Robert Fleming

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.

Inheritance of bone characteristics affecting osteoporosis in laying hens

1. Heritabilities of a range of morphometric, radiological and strength characteristics were measured in the bones of end-of-lay hens. 2. Tibial strength (TSTR), humeral strength (HSTR) and keel radiographic density (KRD) were moderately to strongly inherited and were combined in a Bone Index which was used as a basis for selection. Data are available on 6 generations/cohorts of hens (n = 1306), the last 3 of which are the progeny of divergently selected birds. 3. All bone characteristics used in the Bone Index responded rapidly to divergent selection and were strongly correlated with each other. In the last generation, the lines differed by 25% for TSTR, 13% for HSTR and 19% for KRD. The heritability of the index was 0.40. 4. There were no apparent genotype by environment interactions between birds housed at 2 different locations. 5. The incidence of bone fractures was significantly decreased in the line selected for high bone strength compared to the line selected for low bone strength. Humerus fracture incidence differed by a factor of 6 between the lines in the last generation. There was a strong quadratic relationship between tibia strength and overall fracture incidence (r2= 0.92, P <0.01). 6. The results imply that selection for enhanced bone strength can be used as a long-term strategy for alleviating the problems of osteoporosis in laying hens.

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.

Genetic variation for egg production, egg quality and bone strength in selected and traditional breeds of laying fowl

1. A multi-breed experiment was conducted with 25 commercial and traditional lines of laying fowl to determine the extent of between-breed genetic variation for adult body weight, sexual maturity, rate of lay, egg weight and egg composition to 55 weeks of age. The genetic variability for bone strength and eggshell strength was determined at 55 weeks of age and a comparison of commercially selected and traditional breeds was performed. 2. The proportion of the total variation that was associated with breed or line of origin was high (> 0·8) for body weight, sexual maturity and shell colour; moderately high (0·4 to 0·7) for rate of lay, early and late egg weight, weights of egg yolk, albumen and shell at 55 weeks; and low (< 0·4) for egg weight at 42 to 45 weeks, albumen quality and the number of egg inclusions. 3. There were no detectable differences between breed within category (traditional and commercial lines) for rate of lay, and estimates of breed variation for egg weight and egg components were substantially decreased within category compared with the overall analysis. 4. Commercial lines displayed earlier sexual maturity, greater rates and persistency of lay, and higher egg weights at earlier (32 to 35 weeks) and later (52 to 55 weeks) ages. At 55 weeks, the larger eggs from commercial birds contained more albumen of higher quality and paler yolks of similar weight to those from traditional breeds. The relative weight of the eggshell was similar in both categories. 5. There was considerable genetic variation between commercial lines for bone density and a moderate proportion of genetic variability for bone strength. Commercial lines had very weak bones compared with traditional lines but there was relatively little genetic variation for eggshell strength. The results suggest that eggshell quality is maintained in genetically selected lines at the expense of bone strength and bone radiographic density.

The presence of PHOSPHO1 in matrix vesicles and its developmental expression prior to skeletal mineralization

PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase that has previously been implicated in generating inorganic phosphate (P(i)) for matrix mineralization. In this study, we have investigated PHOSPHO1 mRNA expression during embryonic development in the chick. Whole-mount in situ hybridization indicated that PHOSPHO1 expression occurred prior to E6.5 and was initially restricted to the bone collar within the mid-shaft of the diaphysis of long bones but by E11.5 expression was observed over the entire length of the diaphysis. Alcian blue/alizarin red staining revealed that PHOSPHO1 expression seen in the primary regions of ossification preceded the deposition of mineral, suggesting that it is involved in the initial events of mineral formation. We isolated MVs from growth plate chondrocytes and confirmed the presence of high levels of PHOSPHO1 by immunoblotting. Expression of PHOSPHO1, like TNAP activity, was found to be up-regulated in MVs isolated from chondrocytes induced to differentiate by the addition of ascorbic acid. This suggests that both enzymes may be regulated by similar mechanisms. These studies provide for the first time direct evidence that PHOSPHO1 is present in MVs, and its developmental expression pattern is consistent with a role in the early stages of matrix mineralization.

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.

External validation of nomogram for the decline in serum anti-Müllerian hormone in women: a population study of 15,834 infertility patients

The value of anti-müllerian hormone (AMH) as a marker of the ovarian reserve is becoming clear in a range of clinical contexts.This study reports the external validation of a quadratic model-based AMH–age nomogram using a cohort of 15,834 US women. All models previously investigated for the decline in ovarian reserve (i.e. linear, bi-linear, decay curve, power and quadratic models) tended to overestimate AMH by approximately 11% versus the published nomogram, indicating some between-population heterogeneity. Bootstrapping of 1000 datasets indicated that the quadratic model provided the best fit, confirming the choice of this model in the AMH–age nomogram. This nomogram can therefore be used with confidence for the interpretation of AMH in clinical populations.

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.

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.

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.