A method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines

Abstract

The present invention discloses a method for propagating white-feathered broiler chickens using primordial germ cell stem cell lines, in which the primordial germ cell stem cell lines of white-feathered broiler chickens cultured in a laboratory for a long time are injected into the embryos of laying hens, and white-feathered broiler chickens are obtained from the next generation of laying hens. Compared with the traditional method, the method for propagating white-feathered broiler chickens using primordial germ cell stem cell lines provided by the present invention provides a new breeding platform, and solves the problems of long time consumption, difficult management and animal welfare in the breeding process of white-feathered broiler chickens.

Description

Technical Field

[0001] The invention belongs to the technical field of chicken biological breeding, and in particular relates to a method for multiplying white-feathered broiler chickens by using a primordial germ cell stem cell line. Background Art

[0002] White-feathered broilers have become the most effective way to produce animal protein in the breeding industry due to their fast growth rate and low feed-to-meat ratio. The high productivity of white-feathered broilers is obtained through long-term genetic selection.

[0003] The propagation of white-feathered broiler chickens starts with purebred stock chickens. Through a multi-level amplification method of great-grandparent stock chickens, grandparent stock chickens, parent stock chickens to commercial broiler chickens, one white-feathered stock can produce hundreds of thousands to millions of offspring broiler chickens.

[0004] Long-term genetic selection makes broilers grow faster and faster, while their appetites become bigger and bigger, which leads to excessive obesity and reduced fertility. The production of broiler breeders generally adopts a feed restriction management method, but restricted feeding causes other problems in broiler breeders, such as long-term hunger and stress response, thus becoming a problem of production management and animal welfare. These problems make further genetic selection of broiler breeders more difficult. Under the existing system, white-feathered broiler breeding seems to have reached its limit.

[0005] The existing method of breeding white-feathered broiler chickens is costly, difficult to manage, and time-consuming (it takes 2-3 years for a white-feathered superior breed to reach the market). In addition, the breeder chickens cannot be reused for a long time and need to be constantly re-bred and bred.

[0006] Therefore, there is an urgent need for a new method for rapidly multiplying broiler chickens in the art. Summary of the invention

[0007] As previously mentioned, there is a need in the art for a method of rapidly multiplying broiler chickens.

[0008] The inventor selected suitable primordial germ cells of white-feathered broiler chickens for long-term culture to establish a primordial germ cell stem cell line, then injected a suitable number of cells derived from the primordial germ cell stem cell line into high-yielding laying hen embryos, and then obtained white-feathered broiler chicks from the next generation of fertilized eggs of laying hens. Thus, the present invention was realized.

[0009] Therefore, the present invention provides a method for multiplying white-feathered broiler chickens using a primordial germ cell stem cell line, the method comprising the following steps:

[0010] 1) isolating primordial germ cells from the reproductive crescent tissue of a white-feathered broiler embryo, and culturing the primordial germ cells to establish a primordial germ cell stem cell line;

[0011] 2) injecting cells derived from the primordial germ cell stem cell line into blood vessels of laying hen embryos at stages 13-18;

[0012] 3) continuing to incubate the laying hen embryos until hatching, and retaining the hatched chimera chicks of the same sex;

[0013] 4) raising the chimera chicks of the same sex until sexual maturity, and then performing artificial insemination or natural mating to produce fertilized eggs;

[0014] 5) incubating the fertilized eggs until the chicks hatch, and distinguishing the varieties of the chicks according to their characteristics.

[0015] The method of the present invention utilizes the characteristics of primordial germ cells that can be cultured and proliferated for a long time and can be frozen and preserved at any time, breaking through the limitations on the number and time of white-feathered broiler breeding, so that a white-feathered superior breed can be infinitely propagated for a long time and reused; in addition, it also provides a new platform for white-feathered broiler breeding, solving the difficulties in white-feathered broiler breeding management and animal welfare issues. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 Schematic diagram of the method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines according to the present invention.

[0018] Figure 2 The present invention is a schematic diagram of a specific embodiment of the method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines.

[0019] Figure 3 This is a schematic diagram of another specific embodiment of the method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines of the present invention.

[0020] Figure 4 This is a schematic diagram of yet another specific embodiment of the method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines of the present invention.

[0021] Figure 5 This is a schematic diagram of another specific embodiment of the method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be described clearly and completely below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments that can be obtained by those of ordinary skill in the art based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0023] Before the present invention is described in detail, it should be particularly pointed out that although the method of the present invention is described in this article by using steps 1), 2), etc., it should be understood that the numbering is only used to distinguish the steps, and is not intended to limit the order of the steps in a sequential order. These steps can be performed in the order of the numbering, can be performed in the opposite order of the numbering, can be performed in any other order, or can be performed simultaneously, unless it can be clearly judged from the context that they need to be performed in a specific order. For each action involved in a certain step, similarly, it can be performed in the order of the textual expression, can be performed in the opposite order of the textual expression, or can be performed in any other order, or can be performed simultaneously, unless it can be clearly judged from the context that they need to be performed in a specific order.

[0024] As mentioned above, the existing method of breeding white-feathered broiler chickens is costly, difficult to manage, and time-consuming. In addition, the breeder chickens cannot be reused for a long time and need to be constantly re-bred and bred.

[0025] Therefore, the present invention provides a method for multiplying white-feathered broiler chickens using a primordial germ cell stem cell line, the method comprising the following steps:

[0026] 1) isolating primordial germ cells from the reproductive crescent tissue of a white-feathered broiler embryo, and culturing the primordial germ cells to establish a primordial germ cell stem cell line;

[0027] 2) injecting cells derived from the primordial germ cell stem cell line into blood vessels of laying hen embryos at stages 13-18;

[0028] 3) continuing to incubate the laying hen embryos until hatching, and retaining the hatched chimera chicks of the same sex;

[0029] 4) raising the chimera chicks of the same sex until sexual maturity, and then performing artificial insemination or natural mating to produce fertilized eggs;

[0030] 5) incubating the fertilized eggs until the chicks hatch, and distinguishing the varieties of the chicks according to their characteristics.

[0031] The steps of the method of the present invention are described in more detail below.

[0032] Step 1): isolating primordial germ cells from the reproductive crescent tissue of white-feathered broiler embryos, and culturing the primordial germ cells to establish a primordial germ cell stem cell line.

[0033] In this article, the term "primordial germ cell (Primordial Germ Cell)" is sometimes expressed as "PGC", which refers to the ancestral cell of the diploid germ cells in the embryo, which will eventually develop into haploid gametes, such as sperm or eggs. The term "primordial germ cell stem cell line" refers to a cell line obtained by culturing primordial germ cells for at least 20 days to maintain their reproductive stem cell properties, that is, the ability to eventually develop into sperm or eggs. Primordial germ cells can be cultured in the laboratory, and after at least 20 days of culture, their number can reach more than 2 million.

[0034] The white-feathered broiler embryos can be embryos of white-feathered broilers from the original generation, grandparent generation or parent generation. Those skilled in the art know that white-feathered broilers adopt a four-line breeding method, in which the A and B lines are male lines, and the C and D lines are female lines. Male broilers grow faster and have a lower feed-to-meat ratio, but they lay fewer eggs, less than 100 eggs per year. Maternal breeding takes into account both egg laying and meat growth, and its growth rate and feed-to-meat ratio are not as good as those of the male line. In the present invention, white-feathered broilers of these four systems can be used in combination of male and female, but it is preferred to use male white-feathered broilers.

[0035] Primordial germ cells are isolated from the reproductive crescent tissue of a donor embryo (in the present invention, a white-feathered broiler embryo) at a suitable period (e.g., a period starting from stage 4 (H&H period)). For example, the white-feathered broiler embryo is a white-feathered broiler embryo that is in stage 5-10 (H&H period) after a short period of incubation (e.g., about 32 hours). The volume of primordial germ cells is generally twice that of somatic cells, so it is easy to distinguish and separate the two based on their size. The isolated primordial germ cells are cultured for a sufficiently long period of time to obtain a sufficient number (e.g., 2×10 7The primordial germ cells of the present invention are prepared by adding 10-100 ng / mL acidic fibroblast growth factor (aFGF) and 4-10 ng / mL basic fibroblast growth factor (bFGF) to the primordial germ cell stem cell line, thereby establishing the primordial germ cell stem cell line for injection. In one embodiment, in step 1), the primordial germ cells are cultured for 20-60 days in a culture medium such as KO-DMEM culture medium comprising 10-100 ng / mL acidic fibroblast growth factor (aFGF) and 4-10 ng / mL basic fibroblast growth factor (bFGF), to establish the primordial germ cell stem cell line. The culture medium comprising acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) is also referred to as PGC culture fluid in this article. The acidic fibroblast growth factor and basic fibroblast growth factor can be from any source, for example, derived from humans, or derived from other animals, such as mice, rats, chickens, etc. In addition, the acidic fibroblast growth factor and basic fibroblast growth factor can be directly separated and purified from cells expressing these factors, can also be obtained by recombinant means, and can also be directly commercially available. In one embodiment, the PGC culture medium comprises: KO-DMEM, rat hepatocyte (Buffalo Rat Liver BRL) KO-DMEM conditioned medium, fetal bovine serum, chicken serum, glutamine, non-essential amino acids, sodium pyruvate, β-mercaptoethanol, penicillin / streptomycin, human acidic fibroblast growth factor (hFGFa), and human basic fibroblast growth factor (hFGFb). In a specific embodiment, the PGC culture medium comprises: 46% KO-DMEM, 40% rat liver cell (Buffalo Rat Liver BRL) KO-DMEM conditioned medium, 7.5% fetal bovine serum, 2.5% chicken serum, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 0.1% β-mercaptoethanol, 1% penicillin / streptomycin, 10-100 ng / mL human acidic fibroblast growth factor (hFGFa), 4-10 ng / mL human basic fibroblast growth factor (hFGFb), wherein the percentage is the final concentration volume percentage of each substance. Further preferably, in the culture medium, the concentration of human acidic fibroblast growth factor (hFGFa) is 50 ng / mL, and the concentration of human basic fibroblast growth factor (hFGFb) is 10 ng / mL. In one embodiment, the cell culture is continued for 20-60 days, thereby obtaining a considerable amount (e.g., 2×10 7 above) of the primordial germ cells.

[0036] After the cultivation is completed, the cultured primordial germ cells can be directly injected (such as microinjection) into the recipient embryo to implement the method of the present invention, or the cultured primordial germ cells can be frozen in liquid nitrogen for standby use. In the freezing process, a liquid preparation obtained by adding 10% fetal bovine serum and 10% DMSO to the above-mentioned PGC culture solution can be used as a cryoprotectant. Of course, any other cryoprotectant suitable for freezing primordial germ cells can be used, and there are no special requirements for this. Such primordial germ cells can be frozen for a long time, which is equivalent to turning the PGC cell line in the liquid nitrogen tank into a seed farm for broiler breeders.

[0037] It should be noted that, according to step 3), it is understood that the primordial germ cells need to be sexed (see below for details). Therefore, in the process of isolating or culturing primordial germ cells, male and female cell lines need to be placed and cultured separately.

[0038] Step 2): Injecting cells derived from the primordial germ cell stem cell line into blood vessels of laying hen embryos at stages 13-18.

[0039] First, the primordial germ cells can be cells of a primordial germ cell stem cell line derived from primordial germ cells isolated from the reproductive crescent tissue of a white-feathered broiler embryo and further cultured to obtain the primordial germ cells, or they can be primordial germ cells of the primordial germ cell stem cell line that are cryopreserved and further cultured after recovery.

[0040] Before the primordial germ cell injection, the recipient chicken embryo, i.e., the laying hen embryo, has been incubated for a short time or has undergone preliminary development. Generally speaking, the injection is preferably carried out at the 13th to 18th stage (H&H stage) of the recipient chicken embryo development, and most preferably at the 15th stage. For chickens, according to the corresponding relationship between each stage of its embryonic development and time, the injection time is preferably at the 1st, 2nd, 3rd or 4th day of the chicken embryo development, and most preferably at the 2nd to 2.5th day of the chicken embryo development.

[0041] This step of injecting primordial germ cells can be carried out in advance to the recipient chicken embryo or without having carried out the situation of removing endogenous primordial germ cells.In this article, so-called " removing endogenous primordial germ cells " can be used interchangeably with term " sterilization ", refers to the treatment of removing the primordial germ cells of the recipient chicken embryo itself so that it can not produce its own gametes (comprising sperm and ovum) basically.

[0042] Therefore, in one embodiment of the present invention, the donor primordial germ cells are injected into the recipient embryos from which the endogenous primordial germ cells have been removed in advance. In this way, when the recipient embryo develops to sexual maturity, it can produce gametes or donor gametes that are all or substantially produced by the donor primordial germ cells. The operation of removing the endogenous primordial germ cells can be performed before and / or after step 2), depending on the removal method adopted. In addition, the endogenous primordial germ cells of the laying hen embryo can be removed by the following methods: chemical methods, such as busulfan (see, for example, U.S. Patent No. 6,691,638B2, the disclosure of which is incorporated herein by reference); physical methods, such as X-ray irradiation; or biological methods, such as methods for specifically inducing endogenous germ cells to cause death (see, for example, U.S. Patent No. 20210345592A1, the disclosure of which is incorporated herein by reference). It can be understood that the removal of endogenous primordial germ cells can be carried out by a single method or by multiple methods, depending on the circumstances. In addition, whether the removal of endogenous primordial germ cells is performed before or after the injection of donor primordial germ cells also depends on the circumstances. For physical or chemical removal methods, it needs to be performed before step 2), while for biological removal methods, it can be performed before or after step 2), but it is preferably performed after step 2). In addition, those skilled in the art can also understand that it is difficult to achieve 100% removal of endogenous primordial germ cells by physical or chemical removal methods, so the method of the present invention can also be supplemented by other methods that can identify the offspring breed. For example, laying hens with an appearance different from white-feathered broilers (for example, purebred black laying hens or bare-necked laying hens) can be used as recipient laying hens, so that the next generation of chicks of the recipient laying hens can be simply identified by using the characteristics of the coat color or feather growth state of the next generation of chicks (see details below).

[0043] In another embodiment of the present invention, donor primordial germ cells are injected into recipient laying hen embryos that have not been cleared of endogenous primordial germ cells in advance. It is understood that in this case, since the recipient laying hen also contains its own endogenous primordial germ cells, its offspring include not only purebred white broiler breeds, but also purebred laying hen breeds and hybrids of the two. To this end, in order to simply identify these offspring breeds, as described above, laying hens that are different in appearance from white broilers (e.g., purebred black laying hens or naked neck laying hens) can be used as recipient laying hens, thereby simply identifying the offspring breeds by using the coat color or feather growth status of the next generation of chicks of the recipient laying hen (see details below).

[0044] Regardless of whether the endogenous primordial germ cells in the recipient chicken embryos have been cleared beforehand, preferably, 3×10 4 -5×10 4In contrast, in the prior art, generally only a relatively small number of primordial germ cells are injected into each recipient chicken embryo, for example, only 100-1000 primordial germ cells are injected into each recipient chicken embryo. By injecting a large number of donor primordial germ cells into a laying hen embryo, the number of the donor primordial germ cells can be greatly exceeded by the endogenous laying hen primordial germ cells, so as to achieve a high proportion of broiler offspring produced by laying hens that have not been cleared of endogenous primordial germ cells.

[0045] In the process of injecting donor primordial germ cells, preferably adopt the injection needle of 35-37 micron of needle tip diameter.According to the accurate development period and position of embryo, the needle is injected into the embryo peripheral vein, embryo abdominal aorta or the heart that just begins to beat, stops in the body fluid of chicken top or stops in the chicken itself.Before inserting the needle, can beat or drill a hole or a group of holes on the eggshell, so as not to damage or destroy the needle.After injection finishes, can seal the described ovum with the sealing material that bacteria such as wax cannot penetrate basically, so that stop unwanted bacteria from entering subsequently.

[0046] Step 3): continue to incubate the laying hen embryos until hatching, and retain the hatched chimera chicks of the same sex.

[0047] After the injection is completed, the laying hen embryos are transferred to an incubator for further incubation until chicks are hatched. The laying hen embryos can be incubated using conventional conditions and methods known in the art, and there is no particular limitation as long as the laying hen embryos can eventually hatch chicks.

[0048] Those skilled in the art know that donor primordial germ cells have male and female genders, and recipient egg-laying chicken embryos also have male and female genders, so the hatched chicks will include four situations, namely: (1) male chicks containing male donor primordial germ cells, (2) female chicks containing female donor primordial germ cells, (3) female chicks containing male donor primordial germ cells, and (4) male chicks containing female donor primordial germ cells. Among them, the chicks involved in situations (1) and (2) are defined as same-sex chicks in this article, while the chicks involved in situations (3) and (4) are defined as opposite-sex chicks in this article. Therefore, in this step, only the same-sex chicks involved in situations (1) and (2) are retained, and the opposite-sex chicks involved in situations (3) and (4) are eliminated.

[0049] In order to distinguish the chicks in these cases, it is understood that the gender of the donor primordial germ cells (or donor chicken embryos) and the recipient chicken embryos needs to be identified. For the identification of the gender of the donor primordial germ cells, the remaining chicken embryo tissue after the separation of the primordial germ cells can be used, or the PGC itself separated and cultured can be used, and for the recipient chicken embryos, any suitable sample such as blood can be used to identify the gender after the chicks are hatched, or the chicks gender identification method commonly used in the production process of white-feathered broilers (such as the anus-turning identification method) can be used to save costs. In the inventive method, PCR can be used for gender identification, wherein commercially available PCR primers for identifying the gender of birds are used, such as the primers of SEQ ID NO:1 and SEQ ID NO:2. Of course, those skilled in the art know that any other method that can be used for gender identification is also usable, and is within the capabilities of those skilled in the art.

[0050] Step 4): raising the chimera chicks of the same sex until sexual maturity, and then performing artificial insemination or natural mating to produce fertilized eggs.

[0051] Artificial insemination or natural mating can be performed between a female chimera chicken of the same sex and a male chimera chicken of the same sex, or between a female chimera chicken of the same sex and a parent rooster of a white-feathered broiler chicken. As an example, the parent rooster of a white-feathered broiler chicken can be the offspring of a white-feathered broiler chicken obtained by the method of the present invention.

[0052] Step 5): Incubate the fertilized eggs until they hatch, and distinguish the breeds of the chicks according to their characteristics.

[0053] It is understandable that, when the endogenous primordial germ cells in the recipient chicken embryo are completely removed, the offspring obtained by artificial insemination or natural mating of the same-sex chimera chicks are all white-feathered broilers. Of course, this is an extremely ideal situation. The reality is that it is difficult to completely remove the endogenous primordial germ cells in the recipient chicken embryo, especially when they are removed by physical or chemical methods. In addition, as mentioned above, in some cases, the primordial germ cells of the recipient chicken embryo are not removed before the injection of the donor primordial germ cells. In the above two cases, the offspring inevitably include three varieties: purebred broilers, purebred laying hens, and hybrid chickens.

[0054] To differentiate between the three varieties, a number of different approaches can be considered.

[0055] For example, when both broiler and laying hen breeds have white feathers, the offspring breeds can be distinguished based on the growth rate and breast muscle growth of the chicks. Purebred broilers have the fastest growth rate and breast muscle growth, while purebred laying hens have the slowest growth rate and breast muscle growth, and those in between are hybrid chickens.

[0056] In addition, the method of the present invention can be carried out in other ways that can simply distinguish the varieties. For example, a laying hen whose appearance, such as hair color and feather growth state, is different from that of a white-feathered broiler can be used as a recipient laying hen, so that the characteristics of the hair color or feather growth state of the next generation of chicks produced by the recipient laying hen can be used to simply identify the offspring variety. As an example, a naked-necked laying hen can be used as a recipient laying hen. Therefore, in the newly hatched chicks, the white-feathered broiler is covered with uniform light yellow fluff all over the body, while the neck of the naked-necked laying hen is in a naked state without feather covering, and the appearance of the hybrid chicken is between the white-feathered broiler and the naked-necked laying hen. As another example, a black-feathered laying hen can be used as a recipient laying hen. Therefore, in the newly hatched chicks, the white-feathered broiler is covered with uniform light yellow fluff all over the body, the black-feathered laying hen has black feathers, and the feathers of the hybrid chicken are interspersed with black. In this way, the three varieties of purebred broilers, purebred laying hens and hybrid chickens can be easily distinguished.

[0057] The white-feathered broilers obtained in step 5) can be used as commercial broilers or as parent broilers for further propagation of white-feathered broilers. When further used in propagation of white-feathered broilers, broiler individuals that grow faster and have a lower feed-to-meat ratio than commercial broilers can be further selected, and the fertilized eggs obtained therefrom can be further used in steps 1) to 5) of the method of the present invention to achieve further selection and propagation of broilers.

[0058] Figure 1 is a schematic diagram of a method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines according to the present invention, Figure 2-5 Several specific embodiments of the method of the present invention are shown. The method of the present invention is described in more detail below with reference to these drawings.

[0059] Figure 1Schematic diagram of the method of using primordial germ cell stem cell line to propagate white-feathered broiler chickens of the present invention. As shown in the figure, first, broiler eggs are incubated (for example, at 37.8°C, 60% humidity) for a period of time (32 hours) to allow the broiler eggs to develop into stage 5-10 (H&H) embryos. Subsequently, primordial germ cells are separated from the reproductive crescent tissue of the broiler embryo, and the primordial germ cells are cultured to establish a primordial germ cell stem cell line (i.e., donor primordial germ cells), and then processed according to the specific circumstances. For example, the primordial germ cell stem cell line can be used for injection after culture, or after culture, freezing, recovery, and re-culture. For the fertilized eggs of laying hens as recipients, they can be incubated in advance for a period of time, for example, 2.5 days, so that the embryos develop to stage 13-18, and then the donor primordial germ cells are transported to the blood vessels of the recipient chicken embryo by microinjection. The endogenous PGCs of the recipient chicken embryo can be removed before and / or after the injection of the donor PGCs (not shown). As an example, the endogenous PGC of the recipient chicken embryo can be removed by chemical or physical methods before the injection of the donor PGC, and the endogenous PGC of the recipient chicken embryo can be further removed by biological methods after the injection of the donor PGC. Of course, it is understandable that the endogenous PGC may not be removed before the injection of the donor PGC, but the endogenous PGC may be specifically induced to kill by biological methods directly after the injection of the donor PGC. Of course, the endogenous PGC of the recipient chicken embryo may not be removed, and the offspring variety may be identified by other means. Subsequently, the egg-laying hen embryo is continued to be incubated until hatching, and the hatched chimera chicks of the same sex are retained. The chimera chicks of the same sex are raised to sexual maturity, and then the female chimera chimera chickens of the same sex are produced to produce fertilized eggs by artificial insemination or natural mating. The fertilized eggs are incubated until hatching, and the varieties are distinguished according to the characteristics of the chicks. The obtained purebred white feather broiler breed can be further used in the method of the present invention as a broiler for expanding the breed, or raised and marketed as a commercial broiler.

[0060] Figure 2 A specific embodiment of the method of the present invention is shown, in which white-feathered broilers and recipient laying hens are difficult to distinguish in appearance. In this case, if the endogenous primordial germ cells in the recipient chicken embryo can be 100% eliminated, the offspring of the laying hens are all broilers, which can be further used for broiler expansion or as commercial broilers. Of course, in the case where the endogenous primordial germ cells in the recipient chicken embryo cannot be 100%, it can be considered to distinguish the breeds based on the growth rate of the offspring of the laying hens and the growth status of the breast muscles, as described above.

[0061] Figure 3Another specific embodiment of the method of the present invention is shown, in which naked neck laying hens are selected as recipient laying hens. Since naked neck laying hens and white-feathered broilers have obvious differences in appearance, this scheme does not require the removal of endogenous PGCs in naked neck laying hens as recipient chickens. In this specific embodiment, since naked neck laying hens have appearance characteristics different from those of white-feathered broilers, although the endogenous PGCs of naked neck laying hens are not removed, the appearance of newly hatched chicks can be used to simply distinguish between the three breeds of purebred broilers (light yellow fluff), purebred naked neck laying hens (naked at the neck) and hybrid chickens (in between). Of course, those skilled in the art will appreciate that in this specific embodiment, the naked neck laying hens may also be subjected to endogenous PGC removal treatment.

[0062] Figure 4 Yet another specific embodiment of the method of the present invention is shown, in which black-feathered laying hens are selected as recipient laying hens. Since black-feathered laying hens and white-feathered broilers have obvious differences in appearance, this scheme does not require the endogenous PGC removal of black-feathered laying hens as recipient chickens. In this specific embodiment, since black-feathered laying hens have appearance characteristics different from those of white-feathered broilers, although the endogenous PGC of black-feathered laying hens is not removed, the appearance of newly hatched chicks can be simply used to distinguish between the three varieties of purebred broilers (light yellow fluff), purebred black-feathered laying hens (mainly black fluff) and hybrid chickens (light yellow fluff mixed with a little black fluff). Of course, those skilled in the art will understand that in this specific embodiment, the black-feathered laying hens can also be treated to remove endogenous PGC.

[0063] Figure 5Another specific embodiment of the method of the present invention is shown. In this method, female primordial germ cells are separated from the reproductive crescent tissue of the embryo of a female white-feathered broiler (especially a female white-feathered broiler of the AB line), and cultured to obtain a female primordial germ cell stem cell line, and injected directly into the recipient egg-laying chicken embryo, or injected into the recipient egg-laying chicken embryo after freezing, reviving, and re-culturing. In addition, since the recipient egg-laying chicken only receives female primordial germ cells, the offspring obtained from the recipient egg-laying chicken can only carry female primordial germ cells. Therefore, the egg-laying chicken gonad chimera hen (i.e., chimera chicken of the same sex) and the broiler parent generation cock are artificially inseminated or naturally mated to produce fertilized eggs, and the fertilized eggs can obtain more than 50% purebred white-feathered broilers and less than 50% hybrid 817 broilers after hatching. In addition, Hy-Line brown egg-laying chickens can be used as recipient egg-laying chickens, and in the process of donor PGC injection, 30,000 to 50,000 donor PGCs can be injected into the blood vessels of a recipient egg-laying chicken embryo. Both purebred white-feathered broilers and hybrid 817 broilers have considerable economic value. Here, the parent rooster of the broiler chicken can be the parent rooster used for isolating primordial germ cells in step 1) of the method of the present invention, or the purebred broiler chicken obtained by the method of the present invention.

[0064] The beneficial effects of the present invention include one or more of the following:

[0065] (1) High-yield laying hens can produce 300-340 eggs per year, while broiler parent breeders only produce 150-170 eggs. Using high-yield laying hens to replace broiler parent breeders can double the chick production efficiency.

[0066] (2) In the existing broiler breeding system, it takes 2 years from the grandparent generation to the parent generation and then to the commercial generation, while in the present invention, it only takes 6 months to inject primordial germ cells into commercial generation chicks, thus shortening the broiler breeding cycle;

[0067] (3) The weight of broiler parent breeders is more than twice that of laying hens, and their feed intake is at least 50% more per day, making them difficult to manage. The present invention uses laying hens to save breeder feed and management costs;

[0068] (4) High-quality primordial germ cell lines can be permanently stored in liquid nitrogen tanks (-196°C), which can reduce broiler breeding or import of new breeds; by using primordial germ cell culture and primordial germ cell expansion, the number of offspring produced by a broiler breed can be increased by millions of times; high-quality broiler breeds can be reused without the need for repeated breeding;

[0069] (5) Primordial germ cell expansion completely solves the animal welfare problem of long-term hunger of broiler parent breeders;

[0070] (6) Provide a new breeding platform to accelerate the breeding process of broiler chickens in China.

[0071] Example

[0072] The present invention is described in more detail below in conjunction with the embodiments. The test methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are obtained by purchasing from a conventional reagent store unless otherwise specified. It should be noted that the above summary of the invention and the detailed description below are only for the purpose of specifically illustrating the present invention and are not intended to limit the present invention in any way.

[0073] Example 1

[0074] This example provides a method for multiplying white-feathered broilers using primordial germ cell stem cell lines and high-yielding laying hens (white Leghorn chickens). Figure 1 and Figure 2 The method is described in detail below.

[0075] Establishment of white broiler PGC cell line:

[0076] The culture medium of the PGC cell line (PGC culture medium) is composed of 46% KO-DMEM, 40% rat liver cell (Buffalo Rat Liver BRL) KO-DMEM conditioned medium, 7.5% fetal bovine serum, 2.5% chicken serum, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 0.1% β-mercaptoethanol, 1% penicillin / streptomycin, and the above percentages refer to the final concentration volume percentage. 50 ng / mL recombinant human acidic fibroblast growth factor and 10 ng / mL recombinant human basic fibroblast growth factor were added before use.

[0077] The commercially available Ross 308 fertilized eggs (i.e., broiler eggs) were incubated at 37.8°C and 60% humidity for 32 hours to develop to stage 5-10 (H&H). Part of the germinal crescent tissue was then isolated from the embryos that developed to this stage, and the isolated germinal crescent tissue was transferred to a 1.5 mL centrifuge tube containing 200 μL PGC culture medium. The isolated germinal crescent tissue was then blown 40-50 times with a 200 μL pipette tip to make most of the cells into a single cell suspension, which was then transferred to a 48-well plate for culture, and irradiated rat hepatocytes were added as a trophoblast. From the start of culture to the 10th day of culture, half of the culture medium was removed every 2 days, the remaining half was moved to a new trophoblast, and half of the new PGC culture medium was added. After culturing for more than 20 days, the number of cells reached 2 million, and the number doubled every 1-2 days. The culture can be confirmed as a PGC stem cell line. The cultured PGC stem cell line can be frozen in a liquid nitrogen tank using a PGC culture medium supplemented with 10% fetal bovine serum and 10% DMSO for later use.

[0078] Cell line sex identification by PCR:

[0079] In a 1.5 mL Eppendorf centrifuge tube, more than 1000 cells of a cell line were suspended and lysed with 20 μL 0.1 M TrisHCl (pH 7.5), 20 μL 0.2 M NaOH was added, heated to 99°C and maintained at 99°C for 6 minutes. Then, 55 μL 0.1 M Tris HCl was added for neutralization, and centrifuged at 14,000 rpm for 5 minutes, and 2 μL of each sample was taken as a DNA template for PCR. The PCR primers for bird sex identification were USP1 (5'-CTATGCCTACCACMTTCCTATTTGC-3' (SEQ ID NO: 1), wherein M = A / C) / USP3 (5'-AGCTGGAYTTCAGWSCATCTTCT-3' (SEQ ID NO: 2), wherein Y = T / C, W = A / T, S = C / G) and control primers actin-F3 (5'-ATGCGCATAAAACAAGACGAGATT-3' (SEQ ID NO: 3)) / actin-R3 (5'-GGGGACTGTAAAGCCTTCATTCAC-3' (SEQ ID NO: 4)). The PCR amplification procedure is shown in Table 1.

[0080] Table 1: PCR amplification program

[0081]

[0082] Preparation of gonadal chimera chickens:

[0083] After the frozen PGC stem cell line was thawed, it was cultured for 5-7 days until it resumed growth, with the cell number doubling every 2 days. The PGC culture medium was collected and centrifuged at 500g for 5 minutes, and 2×10 4 -3×10 4 Resuspend the cells at a concentration of 100 / μL.

[0084] Bailaihang laying hens were used as recipient laying hens. Fertilized eggs of Bailaihang laying hens were placed in an incubator for incubation, and 1-1.5 μL of cell suspension was injected into each laying hen embryo on the 2.5th day of incubation, and then continued to incubate until hatching. Then, the chicks were sexed using the same method as the sex identification method described above, and only chicks of the same sex were retained, including male chicks containing male PGCs and female chicks containing female PGCs. Table 2 shows the hatching rate of recipient chicken embryos after injection of donor PGCs, from which it can be seen that the hatching rate can reach an average of more than 50%.

[0085] Table 2: Hatching rate of gonadal chimeras produced by injecting broiler PGC stem cell lines into laying hen embryos

[0086]

[0087] White-feathered broilers hatched from fertilized eggs of Bailaihang laying hens:

[0088] The identified chimera chicks of the same sex are raised to sexual maturity (about 160 days), and then the laying roosters and hens injected with the broiler PGC stem cell line are randomly artificially inseminated, and the fertilized eggs are collected and incubated until the chicks hatch. Since both white Laihang laying hens and white-feathered broilers have white feathers, it is impossible to distinguish between purebred broilers, hybrid chickens or purebred laying hens based on their coat color. Therefore, a feeding test is conducted on the hatched chicks to distinguish them based on their growth conditions. Specifically, all hatched chicks are fed broiler feed, and pure broiler and laying hens are fed with controls under the same conditions. After one week, purebred laying hens can be selected from purebred broilers and hybrid chickens due to their slow growth. The ratio of average gonadal transmission in broilers and the ratio of pure broiler offspring can then be calculated using the following formula:

[0089]

[0090]

[0091] Table 3 shows the proportion of white-feathered broilers produced by male and female chimera laying hens injected with PGCs calculated according to the above formula. It can be seen from the table that the proportion of purebred white-feathered broiler offspring ranges from 61.1% to 79.8%, with an average of 71.1%. It can be seen that the use of laying hens to expand white-feathered broilers has a considerable proportion of purebred white-feathered broiler offspring, which is extremely practical.

[0092] Table 3: Proportion of broiler chicks produced from male and female laying hens injected with PGC

[0093]

[0094] Example 2

[0095] This example provides another method for multiplying white-feathered broilers using primordial germ cell stem cell lines and high-yielding laying hens (black-feathered laying hens). The process can be referred to Figure 5 The method is described in detail below.

[0096] Establishment of PGC cell line from female white broiler chickens:

[0097] Two female PGC cell lines, AB2 and AB9, were cultured from embryos of the commercial Dai Ross 308 white broiler AB male line. The line establishment method was the same as the "Establishment of white broiler PGC cell line" in Example 1, and the sex identification of the cell line was the same as the "Sex identification of the cell line by PCR" in Example 1, and the female PGC cell line of white broiler chicken was retained.

[0098] Preparation of gonadal chimera chickens:

[0099] After thawing the frozen female PGC stem cell lines AB2 and AB9, they were cultured for 5-7 days until they resumed growth, with the cell number doubling every 2 days. The PGC culture medium was collected and centrifuged at 500 g for 5 minutes, and 2 × 10 4 -3×10 4 Resuspend the cells at a concentration of 100 / μL.

[0100] Pure black-feathered laying hens were selected from Jingfen No. 8 laying hens as recipient laying hens. Fertilized eggs of black-feathered laying hens were placed in an incubator for incubation, and 1-1.5 μL of cell suspension (about 3×10 4 Cells / laying hen embryo), followed by continued incubation until hatching. Then, the chicks were sexed using the same method as described in Example 1, and only chimera female chicks were retained.

[0101] Offspring hatched from fertilized eggs of black-feathered laying hens:

[0102] The identified chimera female chicks were raised to sexual maturity (about 160 days), and then artificially inseminated with white broiler parent roosters, and the fertilized eggs were collected and incubated until hatching. The hatched offspring included male broiler chickens and 817 hybrid chickens, and the results are shown in Table 4. As can be seen from the table, the percentage of white broiler chickens is as high as 82%-85%, which shows that the proportion of white broiler chickens obtained is extremely high.

[0103] Table 4: Results of multiplying male white broiler progeny

[0104]

[0105] The method for rapid propagation of white-feathered broilers provided by the present invention is introduced in detail above. Specific embodiments are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea; at the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A method for multiplying white-feathered broiler chickens using primordial germ cell stem cell lines, The following steps are involved: 1) isolating primordial germ cells from the reproductive crescent tissue of a white-feathered broiler embryo, and culturing the primordial germ cells to establish a primordial germ cell stem cell line; 2) Injecting cells derived from the primordial germ cell stem cell line into blood vessels of 13-18 stage laying hen embryos, wherein each laying hen embryo is injected with 3×10 4 -5×10 4 cells derived from the primordial germ cell stem cell line; 3) continuing to incubate the laying hen embryos until hatching, and retaining the hatched chimera chicks of the same sex; 4) raising the chimera chicks of the same sex until sexual maturity, and then performing artificial insemination or natural mating to produce fertilized eggs; 5) incubating the fertilized eggs until the chicks hatch, and distinguishing the breeds of the chicks according to their characteristics.

2. The method according to claim 1, It is characterized in that In step 1), the primordial germ cells are cultured in a culture medium containing 10-100 ng / mL acidic fibroblast growth factor and 4-10 ng / mL basic fibroblast growth factor for 20-60 days to establish the primordial germ cell stem cell line.

3. The method according to claim 2, It is characterized in that The acidic fibroblast growth factor is 50 ng / mL.

4. The method according to claim 2 or 3, It is characterized in that The basic fibroblast growth factor is 10 ng / mL.

5. The method according to claim 1 or 2, It is characterized in that Before and / or after step 2), the endogenous primordial germ cells of the laying hen embryo are eliminated.

6. The method according to claim 5, It is characterized in that The removal is performed by the following methods: chemical method and / or physical method and / or biological method.

7. The method according to claim 6, It is characterized in that The chemical method uses busulfan.

8. The method according to claim 6, It is characterized in that The physical method is X-ray irradiation.

9. The method according to claim 1 or 2, It is characterized in that In step 4), after the same-sex chimera chicks are raised to sexual maturity, the female same-sex chimera adult chickens are naturally mated with the male same-sex chimera adult chickens or artificially inseminated to produce fertilized eggs.

10. The method according to claim 1 or 2, It is characterized in that In step 4), after the chimera chicks of the same sex are raised to sexual maturity, the female chimera chickens of the same sex are naturally mated with the parent roosters of white-feathered broiler chickens or artificially inseminated to produce fertilized eggs.

11. The method according to claim 10, It is characterized in that The white-feathered broiler parent rooster is obtained by the method described in claim 1 or 2.

12. The method according to claim 1 or 2, It is characterized in that In step 5), the traits of the chicks include growth rate, breast muscle growth or feather appearance.

13. The method according to claim 1 or 2, It is characterized in that In step 1), the white-feathered broiler chickens are original generation white-feathered broiler chickens, grandparent generation white-feathered broiler chickens, parent generation white-feathered broiler chickens or commercial generation white-feathered broiler chickens.

14. The method according to claim 1 or 2, It is characterized in that PCR was used to identify the sex of donor broilers and recipient laying hens.

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