the plurality of monozygotic embryos to produce the plurality of blastocysts, the method is as follows: (i) A step of isolating one or more of the plurality of monozygotic embryos produced for use as donor embryos in subsequent proliferation, wherein each donor embryo isolated for subsequent proliferation contains at least two embryonic cells, (ii) The step of separating one or more embryonic cells from one or more donor embryos, (iii) The step of expanding the embryonic cells in vitro under conditions suitable for producing multiple embryos, each containing at least two embryonic cells, (iv) a step of isolating one or more of the multiple embryos produced in step (iii) to be used as donor embryos in subsequent proliferation, and (v) The method according to Appendix 6, further comprising the step of repeating steps (i) to (iv) n times before culturing the multiple embryos under conditions suitable for producing multiple blastocysts.

[0163] (Note 8) n is 2 or greater, as described in Appendix 7.

[0164] (Note 9) n is 3 or greater, as described in Appendix 7.

[0165] (Note 10) n is 4 or greater, as described in Appendix 7.

[0166] (Note 11) The method according to any one of the appendices 1 to 10, wherein the donor embryo is divided into multiple parts, each part containing one or more embryonic cells, thereby achieving the separation of one or more embryonic cells from the one or more donor embryos.

[0167] (Note 12) The method according to Appendix 11, wherein one or more donor embryos are divided using a microsurgical blade or laser.

[0168] (Note 13) The method according to any one of the appendices 1 to 10, wherein the zona pellucida (ZP) is destroyed and one or more embryonic cells are isolated from the one or more donor embryos, thereby achieving the isolation of one or more embryonic cells from the one or more donor embryos.

[0169] (Note 14) The method according to Appendix 13, wherein the ZP is enzymatically or mechanically destroyed, and the embryonic cells are isolated by aspirating one or more embryonic cells from one or more donor embryos using a micropipette.

[0170] (Note 15) The method according to any one of the appendices 1 to 14, wherein the embryonic cells are cultured in the presence of one or more factors that can promote embryonic development.

[0171] (Note 16) The method according to any one of the appendices 1 to 15, wherein the embryonic cells are cultured in the presence of one or more factors that can promote totipotency.

[0172] (Note 17) The method according to any one of the appendices 1 to 16, wherein the donor embryo is derived from a mammalian species.

[0173] (Note 18) The method described in Appendix 17, wherein the mammalian species is a domesticated species.

[0174] (Note 19) The method described in Appendix 18, wherein the livestock species is cattle.

[0175] (Note 20) The method according to any one of the appendices 1 to 19, wherein the one or more donor embryos in step (i) are produced by in vivo fertilization.

[0176] (Note 21) The method according to any one of the appendices 1 to 20, wherein the one or more donor embryos in step (i) are produced by in vitro fertilization (IVF).

[0177] (Note 22) The method according to any one of the appendices 1 to 21, wherein the one or more donor embryos in step (i) are fresh.

[0178] (Note 23) The method according to any one of the appendices 1 to 22, wherein the one or more donor embryos in step (i) are cryopreserved.

[0179] (Note 24) The method according to any one of the appendices 1 to 23, further comprising the step of selecting one or more donor embryos based on one or more genetic screening criteria, genetic diagnostic criteria, and / or one or more morphological criteria, prior to step (i) above.

[0180] (Note 25) The method according to any one of the appendices 1 to 24, wherein one or more donor embryos are genetically modified.

[0181] (Note 26) The method according to Appendix 25, wherein the one or more donor embryos include a unique genetic tag or identifier for traceability of embryos produced therefrom and / or animals produced from such embryos.

[0182] (Note 27) The method according to any one of the appendices 1 to 26, wherein the multiple embryos produced are expanded in vitro to form blastocysts.

[0183] (Note 28) The method according to any one of Appendix 1 to 27, further comprising the step of harvesting the embryo produced by the method described above.

[0184] (Note 29) The method according to Appendix 28, wherein one or more of the harvested embryos are stored in an embryo-holding medium.

[0185] (Note 30) The method described in Appendix 29, wherein one or more of the harvested embryos are stored at approximately 4°C.

[0186] (Note 31) The method described in Appendix 28, wherein one or more of the harvested embryos are cryopreserved.

[0187] (Note 32) The method according to any one of the appendices 1 to 31, further comprising the step of transferring one or more of the embryos produced by the above method into the fallopian tube of one or more recipe embryos.

[0188] (Note 33) One or more embryos produced by the method described in any one of the appendices 1 to 32.

[0189] (Note 34) A method of raising animals, (i) The step of transferring one or more embryos produced by any one of the methods described in Appendix 1 to 32 into the fallopian tube of one or more recipient females to establish pregnancy, and (ii) A method comprising the step of producing an animal by birth from the pregnant recipe female.

[0190] (Note 35) The method described in Appendix 34, wherein the animal is a mammal.

[0191] (Note 36) The method described in Appendix 35, wherein the mammalian species is a domesticated species.

[0192] (Note 37) The method described in Appendix 36, wherein the livestock species is cattle.

Claims

[Claim 1] A method for growing one or more donor embryos, (i) The step of obtaining one or more donor embryos containing at least two embryonic cells, (ii) The step of separating one or more embryonic cells from one or more donor embryos, (iii) A step of expanding the embryonic cells in vitro under conditions suitable for producing multiple embryos, each containing at least two embryonic cells. (iv) a step of isolating one or more of the multiple embryos produced in step (iii) above, which will be used as donor embryos in subsequent proliferation, and (v) A method comprising the step of repeating steps (i) to (iv) "n" ("n" ≥ 3) times.

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