Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos.

The formation of transgenic chimeric chickens for use in developmental studies and as intermediates in the production of transgenic chickens requires the incorporation of stably transfected blastodermal cells into a chimera. To obtain blastodermal cells, area pellucidae of stage X (Eyal-Giladi and Kochav, Dev. Biol. 49:321-337, 1976:E.-G.&K.) embryos were collected from unincubated, freshly oviposited Barred Plymouth Rock eggs and dissociated in 0.25% trypsin/0.04% EDTA (w/v) and 2% (v/v) chicken serum in phosphate-buffered saline (Ca2+ and Mg2+ free) at 4 degrees C for 10 min. The blastodermal cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) and transfected by lipofection with superhelical pmiwZ, a plasmid containing a hybrid lacZ gene encoding bacterial beta-galactosidase (beta-gal) under the control of a chicken beta-actin/Rous sarcoma virus promoter. A mixture of 2.5 micrograms Lipofectin and 1.56 micrograms pmiwZ in 250 microliters DMEM was incubated for 30 min at 37 degrees C and added to 500 microliters of 20-40,000 cells in suspension. Cells incubated with the transfection reagents in the presence or absence of pmiwZ were either plated and cultured for 48 h at 37 degrees C in 5% CO2/95% air, or injected through a shell window into the subgerminal cavity of White Leghorn stage X (E.-G.&K.) embryos previously exposed to 500-600 rads from a 60Co source, after which the window was sealed and the egg incubated at 38 +/- 1 degrees C for 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The interrelationship between progesterone and luteinizing hormone during the ovulation cycle of the hen (Gallus domesticus).

The existence of a circadian rhythm in the sensitivity of the hypothalamus of the laying hen to stimulation by progesterone was investigated by injecting 0.5 mg progesterone subcutaneously during the proposed period of maximum insensitivity. Following this treatment increases in plasma concentrations of both LH and progesterone were observed which were comparable to the spontaneous preovulatory rises in the plasma levels of the hormones. The ability of either progesterone or luteinizing hormone releasing hormone (LH-RH) to induce premature ovulation varied according to the stage of follicular development. Neither hormone was more than 28% effective when injected within 6.5 h of the previous ovulation, whereas both hormones were 100% effective approximately 27 h after the terminal ovulation of a clutch sequence. Failure to ovulate in response to LH-RH given 6.5 h after ovulation was associated with a lack of progesterone secretion. Both LH and progesterone were secreted when ovulation was induced by injections of either LH-RH or progesterone, and LH was secreted in response to progesterone given 6.5 h after ovulation. These results demonstrate that progesterone stimulates the secretion of LH and LH stimulates the secretion of progesterone. The precise physiological role of these two hormones, however, was not established.

Changes in the plasma concentrations of luteinizing hormone, progesterone, oestradiol and testosterone and in the binding of follicle-stimulating hormone to the theca of follicles during the ovulation cycle of the hen (Gallus domesticus).

The changes in the binding of FSH during follicular maturation were examined in the hen using 125I-labelled bovine FSH (bFSH) and unlabelled bFSH. The binding of 125I-labelled bFSH was not inhibited by bovine LH or chicken LH but was inhibited by extracts of chicken pituitary glands. The ovarian stroma, which contained both interstitial tissue and small follicles, bound the greatest amount of FSH. As the follicles progressed through the yolk-filled hierarchy of maturation, they bound decreasing amounts of FSH. In the two largest follicles of the hierarchy, there was a significant increase in the binding of FSH 12-16h before ovulation. There were two peaks in the concentrations of LH; a preovulatory peak occurred 4-6h before ovulation and a second peak occurred 14-16h before ovulation. Plasma concentrations of testosterone, oestradiol and progesterone began to rise 9, 8 and 6h, respectively, before ovulation. These data are consistent with the hypothesis that changes in the gonadotrophin concentration and binding regulate the order of the follicular hierarchy and the onset of preovulatory steroidogenesis in the hen.

The physiological significance of androgen-induced ovulation in the hen.

The ovulation-inducing property of androgens in the laying hen was investigated. In a first experiment, four different androgens were injected subcutaneously into single-comb White Leghorn hens on the day of the last oviposition of a sequence. The hens were killed 10 h later and examined for the presence of an ovum in the oviduct. Testosterone induced ovulation in accordance to the dose injected (median effective dose, 966 +/- 193 microgram/hen) but the responses to 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were not dose-related. The effect of 4-androstene-3, 17-dione was more like that of progesterone since it induced ovulation 2 h earlier than the three other androgens. The physiological significance of the ovulation response to an injection of testosterone was examined in more detail in experiment 2. Seven out of ten hens which were injected with 1 mg testosterone/kg body weight ovulated within 10 h after the injection. Blood samples were taken at hourly intervals and the concentrations of testosterone and progesterone were determined by radioimmunoassay. An injection of testosterone produced an increase in the concentration of testosterone in plasma which was considerably greater and occurred earlier than the preovulatory increase of testosterone in the control birds. The increase in the concentration of progesterone in the hens injected with testosterone was similar in magnitude but occurred earlier than the spontaneous preovulatory increase of progesterone in the control hens. The possible physiological role of testosterone in the ovulation cycle is discussed.

Progesterone, androstenedione and oestradiol content of theca and granulosa tissues of the four largest ovarian follicles during the ovulatory cycle of the hen (Gallus domesticus).

The progesterone, androstenedione and oestradiol contents of the theca and granulosa tissues of the four largest follicles in the ovarian hierarchy of the hen were determined. The granulosa tissue contained significantly (P less than 0.05) more progesterone and less androstenedione and oestradiol than the theca tissue. The content of progesterone was greatest in the granulosa tissue of the first three follicles in the hierarchy and in each of these follicles there was a peak in progesterone content of the granulosa 4 h before ovulation. The theca of the second, third and fourth follicles and the granulosa of the third and fourth follicles contained significantly (P less than 0.05) more androstenedione than either tissue in the largest follicle. The content of androstenedione was maximal approximately 8 h before ovulation in both tissues of the second and third follicles. The content of oestradiol in the granulosa did not vary as follicles changed position within the hierarchy or during the ovulatory cycle. The oestradiol content of the theca tissue remained constant during the third and fourth positions in the hierarchy and declined throughout the second and first positions until a nadir was observed approximately 20 h before ovulation. It was concluded that the synthesis of androstenedione and oestradiol ceases in both follicular tissues after the follicle is exposed to the penultimate preovulatory surge of LH and that progesterone production is stimulated in the granulosa of the three largest follicles at the time of the preovulatory release of LH.

Blood levels of ionized calcium, inorganic phosphorus, 1,25-dihydroxycholecalciferol and gonadal hormones in hens laying hard-shelled or shell-less eggs.

The production of shell-less eggs was induced in hens to measure the effects of the high demands made by shell formation on the blood minerals and hormones whose concentrations change during egg formation. In control hens laying hard-shelled eggs, the concentration of ionized calcium in plasma decreased at the onset of shell formation, but no change was found in hens laying shell-less eggs. Total calcium concentrations in plasma decreased slightly throughout the ovulation cycle in both groups. Concentrations of inorganic phosphorus in the plasma were increased in the control group during the period of shell formation and decreased when calcification was suppressed. Finally, the concentrations of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) in plasma were significantly increased 16 and 20 h following an ovulation compared with 4 h after ovulation, or compared with the concentrations observed in hens laying shell-less eggs. The variations in the plasma concentrations of ionized calcium, inorganic phosphorus and 1,25-(OH)2D3 associated with egg formation were therefore absent in hens laying shell-less eggs demonstrating their direct link with shell calcification. On the other hand, suppression of shell production had no influence on the changes in the plasma concentrations of progesterone, oestradiol and testosterone which are associated with the normal ovulatory cycle. It is concluded that the increases in intestinal and uterine calcium transport and in 1,25-(OH)2D3 production which occur at the onset of egg production in hens are mainly controlled by factors involved in maintaining calcium homeostasis rather than by gonadal hormones.

A hormonal assessment of bovine parturient paresis: evidence for a role of oestrogen.

A comparative assessment was made of the hormonal control of calcium homeostasis in eight dairy cows which developed parturient paresis and in seven normal animals from the same herd. Plasma levels of calcium, phosphorus, magnesium, free hydroxyproline, 25-hydroxycholecalciferol (25-OHD), 1,25-dihydroxycholecalciferol (1,25-(OH)2D), parathyroid hormone, calcitonin, prolactin and oestrogen were monitored from 30 days prepartum to 15 days post partum. Prepartum levels of plasma calcium, hydroxyproline and calcitonin were depressed in the paretic animals, and plasma levels of phosphorus and oestrogen were elevated. Plasma levels of 25-OHD remained stable in both groups, whereas levels of 1,25-(OH)2D, parathyroid hormone and prolactin rose sharply at parturition. Plasma hydroxyproline, an index of bone resorption, began to rise 2 days prepartum in the control cows but not until 2 days post partum in the paretic cows. The data indicate that bone resorption was inhibited in the paretic group at the onset of lactation, and that a decreased capacity for bone resorption is a major factor in the susceptibility of some cows to this disease. The failure of the paretic animals to resorb bone was not associated with an inability to synthesize the calcium-mobilizing hormones parathyroid hormone or 1,25-(OH)2D, or to regulate the production of calcitonin. However, hypocalcaemia in the affected animals was associated with a significantly higher plasma level of oestrogen (a known inhibitor of bone resorption) in the immediate prepartum period. Following parturition, plasma levels of oestrogen fell rapidly and active bone resorption ensued in the paretic animals.

A radioimmunoassay for corticosterone and its application to the measurement of stress in poultry.

A radioimmunoassay for corticosterone was developed using an antibody to corticosterone-21-hemisuccinate:bovine serum albumin. The assay possessed good specificity, sensitivity and reproducibility and required minimal sample preparation. Tests of adrenal function showed that stimulation of the adrenal with exogenous ACTH and with dexamethasone caused an increase and decrease, respectively, in plasma concentrations of corticosterone. Exposure to cold environmental temperatures caused an increase in plasma corticosterone. Handling and the removal of blood samples by venepuncture had no effect upon the concentration of corticosterone. It was concluded that this assay would accurately measure the response to stresses which affect the pituitary-adrenal axis.

Early development of the chick embryo.

The ultrastructure of the early chick embryo was investigated, using scanning (SEM) and transmission electron microscopy (TEM). Eggs were obtained from the shell gland by injecting hens intravenously with a synthetic prostaglandin or arginine vasopressin. Embryos were examined during late cleavage (stages IV-VI, Eyal-Giladi and Kochav, '76), formation of the area pellucida (stages VII-XI), and formation of the hypoblast (stages X-XIV). SEM highlighted the reduction in cell number at the underside of the embryo during formation of the area pellucida although it became apparent that the thickness of the embryo is not reduced to a single layer of cells at stage X. In addition, blastomeres at the perimeter of embryos (stages V-VI) project filopodial extensions onto a smooth membrane that separates the sub-embryonic cavity from the yolk. During hypoblast formation, epiblast cells generate stellate projections at their basal aspect, thus providing a meshwork for the advancing secondary hypoblast cells. By stage XII the epiblast was one cell thick and reminiscent of a columnar epithelium when viewed transversely. Cells of the deep portion of the posterior marginal zone were distinguished morphologically in the stage XII embryo by their many cell surface projections and ruffled appearance. Blastomeres at the perimeter of stage V-VI embryos projected filopodial extensions onto a smooth membrane which separates the sub-embryonic cavity from the yolk. This membrane is presumed to be confluent with the cytolemma. Evidence is presented demonstrating the presence of intracellular membrane-bound droplets which are hypothesised to contain sub-embryonic fluid.

5alpha-Androstenone concentrations in sow plasma during the estrous cycle.

Plasma concentrations of 5alpha-androst-16-en-3-one (5alpha-androstenone), the steroid responsible for boar taint, were measured by radioimmuno-assay in eight sows during the estrous cycle. The steroid concentrations ranged from a low of approximately 500 pg/ml to a high of 2.2 ng/ml during the estrous cycle. Noticeable features in the hormone profile of sows sampled, were the decline in the period preceding estrus, comparatively low levels at estrus and an increase post-estrous. Changes in hormone concentrations over the estrous cycle were significant (P<.05). The timing of the changes in 5alpha-androstenone concentrations during the period preceding estrus among the sows studied was not consistent. The physiological significance of the changes in 5alpha-androstenone concentrations during the period preceding estrus among the sows studied was not consistent. The physiological significance of these changes in 5alpha-androstenone concentration seen during the estrous cycle is not clear. Any bearing these changes may have on the malefemale sexual relationship in pigs needs to be further investigated in view of the known pheromonal function of 5alpha-androstenone.

From chicken coops to genome maps: generating phenotype from the molecular blueprint.

The tools of molecular and cellular biology can be used to precisely describe traits in terms of a sequence of nucleic acids when their molecular and cellular bases are well understood. The entire genome of elite production birds, however, cannot be written as a series of A's, T's, C's, and G's because the interaction between alleles at the same and different loci is too large and there is likely to be many genotypes that encode the same production trait phenotype. A first draft of the genetic map of the chicken is anticipated within the next few years, but a complete molecular description of the genome of birds with elite production characteristics is not anticipated in the near future. Quantitative genetics will remain the cornerstone of breeding programs for production traits. Novel sequences encoding traits such as enhanced nutritional capability (e.g., expression of phytase) and resistance to specific diseases could be introduced into lines of chickens using the tools of molecular and cellular biology. Cloning could be used by the poultry industry to disperse highly desirable genotypes without the need for grandparent and parent flocks for multiplication.

The effects of ahemeral light and dark cycles on growth and sexual maturity in chickens.

Replacement pullets were reared in environmentally controlled chambers in which the lighting schedule was 14L:7D, 14L:10D or 14L:14D. Both temperature and humidity were constant throughout the experiment. Neither body weight nor age at sexual maturity were altered by these lighting schedules. It was concluded that a unifying hypothesis to explain the various effects of photoperiods on growth and sexual maturity would probably not involve circadian rhythms.

Plasma concentrations of progesterone and corticosterone during the ovulation cycle of the hen (Gallus domesticus).

The observation that injections of either ACTH or corticosterone induced ovulation in the hen has prompted speculation which implicates the adrenal gland in the mechanisms controlling ovulation. In this study, the role of the major adrenal steroid in birds was further examined by measuring the plasma concentration of corticosterone during the 24 hr period preceding mid-sequence ovipositions and the 28 hr period preceding terminal ovipositions. Progesterone, which is an accurate index of preovulatory ovarian activity, was also measured in the same samples. An increase in plasma progesterone preceded mid-sequence ovipositions which are accompanied by an ovulation. This preovulatory surge in plasma progesterone began approximately 7 hr before ovulation, reached a maximum of 6.9 ng/ml 3 to 2 hr before ovulation, and returned to baseline concentrations at the time of ovulation. A major preovulatory peak of progesterone was not observed during the interval between sequences, although a small but statistically significant rise in progesterone was observed between midnight and 0600 hr on the day of the last oviposition of the sequence. The concentration of corticosterone increased approximately two-fold during the dark portion of the photoperiod regardless of the position of the oviposition in the sequence. Baseline concentrations during the illuminated portion of the photoperiod ranged between 1 and 4 ng of corticosterone per ml of plasma whereas during the period of darkness, they ranged between 2 and 6 ng of corticosterone per ml of plasma. It was concluded, therefore, that the circulating concentration of corticosterone was regulated by a circadian rhythm which operates independently of follicular maturation.

Effect of pregnant mare serum gonadotropin on plasma prolactin, luteinizing hormone, estradiol, and ovarian growth in incubating and out-of-lay turkeys.

The effect of pregnant mare serum gonadotropin (PMSG) on ovarian growth and estradiol production was assessed in out-of-lay (OL) turkey hens that have low plasma concentrations of prolactin (PRL) and in incubating hens that have high plasma levels of PRL. In OL hens after injection with 400 or 2,000 IU PMSG, plasma concentration of PRL did not change, whereas ovarian weight and plasma concentration of estradiol increased by greater than 20-fold to levels comparable to those of laying hens. Follicles were not arranged in a hierarchy following the injection of either 400 or 2,000 IU PMSG into OL hens. There was a linear relationship between the dose of PMSG injected into incubating hens and the subsequent increase in plasma concentration of estradiol. Plasma levels of PRL were not different among hens injected with 0, 16, 80, or 400 IU PMSG, whereas plasma levels of PRL decreased in incubating hens injected with 2,000 IU PMSG. A significant increase in ovarian weight occurred only in hens injected with 2,000 IU PMSG. None of the hens deserted the nest following the injection of PMSG, indicating that the maintenance of incubation does not require a steroidogenically quiescent ovary. Although the regressed ovaries of both OL and incubating hens are responsive to gonadotropin stimulation, it would seem that the higher levels of PRL in incubating hens may act, in part, to suppress PMSG-induced ovarian growth and steroidogenesis.

Expression of protein tyrosine kinases and stem cell factor in chicken blastodermal cells.

Chicken blastodermal cells (CBC) from Stage X embryos, which were isolated from newly laid, fertile, unincubated eggs, are pluripotent cells and can produce somatic and germline chimeras when injected into recipient stage X embryos. The CBC retain their pluripotential ability for up to 7 d in vitro. The molecular mechanisms that control proliferation and differentiation of CBC are largely unknown, although protein tyrosine kinases (PTK) are known to play important roles in these processes in similar cells. To understand better the molecular mechanisms of proliferation and differentiation in CBC, expression profiles of PTK and stem cell factor (SCF) were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific and degenerate oligonucleotide primers. Seventeen distinct PTK, including 14 receptor-type and 3 nonreceptor-type PTK and SCF were identified by RT-PCR. Expression of all of the genes was confirmed by northern blot analysis. The northern blot analysis showed that all probes hybridized with one or more transcripts at various expression levels. The expression of the 17 PTK and SCF genes in CBC suggests that they might play a role in signal transduction pathways that control the proliferation or differentiation in CBC.

Responses of luteinizing hormone and prolactin plasma concentrations to oral administrations of clomiphene citrate in broody turkey hens.

Plasma levels in luteinizing hormone (LH) and prolactin were measured after oral administration of clomiphene-citrate (CC) at doses of 6 and 12 mg/kg BW/day for 5 consecutive days to broody turkey hens. No significant changes in prolactin levels were measured following treatment. The LH concentrations were significantly decreased (P less than .05) following administration of the 12-mg/kg dose. These results were interpreted as an indication of oestrogenic-like, rather than antioestrogenic-like, activity of CC when administrated to turkey hens at this particular physiological stage.

A homologous radioimmunoassay for turkey prolactin: changes during the reproductive and ovulatory cycle.

A rabbit antiserum to turkey prolactin (tPRL) was used in a homologous radioimmunoassay with 125I-tPRL. This assay did not cross-react with turkey luteinizing hormone (tLH), chicken LH (cLH), turkey follicle stimulating hormone (tFSH), or turkey growth hormone (tGH). The within- and between-assay coefficients of variation were 6.2 and 14.0%, respectively, and the useful range of the standard curve extended from .5 to 10.0 ng/tube. In two separate experiments, the plasma concentrations of prolactin were estimated throughout the reproductive cycle of the turkey hen using a heterologous assay previously described and the current homologous assay. The correlation coefficients between the two estimates of prolactin in nonbroody hens were .84 and .83. In both experiments, the plasma concentrations of prolactin were low when egg production was initiated, rose to maximum concentrations 5 to 7 weeks later, and thereafter declined. In the first experiment, the hens were selected because their plasma contained large amounts of immunoreactive PRL during the first 2 weeks after photostimulation in the heterologous tPRL assay. This immunoactivity was not evident when using the homologous assay. Using the heterologous assay, no differences in the plasma prolactin concentrations were detected between broody and nonbroody hens during the reproductive cycle. However, using the homologous assay, a sustained increase in the concentration of prolactin was noted for several weeks after broody behavior was detected. The maximum levels observed in broody hens were equal to those observed when the rate of egg production was maximal. There were no statistically significant changes in the concentrations of prolactin during the ovulatory cycle.

The effect of hypophysectomy upon corticosterone-induced ovulation in the hen (Gallus domesticus).

When the ovary of a laying hen contains a mature follicle, ovulation can be induced by an injection of corticosterone. The dose which is required to consistently induce a premature ovulation causes the plasma concentration of corticosterone in the peripheral blood to rise to levels which are higher than those normally observed during the ovulation cycle. The accompanying rise and fall in progesterone which follows an injection of corticosterone. It was concluded that the pituitary gland for both ovulation and progesterone secretion to occur.

Finishing broiler toms using an estradiol 17 beta implant together with a high energy-low protein final feed.

Wrolstad Small White toms were implanted with 10 mg of estradiol 17 beta monopalmitate (EMP) at 8 weeks of age. Common corn-soybean meal feeds were given through to 12 weeks, then one-half the birds from control and EMP groups received either an adequate (16% protein, 3166 kcal ME/kg) or high energy-low protein (HE-LP, 12%, 3373 kcal) feed to 14 weeks. No differences in weight gain and feed conversion occurred between EMP and control treatments at 12 weeks but at 14 weeks when the HE-LP diet had been fed the implanted birds performed better than controls. The HE-LP feed led to body weights and feed efficiencies below that of toms given adequate diet. In all cases, EMP elicited male secondary sex characteristics rather than feminization. Processing losses were increased with EMP and when the HE-LP feed had been given. Both treatments also improved finish assessment and were additive to the extent that a substantial increase in grade occurred. Effects on carcass composition, yield of commercial cuts, and cooking loss were small. Implantation, reduced meat yield percentage of breast and thigh. The increase in grade advantage from combining EMP with a feed that forced fat deposition more than compensated for the adverse effects.

The effect of photoperiod and position in the ovulatory sequence on plasma concentrations of luteinizing hormone during the ovulatory cycle of the hen.

The concentration of LH was studied in blood samples removed intermittently from hens. Hens were selected for study in the middle of sequences when ovulations are separated by 24 to 25 hr, before the first ovulation of a sequence when successive ovulations are separated by 40 hr, and in an 14L : 14D photoperiod when all ovulations are separated by 28 hr. A preovulatory surge of LH always preceded ovulation by 4 to 7 hr, and a nadir in the concentration of LH always occurred 10 to 11 hr before ovulation. This nadir followed a gradual decline in the concentration of LH, which began 19 hr before a midsequence ovulation, 27 to 31 hr before the first ovulation of a sequence, and 20 to 28 hr before ovulation in a 14L : 14D photoperiod. It was concluded that this decline in plasma LH terminating at a nadir 10 to 11 hr before ovulation may be involved in the acquisition of ovulability by the largest follicle in the ovarian hierarchy.