Establishment of a pheasant (Phasianus colchicus) spermatogonial stem cell line for the production of interspecies germ line chimeras

Background Spermatogonial stem cells (SSCs) are important for the production of interspecies germ line chimeras. The interspecies germ cell transfer technique has been suggested as a way to conserve endangered birds. Our objective was to develop a technique for restoring endangered birds by developing interspecies germ line chimeras between pheasant (Phasianus colchicus) and chicken (Gallus gallus) with SSCs. Results SSCs were isolated from the surgically removed testis of a pheasant. Growth conditions for pheasant SSCs were established by co-culturing STO (SIM mouse embryo-derived thioguanine and ouabain resistant) cells and pheasant SSCs. The colony-forming cells divided and proliferated stably to yield an established SSC line. Pheasant SSCs showed strong reactivity for GDNF family receptor alpha1 (GFRa1) marker. Finally, production of germ line chimeras was attempted by transferring pheasant SSCs into recipient embryos. Although final embryo survival was 5.6% (20/354), the initial survival rate was 88% (312/354). To measure the percent transfer of donor SSC to gonads, the pheasant SSCs were labeled with PKH 26 fluorescent dye. We observed 30% donor cells and 9.48% c-kit/CD117-positive cells in the gonads of recipient chickens. Donor SSCs were thus stably engrafted in the recipient gonads. Conclusions This study showed that SSCs can be used as a tool for the conservation of endangered birds and the production of germ line chimeras. Our findings yield insights into how we may use the pheasant spermatogonial stem cell line for efficient production of interspecies germ line chimeras and ultimately, to the restoration of endangered birds.

Histological description of morphogenesis of the gastroesophageal mucosa of gallus gallus domesticus (linnaeus, 1758) / Descripción histológica de la morfogénesis de la mucosa gastroesofágica de gallus gallus domesticus (linnaeus, 1758)

The ontogenesis of the gastroesophageal mucosa involves morphological alterations related to its structure and the function of each segment. The present study describes the histogenesis of the mucus-secreting epithelium and glands of the esophagus, gizzard, and proventriculus of the chicken (Gallus gallus), and identifies alterations in the secretion pattern of glycosaminoglycans (GAG's). We analyzed 38 chicken embryos, processed the material collected following the histological routine, and then stained it with hematoxylin-eosin for the analysis of tissue structure and with Gomori's trichrome for the identification of conjunctive tissue and collagen fibers. We used the PAS histochemical technique for the analysis of neutral GAG's and the AB pH 2.5 histochemical technique for the analysis of acid GAG's. The embryos at late stage of development had the esophagus wall composed of four layers: mucosa, submucosa, muscularis, and serosa, whereas the proventriculus and the gizzard were composed of three layers: mucosa, muscularis, and serosa. In all three segments, we identified the superficial epithelium as mucus-secreting; in the esophagus this epithelium was mucus-secreting only at the initial development stages. The proventricular glands began to form at the initial development stages, whereas the tubular glands began to form in the gizzard just after the 15th day. The differences in the production of GAG's in these regions of the digestive tract are related to development stages, functions, and physiological requirements of each segment, and to the gradual adjustment of the body to the post-hatching life.

La morfogénesis de la mucosa gastroesofágica implica alteraciones morfológicas relacionadas con su estructura y la función de cada segmento. El presente estudio describe la histogénesis del epitelio secretor mucinoso y de las glándulas del esófago, molleja y proventrículo del pollo (G. gallus), ademas de identificar los cambios em el patrón de secreción de glicocosaminoglicanos (GAG's). Se utilizaron treinta y ocho embriones de pollo. El material recogido fue procesado de acuerdo a la rutina histológica y posteriormente las secciones se tiñieron con hematoxilina-eosina para su análisis histológico, con tricrómico de Gomori para identificar el tejido conectivo y las fibras de colágeno y con PAS y AB pH 2,5 para el análisis GAG's neutro y ácido. En una etapa avanzada de desarrollo de los embriones, se pudo obervar en la pared del esófago cuatro capas: mucosa, submucosa, muscular y serosa, mientras que el proventrículo y molleja se muestra constituida por tres capas: mucosa, muscular y serosa. En los tres segmentos de la superficie, el epitelio se identificó como mucinoso y en el esófago sólo en la etapa inicial de desarrollo. Las glándulas del proventrículo se empiezan a formar en las primeras etapas de desarrollo, mientras que en las glándulas tubulares de la molleja comienzan su sólo después del día 15. Las diferencias en la producción de GAG's en estas regiones del tracto digestivo están relacionadas con las etapas de desarrollo, las funciones y necesidades fisiológicas de cada segmento del cuerpo y se adapta gradualmente a la vida después de la eclosión.

Temporal expression pattern of the insulin-like growth factor II and fibroblast growth factor transcripts in avian embryogenesis

In this study, the abundance of IGF-II and bFGF transcripts was estimated in the chicken embryos using the competitive RT-PCR analysis. Significant enhancements in the abundance of IGF-II mRNA were observed at stages HH1 and 5, and a new accumulation in these levels was observed at stage HH18 in comparison to the basal levels. The abundance of bFGF mRNA increased significantly at stages HH18 and 20, followed by an upregulation in the expression of these transcripts at stage HH26. These findings provided important information about the temporal expression pattern of IGF-II and bFGF transcripts in the whole chicken embryos during in ovo development.

Fatores de crescimento coordenam múltiplas vias de sinalização durante o desenvolvimento embrionário. Neste estudo, a abundância de mRNA dos genes IGF-II e bFGF foi estimada em embriões de galinha por análises de RT-PCR competitiva. Aumentos na abundância de mRNA de IGF-II foram observados nos estádios HH1, 5. Os níveis de mRNA de bFGF exibiram aumentos a partir dos estádios HH18 e 20, seguido por uma acentuada redução a níveis basais no estádio HH24 e por um segundo pico na expressão destes transcritos no estádio HH26. Tais descobertas proporcionam importantes informações sobre o padrão de expressão destes fatores de crescimento durante a embriogênese de aves

Some observations on the adaptation of Eimeria tenella (local isolates) sporozoites on chicken embryos through chorioallantoic membrane

Eimeria (E.) tenella (local isolate) sporozoites were adapted on the chorioallantoic membrane (CAM) of 10-12 days chicken embryos and completed its life cycle in 6~7 days at 39 degrees C and 70 per cent humidity. Only 23 embryos (4.6%) were found dead from 1~4 day post inoculation of sporozoites with mild lesions on CAM with no gametocytes but few sporozoites in chorioallantoic fluid (CAF). On 5~7 day post inoculation, 432 embryos (86.4%) were found dead with severe haemorrhages on CAM and CAF contained uncountable number of gametocytes. After seven days post inoculation, 45 embryos (9%) were found to be alive. Some oocysts were also detected in the CAF on 6~7 days post inoculation. In the histological sections of the CAM, there were abundant small dark colored rounded bodies of gametes; distributed extensively in tissues of CAM on 5~7 days post inoculation of sporozoites. In some cases, cluster of small mature and immature relatively large bodies were seen in increasing numbers on 5~6 days post inoculation.

Efeitos tóxicos de alfa e ß-asaronas e do óleo de cálamo em camundongos, embriões de galinha e mutagenicidade em Salmonella tryphimurium / Toxic effects of alpha and beta-asarone and of the calamus oil in mices, chick embryos and mutagenicity in Salmonella typhimurium

Foram verificadas as DL50 do óleo de cálamo de variedades diferentes, assim como os seus componentes ativos a e B3-asaronas em camundongos, por via intraperitoneal. A DL50 do óleo de cálamo, variedade indiana, foi de 154,5 mgv'kg, a da ,B-asarona, de 184,0 mg/kg e a da a-asarona, de 226,0 mgv'kg. Para variedade européia e para o extrato oleoso isento de asaro nas, em virtude de suas baixas toxicidades agudas, não foi possível a determinação dos valores de DL50. O estudo da toxicidade em embriões de galinha para verificar eventual atividade teratogênica foi feito também para os óleos de diferentes variedades, assim como os componentes a e B3-asaronas isolados. A mortalidade média variou de 10,0 a 100,0% e não se constataram sinais de malformação nos embriões desenvolvidos. O teste de Ames foi conduzido com o componente B3-asarona nas concentrações de 10, 50, 100, 200 e 500 ,ug/placa. A amostra não apresentou atividade mutagênica em nenhuma das concentrações, frente à cepa de Salmonella typhimurium, com e sem adição de ativador obtido de extrato de fígado de rato após índução com Aroclor 1254 (AU).