Investigation of the insulin-like growth factor system in the avian epiphyseal growth plate.

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.

Comparative assessment of bone among wild-type, restricted ovulator and out-of-production hens.

1. The aim of this study was to assess bone characteristics in restricted ovulator (RO) hens. These hens generally are unable to ovulate due to a point mutation in the oocyte VLDL receptor gene whose protein product mediates the uptake of yolk precursors. Because these hens do not have the cyclic calcium (Ca) metabolism associated with egg formation, they could be a useful model for studying bone metabolism. 2. RO hens had greater humerus, femur and tibia ash concentrations than wild-type (WT) and out-of-production (OP) hens. Bone mineral content and density obtained with dual-energy X-ray absorptiometry (DXA) were highly correlated with the results of conventional bone assays. 3. Gross and histological examination of the femurs confirmed the presence of extremely dense medullary bone deposition in the RO hens. However, the composition of non-collagenous protein extracts of medullary bone was similar for the two genotypes. 4. Analysis of medullary bone extracts for glycosaminoglycans (GAG) confirmed the presence of large amounts of keratan sulphate (KS) in the matrix of medullary bone. 5. Plasma Ca, total GAG and KS concentrations of RO hens were markedly higher than WT and OP hens. The changes in plasma calcium and keratan sulphate are probably a reflection of elevated Ca-binding yolk precursor molecules and intensive medullary bone formation in response to increased plasma oestrogen observed by others in RO hens.

Collagen X expression in oviduct tissue during the different stages of the egg laying cycle.

The purpose of this investigation was to study the expression of type X collagen in the hen's oviduct. Type X collagen is a short-chain collagen that is present in the fibers of eggshell membranes, and there is evidence to suggest that it contributes to structural integrity. In situ hybridization and Northern blot analysis were used to study the expression of this important matrix constituent. The results demonstrated that gene expression was predominantly in the tubular gland cells of the isthmus segment of the oviduct. In contrast to observations with other matrix proteins, such as parathyroid hormone-related peptide and osteopontin, gene expression did not fluctuate with the position of the egg in the oviduct.

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate.

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.

The use of growth factors in the proliferation of avian articular chondrocytes in a serum-free culture system.

The purpose of this research was to develop a serum-free culture system for the proliferation of articular chondrocytes. Various growth factors and hormones were tested for their ability to stimulate avian articular chondrocyte proliferation in a defined, serum-free media. Multiple members of the fibroblast growth factor (FGF) family (FGFs: 2, 4, and 9), insulin-like growth factor-1 (IGF-1) and transforming growth factor beta (TGF-beta) significantly stimulated H-thymidine uptake by chondrocytes grown in an adherent serum-free, culture system. Double or triple combinations of these mitogenic growth factors further stimulated cell proliferation to levels that were equivalent to, or surpassed those of cells grown in serum. Although proliferation was maximally stimulated, chondrocytes grown in the presence of FGF-2, IGF-1, and TGF-beta, began to exhibit changes in morphology and collagen II expression declined. This culture system could be used to rapidly expand a population of articular chondrocytes prior to transferring these cells to a non-adherent culture system, which could then stabilize the chondrocyte phenotype and maximize matrix synthesis and integrity.

Gene expression and tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a skeletal deformity associated with rapid growth in a number of avian species. The disease is the result of a disruption in the cascade of events that occur in the epiphyseal growth plate. Whereas the incidence of TD is susceptible to genetic selection, no specific genetic defect has been identified. Although there are extensive data describing the morphological and biochemical characteristics of the lesion, the mechanism of lesion formation is unknown. However, naturally occurring or induced genetic mutations in other species can provide important clues to possible mechanisms responsible for lesion development. Disruption of normal chondrocyte differentiation by constitutive activation of the parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, inactivation of the fibroblast growth factor receptor-3 (FGFR-3) receptor, and blocking vascular endothelial growth factor (VEGF) signaling all result in lesions that resemble TD. Impairment of vascular penetration due to the ablation of matrix metalloproteinase-9 (MMP-9) or tartrate-resistant acid phosphatase (TRAP) activity also results in similar cartilage abnormalities. We have integrated these observations with our current knowledge of TD to describe a hypothesis for the sequence of events responsible for the development of tibial dyschondroplastic lesions.

Morphometric and histologic changes in the pulmonary system of broilers raised at simulated high altitude.

Broilers were reared in conditions of hypobaric hypoxia (3500 m altitude) to investigate the development of pulmonary nodules in birds reared in hypobaric hypoxia and judged by clinical observation to be developing broiler pulmonary hypertension syndrome (BPHS); in unaffected birds reared in hypobaric hypoxia; and in birds reared at ambient atmospheric pressure. Gross pulmonary morphometric measurements, packed cell volume, electrocardiogram QRS amplitude and body weight also were compared among the three experimental groups. Results indicate that hypobaric hypoxia alone exercised little influence on the development of pulmonary nodules. Nodule numbers per section and nodule area per section were numerically greater in birds reared in hypobaric hypoxia, but there were no consistent significant differences in nodule numbers in birds which developed BPHS. As expected, absolute lung length, inter-rib distance and lung volume increased significantly with increasing age and size; but when these parameters were expressed as a function of body weight, they decreased with increasing age. Again, intergroup differences were inconsistent. Packed cell volume and electrocardiogram QRS amplitude were significantly increased in birds reared in hypobaric hypoxia.

Physiologic and electrocardiographic changes occurring in broilers reared at simulated high altitude.

An experiment was conducted to determine whether differences in the electrocardiograms (EKGs) of broilers reared at simulated high altitude from the day of hatch can be used to predict which birds are developing ascites. In three replicate experiments, conducted with 100 broilers per replicate, birds were reared at a simulated altitude of 3000 meters or at ambient atmospheric pressure. Lead I, II, and III EKGs were obtained from all birds on days 0, 14, 28, and 42. No consistent significant differences were seen on day 0 in the amplitude of the R or S wave or total amplitude of the QRS complex when broilers that developed ascites while being reared at simulated high altitude were compared with unaffected birds reared at simulated high altitude and with birds reared at ambient atmospheric pressure. On days 14 and 28, the average amplitude of the S wave and the total amplitude of the QRS complex were significantly higher in the ascites group than in the two other groups. Packed cell volumes were significantly higher in birds reared at simulated high altitude at all sampling days (days 14, 28, and 42) than in those reared at ambient atmospheric pressure, and they were significantly higher in the ascites group on day 28 than in the two other groups. Birds in the ascites group weighed significantly less than the two other groups by day 14, and this trend persisted.

Responses of laying hens to diets containing up to 2% DL-methionine or equimolar (2.25%) 2-hydroxy-4-(methylthio)butanoic acid.

Diets supplemented with up to .6% DL-Met (DLM) or .68% 2-hydroxy-4-(methylthio)butanoic acid (HMB, Alimet) acidify the urine and reduce the incidence of urolithiasis in pullets and laying hens. Excessive acidification potentially may reduce eggshell quality and bone mineralization by interfering with Ca metabolism and may severely challenge the liver and kidneys, which are the primary organs responsible for attenuating metabolic acidosis. To evaluate these possibilities, 30-wk-old Single Comb White Leghorn hens in full production (five hens per replicate, six replicates per diet treatment) were fed for 30 d a 15.7% CP corn and soybean meal-based control layer ration alone or supplemented with DLM (.5, 1, 1.5, or 2%) or equimolar HMB (.56, 1.13, 1.69, or 2.25%). None of the diets caused mortality or gross hepatic or renal damage. Hens fed diets supplemented with the highest levels of DLM and HMB exhibited significant reductions in feed intake, hen-day egg production, and liver mass and had lower plasma concentrations of alanine amino-transferase and isocitrate dehydrogenase when compared with hens fed the control diet. Kidney mass was not significantly affected by high levels of DLM or HMB, but plasma uric acid was significantly higher in hens fed 2% DLM compared with hens fed the control diet. The highest levels of DLM and HMB did not significantly alter total plasma Ca or inorganic phosphate concentrations, nor were percentage eggshell or femur mineralization (femur ash mass:defatted bone mass, femur ash mass:bone volume) significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute heat acclimation and kidney function in broilers.

Broilers previously exposed to high environmental temperatures (heat-acclimated) are more resistant to heat stress and consume more water during heat stress than nonacclimated controls. Two experiments were conducted to determine whether heat-acclimated broilers conserve body water by reducing urine and solute (Na) excretion. In the first experiment, renal function studies were conducted at an ambient temperature (Ta) of approximately 21 C using anesthetized 7-wk-old male broilers. Control birds reared at a constant Ta of 24 C (Group N: noncycled Ta) were compared with birds that had been heat-acclimated by exposure for 3 to 6 d to a daily sinusoidal cycle of 24 to 35 to 24 C (Group C: cycled Ta). In the second experiment, renal function studies were conducted on anesthetized 5-wk-old control and heat-acclimated male broilers while they were exposed to a Ta of 21 C (Ambient Ta: Groups NA, CA), or to a Ta of 32 C (High Ta: Groups NH, CH). When high intravenous infusion rates (.37 mL/kg body mass per min) were used to simulate the volume expansion caused by thermogenic polydipsia, urine flow rates were significantly lower in Groups C and CA than in Groups N and NA, osmolal clearances were lower in Groups CA and CH than in Groups NA and NH, and all heat-acclimated groups in both experiments (Groups C, CA, CH) had significantly lower glomerular filtration rates (GFR), filtered loads of Na, and tubular Na reabsorption rates than the respective control groups (Groups N, NA, NH). These changes in kidney function potentially would minimize urinary fluid and solute loss when heat-acclimated broilers consume large quantities of water to support evaporative cooling. Reductions in GFR, filtered loads of Na, and tubular Na reabsorption rates also may help heat-acclimated broilers reduce the metabolic heat load associated with active (energy requiring) recovery of solute (Na) from the glomerular ultrafiltrate.

Liquid methionine hydroxy analog (free acid) and DL-methionine attenuate calcium-induced kidney damage in domestic fowl.

To evaluate the possibility that kidney damage may be induced by the commercial practice of feeding high-Ca (HCa) prelayer rations, and to evaluate the protective efficacy of supplementing HCa diets with liquid methionine hydroxy analog free acid or DL-methionine, 12-wk-old female Single Comb White Leghorn pullets were fed one of the following corn-soybean meal-based diets until they reached 22 wk of age: normal-Ca (NC, 1% Ca); HCa (HC, 3.5% Ca); HCa supplemented with .34 or .68% liquid methionine hydroxy analog free acid (HC3A or HC6A); or HCa supplemented with .3 or .6% DL-methionine (HC3DL or HC6DL). The unsupplemented HC diet caused a significant reduction in kidney mass and a significant increase in the incidence of gross kidney damage and urolithiasis in pullets necropsied at 22 wk of age. Calcium-induced kidney damage was attenuated in a dose-response fashion by supplementing the HC diet with liquid methionine hydroxy analog and DL-methionine. None of the diets caused a significant metabolic acidosis. Plasma uric acid concentrations were not predictive of the extent of Ca-induced kidney damage. Analyses of glomerular size distributions indicated that subclinical or "hidden" kidney damage may not progressively develop into urolithiasis as hens mature. When compared with hens reared on the NC diet, rearing hens on the HC, HC3A, HC3DL, HC6A, or HC6DL diets did not consistently affect hen-day egg production, egg mass, eggshell mass, percentage eggshell, or bone mineralization.