In young pullets, long bones elongate by endochondral growth. Growth plate chondrocytes proliferate, then hypertrophy, and are replaced by osteoblasts that form a network of trabecular bone. This bone is gradually resorbed by osteoclasts as the bone lengthens. Long bones widen, and flat bones are formed, by intramembranous ossification in which cortical bone formation by osteoblasts in the periosteal layer is accompanied by osteoclastic resorption at the inner endosteal surface. Growth of structural trabecular and cortical bone types continues up to the onset of sexual maturity in pullets. At this point, the large surge in estrogen changes the function of osteoblasts to forming medullary bone rather than structural bone. Medullary bone is a woven bone that acts as a labile source of calcium for eggshell formation. It lines structural bone and also occurs as spicules within the marrow cavity. It has little inherent strength but can contribute to fracture resistance. Osteoclasts resorb both medullary and structural bone so that during the period the hen remains in reproductive condition there is a progressive loss of structural bone throughout the skeleton, which is characteristic of osteoporosis. The increasing fragility of the bones makes them more susceptible to fractures. The dynamics of bone loss can be affected by a number of nutritional, environmental, and genetic factors. If the hen goes out of reproductive condition, estrogen levels fall, osteoblasts resume structural bone formation, and skeletal regeneration can take place.

The complete definition of changes in a proteome requires information about dynamics and specifically the rate at which the individual proteins are turned over intracellularly. Whilst this can be achieved in single-cell culture using stable isotope precursors, it is more challenging to develop methods for intact animals. In this study, we show how dietary administration of stable isotope-labelled amino acids can obtain information on the relative rates of synthesis and degradation of individual proteins in a proteome. The pattern of stable isotope-labelling in tryptic peptides can be deconstructed to yield a highly reliable measure of the isotope abundance of the precursor pool, a parameter that is often difficult to acquire. We demonstrate this approach using chickens fed a semisynthetic diet containing [(2)H(8)]valine at a calculated relative isotope abundance (RIA) of 0.5. When the labelling pattern of gel-resolved muscle proteins was analyzed, the intracellular precursor isotope abundance was 0.35, consistent with dilution of the amino acid precursor pool with unlabelled amino acids derived from degradation of pre-existing proteins. However, the RIA was stable over an extended labelling window, and permitted calculation of the rates of synthesis and degradation of individual proteins isolated by gel electrophoresis. For the first time, it is feasible to contemplate the analysis of turnover of individual proteins in intact animals.

Osteoporosis in laying hens is a condition that involves the progressive loss of structural bone during the laying period. This bone loss results in increased bone fragility and susceptibility to fracture, with fracture incidences of up to 30% over the laying period and depopulation not uncommon under commercial conditions. A major cause of osteoporosis is the switch in bone formation from structural to medullary bone at the onset of sexual maturity, but structural bone loss is accelerated by the relative inactivity of-caged birds. Allowing birds more exercise, as in aviary systems, results in better bone quality but may not decrease the overall fracture incidence. Good nutrition can help to minimize osteoporosis but is unable to prevent it. Best nutritional practice involves transferring birds to a higher calcium diet at lighting up rather than at first egg, providing a source of calcium in particulate form, and not withdrawing feed some days before depopulation. Breeding may be an effective way of combating ostoporosis. Some bone strength traits have been shown to be heritable, and divergent selection for resistance or susceptibility to osteoporosis has resulted in lines with markedly different bone characteristics. After three generations of selection, the lines differ by 19% for keel bone mineral density, 13% for humerus breaking strength, and 25% for tibia breaking strength and show a sixfold difference in fracture incidence under commercial breeding conditions. The difference in bone quality among the lines is maintained under different housing systems.

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.

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.