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Presently, it is difficult to undertake germ line modification of the chicken with primordial germ cells (PGC) because it has been difficult to efficiently fractionate the PGC from the total somatic cell population. The objective of this study was to develop a method that allows isolation of an enriched population of viable PGC from embryonic blood and embryonic gonadal tissue. Blood was harvested from early chick embryos (stages 13 to 15), and cells were liberated from the gonads of stage 27 chick embryos. Subsequently, viable PGC were labeled with anti-stage-specific embryonic antigen-1 (SSEA-1), which was detected with goat-anti-mouse IgM-fluorescein isothiocyanate. Fluorescently labeled cells were sorted from the unlabeled cells using fluorescence-activated cell sorting (FACS), and the identities of the PGC were confirmed using periodic acid-Schiff (PAS) staining or anti-embryonic mouse antigen-1 (EMA-1) staining followed by microscopic evaluation. Finally, PGC were sorted from somatic cells of sex-identified embryos. Less than 0.1% of the blood cell population was collected as SSEA-1-positive cells. Similarly, approximately 2% of the gonadal cell population were collected as SSEA-1-positive cells. Therefore, fewer (-1,000 to 9,000) PGC were recovered from each isolate. Placing the sorted SSEA-1-positive cells on a glass slide from a microcentrifuge tube resulted in a recovery rate of 53 to 73% relative to the number detected by FACS. Furthermore, the proportions of sorted cells that stained with PAS or anti-EMA-1 following sorting were 92+/-4% PAS positive and 94+/-1% anti-EMA-1 positive. Finally, the sorted SSEA-1-positive cells were maintained in vitro to demonstrate their viability after sorting. It was demonstrated that it is possible to label blood and gonadal chicken PGC with SSEA-1 and subsequently to sort viable SSEA-1-positive PGC from somatic cells.

This study was conducted to determine if ascorbic acid (AA) 1) increases resistance to high environmental temperature in young chickens and 2) alters heat-induced changes in several physiological responses. Groups of male chicks received either a standard ration containing 1,000 mg/kg (ppm) of AA or the ration without AA. Chicks were brooded for 3 wk and then maintained at 22 +/- 0.8 degrees C. At 4 wk of age, both AA-supplemented and control chicks were exposed to 30 min of heating (43 +/- 0.1 degrees C and 40 +/- 2% rh) on each of 3 consecutive h in an environmentally controlled chamber. Chicks were challenged with sheep erythrocytes (1 ml, 10(5) cells, iv) 12 h postheating. Heating reduced plasma potassium, body weight gain, relative bursa and spleen weights, and anti-sheep erythrocyte levels. Heating increased cloacal temperature, plasma protein, corticosteroid levels, and mortality. AA ameliorated many of these stress-related responses.

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.

The present study was conducted to determine if dietary ascorbic acid (AA) would improve growth, feed efficiency, and livability of broilers following an acute heating episode. Supplemental AA was provided in the diets at calculated levels of 0, 250, 500, and 1000 ppm, continuously. Females that received 1000 ppm exhibited significantly greater body weights at 2 and 4 weeks of age. No significant effects due to AA supplementation were observed in body weights of males. At 4 weeks of age, chicks were heated on two consecutive days by increasing the ambient temperature (38.3 C at bird level) in the production facility. Heating significantly reduced body weights in males, but not females, at 5 and 7 weeks of age. Feed conversions were increased after heating, but significant effects due to AA were not found. Ascorbic acid did not improve overall livability significantly in either sex, but heat-associated mortality was reduced in supplemented females.

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.

Four experiments were conducted to determine if supplemental ascorbic acid (AA) would ameliorate the immunosuppression induced by exogenous cortisol (COL). Supplemental AA was provided in the diets at levels of 0 and 1000 ppm. Administration of COL significantly lowered plasma AA at both 0 and 10 days postimmunization, and AA supplementation elevated plasma AA at both times. Cortisol-treated chicks exhibited significantly reduced agglutinins to sheep red blood cells but not to Brucella abortus. Supplemental AA significantly ameliorated the immunosuppression associated with exogenous COL and may serve as an antiimmunosuppressive agent in the chicken.

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