Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken.

1. The aim of the present study was to determine the sex of newly-hatched chicks of Denizli chicken, a local Turkish breed, by polymerase chain reaction (PCR) using DNA extracted from the chorioallantoic membrane (CAM). 2. Fertilised eggs were incubated individually and a total of 20 CAM samples were collected following the hatching process. DNA was isolated from the CAM samples and PCR was performed using W-repeat (W) and 18 S ribosomal gene (R) primers. 3. Screening of the PCR products by agarose gel electrophoresis revealed that males have a single band (256 bp) and females have an extra second band (415 bp) as expected. 4. The present study describes a reliable, rapid, and simple multiplex PCR protocol that can be put into use to sex local breeds of chicken in which phenotypic sexing is impossible, using DNA isolated from the CAM that is discarded and remains attached to the egg shell following the hatching process.

Impact of oxygen concentration on embryonic development of mouse zygotes.

The aim of the present study was to examine the effect of culture under 5 and 20% oxygen on the development, differentiation and viability of zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development. First, zygotes collected in a common pool were cultured in 20% O2 for 0, 23, 46 and 95 h. Zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development were then cultured in 5 or 20% O2. The proportion of embryos reaching the compaction and blastocyst stages of development did not differ between groups regardless of the period of time embryos were cultured in 20% O2 or the stage at beginning of culture. Duration of culture under 20% O2 had a significant effect on total number of blastocyst cells. A stage-specific effect was observed on total and trophectoderm cell numbers in blastocysts resulting from the culture of zygotes and in-vivo-produced embryos under 20% O2. ICM and percent ICM development was significantly decreased by culture in 20% O2 at all stages examined. Oxygen concentration had no effect on implantation rate and fetal weights upon embryo transfer. However, transfer of zygotes grown to the blastocyst stage in 20% O2 resulted in a dramatic decrease in fetal development per blastocyst and fetal development per implantation. These results demonstrate that culture of F1 mouse zygotes in 20% O2 compromises the developmental potential of resultant blastocysts, which appear to be normal on morphological assessment.

Soluble factors and the emergence of chick primordial germ cells in vitro.

Previous observations obtained from a culture of blastodermal cells on a mouse fibroblast feeder layer (STO) suggested that STO cells provide a factor or factors that facilitate development of avian primordial germ cells (PGC) from dispersed embryo cells. The purpose of the current study was to test the hypothesis that soluble factors produced by STO cells are responsible, at least in part, in supporting the development of PGC in culture and to examine the effect of stem cell factor (SCF), ciliary neurotrophic factor (CNTF), and basic fibroblast growth factor (bFGF) in the development of PGC in culture. Blastodermal cells on gelatin-coated plastic or on feeder layers of CV-1 cells yielded a small number of PGC. When blastodermal cells were cultured on STO cells, a marked increase in PGC was observed. The addition of STO cell-conditioned medium (STO-CM) to blastodermal cells cultured on gelatin-coated plastic and on feeder layers of CV-1 cells resulted in a significant increase in the number of PGC, indicating that soluble factors produced by STO cells can enhance the development of chicken PGC in culture. Supplementation of blastodermal cells with SCF (100 ng/mL) or CNTF (2 ng/mL) or with CNTF and SCF together resulted in a significant increase in the number of PGC after 48 h of culture on feeder layers of CV-1 cells. However, addition of bFGF (100 ng/mL) did not increase PGC. We concluded from these observations that the culture of blastodermal cells on feeder layers of STO and CV-1 cells can be used as a useful biological system in examining the regulatory factors that govern the ontogeny of the germ cell lineage in the avian embryo.

The origin of the avian germ line and transgenesis in birds.

The origin of the germ cell lineage in vertebrates is a fundamental question that has preoccupied developmental biologists. Recent work on the origin of the avian germ line has extended and clarified our understanding of the temporal and spatial segregation of primordial germ cells (PGC) during prestreak stages of development. The germ cells first appear at Stage X (Eyal-Giladi and Kochav, 1976) in the ventral surface of the area pellucida in a scattered pattern among polyingressing cells. Subsequently, the PGC gradually translocate from the epiblast to the hypoblast. The entire process appears to be dependent upon the maintenance of an organized area pellucida. Little is known about the regulatory events governing germ cell emergence during this period; however, the culture of dispersed blastodermal cells on a mouse fibroblast feeder layer can compensate for a disorganized area pellucida and offers an in vitro system to examine the molecular basis of germ cell development. Such basic information is valuable for current approaches towards the production of transgenic poultry with targeted changes to the genome through the use of avian embryonic stem cells or primordial germ cells. Refinement of the culture of primordial germ cells or their precursors should allow academic and industrial research laboratories to answer significant biological questions and to improve the genetic potential of commercial poultry stocks. A better understanding of the biology of avian primordial germ cells during early embryo development can only enhance this process.

Origin of primordial germ cells in the prestreak chick embryo.

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII-XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vitelline membranes at stages VII-IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX-XIV with and without on STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII-IX. When individual stage IX-XIV embryos were dispersed and cultured without a feeder layer, 25-45 PGCs/embryo were detected only with stage X-XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX-XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI-XIV.