Innovations in poultry reproduction using cryopreserved avian germ cells.

Meat and eggs from chicken are the major source of animal protein for the human population. The cryopreservation of poultry species is needed to guarantee sustainable production. Here, we describe the existing cryopreservation technologies for avian reproductive cells using embryonic germ cells, spermatozoa and ovarian tissues. We outline strategies to reconstitute chicken breeds from their cryopreserved embryonic germ cells using surrogate hosts and discuss the perspectives for genetic conservation and reconstitution of chicken and wild avian species using surrogate host animals.

Creating resistance to avian influenza infection through genome editing of the ANP32 gene family.

Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape.

The Transcriptome of Chicken Migratory Primordial Germ Cells Reveals Intrinsic Sex Differences and Expression of Hallmark Germ Cell Genes.

Primordial germ cells (PGCs) are germline-restricted embryonic cells that form the functional gametes of the adult animal. The use of avian PGCs in biobanking and producing genetically modified birds has driven research on the in vitro propagation and manipulation of these embryonic cells. In avian species, PGCs are hypothesized to be sexually undetermined at an early embryonic stage and undergo differentiation into an oocyte or spermatogonial fate dictated by extrinsic factors present in the gonad. However, chicken male and female PGCs require different culture conditions, suggesting that there are sex-specific differences, even at early stages. To understand potential differences between male and female chicken PGCs during migratory stages, we studied the transcriptomes of circulatory stage male and female PGCs propagated in a serum-free medium. We found that in vitro cultured PGCs were transcriptionally similar to their in ovo counterparts, with differences in cell proliferation pathways. Our analysis also revealed sex-specific transcriptome differences between male and female cultured PGCs, with notable differences in Smad7 and NCAM2 expression. A comparison of chicken PGCs with pluripotent and somatic cell types identified a set of genes that are exclusive to germ cells, enriched in the germplasm, and associated with germ cell development.

Chicken genome editing for investigating poultry pathogens.

Major advances in pathogen identification, treatment, vaccine development, and avian immunology have enabled the enormous expansion in global poultry production over the last 50 years. Looking forward, climate change, reduced feed, reduced water access, new avian pathogens and restrictions on the use of antimicrobials threaten to hamper further gains in poultry productivity and health. The development of novel in vitro cell culture systems, coupled with new genetic tools to investigate gene function, will aid in developing novel interventions for existing and newly emerging poultry pathogens. Our growing capacity to cryopreserve and generate genome-edited chicken lines will also be useful for developing improved chicken breeds for poultry farmers and conserving chicken genetic resources.

Genome editing of avian species: implications for animal use and welfare.

Avian species are used as model systems in research and have contributed to ground-breaking concepts in developmental biology, immunology, genetics, virology, cancer and cell biology. The chicken in particular is an important research model and an agricultural animal as a major contributor to animal protein resources for the global population. The development of genome editing methods, including CRISPR/Cas9, to mediate germline engineering of the avian genome will have important applications in biomedical, agricultural and biotechnological activities. Notably, these precise genome editing tools have the potential to enhance avian health and productivity by identifying and validating beneficial genetic variants in bird populations. Here, we present a concise description of the existing methods and current applications of the genome editing tools in bird species, focused on chickens, with attention on animal use and welfare issues for each of the techniques presented.

Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer.

The intestinal epithelium plays a variety of roles including providing an effective physical barrier and innate immune protection against infection. Two-dimensional models of the intestinal epithelium, 2D enteroids, are a valuable resource to investigate intestinal cell biology and innate immune functions and are suitable for high throughput studies of paracellular transport and epithelial integrity. We have developed a chicken 2D enteroid model that recapitulates all major differentiated cell lineages, including enterocytes, Paneth cells, Goblet cells, enteroendocrine cells and leukocytes, and self-organises into an epithelial and mesenchymal sub-layer. Functional studies demonstrated the 2D enteroids formed a tight cell layer with minimal paracellular flux and a robust epithelial integrity, which was maintained or rescued following damage. The 2D enteroids were also able to demonstrate appropriate innate immune responses following exposure to bacterial endotoxins, from Salmonella enterica serotype Typhimurium and Bacillus subtilis. Frozen 2D enteroids cells when thawed were comparable to freshly isolated cells. The chicken 2D enteroids provide a useful ex vivo model to study intestinal cell biology and innate immune function, and have potential uses in screening of nutritional supplements, pharmaceuticals, and bioactive compounds.

Avian Primordial Germ Cells Are Bipotent for Male or Female Gametogenesis.

In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW). Here, we investigate the role of chromosomal sex and germ cell competition on avian germ cell differentiation. We recently developed genetically sterile layer cockerels and hens for use as surrogate hosts for primordial germ cell (PGC) transplantation. Using in vitro propagated and cryopreserved PGCs from a pedigree Silkie broiler breed, we now demonstrate that sterile surrogate layer hosts injected with same sex PGCs have normal fertility and produced pure breed Silkie broiler offspring when directly mated to each other in Sire Dam Surrogate mating. We found that female sterile hosts carrying chromosomally male (ZZ) PGCs formed functional oocytes and eggs, which gave rise to 100% male offspring after fertilization. Unexpectedly, we also observed that chromosomally female (ZW) PGCs carried by male sterile hosts formed functional spermatozoa and produced viable offspring. These findings demonstrate that avian PGCs are not sexually restricted for functional gamete formation and provide new insights for the cryopreservation of poultry and other bird species using diploid stage germ cells.

Surrogate broodstock to enhance biotechnology research and applications in aquaculture.

Aquaculture is playing an increasingly important role in meeting global demands for seafood, particularly in low and middle income countries. Genetic improvement of aquaculture species has major untapped potential to help achieve this, with selective breeding and genome editing offering exciting avenues to expedite this process. However, limitations to these breeding and editing approaches include long generation intervals of many fish species, alongside both technical and regulatory barriers to the application of genome editing in commercial production. Surrogate broodstock technology facilitates the production of donor-derived gametes in surrogate parents, and comprises transplantation of germ cells of donors into sterilised recipients. There are many successful examples of intra- and inter-species germ cell transfer and production of viable offspring in finfish, and this leads to new opportunities to address the aforementioned limitations. Firstly, surrogate broodstock technology raises the opportunity to improve genome editing via the use of cultured germ cells, to reduce mosaicism and potentially enable in vivo CRISPR screens in the progeny of surrogate parents. Secondly, the technology has pertinent applications in preservation of aquatic genetic resources, and in facilitating breeding of high-value species which are otherwise difficult to rear in captivity. Thirdly, it holds potential to drastically reduce the effective generation interval in aquaculture breeding programmes, expediting the rate of genetic gain. Finally, it provides new opportunities for dissemination of tailored, potentially genome edited, production animals of high genetic merit for farming. This review focuses on the state-of-the-art of surrogate broodstock technology, and discusses the next steps for its applications in research and production. The integration and synergy of genomics, genome editing, and reproductive technologies have exceptional potential to expedite genetic gain in aquaculture species in the coming decades.

Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics.

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

Direct allele introgression into pure chicken breeds using Sire Dam Surrogate (SDS) mating.

Poultry is the most abundant livestock species with over 60 billion chickens raised globally per year. The majority of chicken are produced from commercial flocks, however many indigenous chicken breeds play an important role in rural economies as they are well adapted to local environmental and scavenging conditions. The ability to make precise genetic changes in chicken will permit the validation of genetic variants responsible for climate adaptation and disease resilience, and the transfer of beneficial alleles between breeds. Here, we generate a novel inducibly sterile surrogate host chicken. Introducing donor genome edited primordial germ cells into the sterile male and female host embryos produces adult chicken carrying only exogenous germ cells. Subsequent direct mating of the surrogate hosts, Sire Dam Surrogate (SDS) mating, recreates the donor chicken breed carrying the edited allele in a single generation. We demonstrate the introgression and validation of two feather trait alleles, Dominant white and Frizzle into two pure chicken breeds using the SDS surrogate hosts.

Livestock 2.0 - genome editing for fitter, healthier, and more productive farmed animals.

The human population is growing, and as a result we need to produce more food whilst reducing the impact of farming on the environment. Selective breeding and genomic selection have had a transformational impact on livestock productivity, and now transgenic and genome-editing technologies offer exciting opportunities for the production of fitter, healthier and more-productive livestock. Here, we review recent progress in the application of genome editing to farmed animal species and discuss the potential impact on our ability to produce food.

The development and maintenance of the mononuclear phagocyte system of the chick is controlled by signals from the macrophage colony-stimulating factor receptor.

BACKGROUND: Macrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model. RESULTS: Based upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP+ donors. In both cases, the transferred cells gave rise to large numbers of EGFP+ tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP+ cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings. CONCLUSIONS: The data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling.