Third Day Of Incubation Research Articles

Drug modification of catecholamine synthesis and uptake in early chick embryos

Using fluorescence histochemistry for catecholamines, the primary trunks in the chick embryo can be seen on the third day of development. It has been reported previously that the enzymes necessary for production of epinephrine and norepinephrine are not present until after the third day of incubation, and that the yolk contains both of these catecholamines. Therefore, the source of catecholamines in the primary trunks has been studied using alpha-methyl-p-tyrosine (AMT) to block synthesis and desmethylimipramine (DMI) to block uptake. Only embryos treated with both AMT and DMI showed an absence of fluorescence in the primary trunks. However, radioisotopic-enzymatic measurements of catecholamines in whole embryos revealed that epinephrine and norepinephrine were depleted by AMT alone. This indicates that the catecholamines in the primary trunks are synthesized rather than taken up from the yolk. Depletion of the catecholamines in the primary trunks did not cause any developmental aberration in either the primary or secondary sympathetic trunks, which may indicate that catecholamines are not necessary for the development of the trunks, or may be attributed to alpha-methylnorepinephrine, which was present in AMT-treated embryos. Blocking uptake alone caused large increases in both epinephrine and norepinephrine. Since these increases were blocked by AMT, they are believed to be due to increased tyrosine hydroxylase activity. This may indicate that there is end-product regulation of tyrosine hydroxylase in the developing primary sympathetic trunks similar to that reported in adults.

The Regulation of IgM Production in the Chick: Roles of the Bursa of Fabricius, Environmental Antigens, and Plasma IgG

Abstract Previous studies by us have shown that preventing development of the bursa of Fabricius by dipping fertile eggs in testosterone propionate (TP) on the third day of incubation results in elevated levels of IgM in plasma beginning 2 to 4 weeks after hatching. In the present study we have found that such chicks repeatedly immunized with sheep red blood cells (SRBC) produce increasing amounts of IgM antibody but no IgG antibody, in contrast to normal birds which show increasing antibody responses only in the IgG class. TP birds raised in a pathogen-free (PF) environment from hatching until 10 weeks of age have significantly lower levels of IgM than TP birds raised in a conventional environment. The IgM levels approach those of the latter birds at 15 weeks, 5 weeks after being returned to a conventional environment. Further experiments with infusion of normal and immune IgM and IgG into normal birds demonstrated some enhancement of IgM levels by IgM but marked depression of levels of immunoglobulin and antibody in both classes by IgG. The results strongly support the hypothesis that elevated levels of IgM in bursaless, TP-treated birds result from immune responses to environmental antigens unregulated by feedback from IgG. They provide further evidence that the bursa is essential for the normal switch from IgM to IgG production but not for the development of lymphoid cells producing IgM.

Limb-somite relationship: origin of the limb musculature

Quail-to-chick grafting experiments performed during the third day of incubation demonstrate that somites can contribute to limb development. In orthotopic recombinations, migrating cells originating from the grafted unsegmented or segmented somitic mesoderm adjacent to the wing or leg field end up in the musculature respectively of the wing or the leg, where they express exclusively myogenic properties. Thus, in these heterospecific recombinations, the anatomical muscle has a double origin: muscle bulk of somitic origin; tendons and connective tissues of somatopleural origin. Similar features are observed in heterotopic recombinations with (segmented or unsegmented) somitic mesoderm located cranially or caudally to the limb levels. In the reverse chick-to-quail grafting experiments, the somitc participation to the limb mesoderm can also be observed. But it is less regular than that obtained in the quail-to-chick recombinations, and the muscle bulk is made up in various proportions of graft-originated somitic cells and of host somatopleural cells. The possible existence of juxtaposed and interdigitated myogenic and tendinogenic compartments is discussed in view of the dissimilarity between the results of the two kinds of heterospecific recombinations.

A DIFFERENTIAL MEDIUM FOR THE ISOLATION OF Aspergillus flavus FROM COTTONSEED

ABSTRACTAn amended Aspergilhrs Differential (BSAD) agar prepared by a modification of Bothast and Fennell's formula [Mycologia (1974) 66: 365] using botran (2,6‐dichloro‐4‐nitroaniline) facilitated the isolation and enumeration of A. flavus from cottonseeds based on the characteristic orange yellow under‐colony pigmentation after the cultures were incubated for 5 days at 28°C. Pigment production by A. flavus cultures on BSAD agar was detected by the third day of incubation. An incubation period of 5 days at 28°C is recommended for routine screening of cottonseed samples for contamination by A. flavus. At the concentrations used, botran (10 mg/liter) and streptomycin sulfate (50 mg/liter) did not interfere with the pigment production by A. flavus but decreased the numbers and colony size of other fungi and bacteria. Decrease in pH of the medium from 6.5 to 5.5, 4.5 and 3.5 resulted in decrease of the intensity of pigmentation while the sporulation of A. flavus colonies increased. When ferric citrate was omitted from ADM containing botran or was replaced with manganous sulfate, zinc sulfate or copper sulfate, orange yellow pigmentation was not produced. Kojic acid produced by strains of Aspergillus reacts with ferric citrate in the medium to produce orange‐yellow pigmentation. When esculin hydrate (6,7‐dihydrocoumarin‐6‐glucoside) was added to BSAD agar at a 1% level, deep reddish brown pigment was produced by all isolates of A. flavus and A. parasificus tested. Similarities between pigment production and nitrification by the A. flavus group of fungi was observed. Few isolates of A. oryzae produced pigmentation similar to that produced by A, flavus.

Putative neurotransmitters of the avian visual pathway

The ability of homogenates of the chick optic lobe to accumualt a series of possible neurotransmitters has been studied. High affinity uptake of several possible neurotransmittes was examined in optic lobes of 21-day-old embryos that had a single eye removed on the third day of incubation and in 23-day-old chicks that had an eye removed at hatch. Embronic enucleation resulted in severe reduction of development of the ability of the contralateral optic lobe to take triated GABA, dopamine, choline, serotonin and glutamate from solutions around 10 −8 M. Unilateral eye removal of new-hatched chicks caused failure of the denervatd optic lobe to grow, but only the uptake capacity for glutamate was significantly reduced. This deficit was apparent as early as 4 days after enucleation. The transport of other compounds was unimpaired. The uptake of glutamate by homogenates of the optic tract was 43% of that of the optic lobe. This was a much greater fraction than the corresponding value for other postulated neurotransmitters. These data suggest that glutamate may be the primary neurotransmitter of the fibers of the optic tract originating in the retinal ganglion cells.

The time of origin and the pattern of survival of neurons in the isthmo-optic nucleus of the chick.

The time of origin of the cells in the isthmo-optic nucleus (ION--the nucleus of origin of centrifugal fibers to the avian retina) has been determined in the chick by a variant of the cumulative labeling method, using 3H-thymidine autoradiography. All the neurons of the ION are generated (i.e., pass through their last phase of DNA synthesis) over a 50-hour period between the latter part of the fifth and the seventh days of incubation (stages 28--31 of the Hamburger and Hamilton ['51] series) but the cells come to be assembled within the nucleus along a distinct temporo-spatial gradient. The earliest-formed cells occupy the ventrolateral part of the nucleus while the last neurons to be generated come to lie along its dorsomedial margin. When the nucleus is numerically complete, around the eleventh day of incubation, it contains about 22,000 neurons, but between the thirteenth and seventeenth days (stages 39 through 43) this number is produced by nearly 60% to about 9,500 cells. Following the radical extirpation of the one optic cup or the circumscribed removal of the neural retina early on the third day of incubation, the cell loss in the contralateral ION is greatly accentuated, so that by the eighteenth day of incubation no cells remain in the nucleus. Serial counts of the numbers of cells in the nucleus on the side of the eye (or retinal) removals show that it, too, undergoes additional cell degeneration, so that by the end of the phase of naturally occurring neuronal loss, only about half the normal number of cells persist in these experimental animals.

The specification of the retino-tectal projection in the chick.

AbstractThe specificity of the connections between the retina and the optic tectum has been studied in the chick by ablating between 15% and 75% of the optic cup (usually during the third day of incubation) and subsequently determining the distribution, within the tectum, of the synapses formed by the axons of the surviving ganglion cells. This was done towards the end of the incubation period, or shortly after hatching, either autoradiographically following the injection of a tritiated amino acid into the eye, or using a variant of the Nauta‐Gygax method after sectioning the optic nerve. In every case in which the initial retinal lesion was placed after Stage 12/13 (i.e., late on the second day of incubation) the surviving ganglion cells could be shown to have formed synapses in only a limited region of the contralateral optic tectum; and as far as could be determined from an examination of the cell loss in the isthmo‐optic nucleus (in which the centrifugal fibers to the retina have their origin) the remaining portion of the neural retina consistently projected only to the homotopic region of the tectum (i.e., the region to which it would normally have been expected to project). In several cases it was found that the axons had passed over a heterotopic region of the tectum in order to reach a more distant region in which they had formed synapses. After a lesion of the optic vesicle before Stage 11 the surviving ganglion cells appeared to innervate all parts of the tectum; since the earliest retinal ganglion cells are formed at Stage 11/12 it would seem that in the chick, as in Xenopus, there is a clear temporal co‐incidence between the withdrawal of the first ganglion cells from the cell‐cycle and the establishment of the topographic specification of the retino‐tectal projection.Preliminary studies of the projection of the retina upon the optic tectum, and of the tectum upon the isthmo‐optic nucleus, in four to five week old chicks, indicates that the organization of these connections (and hence, presumably, that of the centrifugal fibers from the isthmo‐optic nucleus to the retina) is essentially the same as that in the pigeon. However, there is a significant difference in the overall morphology of the isthmo‐optic nucleus in the two species: as can best be seen in parasagittal sections, the isthmo‐optic nucleus of the chick lacks a well‐developed “anterior limb,” so that the representation of the horizontal meridian lies close to the junction of the “body” of the nucleus and its “posterior limb”.

Hypothalamic Control of Sexual Behavior in the Chicken

This study was an attempt to determine if testosterone propionate (TP) influences sexual behavior in male chickens by way of its action on one or more specific areas of the hypothalamus. The three mating behavior responses observed were waltz, attempts to mate, and mating. Electrical lesions selectively placed in the preoptic and mammilary regions caused mating behavior to decrease in non-testosterone treated males (Controls, C) and failed to increase mating behavior in males from eggs dipped in a TP solution on the third day of incubation (TP) males. Non significant increases in attempts to mate were observed in C males lesioned in the areas of the ruber nuclei (Ru), nuclei mesencephalicus (MPV) and nervus oculomotorius (NOC). Lesions in the optic chiasma (CHI) did not alter sexual behavior. Histological data of testes and vas deferens from TP males showed impaired development at 14 weeks of age but little difference from C males at 54 weeks of age.

Studies on the development of the chick optic tectum. III. Effects of early eye removal

The effects of early deafferentation upon the development of the optic tectum have been studied in a series of chicks from which the right eye had been removed during the second or third day of incubation. No change could be observed in the tectum contralateral to the eye removal (apart from the absence of the stratum opticum in which the retinal fibers are normally found) until the twelfth day of incubation. From this it is concluded that deafferentation has no effect upon cell proliferation, neuronal differentiation or the initial migration of neurons in the tectum, since these morphogenetic processes are largely completed by day 12. Beyond the fourteenth day of incubation there was a progressive atrophy of the tectum, so that by the time of hatching the affected side was markedly reduced in volume and had undergone a considerable degree of cell loss. In addition, the cytoarchitectonic differentiation of the layers in which the retinal afferents terminate was disrupted, and in certain of the deeper layers there was a conspicuous shrinkage of the surviving neurons. These findings have been compared to the effects of eye removal some time after retino-tectal connections were established. Deafferentation at this time results in a qualitatively similar, but quantitatively less severe, atrophy of the tectum. A consideration of these observations, and the findings of similar studies in the literature, leads to the general conclusion that in the avian nervous system the principal effect of early deafferentation is an atrophy, or degeneration, of differentiated neurons; this may, in time, lead to a disruption of the normal pattern of growth and cytoarchitectonic structure.

A histochemical and fine structural study of early extracellular connective tissue in the chick embryo.

AbstractThe histochemistry of developing connective tissues and its relation to early connective tissue fibrils was investigated in the perinotochordal area of the chick embryo. Chick embryos were sacrificed at one, two, three, four, six and ten days of incubation and were prepared for both light and electron microscopy. Five histochemical stains (PAS, alcian blue, Hale colloidal iron, metachromatic toluidine blue, methenamine silver) were used to demonstrate polysaccharides, mucoproteins and mucopolysaccharides. Mature connective tissue elements were demonstrated by Mallory's connective tissue stain and Weigert's resorcin‐fuchsin stain. Routine electron microscopic techniques served to demonstrate extracellular connective tissue fibrils.The first positive response for all histochemical stains occurs on the third day of incubation. Moderate microfibrillar growth precedes this by one day. Light microscopic staining patterns differ from electron microscopic fibrillar arrangements. In the perinotochordal area microfibrils later contribute to the cartilaginous model of the future vertebral body. By the sixth day, dense microfibrillar concentrations appear in the precartilage area where acid mucopolysaccharides are intensely concentrated. Staining for mature connective tissue fibrils does not occur until the tenth day.

Resting membrane potential in chick muscle cells during ontogeny.

AbstractResting membrane potentials were recorded from developing thing muscle cells of the chick at different stages from the third day of incubation. During the early stages the membrane potentials were lower than in the adult animal. No significant changes in potentials were observed until after the fifteenth day. There was a marked increase between the fifteenth and nineteenth days, and the membrane potential reached values comparable to those of the adult. In a study of the concomitant morphological development, the increase in membrane potentials was found to coincide with the transformation of the majority of the cells from myotubes to myocytes.

The fine structure of the trout blastoderm, Salvelinus fontinalis (Mitchill).

AbstractThe Speckled Trout blastoderm at the late high blastula stage is characterized by two different cell populations. The “light” blastomeres comprise one cell type while the “dark” and “medium” blastomeres appear to differ from one another only in degree and thus may be considered as the second type.“Dark” and “medium” blastomeres are irregular in shape, are located centrally and deep in the blastoderm, have an abundance of smooth endoplasmic reticulum with associated 520 Å glycogen particles and a single mitochondrial profile. The “light” blastomeres have randomly arranged glycogen particles in minimal quantities in contrast to the “medium” and “dark” blastomeres and in addition exhibit three mitochondrial profiles, which could however represent artifacts.It is postulated that in the Speckled Trout cellular differentiation has commenced by the third day of incubation at 10°C and that this is manifested visually by the appearance of two different cell populations; the more differentiated “dark” and “medium” blastomeres possibly destined to give rise to the hypoblast and the less differentiated “light” blastomeres.

Testosterone’s Influence on Brain Enzymes in the Developing Chick , ,

MALE birds that have been exposed to testosterone propionate in the early embryo stage manifest less reproductive efficiency than normal birds (Glick, 1961, 1965, 1966). Evidently a neural mechanism necessary for the expression of the male mating pattern is affected during the period or organization and differentiation of sexual function (Glick, 1966).An avian model was produced by dipping eggs on the third day of incubation in a 2.56% solution of testosterone propionate in alcohol. The chicks or embryos from these eggs will be referred to as T.P. chicks or T.P. embryos. The following study was designed to investigate the possibility that the abnormal mating behavior of male T.P. birds might be related to a modification of brain enzymes during embryonic development.Since alkaline phosphatase (A.P.) activity in the developing embryo is associated with differentiation (Krugelis, 1950; Lovtrup, 1955; Moog, 1944; and Rogers et al., 1960), the activity of this…

D-amino acid oxidase in extra-embryonic membranes of the chick

D-amino acid oxidase is present in the extra-embryonic membranes of the developing chick, and can be detected on the third day of incubation (37°) four days prior to its earliest appearance in the hepatic tissue and mesonephros. As the membranes grow, those which form the yolk sac continue to show enzymatic activity. After the sixth day the mesonephros and hepatic tissue both contain activity and on the thirteenth day of development the enzyme appears in the metanephros. Post-hatch, when the mesonephros disappears, the metanephros maintains a specific activity higher than the liver. Enzymatic activity persists in the yolk sac membranes until several days after hatch.

Sensitivity of chick embryo to radioactive phosphorus-32P.

SummaryThe development of the chick embryo has been studied as a function of 32P activity level injected into the egg. 50, 100 and 200 μCi of 32P proved to be lethal, 10 or 25 μCi did not inhibit the development of the embryo; however, even these latter doses reduced the hatching rate by about 30 per cent if the radionuclide was injected on the third day of incubation. Embryonic death is preceded by an appreciable retardation in weight. We examined to what extent the biological effects should be attributed to the absorption by the embryo of the radiation emitted by the injected radionuclide when decaying and/or to the transmutation of the radionuclide. The distribution of the injected radionuclide within the egg, radioisotope concentration in the embryo and its surroundings have been studied. In addition, the radiation dose absorbed by the embryo at different 32P levels was measured in chick embryo models by chemical dosimetry. The dose—effect relations so obtained prove that 32P transmutation plays an ap...

Ultrastructural changes in the developing chick liver: I. General cytology

The cytological changes accompanying the differentiation of the liver of the the chick have been investigated from its first appearance on the third day of incubation, through hatching, and up to the fifteenth day after hatching. The appearance of glycogen and lipid are discussed in the light of recent findings of β granule formation in the pancreas and other available data. Activation of the Golgi complex takes place on the fourth to fifth day of incubation as judged by its expansion and the formation of a variety of vesicles. Mitochondria are sparse and have an average diameter of 0.2 μ at 4 days of incubation which increases to over 1 μ at hatching and then decreases to approximately 0.5 μ . The endoplasmic reticulum (ER) 0072 0154 is very sparse at 3 days of incubation and appears first mainly in a vesicular form which eventually changes to a cisternal form and becomes closely associated with the plasma membrane and mitochondria, a single cisterna surrounding almost every mitochondrion. The rough ER gives rise to the smooth vesicular ER associated with glycogen deposition. Polyribosomal groups are frequently seen in the cytoplasm from the eighth day on. Microbodies become more numerous at hatching and are frequently located in the glycogen-lipid areas.

Cytodifferentiation of the chick pancreas: I. Ultrastructure of the islet cells and the initiation of granule formation

Electron microscopic observations have shown that the characteristic granules of the alpha and beta cells of the chick (White Leghorn) pancreatic endocrine tissue are present on the third day of incubation. Granule formation in these cells is initiated concomitantly with or shortly after evagination of the intestinal endothelium to form the pancreatic diverticula. Evidence is also presented for the secretion of both types of granules on the fifth day of incubation. The sequelae of ultrastructural changes leading to the differentiation of the alpha and beta cells was difficult to assess. The islet tissue originated from primordial duct epithelium and differentiated before the acinar components. The elaboration of biosynthetic mechanisms to facilitate the production of secretory products was evidenced morphologically in the beta cells by an enlarged Golgi, ergastoplasm of four to eight members, and the synthesis of numerous smooth membrane vesicles. A locus of fine fibrillar material and one cilium per cell were present. Stages in the formation of the beta granules with their characteristic sheaves of material were also noted. Similary, the differentiation of alpha cells included enlargement of the Golgi and synthesis and accumulation of numerous vesicles with varied amounts of enclosed particulate material. Fine fibrillar material was near the Golgi and the rough reticulum appeared augmented.

Temperature Control of Testosterone Propionate Solutions Used for Reducing Embryonic Development of the Bursa of Fabricius ,

THE bursa of Fabricius is a central lymphoid tissue that influences the immunological competence of the chicken, Glick (1964). Embryonic development of the bursa can be inhibited by injecting testosterone propionate (TP) into the egg (Aspinall et al. 1961; and Warner and Burnet, 1961), or by dipping the egg in ethyl alcohol (EA) solutions containing TP, Glick and Sadler (1961) and Sato and Glick (1964). The efficiency of the dipping procedure depends on the temperature of the TP-EA solution and, within limits, does not seem to be affected by the number of eggs dipped in a given solution. Data will be presented to support these conclusions.MATERIALS AND METHODChickens used in these experiments were from a strain of the New Hampshire breed developed by Professor L. J. Dreesen at the Mississippi Agricultural Experiment Station.The eggs were dipped on the third day of incubation. Approximately 1½ inches of the …

Responses of Heterotrophic Cultures of Chlorella vulgaris Beyerinck to Darkness and Light. II. Action Spectrum for and Mechanism of the Light Requirement for Heterotrophic Growth

Chlorella vulgaris Beyerinck (Emerson's strain), fails to grow in the dark even when sugars are provided. This phenomenon was clearly demonstrated in the alga, C. vulgaris, for which the growth rate in darkness on a glucose medium remained constant for 2 days and then declined to approach zero. Pigment concentrations also declined in darkness. Changes in flow rate of 1% CO(2)-in-air from zero to 7 ml per minute caused a progressive increase in the dark growth rate over a 5-day period, but did not maintain growth in the dark. Rates above 7 ml per minute produced no changes in growth rates.White light intensities below the compensation point of the alga maintained heterotrophic growth. The saturation value for this response was 0.8 muw/cm(2). White light also initiated growth in nongrowing cultures transferred from darkness to light.The action spectrum for heterotrophic growth indicated a porphyrin as the active pigment. Light in the 425 mmu region was 4 times as effective as white light in stimulating heterotrophic growth. A secondary peak of growth stimulation occurred in the 575 mmu region.The respiration of glucose by the alga was stimulated by low intensities of white light. This response was not immediate, but was clearly present after the third day of incubation.Malonate and cyanide were inhibitory to growth of C. vulgaris on inorganic medium or glucose medium under 300 ft-c of white light. These data suggested that succinic dehydrogenase and cytochrome oxidase systems were present.Substances inhibitory to growth were excreted into the medium under dark-growth conditions, and 2 of these substances were indentified as formic and acetic acids.The evidence suggested that respiration of glucose cannot proceed for an extended period of time in darkness. The reason for this is postulated to be the lack of a cytochrome or a cytochrome precursor.

Weight of the Bursa of Fabricius and Antibody Response of Chicks Hatched from Eggs Dipped in Varying Concentrations of Testosterone Propionate ,

SELTZER (1956) patented a method for controlling the sex of chicks by dipping hatching eggs in solutions of sex hormones. A procedure similar to Seltzer’s has been employed by Glick and Sadler (1961) and Glick (1961, 1962, 1963a) to introduce testosterone propionate (TP) into fertile eggs. These workers reported a significantly smaller bursa of Fabricius or no bursa in birds hatched from eggs dipped in 0.67 to 3.0 percent solutions of TP. The immunological competence of birds hatched from the TP dipped eggs was markedly reduced or eliminated.The primary purpose of this paper is to report the quantitative changes occurring in the weight of the bursa of Fabricius and antibody response in birds hatched from eggs dipped in varying levels of TP. PROCEDUREEggs were removed from the incubator on the third day of incubation. One and one-quarter inches of the small end of the egg was immersed in…