Concentration and total number of circulating primordial germ cells in Green-legged Partridgelike chicken embryos.

The Green-legged Partridgelike fowl is an old Polish indigenous breed of chicken. Primordial germ cells (PGCs) are one of the best sources of precursor cells that can be used for the conservation and proliferation of the endangered breeds of bird. Initially, the chicken PGCs colonize at the anterior extraembryonic region called "germinal crescent," and after the establishment of blood vascular circulation, they temporally circulate via the embryonic blood vascular system along with embryonic blood cells. They further colonize at the microcapillary networks of both right and left future gonadal regions. Subsequently, they migrate interstitially to reach gonadal anlages, where they begin to differentiate and eventually develop into the future ova or sperm. The basic knowledge regarding the concentration and the total number of circulating PGCs (cPGCs) throughout their circulating phase in the early embryonic stages is crucial for providing an insight into the mechanisms by which they circulate and colonize at the capillary networks of left and right future gonadal regions in each developmental stage. The present study aims to determine the most efficient developmental stage that is suitable to collect cPGCs. The concentration of cPGCs was directly measured, and total volume of embryonic blood was calculated based on the concentration of PKH26-stained embryonic blood cells which were injected 10 min before the blood sampling process in the same embryo during each stage of embryonic development from stage 13 Hamburger and Hamilton (HH; Hamburger and Hamilton, 1951) to 16 HH. Analysis of whole embryonic bloodstream revealed that at stage 14 HH of embryonic development, peak total number of cPGCs (386.3 cells/µL) and peak concentration of cPGCs (18.6 cells/µL) were observed. Later, there was a decrease in concentration, suggesting that the cPGCs might be trapped gradually by the capillary networks at the future gonadal regions after stage 15 HH.

In Vitro Culture of Chicken Circulating and Gonadal Primordial Germ Cells on a Somatic Feeder Layer of Avian Origin.

The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo's feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture.

Dynamics of the transcriptome during chicken embryo development based on primordial germ cells.

OBJECTIVE: Regulation of gene expression during embryo development on the basis of migration of primordial germ cells (PGCs) in vivo has been rarely studied due to limited cell number and the necessity to isolate PGCs from a large number of embryos. Moreover, little is known about the comprehensive dynamics of the transcriptome in chicken PGCs during early developmental stages. The current study investigated transcriptome dynamics of chicken PGCs at key developmental stages: 4.5, 8 and 12 days of embryo incubation. PGCs were collected, and RNA was isolated using a commercial kit for single cells. The isolated RNA was subjected to microarray analysis (Agilent Technologies). RESULTS: Between 8 and 12 days of incubation, the highest number of genes was regulated. These data indicate that the most intense biological activity occurs between 8 and 12 days of embryo development. Heat map showed a significant decrease in gene expression on day 8, while it increased on day 12. The development of a precise method to isolate bird PGCs as well as the method to isolate RNA from single cells isolated from one embryo allows for early molecular analysis and detection of transcriptome changes during embryonic development.

Generation of transgenic chickens by the non-viral, cell-based method: effectiveness of some elements of this strategy.

Transgenic chickens have, in general, been produced by two different procedures. The first procedure is based on viral transfection systems. The second procedure, the non-viral method, is based on genetically modified embryonic cells transferred directly into the recipient embryo. In this review, we analyzed the effectiveness of important elements of the non-viral, cell-based strategy of transgenic chicken production. The main elements of this strategy are: isolation and cultivation of donor embryonic cells; transgene construction; cell transfection in vitro; and chimera production: injection of cells into recipient embryos, raising and identification of germline chimeras, mating germline chimeras, transgene inheritance, and transgene expression. In this overview, recent progress and important limitations in the development of transgenic chickens are presented.

The influence of in ovo injection with the prebiotic DiNovo® on the development of histomorphological parameters of the duodenum, body mass and productivity in large-scale poultry production conditions.

BACKGROUND: Among various feed additives currently used in poultry nutrition, an important role is played by bioactive substances, including prebiotics. The beneficial effect of these bioactive substances on the gastrointestinal tract and immune system give rise to improvements in broiler health and performance nutrition, thus increasing the productivity of these birds. An innovative method for introducing bioactive substances into chickens is the in ovo injection into eggs intended for hatching. The aim of the study was to evaluate the development of histomorphological parameters of the duodenum and productivity in chickens injected in ovo with the prebiotic DiNovo® (extract of Laminaria species of seaweed, BioAtlantis Ltd., Ireland) on d 12 of incubation, under large - scale, high density poultry production conditions. RESULTS: There was no significant impact of the injection of DiNovo® prebiotic on the production parameters of broiler chickens (body weight, FCR, EBI and mortality) obtained on d 42 of rearing. No significant impact of the DiNovo® injection on the duodenum weight and length was observed, as well as on the CSA, diameter and muscular layer thickness of the duodenum. The in ovo injection of DiNovo® significantly increased the width of the duodenal villi (P < 0.05) and crypt depth (P < 0.01) of chickens on d 21 of rearing. Other histomorphological parameters of duodenal villi at d 42 of chickens rearing such as: the height, width, and cross section area of villi were significantly greater in chickens from the control group compared to those from the DiNovo® group (P < 0.05 and P < 0.01). CONCLUSIONS: In conclusion, this study demonstrates that injection of DiNovo® prebiotic into the air chamber of egg significantly influences the histomorphological parameters on d 21 of rearing without negatively affecting productivity in chickens at the end of rearing.

Effects of Prebiotics and Synbiotics Delivered In Ovo on Broiler Small Intestine Histomorphology During the First Days After Hatching.

The objective of the study was to determine the effect of prebiotics and synbiotics administered in ovo on the 12th day of incubation, on the development of the intestinal villi and the number of goblet cells in the small intestine of broiler chickens on the Ist and the 4th days of life of chicks. Two prebiotics: inulin (PI) or Bi2tos (PB) and two synbiotics: inulin + L. lactis subsp. lactis (SI) or Bi2tos + L. lactis subsp. cremoris (SB) were injected in ovo on the 12th day of embryonic development. The control group of the embryos was injected with physiological saline (C). On the 1st day of life, an increase in the height of the villi in the jejunum was reported as a result of the injection of pre- and synbiotics, moreover an increase in the surface area of the villi in the jejunum and the duodenum in chicks from the SB group was also observed. A stimulatory effect of synbiotics on the morphology of the duodenum and thejejunum was also observed on the 4th dh day after hatching. Conversely, in the ileum, in the SB group, a reduction in the height of villi was found both on thse 1t and thte 4h days of life. In contrast, injection of inulin and synbiotic with the addition of inulin resulted in an increase in the number of goblet cells in the duodenum and the jejunum on thst 1" day of life, and caused a significant decrease on the 4th day after hatching.