Effect of Hydrogen Dioxide Treatment on the Osteogenic Potential of Duck-beak Bone-derived Natural Bioceramic Microparticles.

BACKGROUND/AIM: As an alternative material to the autogenous bone, duck-beak bone particle for bone substitute have been attracting great attention due to their biological properties. To deliver the most favorable outcome of medical treatment, it is essential to study the effect of various processing methods of the duck-beak bone. In this study, we compared the two deproteinizing agents for manufacturing duck-beak bone. Group 1 was treated by a conventional chemical agent (ethylenediamine) and Group 2 by hydrogen dioxide (H2O2). In vitro and in vivo experiments were conducted in parallel to compare the cytocompatibility and osteogenic capability between two processing methods. For in vitro tests, human adipose-derived mesenchymal stem cells (hAD-MSCs) were planted onto each sample and their attachment and growing were evaluated. For in vivo biocompatibility and osteogenic properties, the samples were applied on the critical-sized calvarial bone defect of rats. Group 2 showed significantly higher cell attachment but Group1 showed slightly higher cell proliferation. In in vivo tests, all groups have shown biocompatibility and increased level of osteogenic potential. However, Group 2 had significantly higher bone regeneration (p<0.05). This experiment confirmed that H2O2 can be an optimal processing method for duck-beak bone particle.

Environmental contaminants and chromosomal damage associated with beak deformities in a resident North American passerine.

A large cluster of beak abnormalities among black-capped chickadees (Poecile atricapillus) in Alaska raised concern about underlying environmental factors in this region. Metals and trace elements, organochlorine pesticides, polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-dioxins and polychlorinated dibenzofurans (PCDD-Fs) were analyzed in adults, nestlings, and eggs of the affected population; local bird seed was also tested for organochlorine pesticides. The results offered no support for the hypothesis that selenium or any other inorganic element was responsible for beak deformities among chickadees, but some evidence that organochlorine compounds may be contributing factors. Adults with beak deformities had an elevated level of chromosomal damage, which was correlated with lipid level and concentrations of several organochlorine compounds. Multivariate analyses of pesticides and PCBs did not distinguish abnormal from normal adults, but subsequent univariate analysis demonstrated higher concentrations of heptachlor epoxide and PCB-123 in abnormal adults. Concentrations of all organochlorine compounds were low, and none is known to cause beak or keratin abnormalities. Patterns of PCB congener concentrations differed between nestlings with normal and abnormal parents. Eggs from clutches with low hatchability had higher concentrations of hexachlorobenzene and PCDD-Fs than those with high hatching success, and hexachlorobenzene was found in seeds. Additional testing for PCDD-Fs, polycyclic aromatic hydrocarbons, and other emerging contaminants, including brominated compounds, is needed to rule out environmental contaminants as a cause of beak deformities in chickadees in Alaska.

Bare-part color in female budgerigars changes from brown to structural blue following testosterone treatment but is not strongly masculinized.

Whereas several studies have shown that experimentally increased levels of the androgenic steroid testosterone can affect female behavior, fewer studies have focused on the activational effects of exogenous testosterone on female morphology. With respect to colorful displays in birds, almost exclusively the effects of testosterone manipulation on female carotenoid-based colorations have been studied. Other color types such as structural colors (i.e. UV, blue and violet colors that result from differential light reflection in the nanostructures of the tissue) remain largely unstudied. Here, we investigated the short- and long-term effects of exogenous testosterone on the expression of structural bare-part coloration in female budgerigars, Melopsittacus undulatus. In this parrot species, bare-part coloration is expressed in the cere, a structure over the beak which is brown in females and structural blue in males. We experimentally increased plasma testosterone levels in testosterone-treated females (T-females) compared to controls (C-females) and we performed weekly spectrophotometric measurements of the cere for five weeks after implantation and one measurement after ten weeks. We also estimated the extent to which testosterone masculinized female cere color by comparing the experimental females with untreated males. We found significant effects of testosterone on cere color from week four after implantation onwards. T-females expressed significantly bluer ceres than C-females with higher values for brightness and UV reflectance. T-female cere color, however, remained significantly less blue than in males, while values for brightness and UV reflectance were significantly higher in T-females than in males. Our quantitative results show that exogenous testosterone induces the expression of structural blue color in females but does not strongly masculinize female cere coloration. We provide several potential pathways for the action of testosterone on structural color.

Short-term exposure to testosterone propionate leads to rapid bill color and dominance changes in zebra finches.

Testosterone (T) can influence both male-male competition and mate choice displays. In zebra finches, female mate choice is based in part on bill color, and bill color has been shown to be enhanced by long-term testosterone supplementation. However, it is not clear whether bill color plays a role in male-male interactions and how bill color responds to shorter-term changes in T. We tested whether a single injection of testosterone propionate (TP) would influence male-male dominance interactions and lead to rapid (over a three-day period) changes in bill color. In addition, we tested whether bill color predicted aggression and dominance. We allowed birds in triads to establish hierarchies and then injected either dominant or subordinate individuals with TP, in addition to establishing sham control triads. We found that red chroma, but not hue, predicted aggressiveness of males. Exposure to TP led both dominant and subordinate birds to increase dominance scores over three days, longer than the <24h period in which injected TP stays active. In addition, exposure to TP increased red chroma and hue in three days showing the dynamic nature of allocation of pigments to the bill. Our results suggest that zebra finches can modulate T and bill color levels over short time periods and these changes may occur through positive feedback between T-levels and dominance.

Dynamics of PHA-induced immune response and plasma carotenoids in birds: should we have a closer look?

Allocation trade-offs of limited resources are thought to ensure the honesty of sexual signals and are often studied using controlled immune challenges. One such trade-off between immunity and ornaments is that involving carotenoids. Phytohemagglutinin (PHA)-induced immune response is a widely used immune challenge, yet more details on the underlying physiological mechanisms and potential costs are needed. We investigated the temporal dynamics of PHA-induced immune response and associated changes in blood carotenoids, body mass and a carotenoid-based coloured signal. We found variation in individual response patterns to PHA after peak swelling was reached, with birds showing either a rapid or a slow subsequent decrease in swelling, suggesting variation in the duration of the immune response and/or inflammation. Body mass did not affect immune response. Plasma carotenoids followed a transient decrease closely matching the dynamics of the swelling. The peak of the immune response was negatively related to initial plasma carotenoid levels and positively correlated to the relative decrease in plasma carotenoids. Individual variation in duration of the swelling could be partly explained by plasma carotenoids; high initial carotenoid levels were associated with a slower decrease of the swelling. These contradictory effects of carotenoids suggest a complex role in the immune response. Bill colour was positively correlated to initial plasma carotenoid concentration but it did not predict or change as a consequence of immune response to PHA. Bill colour thus reflects medium- or long-term quality rather than immediate quality. Taking into account the dynamics of the immune response and that of associated physiological parameters would thus yield new insights into our interpretation of variation in PHA response.

A multifactorial test of the effects of carotenoid access, food intake and parasite load on the production of ornamental feathers and bill coloration in American goldfinches.

It has been well established that carotenoid and melanin pigmentation are often condition-dependent traits in vertebrates. Expression of carotenoid coloration in birds has been shown to reflect pigment intake, food access and parasite load; however, the relative importance of and the potential interactions among these factors have not been previously considered. Moreover, carotenoid and melanin pigmentation have been proposed to signal fundamentally different aspects of individual condition but few data exist to test this idea. We simultaneously manipulated three environmental conditions under which American goldfinches (Cardeulis tristis) grew colorful feathers and developed carotenoid pigmentation of their bills. Male goldfinches were held with either high or low carotenoid supplementation, pulsed or continuous antimicrobial drug treatment, or restricted or unlimited access to food. Carotenoid supplementation had an overriding effect on yellow feather coloration. Males given more lutein and zeaxanthin grew yellow feathers with hue shifted toward orange and with higher yellow chroma than males supplemented with fewer carotenoids. Parasites and food access did not significantly affect yellow feather coloration, and there were only minor interaction effects for the three treatments. By contrast, bill coloration was significantly affected by all three treatments. Carotenoid supplementation had a significant effect on yellow chroma of bills, drug treatment and food access both had a significant effect on bill hue, and food access had a significant effect on the yellow brightness of bills. Neither the size nor blackness of the black caps of male goldfinches was affected by any treatment. These results indicate that pigment intake, food access and parasite load can have complex and variable effects on color displays, and that feather and bill coloration signal different aspects of male condition.

A novel lipoprotein-mediated mechanism controlling sexual attractiveness in a colorful songbird.

Sexually selected traits like complex vocalizations or vibrant colors communicate reliable information about mate quality when they are costly to display. Although several general condition-dependent mechanisms underlying the acquisition of mating advertisements have been identified, we rarely know the precise physiological and molecular challenges that animals must meet to develop their sexual ornaments. The flashy pigment-based colors commonly displayed by birds are ideal candidates for investigating the pathways and demands of sexual-signal expression, because we know the biochemical currency with which the trait is produced. Carotenoid colors in birds, for example, are derived from pigments that are acquired from the diet and assimilated into feathers and bare parts. In previous work, we showed that variation in the sexually attractive red carotenoid-colored beak of male zebra finches (Taeniopygia guttata) was predicted not by the amount of food or pigments ingested, but by the levels of carotenoids that birds circulated in blood. Here we elucidate a novel physiological mechanism by which birds are able to accumulate high levels of carotenoids in the body and develop a colorful bill. Carotenoids are transported through the bloodstream bound to lipoproteins. We assayed a critical component of lipoprotein particles-cholesterol-and found that males with higher cholesterol levels circulated more carotenoids and displayed redder beaks. Experimental supplementation of dietary cholesterol elevated carotenoid levels in the blood and beak hue. Experimental reductions in blood cholesterol, using the human lipid-lowering agent atorvastatin, diminished blood carotenoids and faded the beak; carotenoid and cholesterol levels were restored, however, by subsequent addition of dietary cholesterol. These results suggest that the production of circulating lipoproteins critically regulates the development of a colorful sexually selected trait in zebra finches.

Cleft beak induced by hydrocortisone in the chick is prevented by increased cell division after experimental reduction of amniotic fluid.

Hypoplasia of the medial nasal process has been reported in chick embryos on embryonic day (ED) 5, 24 h after their exposure to hydrocortisone (HC). As a result, the cleft beak occurs in 80-100% of specimens on ED 9. In order to analyze its influence on cell proliferation, HC was injected intra-amniotically into embryos on ED 4, and the mitotic index and number of BrdU-positive cells were evaluated 24 h later, both in the epithelium and mesenchyme of the medial nasal processes, on serial frontal histological sections. Two hours after BrdU administration, there were 50% of labeled mesenchymal cells in the embryos exposed to HC and only 23% in the control group. The mitotic index of mesenchymal cells was significantly lower in the HC group than in the controls. The epithelium showed no significant difference. HC seemed to prevent the mesenchymal cells from entering mitosis. The cleft beak in the embryos exposed to HC on ED 4 was totally eliminated by tearing open the amnion (amniotomia) and allowing fluid to leak out on ED 5. In some of specimens exposed to HC, the mitotic index was investigated at six time intervals from 15 to 120 min after amniotomia. A significant increase in the mitotic index was detected in the mesenchymal cells of the medial nasal processes during the first hour after amniotomia. Such a prompt increase of the mitotic activity may be hypothetically explained by release of the HC from its receptor binding as a consequence of outflow of the amniotic fluid together with the HC pool, and freeing of the mesenchymal cells, blocked in the G2 phase, to enter mitosis. As a result, the hypoplasia of the medial nasal process might be compensated and the development of the cleft beak prevented.

Dietary supplementation with xanthophyll as an effective way of identifying low-producing broiler breeder hens.

The relationship between dietary levels of xanthophyll, the degree of pigmentation in the hen, and egg production rate was studied in commercial broiler breeders (Anak 2000). In the first study, the degree of shank and beak coloration, measured with a 15-grade Roche yolk color fan, was determined in broiler breeder pullets until 34 wk of age. Although overall body coloration decreased with age following initiation of egg production, the degree of shank coloration was two- to threefold higher than that of the beak. In the second study, Roche Carophyll-red (canthaxanthin, 10%) was supplemented at levels of 0, 10, and 20 mg/kg in a corn and soybean diet containing xanthophyll, and given to hens for a period of 4 wk beginning at 55 wk of age. Increasing dietary canthaxanthin levels increased pigmentation of beak and egg yolk in a quadratic manner, whereas pigmentation increased linearly in the shank. The production rate was inversely related to the degree of shank and beak pigmentation. In a third study, the relationship between hen pigmentation (using Carophyll-red, 30 mg/kg of diet) and production rate was examined in a commercial flock of broiler breeder hens. Hens were sampled according to shank coloration (Grades 1 to 3) and egg production was monitored. The production rate of hens with high coloration was significantly lower (by approximately 33%) than the flock average. At peak production, shank pigmentation was too low to differentiate visually between degrees of coloration. Dietary supplementation of 5% corn gluten meal increased shank pigmentation and enabled efficient identification of the nonlaying hens.

Late afternoon administration of melatonin is prosomatotrophic and exerts androgen independent effects on erythropoiesis in male house sparrow Passer domesticus.

In the third week of September 1989, birds were purchased locally and acclimated to their housing conditions in a room fully exposed to natural day length (average: 11.96 hr) and temperature (26 degrees +/- 2 degrees C) for 2 weeks. Birds were in the regressive phase of their annual gonadal cycle. In the first experiment 24 birds were selected randomly and were divided into 3 groups of 8 birds each. Initial body weight and bill color score were recorded. The birds of group-I and group-II were injected daily with 5 and 10 micrograms of melatonin in 0.1 ml of vehicle, respectively. The birds of group-III were injected with vehicle only and treated as control. Injections were given daily between 1700 and 1730 hrs over a period of 10 days. At the termination of the experiment, the birds were weighed, sacrificed, bill color scored, blood collected and immediately processed to determine the number of erythrocytes and hemoglobin concentration. The mean body weight loss amounted to 9.6% in vehicle-treated house sparrow. Birds receiving low and high doses of melatonin maintained their initial body weight. Melatonin significantly accelerated the rate of bleaching of bill color. Results clearly indicate that in house sparrow, melatonin produces prosomatotrophic and antigonadotrophic effects. The low dose of melatonin stimulated erythropoiesis significantly. In the second experiment, melatonin nullified the castration-induced decline in the number of circulating red cells. This clearly suggests that the influence of melatonin on erythropoietic machinery appears to be independent of testicular hormone(s).

Quantitative analysis of the effect of retinoids on facial morphogenesis.

Retinoids have been applied to stage 20 chick embryos by using beads that act as controlled release carriers. With beads soaked in high concentrations of all-trans-retinoic acid, the face, in addition to the wing, is affected. Severe bilateral clefting of the primary palate results; the upper beak is completely missing, whereas the lower beak is unaffected. By using a scoring system that quantitates the severity of the upper beak defect, dose-response curves have been obtained. With beads soaked in progressively higher concentrations of retinoic acid, the upper beaks are increasingly truncated. The synthetic retinoid TTNPB also causes beak defects and is 30 times more potent than all-trans-retinoic acid. By removing beads soaked in retinoids at different times after implantation, the effect of varying the length of exposure of the developing face to retinoids has been examined. The production of beak defects is a two-step process and only a short exposure to retinoid is required to produce clefting. There are striking similarities in the dose-time relationships of retinoid treatments that are required to bring about beak defects and pattern changes in the limb. The outgrowth and development of spatially defined patterns of connective tissue within the upper beak appear analogous to processes involved in limb morphogenesis. We propose that retinoids may act by the same mechanisms in both systems. An unsolved puzzle is why retinoids specifically affect the development of the upper beak.

The effects of zinc deficiency on oral behaviour and taste bud morphology in chicks.

The number of beak and tongue movements was measured in chickens (Gallus domesticus) following oral stimulation with different concentrations of quinine hydrochloride (0.01 to 0.25 M), acetic acid (0.25 to 5.0 M), sodium chloride (0.5 to 5.0 M) and distilled water. The birds showed an increase in behaviour with increasing concentration up to a certain point, when the response showed no further increase. 2. Two groups of chicks, one fed on a normal diet and one fed on a diet deficient in zinc, were stimulated orally every 3 to 4 d with 0.1 ml of sodium chloride (2 M), acetic acid (2 M), quinine hydrochloride (0.1 M) and water, and the numbers of beak and tongue movements were recorded. 3. All the birds fed on the zinc-deficient diet showed significant increases in beak and tongue movements compared with the control birds. The time of onset of this increase in behaviour was variable: with quinine hydrochloride it was 3 d whereas with acetic acid, sodium chloride and water it was between 6 and 9 d. 4. The morphology of the taste buds in zinc deficiency was investigated and there was no evidence of primary or preferential involvement of the taste buds; these structures were either morphologically normal or were involved in a generalised degeneration of the epithelium. 5. The increase in oral behaviour is discussed in relation to the possible ageing of the taste cells and to the general degeneration of the oral epithelium.

The effects of vitamin A deficiency of oral gustatory behaviour in chicks.

1. Two groups of chicks (Gallus domesticus) were fed on a vitamin A-deficient diet for 24 d. Birds in one group, which acted as a control, were dosed orally with retinyl palmitate in corn oil, whereas the other group (experimental) was given corn oil only throughout the experiment. 2. At regular intervals birds in both groups were stimulated, orally with solutions of acetic acid, quinine hydrochloride and sodium chloride and the number of beak and tongue movements counted. 3. By the end of the experiment the vitamin A-deficient birds showed a significant decrease in their response to the oral stimulants compared with the control birds.

The influence of propanediol-1,3 on the development of the legs, wings and lower beak of the chick embryo.

In seven series of 31-34 chick embryos each, the influence of the position of the egg during incubation and of the administration of 0.05 ml propanediol-1,3 on the 5th day of incubation on the development and growth of the limbs and beak were studied. Isotonic saline injected embryos served as controls. The embryos were sacrificed at the age of 15 days. Incubation of the egg in a vertical position, with the air chamber pointing upward, proved to result in a slight, through significant reduction in the length of the limbs and beak. Propanediol was highly toxic if injected in the immediate vicinity of the embryo. If injected at some distance from the embryo, either into the air chamber or into the yolk, the agent proved to be teratogenic: measurements showed a considerable retardation of the longitudinal growth of the limbs and beak, the latter being a parrot beak in 60 per cent of the cases, whereas both the tibia and the metatarsals were, moreover, bent posteriorward. Histological studies of the tibia showed an underdevelopment of the periosteal bone collar and, at the angle of the bone, a replacement of the posterior part of this collar by endochondral bone trabeculae. The abnormalities observed are dissimilar to those demonstrated in hereditary congenital chondrodystrophy and in micromelic conditions induced by insulin or sulfonamides.