Tetherin/BST2 is an antiviral restriction factor initially described in mammals. It is active against multiple enveloped viruses at the budding phase, where it is able to physically link the budding virions to the virus-producing cell. We and others have previously identified tetherin orthologs in birds, and characterized the antiviral activity and interferon-inducibility of chicken tetherin. In this work, we have generated an in vivo model of tetherin absence in chicken by CRISPR/Cas9 modification of chicken primordial germ cells (PGC). The modified PGCs were transplanted into roosters with suppressed endogenous spermatogenesis, and transgenic (tetherin knockout) progeny was obtained by further crosses. The viability and phenotype of tetherin knockout animals did not differ from wild type chicken. In more detailed investigation, flow cytometry based differential white blood cell count revealed an increased number of heterophils in tetherin knockouts. Upon challenge with avian sarcoma and leukosis virus (ASLV), a prototypic avian retrovirus, we detected increase in viremia at days 6 and 13 post infection in tetherin knockout animals. The increased virus susceptibility is consistent with absence of antiviral tetherin. In summary, we introduce a new in vivo knockout model of chicken antiviral gene tetherin. These animals can be used in further characterizations of avian antiviral defenses and also to define thus far unknown physiological effects of tetherin in birds. ### Competing Interest Statement The authors have declared no competing interest.

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva −/− chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/− siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

Avian sarcoma and leukosis virus (ASLV), diversified into seven phylogenetically relativesubgroups (A, B, C, D, E, J, and K), present as either exogenous or endogenous viruses in domesticchicken. [...]

ABSTRACT In order to find out the main factors influenced the hatchability and improve the hatchability of the windowed chicken eggs at stage X, several experiments were made on the basis of the former patent of eggshell windowing methods on equatorial plane, such as cutting and sealing techniques, air cell recovering, laying position immediately after sealing, as well as the injection volumes into the subgerminal cavity of the blastoderm. The result showed that:1) the best sealing material combination was straw powder (SP) and instant glue (IG); 2) there was a highly positive correlation between air cell rate and hatchability; 3) the highest hatchability increased to 71.6% when the eggs were windowed and sealed with IG dropped firstly and then SP sprinkled, finally lay down with the blunt end upward immediately after being sealed; 4) the hatchability was significantly reduced as injection volume (DMEM) was increased (p<0.05 or p<0.01) from 1 µL to 10 1 µL, and the group of injecting 1 µL was the highest (48.4 %). The hatchability and efficiency with such method of windowing, injecting, and sealing was the highest at the present time (more than 30 eggs per hour per person), and it might be broadly used in the fields of avian transgenesis, genetic resources preservation, and embryonic development model of human medicine.

Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

A polymer probe based on N-(2-hydroxypropyl)methacrylamide copolymers labelled with a fluorescent dye Dy-633 or Cy-7 and decorated with targeting oligopeptides GE-7 or GE-11, specific targeting ligands binding to epidermal growth factor receptor (EGFR) highly expressed on surface of tumour cells, was designed, synthesised and characterised. Specific accumulation of the polymer probe in the tumour mass is a prerequisite for successful fluorescence-guided endoscopic surgery as the fluorescence signal from the malignant cells enables more precise resection of the tumour without damaging the healthy tissue. Flow cytometry and confocal microscopy was used to assess the binding efficacy of the oligopeptide conjugates to EGFR on the cell membranes of the malignant cells. The results showed that the highest binding efficacy was achieved with polymers bearing the GE-11 targeting oligopeptide in human EGFR-positive hypopharyngeal carcinoma cells (FaDu) and in breast adenocarcinoma cells (MDA-MB-231). Similarly, the polymer probes targeted by the GE-11 oligopeptidewere found in vivo as highly effective in tumour accumulation, as determined from fluorescence imaging. Indeed, the ex vivo cross-section of the tumours showed significant tumour border fluorescence proving the potential of the studied polymer probes. Moreover, the presence of the active targeting moiety on the polymer-drug conjugate should enable the use of such a conjugate as a targeted polymer system for treatment of solid tumours. Replacement of the fluorescent probe with a cytostatic drug provides a targeted polymer nanocancerostatic for advanced treatment of neoplastic diseases, thus the polymer probes have multiple functions.

Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15-18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.

We obtain analytical formulas for the computation of integrals (moments) of all the most common Radial Basis Functions (usually shortened as RBFs) on polygonal regions that may be nonconvex or even multiply connected. With RBFs of finite regularity, such as Thin-Plate Splines, Wendland functions and Radial Powers, our Matlab codes, based on standard linear solvers for the corresponding moment matching systems, provide cubature rules that are reasonably accurate and numerically stable.

Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.

A chicken multiplex cytokine assay (Bio-Plex) to detect four different cytokines (IL-2, IL-12, IL-10, and interferon gamma) simultaneously in plasma samples was designed. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL, except for IFNγ with the range of 50 to 25,000 pg/mL. Such a chicken multiplex assay proved to be fast and reliable, and comparable in sensitivity, accuracy, and reproducibility to conventional enzyme-linked immunosorbent assays. Comparison of the multiplex assay with the ELISA technique using the same clones of detection and capture antibodies resulted in correlation coefficients for all cytokines ranging from 0.95 to 0.99. Lower limit of detection and limit of quantification values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. To reduce the risk of cross-reaction with other proteins, the Bio-Plex system was used, combining the principle of sandwich immunoassay with the Luminex bead-based technology. The cytokine standard recoveries for each cytokine varied between 86 and 118% in dynamic concentration ranges. A chicken multiplex cytokine assay (Bio-Plex) provided a more complete picture of differences between the Th1/Th2 cytokine profiles of the immunized via a new system of antigen delivery into chicken antigen-presenting cells and control groups. This multiplexed fluorescent-bead-based detection assay can be used as a quantitative or comparative tool for the study of the chicken ex vivo cellular immune response.

The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G 0 roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G 1 offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G 0 roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.

Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.

This review discusses various techniques of storage of avian genetic resources. Semen cryopreservation still seems to represent one of the most effective and feasible methods for preservation of genetic resources. Various cryoprotective agents, cooling rates and semen packaging methods are discussed. The use of ovarian tissue, embryonic cells, such as primordial germ cells (PGCs) or blastodermal cells, is the most promising approach because both male and female genetic information can be preserved. A relatively innovative technique; transplantation of cryopreserved testicular cells into adult animals with subsequent production of donor-derived progeny is also mentioned. This transplantation method may contribute to the preservation of endangered avian species and to maintaining their genetic variability.

Soybean lecithin-based extenders represent an alternative to extenders containing egg yolk, but there has been discussion about their cryoprotective efficacy. Low-density lipoprotein (LDL) was proved as a cryoprotective agent, which can replace egg yolk in extenders. The aim of this study was to investigate the effects of LDL addition to the soybean extenders on their cryoprotective properties. The effect of the LDL of our production was verified using commercial egg yolk extender BULLXcell®, 6%, 8%, and 10% LDL (v/v) as an egg yolk replacement. The effects of LDL addition to the soybean lecithin-based extenders in concentrations of 4%, 6%, and 8% (v/v) were tested using extenders AndroMed® and Bioxcell®. In total, 64 samples from eight bulls were evaluated. Kinematic parameters of spermatozoa, resulting from Computer Assisted Sperm Analysis, and their viability, evaluated by fluorescent technique, were assessed immediately after thawing and after 2 hours. The quality of LDL compared to other studies was confirmed, and the beneficial effects of egg yolk replacement by LDL were proved in extender BULLXcell®. 8% LDL provided the best values for the majority of kinematic parameters (P < 0.05), without effect on total motility (P > 0.05). Furthermore, addition of 4%, 6%, and 8% LDL to the soybean lecithin-based extender Bioxcell® showed a positive effect on the majority of kinematic parameters of spermatozoa (P < 0.05) at both times of incubation. However there was no significant influence on total motility (P > 0.05). Viability was higher after thawing in the case of 8% LDL (P < 0.05). However, there was no consistent effect of LDL addition to the AndroMed® extender. In conclusion, cryoprotective properties of the semen extenders based on a soybean lecithin can be improved by the addition of LDL.

1. The aim of this study was to evaluate the ability of frozen-thawed testicular cells transplanted into infertile cocks to restore spermatogenesis and to compare two cryoprotectants (CPA) (dimethylsulfoxide (DMSO) and Biofreeze). 2. A total of 24 infertile White Leghorn (WL) cocks were transplanted with cryopreserved testicular cells from fertile adult donor cocks. Both genetically close and phylogenetically distant chicken breeds were used as donor cocks. 3. Twelve out of 24 WL recipient cocks with cryopreserved testicular cells restored spermatogenesis within 2 months after the transplantation. Six out of 12 recipient cocks with restored spermatogenesis successfully produced progeny expressing the donor phenotype. 4. There was no difference between the CPA in cell viability after thawing or in the number of offspring produced from cryopreserved testicular tissue. 5. The present work represents the first report of production of a donor-derived healthy progeny following frozen-thawed testicular cell transplantation in adult birds. The described results may contribute to preservation of endangered avian species and to maintaining their genetic variability.

The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFRα1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFRα1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFRα1 proteins. Using specific polyclonal mouse anti-cGFRα1 serum, a total of 2.8% cells were recognized as cGFRα1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFRα1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFRα1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFRα1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipient's testes with transplanted cGFRα1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.

Chicken antibodies isolated from egg yolks (IgY) are a suitable alternative to conventional antibodies from blood of experimental animals. Main advantages of IgY are: (i) larger amounts of IgY could be prepared from repeatedly laid chicken eggs, (ii) collection of eggs is a non-invasive procedure contrary to bleeding of mammals, and (iii) a better response of chicken to mammalian antigens due to the larger evolutionary distance. Here, we report a cost- and labor-effective two-step procedure consisting of yolk extraction by tap water (8-fold dilution, freezing, and filtration) followed by a specific precipitation of IgY at pH 4 with sodium chloride in the final concentration of 8.8%. Using this procedure, the highly purified antibody (97%) was prepared. Resulting IgY preparations are acceptable for a wide range of applications as it was prepared using only bio-compatible chemicals. The purification protocol was developed and optimized in terms of the time, materials and the necessary manipulations.

It was the most economic way to preserve chicken breed The procedures relative to chicken semen freezing were reviewed,such freezing semen,equilibrium time,freezing rate,thawing rate,semen quality addition the ＂problem which hindered the applying of chicken semen in the ideas to solve these problems in the future were discussed by cryopreservation semen. as diluents,packing type of testing and so on. In practical was analyzed and

There were two kinds of pluripotent stem cells in the fertile chicken eggs,one was blastodermal cells(BCs) and the other was primordial germ cells(PGCs) and both of them were the main methods of transgenic chicken.The White Leghorn(WL) and Isa Brown(IB) fertile eggs at the stage of BCs and PGCs,were windowed equatorially,and the Lentivrial vector pLenti6/v5-DEST-EGFP was microinjected into the eggs.For BCs fertile eggs from WL,the positive embryos was 64.7%(11/17)when the 13-day embryos were checked by PCR.For the PGCs(eggs incubated for72h) stage fertile eggs from IB,the hatchability was 35%,three chickens died little time after hatching,which were all positive when checked using the liver DNA by PCR at the age of 1 month and 6 month.There were three chickens among four alive ones(75%) was positive when checked the blood DNA by PCR,however they were very weak positive at the age of 12 month.For the PGCs(eggs incubated for 72~79h) stage fertile eggs from WL,the average hatchability was 21.1%,the ratio of being able to injected eggs through the window was 75.0%~92.9%,which was highest and covered the incubated time from 73h to 77h.The ratio was 44.4% when the hatched chickens were checked using the blood DNA by PCR.Comparing BCs method,the PGCs method was much better,such as on the hatchability,embryo locating on the equatorial window and so on.