Expression patterns of candidate genes for tibial dyschondroplasia in chicken growth plate chondrocytes separated on percoll density gradients.
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Animals↗
Biomedical subjects
C C Whitehead
Publications and source records attributed to C C Whitehead.
Vitamin E can alleviate the depressed egg production of heat-stressed laying hens.
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Animals↗
Digitised fluoroscopy (DF) predicts breaking strength in osteoporotic avian bone in vivo.
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Animals↗
Bone structure and strength at different ages in laying hens and effects of dietary particulate limestone, vitamin K and ascorbic acid.
1. A range of bone structural and strength characteristics was determined in laying hens at 15, 25, 50 and 70 weeks of age. The birds were fed up to 25 weeks on diets supplemented with additional vitamin K (10 mg menadione/kg) or ascorbic acid (250 mg/kg) or up to 70 weeks on diets containing limestone in powder or particulate form. 2. There were important effects of age on all bone characteristics. Between 15 and 25 weeks there was a rapid loss of cancellous bone and a rapid accumulation of medullary bone in the proximal tarsometatarsus (PTM). These changes continued at a slower rate up to 70 weeks. Cancellous bone content of the free thoracic vertebra (FTV) also declined after 15 weeks. 3. Breaking strengths of tibia and humerus did not change between 15 and 25 weeks but decreased later in lay. 4. None of the nutritional treatments affected bone characteristics at 15 weeks of age. 5. Increasing the dietary vitamin K supplement from 2 to 12 mg menadione/kg increased cancellous bone volume in the PTM at 25 weeks. 6. Dietary ascorbic acid did not affect any of the bone characteristics measured up to 25 weeks. 7. Particulate limestone resulted in a smaller loss of cancellous bone between 15 and 25 weeks and increased accumulation of medullary bone in the PTM. Breaking strength of the tibia and radiographic densities of tibia and keel were also improved. 8. It is concluded that patterns of bone loss over the lifetime of laying hens vary, depending upon the bone type. Feeding a particulate source of calcium can help to alleviate some of the characteristics of osteoporosis. Supplementation with extra vitamin K may also be beneficial.
Aging↗
Influence of high dietary vitamin E supplementation on egg production and plasma characteristics in hens subjected to heat stress.
1. The effects of different dietary concentrations of vitamin E (alpha-tocopherol acetate) were investigated in 2 experiments on laying hens exposed to chronic heat stress at 32 degrees C. 2. In the first experiment, egg production and plasma concentrations of calcium and egg yolk precursors were measured in 24 hens before, during and after a stress period of one week and fed on diets containing 10 or 500 mg vitamin E/kg. 3. In the second, larger experiment, egg production and food intake were measured in 300 hens housed in 2 temperature-controlled rooms and fed on diets containing 10, 125 or 500 mg vitamin E/kg. Birds in room 1 were stressed from 24 to 28 weeks of age and those in room 2 from 32 to 36 weeks. 4. In experiment 1, egg production and egg weight were significantly higher (72.6 vs 51.2%, P < 0.05 and 66.6 vs 63.1 g, P < 0.005 respectively) during and after the period of stress in the group given 500 mg vitamin E/kg. Plasma concentrations of calcium, vitellogenin (zinc) and VLDL (triglyceride) were also higher in this group. 5. In experiment 2, egg production was significantly higher (65.4 vs 56.2%, P < 0.05) during and after the period of heat stress in birds in room 1 fed on the diet containing 500 mg vitamin E/kg. Egg production was also higher (49.9% vs 44.7%) on this treatment during the stress period in room 2, though the difference was not significant (P < 0.10). Egg weight and food intake were unaffected by treatment in either room. 6. It is concluded that dietary supplementation with extra vitamin E can, at least in part, alleviate the adverse effects of chronic heat stress in laying hens, perhaps by maintaining the supply of egg precursors in plasma.
Animal Feed↗
Dyschondroplasia in poultry.
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Animal Nutritional Physiological Phenomena↗
Studies on effects of nutritional factors on bone structure and osteoporosis in laying hens.
1. A modern hybrid strain of laying hen (Hisex) was fed from point of lay to 68 weeks on a control diet and diets containing oystershell, fluoride, 1,25-dihydroxycholecalciferol, ascorbic acid, a lower concentration of phosphorus and a combination of a lower concentration of crude protein and higher concentration of vitamin K. Hens from a much older strain (Brown Leghorn J-line) were fed on the control diet. 2. Plasma variables were measured during lay. End-of-lay trabecular and medullary bone volumes in the proximal tarsometatarsus and free thoracic vertebra were measured by histomorphometry. 3. The majority of Hisex hens were considered to be osteoporotic by the end of lay. In contrast, none of the J-line were osteoporotic. 4. None of the nutritional treatments affected trabecular bone volumes. Medullary bone volumes were increased significantly by feeding oystershell or fluoride. 5. There was no phenotypic correlation between egg production and trabecular bone volume in the Hisex hens. 6. The experiment provided evidence that osteoporosis in laying hens, as assessed by trabecular bone volumes, is not caused by calcium deficiency and could not be prevented by any of the nutritional treatments studied.
Animal Nutritional Physiological Phenomena↗
Further observations on programmed cell death in the epiphyseal growth plate: comparison of normal and dyschondroplastic epiphyses.
The objective of the investigation was to provide information on apoptosis in the normal epiphysis and to assess apoptosis in the plate of the dyschondroplastic chick. Apoptosis was evaluated using two terminal deoxynucleotide transferase end-labeling procedures, DNA fragmentation and nuclear morphology. We found that there was a minimal level of apoptosis in the dyschondroplastic cartilage. In the tibial dyschondroplastic (TD) lesion itself, only about 3% of cells are positive in the articular and proliferative regions; 11% of prehypertrophic chondrocytes are stained by the end-labeling procedure, and most of the cells are localized around vascular channels at the calcifying front. This finding suggests that dyschondroplasia is linked to impairment of apoptosis, and as a result the tissue contains immature cells that have outlived their normal life span. In contrast, in the normal plate, we noted that when the proliferative period was complete, the cells became terminal transferase positive; in addition, chondrocytes in the normal plate exhibited DNA fragmentation. Semiquantitative analysis of stained chondrocytes in the growth plate indicate that in the proliferative zone 15.5% of cells are terminal deoxynucleotidyl transferase (TUNEL) positive; in contrast, 44% of postmitotic chondrocytes are stained by the TUNEL procedure. The presence of a sharp border between the pre- and postmitotic zones suggests that the stimulus for apoptosis is maturation dependent and reflects local metabolic control. We also examined apoptosis in metaphyseal osteoblasts. We found that adjacent to the epiphysis, many osteoblasts were undergoing apoptosis. In more mature sites in the metaphysis, there was less cell death, indicating that osteoblast apoptosis was delayed and cells were completing their normal life cycle. Although terminal transferase end-labeled cells were not seen in articular cartilage, we noted that fibroblasts, in the perichondrial ligament surrounding the articular as well as the epiphyseal regions of the plate, were undergoing apoptosis. Apoptosis at this site may be related to lateral expansion of the cartilages, reflect a high cell turnover rate at the junction between the tissues, and result from paracrine signals received from the underlying cartilage.
Animals↗
Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate.
In order to assess alterations in the collagen network during endochondral ossification the pyridinium cross-links of collagen were quantified in sequential transverse sections through the chick growth plate. This was accomplished using both morphological (alkaline phosphatase (ALP) histochemistry and collagen type X immunostaining) and analytical (HPLC) analyses. In articular cartilage, pyridinoline concentrations were maximal in the deep mature zones. In contrast, the proliferating chondrocyte zone of the growth plate had approximately a 10-fold greater pyridinoline cross-link concentration than the mature hypertrophic zone. Deoxypyridinoline was first found in the prehypertrophic zone of the growth plate cartilage that reacted positively for ALP activity but before collagen type X was detected. However, deoxypyridinoline concentrations were highest in the most differentiated regions of the growth plate where it was the principal pyridinium cross-link. In tibial dyschondroplasia, where chondrocyte differentiation is arrested in the prehypertrophic zone, higher concentrations of both cross-links were found with increasing distance down the lesion. We conclude that the decrease in pyridinoline cross-link concentration down the growth plate may be an essential adaptation (via increased collagenase activity and collagen turnover) of the matrix for vascular invasion and osteoclastic resorption to occur.
Animals↗
Growth plate chondrocyte vitamin D receptor number and affinity are reduced in avian tibial dyschondroplastic lesions.
Tibial dyschondroplasia (TD) is a condition of rapidly growing poultry in which a mass of unmineralized cartilage extends distally from the tibiotarsal growth plate, leading to deformity and lameness. The lesion is characterized by the accumulation of prehypertrophic chondrocytes, probably because the maturing chondrocytes are unable to differentiate fully. The condition can be prevented by feeding 1,25-(OH)2D3. We have investigated the possibility that vitamin D receptors (VDR), through which 1,25-(OH)2D3 exerts its differentiating effects on chondrocytes, may be defective in TD birds. Chondrocytes were isolated from the proliferating and hypertrophic zones of normal tibiotarsi and from the proliferating zone and lesion of affected birds and receptors were characterized by Scatchard analysis. Results showed that, while cells from the proliferating zone in TD birds had normal receptors, those from the TD lesion had significantly lower numbers and affinity for 1,25-(OH)2D3 compared to all other zones. Lesion VDR had low affinity; Kd 83.9 +/- 20.6 pM compared to 30.0 +/- 2.8, 37.8 +/- 3.1, and 33.0 +/- 4.0 pM (p < 0.001), and low receptor number per cell, 920 +/- 74, compared to 1329 +/- 151, 1664 +/- 167, and 1360 +/- 104 (p < 0.01) in the normal proliferating, normal hypertrophic, and TD proliferating cells, respectively. These findings were confirmed by immunohistochemical localization of VDR in sections of normal and TD growth plates using monoclonal antibody 9A7 gamma. In normal growth plate, most cells were VDR positive with intense staining in the mature hypertrophic chondrocytes; in TD growth plates, proliferating zone cells stained well but signal was largely absent from chondrocytes in the lesion. Image analysis showed integrated nuclear staining density per cell of 168.2 +/- 36.9 arbitrary units in normal hypertrophic cartilage compared to 98.8 +/- 60.2 units in the top of the lesion and 2.2 +/- 2.0 units in the midlesion. We conclude that both numbers and affinity of VDR are reduced in TD and this may explain the failure of chondrocytes to differentiate to the mature form. The adverse consequences of defective receptors may be partly overcome by treatment with 1,25-(OH)2D3.
Animals↗
Effectiveness of dietary 25- and 1-hydroxycholecalciferol in combating tibial dyschondroplasia in broiler chickens.
1. Three experiments were carried out to determine the effects of feeding diets containing different concentrations of cholecalciferol, 1 alpha-hydroxycholecalciferol (1-HCC), 25-hydroxycholecalciferol (25-HCC), 1,25-dihydroxycholecalciferol (1,25-DHCC) and ascorbic acid on the incidences and severities of tibial dyschondroplasia (TD) at 3 weeks of age in male broiler chicks. 2. In experiment 1, replacing 75 micrograms cholecalciferol/kg with the same weight of 25-HCC decreased significantly (P < 0.01) the incidence of TD from 65 to 10%. 3. In experiment 2, the incidence of TD in the control group was lower, but feeding amounts of 25-HCC up to 250 micrograms/kg had a linear effect on the incidence of TD that was significant at P = 0.06. There was no effect or interactions with dietary addition of 250 mg ascorbic acid/kg. Dietary addition of 5 micrograms 1-HCC/kg decreased TD incidence from 21 to 5%, though the effect was not significant (P > 0.1). 4. TD incidence in experiment 3 was too low to determine an effect of25-HCC or 1,25-DHCC on TD incidence, though in this, as in both other experiments, the severities of TD lesions were always lower with diets containing cholecalciferol metabolites. 5. Hypercalcaemia was not observed after feeding up to 250 micrograms 25-HCC/kg in either experiments 2 or 3. 6. It is concluded that 25-HCC may be an effective practical means of improving broiler leg health by alleviating the incidence and severity of TD.
Animals↗
In vivo and in vitro effect of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxy-16-ene-23-yne-vitamin D3 on the proliferation and differentiation of avian chondrocytes: their role in tibial dyschondroplasia.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is regarded as the most biologically active metabolite of cholecalciferol. It prevents tibial dyschondroplasia (TD) in chicks where inhibition of chondrocyte differentiation within the growth plate occurs. However, it is unclear whether its mode of action is through direct interaction with its chondrocyte receptor and its known regulatory role in cell differentiation or is mediated by increased calcium absorption and mobilisation. Synthetic analogues of 1,25(OH)2D3 such as 1,25-dihydroxy-16-ene-23-yne cholecalciferol (RO 23-7553) with increased differentiation properties but reduced calcaemic activity have been synthesised. In this study, the in vitro and in vivo effects of 1,25(OH)2D3 and RO 23-7553 on chick chondrocyte growth and differentiation were examined. In addition, the in vivo effectiveness of these steroids in preventing TD in chicks was assessed. 1,25(OH)2D3 and RO 23-7553 (10(-12)-10(-7) M) displayed biphasic concentration effects and had similar potencies in vitro in regulating chondrocyte proliferation and differentiation. However, while the incidence of TD in birds dosed with 1,25(OH)2D3 was lower (10%) than in control chicks (55%), RO 23-7553 was ineffective (50%). This may be the result of its reduced affinity (1000 times less) for the plasma vitamin D binding protein (DBP) and the chondrocyte receptor in comparison to that of 1,25(OH)2D3. A reduction in calcium supply to the chondrocyte may also result in decreased chondrocyte differentiation but blood ionised and plasma total calcium were normal in birds dosed with RO 23-7553. These data suggest that RO 23-7553 and 1,25(OH)2D3 regulate chondrocyte proliferation and differentiation similarly in vitro but not in vivo. This may be caused by differences in DBP binding and clearance rates of the two steroids in vivo.
Animals↗
The expression of transforming growth factor-beta by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-beta (TGF-beta) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-beta 1 to -beta 3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-beta mRNA and conditioned medium was assayed for TGF-beta activity and isoform composition. Active TGF-beta was only detected in 10(-8) M 1,25(OH)2D3-treated cultures (8.37 ng active TGF-beta/mg protein). There was a significant decrease in total (latent-active) TGF-beta activity in conditioned medium of 10(-12) M (23.4%; P < 0.05) and 10(-10) M (20.7%; P < 0.05) 1,25(OH)2D3-treated cultures but 10(-8) M 1,25(OH)2D3 significantly increased (30.9%; P < 0.01) TGF-beta activity. The amounts of TGF-beta 1, -beta 2 and -beta 3 isoforms produced were similar in control, 10(-10) or 10(-12) M 1,25(OH)2D3-treated cultures but the conditioned medium of 10(-8) M 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-beta mRNA demonstrated differential control of TGF-beta gene expression with TGF-beta 1 and -beta 3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10(-8), 10(-10) and 10(-12) M) whilst TGF-beta 2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-beta secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth.
Animals↗
Biochemical changes in the collagenous matrix of osteoporotic avian bone.
No detailed biochemical analysis has been carried out of the compositional changes in the collagen matrix of avian bone in relation to increased bone fragility in osteoporosis. We have shown that osteoporosis in avian bone is certainly not just a simple loss of apatite and collagen, but involves significant changes in the biochemistry of the collagen molecule and consequently in the physical properties of the fibre. The decreased mechanical strength and the change in the thermal stability can be directly related to changes in post-translational modifications, i.e. lysine hydroxylation and the intermolecular cross-link profile. The increased hydroxylation and change in cross-linking are consistent with increased turnover of the collagen, possibly in an attempt to initiate a repair mechanism which, in fact, leads to an acceleration in the increase in fragility of the bone. Clearly there are post-translational modifications of the newly synthesized collagen in avian osteoporosis, and these changes may play a role in the pathogenesis of the disease.
Animals↗
Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: a model for endochondral ossification.
Chondrocytes isolated from the proliferative and differentiating zones of 3-wk-old chick growth plates were cultured in the presence of 10% fetal bovine serum (FBS) and ascorbic acid for up to 21 d in a high cell density culture within Eppendorf tubes. The proliferative, differentiating, and calcification properties of the chondrocytes were examined by immunolocalization and by enzyme histochemical and biochemical methods. The cells maintained a chondrocyte phenotype throughout culture: they were round in shape and synthesized both collagen type II and proteoglycans. The expression of a hypertrophic phenotype was evident by Day 3 of culture and from this time onwards characteristics of terminal differentiation were observed. The cells were positive for both alkaline phosphatase (ALP) activity and c-myc protein and the surrounding matrix stained strongly for collagen type X. Small foci of mineralization associated with individual chondrocytes were first evident by Day 6 and more widespread areas of mineralization occupying large areas of matrix were present by Day 15. Mineralization occurred without the addition of exogenous phosphate to the medium. This culture system displays characteristics that are similar in both morphological and developmental terms to that of chick chondrocyte differentiation and calcification in vivo and therefore offers an excellent in vitro model for endochondral ossification.
Alkaline Phosphatase↗
Regulators of chondrocyte differentiation in tibial dyschondroplasia: an in vivo and in vitro study.
Tibial dyschondroplasia (TD) is a disorder of endochondral bone growth and results in the retention of a mass of unmineralized, avascular cartilage extending into the metaphysis. We have studied various parameters of chondrocyte differentiation, both in isolated chick chondrocytes and growth plate sections, in an attempt to determine whether the inhibition in chondrocyte differentiation seen in TD is a consequence of an inherent incapability of chondrocytes to differentiate terminally and mineralize. Results from in vitro experiments indicated that both normal and lesion chondrocytes synthesized a matrix that stained with antibodies to types II and X collagen and displayed foci of mineralization. Alkaline phosphatase activity in lesion chondrocytes was significantly increased in comparison to that in normal hypertrophic chondrocytes. In addition, normal and lesion chondrocytes in culture synthesized transforming growth factor-beta and 24,25(OH)2D3 but not 1,25(OH)2D3. There was no significant difference in the production rate of these growth regulators between normal and lesion chondrocytes. In contrast, in growth plate sections, alkaline phosphatase activity was markedly reduced in the lesion chondrocytes and sites of mineralization were not evident. Type II collagen was located throughout the growth plate and lesion, but type X collagen was not present within the lesion except at sites of vascularization. These results indicate that, in culture, lesion chondrocytes have the ability to differentiate terminally and mineralize, and suggest that the primary abnormality in TD is related to a developmental fault which is only operative in vivo. This may include a defect in cartilage vascularization and/or impairment of chondrocyte differentiation by mechanisms that have not yet been elucidated but may involve the abnormal production of regulatory factors.
24,25-Dihydroxyvitamin D 3↗
Folic acid requirements of broilers.
1. Dietary folic acid requirements of broilers were studied in three experiments using wheat- and maize-based practical diets. Requirements were assessed on the basis of performance and metabolic criteria. 2. Growth and food conversion efficiencies were optimised with supplements of 1.5 mg folic acid/kg added to basal mash starter diets. The dietary folic acid requirement of broilers was estimated to be in the range of 1.7 to 2.0 mg/kg. 3. Red blood cell phosphoribosylpyrophosphate concentrations and dihydrofolate reductase activities did not show consistent changes over the range of dietary folate concentrations studied but plasma folate concentrations responded markedly to dietary folate supplementation. 4. Adding choline to diets in amounts greater than the normal requirement did not spare the requirement for folic acid. 5. It is suggested that minimum folic acid supplements for pelleted practical diets should be in the order of 2.5 to 3 mg/kg.
Animals↗
Interaction between dietary 1,25-dihydroxycholecalciferol and calcium and effects of management on the occurrence of tibial dyschondroplasia, leg abnormalities and performance in broiler chickens.
1. Two experiments were performed to compare the relative effectiveness of feeding 1,25-dihydroxycholecalciferol (1,25-DHCC) in minimising leg abnormalities in broilers with other methods and to investigate interactions between dietary 1,25-DHCC and calcium. 2. Adding 5 micrograms 1,25-DHCC/kg to a diet containing 12 g calcium/kg was more effective than early food restriction or meal feeding in preventing leg abnormalities but was found to cause a growth depression. 3. The second experiment, which had a factorial design, with diets containing 7.5, 10.0 and 12.5 g calcium and 0, 2.0, 3.5 and 5.0 micrograms 1,25-DHCC/kg, showed linear and quadratic interactions between these dietary factors. Diets with higher concentrations of both 1,25-DHCC and calcium resulted in growth depression associated with hypercalcaemia. 4. The incidence of tibial dyschondroplasia (TD) at 3 weeks of age was highest with the basal diet containing 7.5 g calcium/kg and was markedly reduced by addition of 1,25-DHCC and/or calcium. The incidence was very low or non-existent when 1,25-DHCC was fed at 3.5 micrograms/kg or greater. 5. Feeding 5 micrograms/kg 1,25-DHCC had no effect on plasma 1,25-DHCC concentrations, although at the higher dietary calcium contents plasma concentrations of 25-hydroxy- and 24,25-dihydroxy-cholecalciferol were lower in those birds fed 1,25-DHCC. 6. It is concluded that 1,25-DHCC is most effective in preventing TD without accompanying growth depression when it is fed in conjunction with diets containing less than 10 g calcium/kg.
Animal Feed↗