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.

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.

Vent sexing of one-day-old chicks in commercial hatcheries has long been common practice and can be highly accurate.However, there are circumstances when this technique is not applicable such as smaller breeds, non-domestic birds, or where is the necessity of precise sexing.In this study we present a simple and reliable method for fast gender determination in selected Galliformes for which phenotypic determination of sex is difficult until maturity.Four species were tested: two commercial species -chicken (Gallus gallus) and turkey (Meleagris gallopavo), and two game birds -common pheasant (Phasianus colchicus) and wood grouse (Tetraro urogallus).DNA was tested with universal single-pair primers polymerase chain reaction (PCR) detecting W chromosome specific sequence yielding a single band of length specific for each species.The method was developed with regards to time consumption and cost-effectiveness giving results in less than two hours.The method may also be used for early sexing in commercial chicken and turkey flocks as well as sexing of smaller game birds flocks or for research laboratories when rapid sexing of selected Galliformes cells is required.

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in single-cell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.

The objectives of this study were to assess post-hatch development of chickens treated in ovo with the aromatase inhibitor YM511. A total of 137 eggs coming from artificially inseminated hens were at first injected in the albumen with either DMSO alone (54 eggs injected, control group) or with DMSO + aromatase inhibitor (YM511, 1 mg/egg, 83 eggs injected, treated group) and then incubated under standard conditions. Out of the 24 chicks hatched in the treated group, 16 were genetic males (ZZ) and 8 were genetic females (ZW). By 26 weeks of age, secondary sex characteristics of females (cloaca, comb, wattles, song, feathers of hackle and tail) progressively transformed into a male phenotype. Using CT-scanner technology in these 8 birds, the presence of irregular testis-like masses positioned in the antero-ventral portion of the kidneys was observable, an indication that reproductive organs had also been affected by the treatment.

The effect of intramuscularly administered soluble derivatives of beta -1,3-D-glucan, obtained from the culture filtrate of the yeast Saccharomyces cerevisiae, on the immunological activity of neutrophils and monocytes in piglets after weaning was examined. The single intramuscular administration of carboxymethylglucan at doses 0.5 and 5.0 mg/kg b.w. and sulfoethylglucan at a dose of 0.5 mg/kgb.w. enhanced the reductase activity of neutrophils. The greatest enhancing effect was observed at 0.5 mg/kg b.w. of carboxymethylglucan. Bactericidal activity of peripheral phagocytes and LPS- or PHA-induced proliferation of peripheral blood monocytes were also enhanced after the administration of carboxymethylglucan and sulfoethylglucan by the intramuscular route. These results demonstrate that immunocompetent cells were activated after a single intramuscular administration of all soluble derivatives of beta -1,3-D-glucan tested. It is probable that an adverse influence of stress on immune functions in animals after weaning may be reduced by the administration of soluble derivatives of beta -1,3-D-glucan.

The effect of orally or intraperitoneally administered particulate 1,3-β-d-glucan (PBG), carboxymethylglucan (CMG) or sulfoethylglucan (SEG), obtained from the culture filtrate ofSaccharomyces cerevisiae, on the functions of murine peritoneal adherent cells (PC) (peroxidase activity, nitric oxide synthesis), on relative organ mass and on proliferation of splenocytes was determined. The modulating activities after parenteral and non-parenteral administration of these polysaccharides were compared. Significant enhancement of NO production was observed only afterin vitro cultivation of PC in the presence of lipopolysaccharide (LPS) in groups of mice treated repeatedly orally with CMG, PBG and SEG at a dose of 50 mg/kg body mass. Peroxidase activity increased significantly after repeated oral administration of CMG and PBG at doses 150 and 50 mg/kg, SEG 150 mg/kg body mass. The peroxidase activity and NO synthesis in mice given a single intraperitoneal injection of glucans (15 mg/kg body mass) were slightly higher than those after oral administration. Neither a significant enhancement of relative organ mass nor enhancement of the proliferative response of splenocytes toin vitro added stimuli (LPS, phytohemagglutinin) after repeated oral or single intraperitoneal administration of β-glucans was observed.