C C Whitehead

Bone structure and breaking strength in laying hens housed in different husbandry systems

1. Bone structure and breaking strength were measured in hens that had been housed throughout a laying year in battery cages or in Perchery, Naturel or Litter and Wire husbandry systems. 2. Battery caged hens had the poorest bones, as assessed by measurements of cancellous bone volume, radiographic density, cortical thickness and three-point breaking strength. 3. Humeri from birds in the Litter and Wire system were less dense radiographically and weaker than those from Perchery or Naturel birds but leg bone characteristics were similar with these three systems. 4. There were no differences in bone characteristics between birds in Perchery and Naturel systems. 5. There were strong correlations between radiographic densities and strengths of contralateral humeri and tibiae over all husbandry systems. Humerus structural and strength characteristics may be the best criteria of osteoporosis in hens. 6. It is concluded that the extent of movement allowed by different husbandry systems affects structural bone loss and bone strength in laying hens. 7. It is further concluded that the breaking strength of a hens bone is closely related to morphometric measures and radiographic density of its structural components.

Avian tibial dyschondroplasia: The interaction of genetic selection and dietary 1,25 ‐dihydroxycholecalciferol

The effects of dietary 1,25-dihydroxycholecalciferol on the incidence and severity of tibial dyschondroplasia (TD) were assessed in broiler fowl selected for TD (high-TD line) and against TD (low-TD line). Assessment was by examination with a portable Lixiscope to identify lesions in live birds, gross pathology and histopathology. Eighty broilers in four groups of 20 were examined. When fed on standard diets the low-TD line had a 15% incidence whereas the high-TD line had a 63% incidence. The chicks in the high-TD line, when fed on a diet supplemented with 5 mug or with 10 mug 1,25-dihydroxycholecalciferol/kg diet, showed a marked reduction in the incidence of TD, to 21% and 0% respectively. The growth plate in the proximal tibiotarsi of the high-TD line showed accumulations of transitional chondrocytes, supporting the hypothesis that a failure of chondrocyte differentiation is the cause of dyschondroplasia. This hypothesis is further supported by studies which indicate 1,25-dihydroxycholecalciferol has a major role in chondrocyte differentiation.

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.

High vitamin D3 requirements in broilers for bone quality and prevention of tibial dyschondroplasia and interactions with dietary calcium, available phosphorus and vitamin A.

1. Two experiments were carried out to investigate responses in performance and bone compositional and structural characteristics in broilers fed diets containing 4 concentrations of vitamin D3 (5, 20, 125 and 250 µg cholecalciferol/kg) at different concentrations of calcium, available phosphorus and vitamin A. 2. In experiment 1, body weight and tibia breaking strength were maximised at 14 d with 250 µg vitamin D3/kg, tibia ash was maximised with 125 µg vitamin D3/kg. A high incidence of tibial dyschondroplasia (TD) was decreased to very low levels with 125 µg vitamin D/kg. 3. At 42 d, performance and bone characteristics showed no response to vitamin D3 concentrations above 20 µg/kg. 4. Dietary vitamin A within the range 2·4 to 4·5 mg retinol/kg did not show any interaction with vitamin D3 status at either age. 5. In experiment 2, responses to vitamin D3 were strongly influenced by dietary calcium/available phosphorus. With 13 g calcium and 5 g available phosphorus/kg, performance and bone characteristics responded to vitamin D3 concentrations up to 125 µg/kg but more was needed at less optimal concentrations of calcium and available phosphorus. TD incidence was minimised with 250 µg/kg. 6. This study shows that high dietary concentrations of vitamin D3 can prevent TD. It is concluded that the vitamin D3 requirement of broilers up to 14 d of age at optimal dietary calcium and available phosphorus concentrations may be in the range 35 to 50 µg/kg for cortical bone quality and up to 250 µg/kg for prevention of TD. The vitamin D3 requirement for cortical bone quality after 14 d is not higher than 20 µg/kg. These requirements are much higher than earlier estimates and may be related to higher calcium requirements of modern broiler genotypes. Current regulations limiting maximum vitamin D3 concentrations in broiler starter diets may need to be reviewed.

Medullary bone and humeral breaking strength in laying hens

To test the hypothesis that large amounts of medullary bone in the humeral diaphysis may increase breaking strength, various parameters of bone quality and quantity were examined in two large flocks of hens near end of lay. We conclude that the amount of medullary bone in the humerus of hens during the laying period influences bone strength. This medullary bone may not have any intrinsic strength, but may act by contributing to the fracture resistance of the surrounding cortical bone. Using a quantitative, low dose, radiographic technique, we can predict, from early in the laying period, those birds which will develop large amounts of medullary bone in their humeri by the end of the laying period. The formation of medullary bone in the humeral diaphysis is not at the expense of the surrounding radiographed cortical bone.

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.

Relationships between genetic, environmental and nutritional factors influencing osteoporosis in laying hens

1. The effects upon bone quality of feeding limestone in flour or particulate form and housing type (cage or aviary) in lines of hens divergently selected for high (H) or low (L) bone strength over 7 generations were investigated. 2. As in previous generations, highly significant phenotypic differences between lines were observed in all measured bone traits at peak egg production (25 weeks) and towards the end of production (56 weeks) in both cage and aviary systems. 3. At 25 weeks there were no significant effects on bone variables of feeding particulate limestone although a significant reduction in osteoclast number was observed at this age. By 56 weeks osteoclast numbers were further reduced in hens fed particulate limestone and beneficial effects on some bone variables were observed in this treatment group. 4. The genotypic and dietary improvements upon bone quality were independent and additive at both ages. There were very few interactive effects. 5. Hens with the freedom to move in an aviary environment during the laying period had improved bone status compared to caged siblings. Environmental and genotypic effects were additive. 6. There were no effects of line on egg production although H line hens had slightly higher egg production by 56 weeks. Egg numbers were unaffected by diet. Eggshell thickness and strength were unaffected by line but hens fed particulate limestone had thicker- and stronger-shelled eggs over the production period as a whole. 7. We conclude that; (a) genetic selection is extremely effective in improving bone strength and resistance to osteoporosis; (b) allowing hens freedom to exercise can also improve bone strength but may increase the risk of keel damage if they do not have genetically-improved bone status; (c) feeding hens a particulate form of limestone from 15 weeks onwards can also increase bone strength and eggshell quality; (d) genetics, environment and nutrition all have independent and additive effects on bone status in laying hens but the relative effectiveness of these factors is genetics > environment > nutrition.

Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes

Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes.

The Subunit Structure and Dynamics of the 20S Proteasome in Chicken Skeletal Muscle

We have succeeded in purifying the 20S core proteasome particle from less than 1 g of skeletal muscle in a rapid process involving two chromatographic steps. The individual subunits were readily resolved by two-dimensional PAGE, and the identities of each of the 14 subunits were assigned by a combination of peptide mass fingerprinting and MS/MS/de novo sequencing. To assess the dynamics of proteasome biogenesis, chicks were fed a diet containing stable isotope-labeled valine, and the rate of incorporation of label into valine-containing peptides derived from each subunit was assessed by mass spectrometric analysis after two-dimensional separation. Peptides containing multiple valine residues from the 20S proteasome and other soluble muscle proteins were analyzed to yield the relative isotope abundance of the precursor pool, a piece of information that is essential for calculation of turnover parameters. The rates of synthesis of each subunit are rather similar, although there is evidence for high turnover subunits in both the α (nonproteolytic) and β (proteolytic) rings. The variability in synthesis rate for the different subunits is consistent with a model in which some subunits are produced in excess, whereas others may be the rate-limiting factor in the concentration of 20S subunits in the cell. The ability to measure turnover rates of proteins on a proteome-wide scale in protein assemblies and in a complex organism provides a new dimension to the understanding of the dynamic proteome.

Incidence, pathology and prevention of keel bone deformities in the laying hen

1. As a baseline study of the nature and incidence of keel deformities in laying hens, keel condition was examined in three different strains of hen from a total of 4 different caged environments (two commercial farms and two experimental farms). Incidence of keel deformity on farms in end of lay hens ranged from 2·6 to 16·7%. Only 0·8% of younger 15-week-old pullets had deformed keels. 2. Incidence of keel deformities was unchanged in 100 birds sampled from a free-range system compared to conventional caged siblings at the same farm. 3. Keel condition was also examined in 5 selected generations of a study involving the use of a body-weight-restricted selection index for skeletal improvement. Divergent selection for skeletal characteristics decreased incidence of keel deformity and improved radiographic density (RD) in high bone index (BI) hens compared to low BI hens in all selected generations. Male high BI keels were also improved compared to low BI. Shear strength measured in normal keels in generation 6 (G6) of the genetic study was improved in high BI hens compared to low BI hens. For all hens in the genetic study, those with normal keels had stronger tibiotarsus and humerus breaking strengths than hens with deformed keels. 4. Histopathology of keels representative of different deformities showed the presence of fracture callus material and new bone in all cases. This establishes that deformities are a result of trauma and are not developmental in origin. 5. Ash contents of keels, tibiae and humeri showed no differences between hens with normal and deformed keels. There were no differences in indicators of collagen cross-linkage in other bones between hens with normal keels and those with deformed keels. 6. It is concluded that lack of bone mass is the underlying cause of keel fracture and deformity in laying hens, rather than qualitative changes in bone, and that genetic selection can improve keel quality and prevent deformity.