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Medullary bone is a specialized bone found in the marrow cavity of laying birds. It provides a significant contribution to the calcium supply for egg shell formation. Medullary bone is distinguished from cortical bone by the presence of large amounts of a keratan sulfate proteoglycan (KSPG). The aims of the present experiment are to confirm the identity of the core protein of KSPG, identify a marker of medullary bone metabolism, and determine whether changes in keratan sulfate (KS) concentration in blood are associated with the egg-laying cycle. Using two different isolation techniques- one specific for bone and another for blood- we have identified bone sialoprotein (BSP) to be the core protein of this KSPG. We also determined that the amount of keratan sulfate (KS) in laying hen blood fluctuates in synchrony with the egg-laying cycle, and thus can serve as a specific marker for medullary bone metabolism. During the course of this investigation, we also found FGF-23 (phosphatonin) to be expressed in medullary bone, in synchrony with the egg-laying cycle. Western blotting was used to demonstrate the presence of this peptide in both laying hen blood and medullary bone extracts. The importance of FGF-23 (phosphatonin) and parathyroid hormone in normalizing the dramatic changes in plasma calcium and phosphorus during the 24h egg-laying cycle is discussed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Longitudinal bone growth depends upon the execution of an intricate series of cellular activities by epiphyseal growth plate chondrocytes. In order to better understand these coordinated events, microarray analysis was used to compare gene expression in chondrocytes isolated from the proliferative and hypertrophic zones of the avian growth plate. RT-PCR was used to confirm the identity of a select number of genes. The expression of 745 genes was found to differ 3-fold or greater at the 0.05 level of probability. Transferrin was the most highly up-regulated (321-fold) gene associated with chondrocyte hypertrophy. Immunohistochemistry localized this peptide adjacent to the penetrating blood vessels in the growth plate of 3-week-old chicks. Fibulin, OC-116, DMP-1 and PHEX were among the expanded number of genes associated with extracellular matrix metabolism. The presence of NELL2, ATOH8 and PLEXIN suggests a neuronal involvement in growth plate physiology. In addition, the expression of a large number of genes associated with angiogenesis and cellular stress was up-regulated. These processes are important to the physiology and survival of chondrocytes in the unique and stressful environment of the epiphyseal growth plate., (Copyright 2009 Elsevier Inc. All rights reserved.)

Ovocleidin (OC-116), a protein identified in eggshell matrix, was found to be expressed in avian growth plate chondrocytes. Because OC-116 has been reported to be a member of a family of related phosphoprotein genes clustered on avian chromosome 4, we expanded our search to other skeletal tissues. Using Western blotting, we found OC-116 in the matrix of chick cortical bone and laying hen medullary bone as well as in hypertrophic chondrocyte lysates. Furthermore, other members of this family (bone sialoprotein, dental matrix protein-1, and osteopontin) were also present in the eggshell matrix. Reverse transcription-PCR was used to confirm the presence of the OC-116 gene in bone tissues as well as the expression of bone sialoprotein and dental matrix protein-1 in uterine tissue. These results, in combination with those of other laboratories, show that this family of phosphoproteins is found in a wide variety of avian mineralized tissues.

Tibial dyschondroplasia is a skeletal abnormality characterized by an avascular plug of cartilage in the metaphysis of the long bones of rapidly growing birds. An avascular, thickened growth plate also occurs in vitamin D/calcium-deficient rickets. This research was conducted to evaluate tibial dyschondroplasia and rachitic tissue for factors shown to be important in normal growth plate vascularization. Immunohistochemical analysis demonstrated the presence of vascular endothelial growth factor, its receptor Flk-1, and matrix metalloproteinases MMP-9 and MMP-13 to be present in all tissues examined. In most cases, immunostaining was intracellular, except near blood vessels where the matrix was also stained. The results suggest that, for the four proteins examined, disruption of their expression is not a key factor in the development of these avascular cartilage lesions in avians. The relationship of these observations to the pathology of tibial dyschondroplasia is discussed.

Tibial dyschondroplasia is a disease of rapid growth rate that occurs in many avian species. It is characterized by an avascular lesion in which the life span of the growth plate chondrocyte is essentially doubled. A characteristic pattern of gene expression and gene product localization has emerged that mimics the pattern observed with endoplasmic reticulum (ER) stress in growth plate chondrocytes. This activates a cell-survival mechanism called autophagy. The initial phases of this mechanism appear to originate in the avascular transition zone of the growth plate. Because specific genes and gene products are associated with autophagy and ER stress, it should now be possible to identify the mechanisms involved in the development of this cartilage abnormality. The potential biochemical pathways responsible for initiating ER stress are discussed.

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.

Northern blotting, RT-PCR, and Western blotting techniques were used to characterize the matrix constituents of avian cortical and medullary bone. Extracts of bone tissue were found to contain multiple isoforms of bone sialoprotein (BSP), osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and dentin matrix protein-1 (DMP-1). Only single transcripts were observed with Northern blotting; therefore it was concluded that the isoforms were due to differences in post-translational modifications. Since medullary bone is rich in keratan sulfate (KS), RT-PCR was used to investigate the expression of known keratan sulfate-containing proteoglycans (KSPGs). Although this tissue was found to express lumican and osteoglycin/mimecan, there was little evidence to suggest that these proteoglycans were a major source of the keratan sulfate glycosaminoglycans. Treatment of medullary bone extracts with keratanase resulted in the appearance of a BSP immunoactive band of approximately 59 kDa. However, it was not possible to isolate and identify the intact keratan sulfate proteoglycan.

The purpose of this study was to investigate the effect of zinc deficiency on chondrocyte proliferation, differentiation and apoptosis in the epiphyseal growth plate of juvenile chickens. Newly hatched broiler chickens were fed either a low zinc (10 mg/kg diet) or a zinc-adequate (68 mg/kg diet) soy protein-based purified diet. Cell proliferation in the growth plate was evaluated with bromodeoxyuridine (BrdU) labeling. Apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Chondrocyte differentiation was evaluated with immunostaining of osteonectin as a marker of maturation. As early as d 3 of feeding, zinc deficiency significantly inhibited chondrocyte proliferation, promoted cell differentiation and induced cell apoptosis in the growth plate. These effects were manifested primarily in areas remote from the blood supply. Immunostaining for local growth factors such as insulin-like growth factor-1 (IGF-1), parathyroid hormone-related protein (PTHrP) and fibroblast growth factor-2 (FGF-2) did not reveal any differences between growth plates of zinc-deficient and zinc-adequate chickens after 3 d of feeding. By d 7, severe growth plate lesions characterized by reduced cellularity and abnormally shaped cells were formed in areas remote from blood vessels. Immunoreactive IGF-1, PTHrP and FGF-2 were all greatly reduced in the lesion. However, the growth rate and food intake of zinc-deficient chickens were not different from those of the controls during the 7-d experiment. Therefore, a direct effect of zinc deficiency on proliferation, differentiation, and apoptosis of growth plate chondrocytes was indicated.

Tibialdyschondroplasia (TD) is a disease characterized by the formation of an avascular, non-mineralized lesion along the mature face of the epiphyseal growth plate in rapidly growing chickens. In the normal growth plate, cells progress from a proliferative phase to hypertrophy where the tissue is vascularized and replaced by trabecular bone. In TD, cells apparently cease their development early in the transition to hypertrophy. These diseased cells are not removed by vascularization nor does mineralization occur. The resulting lesion increases in size as proliferative cells continue to divide in the absence of removal and replacement of cartilage by bone. This laboratory has previously reported that cells of the TD lesion have the morphological appearance of necrotic cells or in some cases apoptotic cells. In this study we examine in more detail the status of cells comprising the TD lesion using molecular techniques. Genomic DNA isolated from cells of severe TD lesions show the nucleosomal laddering indicative of apoptosis, while DNA isolated from proliferative and hypertrophic cells does not. This result was confirmed by the use of the Cell Death Detection ELISA which shows quantitatively that cells from severe TD lesions contain nearly twice as many nucleosomal fragments as cells from the hypertrophic zone while proliferative chondrocytes do not have significant fragmentation. In situ examination of the epiphyseal growth plate with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) clearly shows that the cells of the severe TD lesion are apoptotic. Cells from smaller lesions are stained to a lesser extent or not at all by TUNEL. We believe that the apoptosis seen in TD is a secondary effect of the disease and not its primary cause.

Recent evidence clearly indicates that basic fibroblast growth factor (bFGF) signal transduction is essential for normal skeletal development. Here, we report that bFGF and its receptor are specifically localized in the terminal hypertrophic chondrocytes of the porcine epiphyseal growth plate, the tissue responsible for longitudinal bone growth. Similar observations were obtained with the chondrocytes immediately adjacent to resorbing blood vessels in the secondary center of ossification of the epiphysis. These results are consistent with a recent proposal that bFGF functions in coupling osteogenesis with chondrogenesis by attracting the vascular invasion of cartilage from adjacent trabecular bone.

A series of experiments was conducted to test the hypothesis that vitamin D utilization may not be as efficient in chicks with tibial dyschondroplasia (TD). The basal diet contained 1.0% Ca and 0.45% available P with no supplemental cholecalciferol (D3). Chicks from low TD (LTD) and high TD (HTD) selected lines were fed diets supplemented with various levels of vitamin D compounds and examined for rickets and TD. When chicks were fed a D3-deficient diet containing only 1.25 micrograms/kg added D3, HTD chicks had a greater incidence of severe rickets than LTD chicks (P < 0.05). The LTD chicks did not exhibit TD when fed a diet containing adequate (20 micrograms/kg) D3. The LTD chicks fed a diet supplemented with 5 micrograms/kg D3, however, had 22% incidence of TD. When HTD chicks were fed diets supplemented with 5 micrograms/kg D3 [control diet that meets NRC (1994) requirement for D3], 20 micrograms/kg D3, 5 micrograms/kg 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] or the combination of both D3 (20 micrograms/kg) and 1,25-(OH)2D3 (5 micrograms/ kg), TD incidence was highest in HTD chicks fed the control diet. When HTD chicks were fed diets with an increased dietary level of 1,25-(OH)2D3 (10 micrograms/kg) further reduction of TD incidence (P < 0.05) occurred. A potentially toxic level (Soares et al., 1983) of 1,25-(OH)2D3 (15 micrograms/kg) fed to HTD chicks resulted in still greater suppression of incidence of TD even though growth and feed intake in HTD chicks was greater than those of LTD chicks. It is concluded that the development of TD in HTD chicks is associated with subnormal ability to metabolize vitamin D.

Osteonectin is an acidic calcium-binding protein found in cartilage, bone matrix, vascular endothelium, and areas of tissue repair. Using immunocytochemistry, osteonectin has been localized in all zones of the normal avian epiphyseal growth plate with notably high amounts in the hypertrophic zone. In the proximal portion of this zone the staining was intracellular, while in the distal calcifying portion of the hypertrophic zone staining was both intracellular and extracellular. Osteonectin was also detected in the growth plate associated with lesions of chickens with tibial dyschondroplasia (TD). Intense intracellular staining was observed in hypertrophic chondrocytes proximal to the lesion; staining was markedly diminished in the TD lesion; extracellular matrix was devoid of staining. Staining intensity was high along the peripheral edges of the lesion that were undergoing vascularization and resorption. This was the only area in the dysplastic cartilage where staining was observed in the extracellular matrix as well as intracellularly. Similar patterns were viewed in all TD lesions examined, whether they were spontaneous or induced by dietary treatments or genetic selection.

Basic fibroblast growth factor (bFGF) is a permissive mitogen for cultured chondrocytes and has been localized in the specific zones of the epiphyseal growth plate. In this study, we demonstrate that bFGF present in cartilage originates from within the cellular constituents of this tissue. Utilizing reverse transcription coupled to the polymerase chain reaction (PCR), bFGF mRNA was found in extracts of cartilage tissue. Immunocytochemical studies revealed that bFGF was present intracellularly in freshly isolated proliferative chondrocytes and in the extracellular matrix (ECM) after 24 h of culture. Western blot analysis of protein extracts from isolated proliferative chondrocytes identified a bFGF immunoreactive species with a molecular weight of approximately 18 kDa. In situ hybridization confirmed the presence of bFGF mRNA in freshly isolated proliferative chondrocytes. The bFGF in the ECM seemed to be sequestered and not available for biological activity, since these cells still required exogenous bFGF for cell proliferation. This sequestered bFGF could be released to stimulate cell proliferation when cultures were treated with plasmin, a proteolytic enzyme. These data support the hypothesis that bFGF is synthesized by chondrocytes and functions as an autocrine/paracrine mitogen via its deposition into the ECM with subsequent release from the ECM of cartilage being a critical step in biological activity. In addition, the study provides further evidence that locally produced bFGF plays an important role in normal growth and development of cartilage tissue.

Recent research in our laboratory has demonstrated that basic fibroblast growth factor (bFGF) is a permissive mitogen for epiphyseal growth plate chondrocytes. Immunocytochemistry demonstrated the presence of bFGF in the proliferative and hypertrophic zones of normal epiphyseal growth plates of 4-wk-old broiler chickens. The purpose of this investigation was to extend this research to include examination of the status of bFGF in the cartilage lesion associated with tibial dyschondroplasia (TD). Immunocytochemistry revealed that the distribution of bFGF in the growth plate proximal to the TD lesion was similar to that observed with normal growth plate. However, the intensity of immunofluorescence was greatly diminished in the TD lesion. The number of chondrocytes staining positive for bFGF was also reduced. In the peripheral edges of the lesion where cartilage was being actively resorbed, the staining intensity was greatly increased when compared to the rest of the TD lesion. Similar patterns were observed in all TD tissues examined whether the lesions were spontaneous or induced by dietary treatments or genetic selection. It is hypothesized that the decrease in bFGF, a potent angiogenic factor, may be responsible for the poor vascularization of the TD lesion.

Previous research in our laboratory has shown basic fibroblast growth factor (bFGF) to be a permissive mitogen for isolated avian growth plate chondrocytes. The present study was conducted to determine whether bFGF is present in avian growth plate and, if present, to determine its localization within the tissue. Immunohistochemical studies revealed that bFGF is present in the resting proliferative and hypertrophic calcifying zones of the growth plate but is absent from the prehypertrophic zone. Basic FGF appears to be associated with the extracellular matrix of the proliferative zone, but it is predominantly intracellular in the hypertrophic and mineralizing zone chondrocytes. Partial purification of cartilage-derived bFGF was performed on crude extracts of cartilage using heparin-Sepharose affinity chromatography. The presence of bFGF in the heparin-Sepharose column fractions was confirmed by immunoblotting and radioimmunoassay. Furthermore, western blot analysis of the extracts showed multiple protein bands having bFGF immunoreactivity, in the molecular weight range 14.4-18 kD. The data support the hypothesis that bFGF has a dual role in the growth plate. In the proliferative zone it acts as a chondrocyte mitogen, whereas when released from terminal hypertrophic chondrocytes, bFGF may serve as a chemotactic signal for metaphyseal blood vessel proliferation.

The need for manganese for normal skeletal development appears to be related to its role in proteoglycan biosynthesis. The purpose of this research was to characterize the proteoglycans synthesized under conditions of manganese deficiency. The proteoglycans were extracted from epiphyseal growth plate cartilage and the monomers separated by cesium chloride density gradient centrifugation followed by column chromatography. The proteoglycan monomers from normal cartilage contained primarily (92%) chondroitin sulfate side chains with keratan sulfate being a minor (8%) component. Manganese deficiency reduced the total amount of cartilage proteoglycans. Of the monomers present in deficient cartilage, the majority (75%) were similar to those found in normal cartilage. Cartilage from deficient chicks also contained a second monomer fraction (25%) characterized by a reduced carbohydrate content. Thus, in addition to a reduction in total proteoglycan content, manganese deficiency results in qualitative changes in the proteoglycans present in epiphyseal growth plate cartilage.

The purpose of this research was to determine the effect of vitamin K deficiency on indices of skeletal metabolism in laying hens, developing embryos and young growing chickens. Laying hens were fed a vitamin K-deficient diet for 28 wk, which resulted in impaired blood clotting and reduced bone gamma-carboxyglutamic acid (Gla) concentration compared with vitamin K-sufficient hens. However, this treatment did not influence egg production, eggshell deposition or other reproductive performance criteria. Vitamin K-deficient embryos were able to mobilize sufficient quantities of calcium for normal skeletal development, although they exhibited severe reduction in blood clotting and bone Gla concentration. Similar results were obtained from progeny of both vitamin K-sufficient and -deficient hens fed deficient diets for 4 wk after hatching. These results indicate that a severe reduction in skeletal protein Gla concentration does not interfere with normal development of this tissue.

Many cadmium-contaminated environments contain high levels of zinc. The effects of plant Zn and plant species on plant Cd bioavailability were tested in Japanese quail fed lettuce and spinach. Four groups of birds received 10% of their diets as lettuce or spinach leaves intrinsically labeled with 109Cd and containing low or high intrinsic Zn. Two other groups were fed control diets containing 109Cd as CdSO4 and low or high Zn as ZnCO3. Cadmium concentrations in diets ranged from 0.857 to 1.05 micrograms/g dry wt. Zinc concentrations in low-Zn diets ranged from 21.2 to 22.8, and in high-Zn diets from 56.0 to 63.3 micrograms/g dry wt. Increased lettuce and spinach Zn decreased plant Cd retention in kidney, liver, and jejunum-ileum of Japanese quail. Spinach Cd was less absorbed than lettuce Cd at both Zn levels. Inorganic Zn produced a lesser decrease in Cd retention in kidney, liver, and jejunum-ileum than did plant Zn. We conclude that (1) crops that transport Zn and Cd readily into edible tissues show lower Cd bioavailability when grown in Zn-Cd contaminated environments than in Cd-only polluted sites, (2) plant species differ in Cd bioavailability for identical concentrations of Zn and Cd in edible tissues, and (3) toxicological studies with animals exposed to Cd salts and Zn supplements do not assess Cd bioavailability of Zn-Cd contaminated crops.

The phylogenetic conservation of the primary structure of PTH-related protein (PTHrP) supports an important, yet undetermined, role(s) for this molecule in the biology of birds and mammals. As an initial step toward understanding the function of PTHrP in birds, we investigated the expression of PTHrP mRNA in tissues of the egg-laying hen. This analysis revealed that PTHrP mRNA is expressed at various levels in lung, brain, heart, and tissues of the digestive tract, including the proventriculus (secretory stomach), gizzard, and small intestine. In the oviduct tissues of adult birds, PTHrP mRNA was detected in the isthmus (membrane-secreting) and shell gland (calcium-secreting) portions, but not in magnum (albumin secreting) tissue. During oviduct development, high levels of PTHrP mRNA present in the oviducts of the 12-week-old bird suggest a role for PTHrP in oviduct development. Interestingly, as the oviduct matures, relatively high levels of PTHrP mRNA segregate with the distal tissues that ultimately differentiate into the isthmus and shell gland (uterus). To address a possible role for PTHrP in the differentiated function of the shell gland, we followed the expression of PTHrP in the shell gland at different times in the laying cycle and found levels of PTHrP to transiently increase as the egg moves through the oviduct, gradually returning to basal levels in the 15-h calcification period. We localized the cycle-associated fluctuations in PTHrP mRNA levels to the shell gland serosa and smooth muscle layer. Immunoreactive PTHrP was localized to the serosal membrane as well as the smooth muscle layer of serosal arterioles, suggesting that PTHrP may modulate vascular smooth muscle activity. In support of this hypothesis, synthetic chicken PTHrP (1-34)NH2 was found to relax the resting tension of isolated shell gland blood vessels in a dose-dependent manner. Together, these data indicate that the expression of the PTHrP gene in the avian oviduct is both temporally and spatially regulated during the egg-laying cycle and that PTHrP may function as an autocrine/paracrine modulator of shell gland smooth muscle activity of both ductal and vascular origins. The vasorelaxant property of N-terminal fragments of PTHrP supports a role for this molecule in the temporal increase in blood flow to the shell gland during egg calcification.

Model-based compartmental analysis was used to describe data on the whole body kinetics of copper metabolism in rats. Data were collected on recovery of radio-copper in plasma, liver, skin, skeletal muscle, bile, and feces for 3 days after the injection of 10 micrograms iv of copper labeled with 64Cu. Data on copper masses and on 64Cu kinetics were analyzed by the stimulation, analysis, and modeling computer programs. Observed data were fit to a 16-component "working hypothesis" model; fractional transfer coefficients were estimated, and steady-state compartment masses and copper transfer rates were calculated. The model postulates a rapid turnover of plasma nonceruloplasmin (NCp) copper (8.5 h-1), which exchanged a large amount of copper with extrahepatic tissues (16 micrograms/h) as well as liver (13 micrograms/h). In comparison, ceruloplasmin (Cp) transferred relatively little copper to tissues (less than 1 microgram/h). NCp copper is postulated to be the precursor for biliary copper. Regarding the intracellular metabolism of copper in hepatic and extrahepatic tissues, the data were compatible with a faster turning-over compartment that exchanged copper with NCp and a slower turning-over compartment with input from Cp. In the liver, the slower turning-over compartment was the copper present in peak II (mol mass 30,000-40,000 Da); this received much of its input from the faster turning-over compartment (peak III), which was hypothesized to represent, in large part, copper in metallothionein.

Young broiler chicks were more sensitive to copper toxicity when they were fed diets deficient or marginal in calcium content. Growth rate was depressed and liver copper concentration was increased under these conditions. Chicks fed a casein-gelatin diet were more sensitive to copper toxicity than those fed a corn-soybean meal diet. Addition of phytic acid to the casein-gelatin basal diet enhanced copper toxicity as evidenced by effects on growth rate and liver copper content. Measurements of intestinal and biliary copper content suggested that the influence of calcium on copper toxicity was mediated via intestinal absorption rather than through influences on copper excretion.

An experiment with a factorial arrangement of treatments was conducted using five levels of dietary calcium and two levels of zeolite A. When dietary calcium was deficient or marginal, zeolite A improved calcium utilization, as evidenced by improved growth rate and a reduction in rachitic lesions. There was little benefit from adding zeolite A to diets adequate in calcium. The effect of zeolite A on the incidence of tibial dyschondroplasia was inconsistent.

The influence of the sex-linked dwarfing gene (dw) on growth and skeletal development in young male broiler chicks was investigated. Chickens that were homozygous (dw/dw) for the dwarfing gene had significantly lower BW and tibiotarsus lengths when compared with the heterozygous (Dw/dw) or normal (Dw/Dw) genotypes. All genotypes exhibited similar incidences of tibial dyschondroplasia. In contrast to several other types of dwarfism, there was no effect of the dw gene on the width of the epiphyseal growth plate or the proteoglycan content of this tissue.

Lysyl oxidase activity was found in the isthmus (the membrane-forming region) of the hen's oviduct in a copper-rich region proximal to the shell gland. Desmosine and isodesmosine, cross-linking compounds associated with mature elastin, were found in hydrolysates of the shell membrane, confirming the necessity for lysyl oxidase in its biosynthesis. Shell membranes from hens fed a copper-deficient diet or a diet supplemented with beta-aminopropionitrile had a reduced content of desmosine and isodesmosine.

Three experiments were conducted to determine the affect of dietary manganese on cholesterol and lipid metabolism in avian species. In the first experiment, day-old chicks were fed a manganese-deficient (4.8 micrograms/g) and a manganese-supplemented (104.8 micrograms/g) diet for 4 weeks after which time they were injected with estrogen (5 mg diethylstilbestrol per kilogram body weight). Manganese deficiency did not significantly alter plasma or liver cholesterol in either group. Estrogen administration significantly increased plasma cholesterol concentration in both dietary groups and liver cholesterol in the manganese-deficient group. In the second experiment, 15-week-old White Leghorn pullets were fed a manganese-deficient (4.5 micrograms/g) diet for 10 weeks. Although dietary manganese deficiency significantly decreased hepatic manganese and cholesterol concentrations, it did not affect hepatic cholesterol and fatty acid synthesis, liver lipid, plasma or egg yolk cholesterol. Similar results were obtained in the third experiment with older (36-week-old) laying hens given similar manganese-deficient and adequate diets. These results indicate that dietary manganese deficiency in the avian species does not result in a significant alteration of cholesterol and lipid metabolism.

Nickel toxicity was studied in young chicks fed a semi-purified diet. Dietary nickel concentrations of 300 mg/kg and higher resulted in significant reduction in growth rate. Mortality and anemia were observed in chicks receiving 1100 mg/kg nickel. Dietary nickel content of 300 mg/kg resulted in a significant increase in kidney nickel content while higher dietary levels were required to affect the nickel content of other body tissues. Supplementation of nickel toxic diets (500 mg/kg) with 100 mg/kg of cobalt, iron, copper, and zinc did not alleviate the symptoms of nickel toxicity or consistently affect tissue nickel accumulation. The addition of cobalt resulted in a further depression in growth rate when added to the nickel toxic diet. However, subsequent studies showed that this was due to the toxicity of cobalt and no evidence was found for an interaction between these two elements. The lack of interaction of nickel with copper, iron, and zinc is in contrast to the results observed by other investigators at low dietary concentrations of nickel.

The effect of inherited chondrodystrophy on the composition of embryonic turkey cartilage was studied. Cartilage from embryos homozygous for the mutant gene contained less than one-half the normal amount of galactosamine containing mucopolysaccharides. Histological examination also showed that there was a substantial decrease in extracellular matrix content of the chondrodystrophic cartilage.

Growth hormone status of normal and tibial dyschondroplastic (TD) birds was determined in 25 d old male chicks genetically selected for high and low incidence of TD. Birds were surgically prepared with indwelling venous catheters and blood samples remotely removed at 20 min intervals for 6 h to establish secretory patterns. Birds were maintained under a 16L:8D cycle, with free access to feed and water at all times. In a second experiment, secretory capacity was evaluated by administering a 10 micrograms/kg body weight dose of thyrotrophin releasing hormone (TRH). Blood samples were removed at 0, 5, 10, 20, 30, 60 and 120 min post-infusion of either TRH or saline (control). All birds displayed pulsatile patterns of GH secretion, with an average peak duration of 60 min and a 90 min inter-peak interval. Dyschondroplastic birds exhibited 50% higher mean peak amplitudes than normal birds (P less than .06), however, this difference was not translated into overall mean or total (curve area) differences. The magnitude of response to a TRH challenge was greater (P less than .10) for TD than for normal birds. In view of the relationships observed in other species between secretory pattern characteristics such as peak amplitude, and growth characteristics, it is suggested that differences in GH status of dyschondroplastic relative to normal birds may be related to initiation of the TD lesion.

Selection for abdominal fat pad size in mature hens has yielded two divergent strains of lean and obese chickens. The females in the obese line have been shown to have a greater amount of total body lipid by six weeks of age. Liver slices from 6- and 17-week old obese chickens incorporated significantly more 3H2O into extracted fractions of total lipid and saponified fatty acids. Palmitate esterification by abdominal fat pad slices from 12-week old pullets was the same for both strains when activity was expressed per unit weight of tissue. The twofold difference in size between the lean and obese fat pads at this age, however, indicates a significantly greater degree of total esterification in the obese birds. At 6-, 12-, and 17-weeks of age, the two lines exhibited similar concentrations of plasma glucose, total protein, and triglycerides.

This study was conducted to assess the effects of age and dietary levels of copper and zinc on the intracellular distribution of these metals in sheep, the domestic species most susceptible to copper toxicity. Hepatic copper concentration was lower in newborn lambs than in 30- and 60-day old lambs and its distribution in neonates differed significantly from that in the older animals and from that observed in newborn and adult rats. Sheep previously maintained on a low-copper diet for 50 days were then fed diets containing 2.2, 11.3 or 47.0 microgram Cu/g diet with and without zinc supplementation (543 or 46 microgram Zn/g diet, respectively) for 60 days. Ceruloplasmin activity, total plasma copper and hematocrit were lower in zinc-supplemented sheep. Hepatic copper concentration was not reduced by zinc supplementation but was increased with each increase in dietary copper; the distribution pattern was significantly altered as hepatic copper increased. Hepatic zinc concentration and distribution were not affected by diet. Sheep fed the highest level of copper had higher copper concentrations in the mucosa of the small intestine and in kidney cortex. The concentrations of zinc in the kidney and of copper and zinc in diaphragm muscle and bile were not affected by diet.

The cadmium content of body tissues and eggs was studied in broiler chicks and laying hens fed diets supplemented with 3, 12, and 48 microgram/g of cadmium. The 48 microgram/g level was selected as a slightly toxic level while the lower levels were felt to be representative of the amounts of cadmium which would occur in feedstuffs due to environmental contamination. All levels of cadmium resulted in increased cadmium content of kidney while only 12 and 48 microgram/g resulted in increases in the cadmium content of liver and muscle. As little as 3 microgram/g cadmium consistently increased the cadmium content of liver and muscle but this did not prove to be statistically significant. The transfer of dietary cadmium to the egg was found to be very low. Only the 48 microgram/g level resulted in an increase in cadmium content of the egg. This dietary treatment also resulted in reduced egg production and egg shell thickness.

The avian epiphyseal growth plate plays an important role in skeletal development. Compared with the mammalian species, the avian growth plate contains more cells, is less well organized and appears more susceptible to malformation. Abnormal cartilage development is associated with chondrodystrophy, tibial dyschondroplasia and rickets. Many nutrient deficiencies result in chondrodystrophy, which is characterized by shortened, thickened bones and a narrowing of the epiphyseal growth plate. Manganese, the most extensively studied of these deficiencies, is necessary for proteoglycan biosynthesis. Tibial dyschondroplasia is a condition in which the prehypertrophic cells fail to hypertrophy and vascularization is aborted. This abnormality is found in genetically predisposed animals and its occurrence is altered by subtle changes in calcium, phosphorus and electrolyte content of the diet. Calcium and vitamin D deficiencies cause rickets, which is characterized by an increase in the width of the prehypertrophic zone of the epiphyseal growth plate. Most of the biochemical and histological changes can be related to the need for calcium for chondrocyte hypertrophy and maturation, although there is some evidence that chondrocytes require specific vitamin D metabolites. Phosphorus deficiency increases the zones of hypertrophy and metaphyseal primary spongiosa.

Liver cytosol from sheep fed diets containing 2.2, 11.3 or 47 microgram Cu/g diet with or without supplemental zinc (543 or 46 microgram Zn/g diet), fractionated on Sephadex G-100, yielded three main copper- and zinc-containing proteins with approximate molecular weights of greater than 150,000, 27,000 and 10,000. Amino acid analysis of the 10,000-molecular-weight proteins were of the metallothionein type. Copper-chelatin was not present in sheep liver cytosol. Copper concentration of the metallothionein fraction increased (P less than 0.01) as dietary copper increased from 2.2 to 11.3 microgram Cu/g, but did not increase further when dietary copper increased to 47 microgram Cu/g in unsupplemented sheep. A low-molecular-weight (approximately 3,500) copper-, but not zinc-containing fraction appeared at this highest level of copper. Zinc supplementation of the diet increased not only the zinc content of the metallothionein fraction but also its copper content, most dramatically in sheep fed the highest copper level. In intestinal mucosal cytosol, no copper and little zinc was associated with the metallothionetin fraction which was not affected by dietary treatment. Evidence from this study suggests that sheep have limited capacity to synthesize metallothionein in response to increased dietary copper.

Copper deficiency in the laying hen resulted in anemia and the production of eggs which were abnormal in size and shape. Many of the eggs had shells which were wrinkled and rough in texture. There was also an increase in the number of shell-less eggs. Examination of malformed egg shells using the scanning electron microscope revealed ultrastructural changes in the mammillary layer of the shell. The effect of copper deficiency on shell formation was attributed to the shell membranes which were altered in color, appearance, and physical consistency. Amino acid analysis of the membranes indicated that the membranes from copper deficient hens were characterized by an increase in lysine content. This suggests that copper is necessary for the formation of lysine derived cross-links in a manner similar to that which occurs in connective tissue. The exact nature of these cross-links is unknown at this time.

Manganese deficiency in the laying hen results in decreased egg production and eggshell thickness. Eggshells from deficient hens show specific changes in appearance characterized by translucent areas. Examination of these translucent areas with the scanning electron microscope revealed changes in the ultrastructure of the shell. The most prominent feature was large irregular mammillary knobs probably due to the fusion of several mammillary cores during the early phases of eggshell formation. Chemical analysis of the organic matrix of the eggshell showed a decrease in hexosamine and hexuronic acid content consistent with the known role of manganese in polysaccharide synthesis. Studies on the characteristics of the polysaccharide chains isolated from normal eggshells suggested that these polysaccharides were considerably different from those isolated from cartilage proteoglycans.

Similarities in morphology between copper-deficient cartilage and abnormal cartilage associated with tibial dyschondroplasis (TD) led to studies dealing with copper metabolism and its possible relation to TD. Abnormal cartilage and copper deficient cartilage cells both oxidize significantly less glucose to CO2 and water when compared to normal epiphyseal and day-old hypertrophic cartilage cells. Plasma ceruloplasmin levels and cartilage copper content were not different between normal birds and those affected wth TD, which seemed to rule out a genetic defect in copper metabolism as being partly responsible for the abnormal cartilage occurrence. Mitochondrial marker enzyme activities were investigated, and abnormal cartilage showed a significant decrease in activity of both cytochrome oxidase and citrate synthase. The yield of mitochondria on a percent of total activity basis was quite low from both normal and abnormal cartilages, and, thus, an absolute conclusion with regard to mitochondrial impairment cannot be made at this time.

Two experiments were conducted to determine the effect of dietary manganese on cholesterol and lipid metabolism in the Wistar rat and the genetically hypercholesterolemic RICO rat. Weanling animals were placed on a manganese-deficient (0.12 microgram/g) and a supplemented diet (100.12 micrograms/g). Mean body weights, hepatic fatty acid synthesis and liver manganese concentration significantly decreased in the deficient group of Wistar rats. Plasma cholesterol, VLDL (very low density lipoprotein) and HDL (high-density lipoprotein) cholesterol, hepatic cholesterol synthesis, liver cholesterol and lipid concentrations were not significantly affected by manganese deficiency. Mean body weights and hepatic manganese content were lower in the manganese-deficient group in both normal and hypercholesterolemic RICO rats. Manganese deficiency significantly decreased LDL cholesterol concentration in the hypercholesterolemic RICO rats. Manganese deficiency had no significant effect on hepatic cholesterol and fatty acid synthesis, plasma cholesterol, VLDL and HDL cholesterol concentrations, liver lipid and liver cholesterol concentration in either group of RICO rats. These results indicate that dietary manganese deficiency does not result in significant alterations in cholesterol and lipid metabolism in the rat.

Two distinctly different basal diets were used to test the influence of supplementary ascorbic acid on the occurrence of tibial dyschondroplasia. Addition of either .1 or .25% ascorbic acid to these diets did not alter the occurrence of tibial dyschondroplasia in 25-day-old broiler chickens. However, supplementary ascorbic acid did increase the amounts of ascorbic acid present in blood plasma. Also, individual differences in circulating ascorbic acid were not associated with this disease. Furthermore, two strains of chickens selected for high or low incidence of tibial dyschondroplasia had similar amounts of this vitamin in blood plasma. In contrast to the results reported for the Willow Ptarmigan, ascorbic acid does not appear to be involved in the development of tibial dyschondroplasia in the young broiler chicken.

Genetic selection for mature abdominal fat pad size has resulted in establishing lean and obese lines of chickens. Although the selection was made with mature hens, studies with the progeny showed that obesity could develop as early as 6 weeks of age. Body weights in the two lines diverged by 7 weeks of age although significant differences in feed intake were not seen until the fourteenth week. A pair-feeding study was conducted from 7 through 17 weeks of age. The results showed that pair-feeding greatly reduced the lipid deposited in the obese line. The two strains differed substantially in carcass lipid content, but the proportion of total body lipid contributed by the carcass, viscera, and abdominal fat pad was similar in both lines.