Integument, mortality, and skeletal strength in extended production cycles for laying hens - effects of genotype and dietary zinc source.

1. This study on long-life layers, covering the period 20-100 weeks of age, investigated longitudinal effects on mortality, layer integument, and skeletal properties in Bovans White (BoW) and Lohmann Selected Leghorn Classic (LSL), with or without supplementation with dietary organic zinc (Zn).2. Two experiments, using 1440 layers in furnished small group cages (FC) and 1836 layers in a traditional floor housing system (Floor), were run in parallel. Each replicate consisted of five adjacent cages containing eight hens in each FC, or a pen with 102 layers in the Floor group.3. Mortality was recorded daily. Integument and keel bone condition were scored at 35, 55, 85, and 100 weeks of age on 20% of the layers. Tibial strength was recorded from 933 layers at 100 weeks. Statistical analyses were performed on replicate means, with four to five and nine replicates per combination of hybrid and diet in Floor and FC groups, respectively.4. Cumulative mortality was 9.6% and 16.3% in FC and Floor, respectively, and increased in the latter part of the production cycle, particularly in the Floor group.5. In FC, LSL had inferior feather cover, less keel bone deviation, and shorter claws than BoW. In Floor, LSL had superior feather cover, less severe vent wounds, more bumble foot, and cleaner plumage than BoW. In both production systems, claws grew longer and keel bone deviation became more severe with age.6. In FC, layers fed organic Zn had lower body weight and less keel bone deviation at 100 weeks of age.7. In conclusion, keel bone integrity, claw length, and mortality rate are potential threats to welfare in long-life layers. Feather pecking is a problem that needs addressing at an early stage in the production period. On the whole, organic Zn did not improve welfare conditions in long-life layers.

Comparative analysis of the morphology, chemistry and structure of the tibiotarsus, humerus and keel bones in laying hens.

1. Bone properties are adapted to their specific functions in the animal, so various types of bones develop different characteristics depending on their location in the skeleton.2. The aim of this research was to compare the chemical composition, mineral characteristics and structural organisation in tibiotarsus, humerus and keel bones as representatives of hen skeletal mineralisation. Complementary analytical techniques, such as X-ray radiography, optical and electron microscopy, thermogravimetry and 2D X-ray diffraction, were used for characterisation.3. The humerus had a thinner cortex and cortical bone mineral had higher crystallinity and a greater degree of crystal orientation than the tibiotarsus. The humerus generally lacks medullary bone although, when present, it has a higher mineral content than seen in the tibiotarsus. These differences were attributed to the different forces that stimulate bone formation and remodelling.4. The keel cortical bone had a lower degree of mineralisation than the tibiotarsus or humerus. Its degree of mineralisation decreased from the cranial to the distal end of the bone. This gradient may affect keel mechanical properties, making it more prone to deformation and fractures.5. Data from studying different bones in laying hens can help to understand mineralisation as well as finding solutions to prevent osteoporosis-related fractures.

Investigation of a gamasid mite infestation in a UK textile mill caused by Dermanyssus gallinae (DeGeer, 1778) (Mesostigmata: Dermanyssidae) special lineage L1.

A recurrent mite infestation affecting a room used to inspect fabric in a UK textile mill was investigated to allay concerns of any potential health risks to factory staff, and to inform the unknown risk of downgrading of the product. The approach integrated conventional morphological examination of adult female mites by referring to published identification keys, with molecular speciation based on amplification of a 16S ribosomal DNA fragment. The methods enabled the mites to be unambiguously identified as Dermanyssus gallinae 'special lineage L1'. Subsequent investigations showed the source of infestation to be pigeons nesting in the air ducts, with the gamasid mites moving into the room once the young birds had fledged. This is the first report of D. gallinae 'special lineage L1' in northern Europe. Previous reports of nosocominal gamasoidosis caused by D. gallinae 'special lineage L1' originating from feral pigeon populations have been from southern Europe. Confirmation of the mite identity was important in allowing the mill to take remedial and preventive action. In this clinical communication, we provide images of the key morphological features used to identify D. gallinae and describe a molecular protocol to confirm 'special lineage L1'.

Investigation of a gamasid mite infestation in a UK textile mill caused by dermanyssus gallinae (DeGeer, 1778) (Mesostigmata: Dermanyssidae) special lineage L1

A recurrent mite infestation affecting a room used to inspect fabric in a UK textile mill was investigated to allay concerns of any potential health risks to factory staff, and to inform the unknown risk of downgrading of the product. The approach integrated conventional morphological examination of adult female mites by referring to published identification keys, with molecular speciation based on amplification of a 16S ribosomal DNA fragment. The methods enabled the mites to be unambiguously identified as Dermanyssus gallinae ‘special lineage L1’. Subsequent investigations showed the source of infestation to be pigeons nesting in the air ducts, with the gamasid mites moving into the room once the young birds had fledged. This is the first report of D. gallinae ‘special lineage L1’ in northern Europe. Previous reports of nosocominal gamasoidosis caused by D. gallinae ‘special lineage L1’ originating from feral pigeon populations have been from southern Europe. Confirmation of the mite identity was important in allowing the mill to take remedial and preventive action. In this clinical communication, we provide images of the key morphological features used to identify D. gallinae and describe a molecular protocol to confirm ‘special lineage L1’

Investigation of a gamasid mite infestation in a UK textile mill caused by dermanyssus gallinae (DeGeer, 1778) (Mesostigmata: Dermanyssidae) special lineage L1

A recurrent mite infestation affecting a room used to inspect fabric in a UK textile mill was investigated to allay concerns of any potential health risks to factory staff, and to inform the unknown risk of downgrading of the product. The approach integrated conventional morphological examination of adult female mites by referring to published identification keys, with molecular speciation based on amplification of a 16S ribosomal DNA fragment. The methods enabled the mites to be unambiguously identified as Dermanyssus gallinae ‘special lineage L1’. Subsequent investigations showed the source of infestation to be pigeons nesting in the air ducts, with the gamasid mites moving into the room once the young birds had fledged. This is the first report of D. gallinae ‘special lineage L1’ in northern Europe. Previous reports of nosocominal gamasoidosis caused by D. gallinae ‘special lineage L1’ originating from feral pigeon populations have been from southern Europe. Confirmation of the mite identity was important in allowing the mill to take remedial and preventive action. In this clinical communication, we provide images of the key morphological features used to identify D. gallinae and describe a molecular protocol to confirm ‘special lineage L1’

Influence of physical activity on tibial bone material properties in laying hens.

Laying hens develop a type of osteoporosis that arises from a loss of structural bone, resulting in high incidence of fractures. In this study, a comparison of bone material properties was made for lines of hens created by divergent selection to have high and low bone strength and housed in either individual cages, with restricted mobility, or in an aviary system, with opportunity for increased mobility. Improvement of bone biomechanics in the high line hens and in aviary housing was mainly due to increased bone mass, thicker cortical bone and more medullary bone. However, bone material properties such as cortical and medullary bone mineral composition and crystallinity as well as collagen maturity did not differ between lines. However, bone material properties of birds from the different type of housing were markedly different. The cortical bone in aviary birds had a lower degree of mineralization and bone mineral was less mature and less organized than in caged birds. These differences can be explained by increased bone turnover rates due to the higher physical activity of aviary birds that stimulates bone formation and bone remodeling. Multivariate statistical analyses shows that both cortical and medullary bone contribute to breaking strengthThe cortical thickness was the single most important contributor while its degree of mineralization and porosity had a smaller contribution. Bone properties had poorer correlations with mechanical properties in cage birds than in aviary birds presumably due to the greater number of structural defects of cortical bone in cage birds.

Quantifying genetic (co)variation and effects of genetic selection on tibial bone morphology and quality in 37 lines of broiler, layer and traditional chickens.

1. Tibial bone morphology and quality was assessed at 6, 8 and 10 weeks of age in 12 broiler, 12 layer and 13 traditional lines of chickens. 2. High intraclass correlations (0.65-0.96) were estimated for tibial weight, length, width, strength, stiffness, stress radiodensity, plateau angle but not for cortical width and tibial torsion at 6 and 10 weeks of age and for weight, length, width, strength radiodensity and plateau angle at 8 weeks of age. Lower intraclass correlations (0.4-0.8) were estimated within category (broiler, layer and traditional) for weight, length, width at 6, 8 and 10 weeks, and for stiffness, stress and radiodensity at 6 and 10 weeks, and were lower (<0.33) at 8 weeks of age. Intraclass correlations for tibial torsion angles were low (< or =0.4) at all ages. 3. Tibia from layers and traditional lines were similar suggesting that intensive genetic selection for high rates of egg laying has not changed bone size, shape or quality. Tibia from broilers, as expected, were heavier and larger and the differences were greatest at 6 weeks of age suggesting that the broilers were earlier maturing than layers and traditional lines. Broiler tibiae were more radiodense, stronger and stiffer and had lower stress values than bones from layers and traditional lines. Plateau angles were higher in broilers and torsion angles had higher external rotation compared with layers and traditional lines.

A QTL for osteoporosis detected in an F2 population derived from White Leghorn chicken lines divergently selected for bone index.

Osteoporosis, resulting from progressive loss of structural bone during the period of egg-laying in hens, is associated with an increased susceptibility to bone breakage. To study the genetic basis of bone strength, an F(2) cross was produced from lines of hens that had been divergently selected for bone index from a commercial pedigreed White Leghorn population. Quantitative trait loci (QTL) affecting the bone index and component traits of the index (tibiotarsal and humeral strength and keel radiographic density) were mapped using phenotypic data from 372 F(2) individuals in 32 F(1) families. Genotypes for 136 microsatellite markers in 27 linkage groups covering approximately 80% of the genome were analysed for association with phenotypes using within-family regression analyses. There was one significant QTL on chromosome 1 for bone index and the component traits of tibiotarsal and humeral breaking strength. Additive effects for tibiotarsal breaking strength represented 34% of the trait standard deviation and 7.6% of the phenotypic variance of the trait. These QTL for bone quality in poultry are directly relevant to commercial populations.

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.

Histological assessment of bioengineered new bone in repairing osteoperiosteal mandibular defects in sheep using recombinant human bone morphogenetic protein-7.

UNLABELLED: Numerous experimental studies have been published about osteoinductive bone morphogenetic proteins (BMPs). However, to our knowledge there has been no detailed histological study of a mandibular defect in a large mammal, reconstructed using BMPs. We describe here the histological features of rhBMP-7-induced bone in mandibular defects in sheep. METHODS: A 35 mm osteoperiosteal defect was created at the parasymphyseal region of the mandible in six adult sheep. The continuity of the mandible was maintained using a bony plate, and rhBMP-7 was applied on a type I collagen carrier. Bone labels were injected at selected time intervals during the follow-up period. The animals were killed after 3 months and bone samples were examined histologically, histomorphometrically, and by fluorescence microscopy. RESULTS AND CONCLUSIONS: We found a mixture of woven and lamellar bone that contained many cells with large nuclei. This had not reorganised to form cortical bone and the rhBMP-7-induced bone was more porous than the native bone. The newly-formed bone restored both endosteal and periosteal layers. rhBMP-7-induced bone was biocompatible and induced no ossification of soft tissue or abnormal growth of nearby vital structures. The mineral apposition rate was 1.98 microm/day (range 0.62-5.63 microm/day), a value close to that reported in humans. This suggests that BMPs have a limited effect in accelerating the rate of mineralisation, but promote the pre-mineralisation processes, and perhaps the formation of woven bone.

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.

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 microg 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 14d with 250 microg vitamin D3/kg, tibia ash was maximised with 125 microg vitamin D3/kg. A high incidence of tibial dyschondroplasia (TD) was decreased to very low levels with 125 microg vitamin D/kg. 3. At 42d, performance and bone characteristics showed no response to vitamin D3 concentrations above 20 microg/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 microg/kg but more was needed at less optimal concentrations of calcium and available phosphorus. TD incidence was minimised with 250 microg/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 microg/kg for cortical bone quality and up to 250 microg/kg for prevention of TD. The vitamin D3 requirement for cortical bone quality after 14 d is not higher than 20 microg/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.

Assessing bone mineral density in vivo: digitized fluoroscopy and ultrasound.

The genetic component of osteoporosis in caged laying hens is large, and a method for detecting hens susceptible to fracture could be useful in breeding programs. A radiographic absorptiometry film method was modified by video digitization from an image intensifier and computer analysis and termed digitized fluoroscopy (DF). Humeral and ulnar DF values were measured in 165 hens during lay. Relationships (P < 0.001) were seen between DF assessments from 25 wk onward and postmortem measurements at 70 wk. We conclude that DF can detect poor bones in hens early but is problematic. Quantitative ultrasound was also investigated. We measured amplitude-dependent speed-of-sound (Ad-SoS) in the third toe in hens. Nutritional studies revealed Ad-SoS values correlated with postmortem peripheral quantitative computerized tomography, (control group, r = 0.48, P < 0.001; treatment group, r = 0.39, P < 0.001). In caged and free-range hens, Ad-SoS correlated with shear strength (r = 0.33, P < 0.001, all hens) and radiographic density values (r = 0.53, P < 0.001, all hens) measured postmortem. The Ad-SoS values were higher in free-range than in caged hens (1,904 vs. 1,850 m/s, P < 0.001). Ad-SoS measurements were made in hens from a study where divergent genetic selection has produced high and low bone index lines with 92% difference in tibia strength. The value in high bone index hens was higher than in low bone index hens at 32 (P < 0.001), 42 (P < 0.001), 52 (P < 0.05), and 62 wk (P < 0.001) in generation 8. In an Ad-SoS heritability study, heritability estimates ranged from 0.15 to 0.39. We conclude that Ad-SoS is a heritable trait, reflects other bone measurements, and rapidly detects poor bone quality in hens.

Effects of dietary particulate limestone, vitamin K3 and fluoride and photostimulation on skeletal morphology and osteoporosis in laying hens.

1. Female chicks of a White Leghorn strain were fed three different diets from one day old: control, additional vitamin K3 (10 mg/kg), and a diet containing a combination of additional vitamin K3, sodium fluoride (10 mg/kg) and limestone in particulate rather than powdered form. At 16 weeks photoperiod was increased for half the birds from 8:16 L:D to 16:8 L:D immediately or by one hour per week to the same ultimate photoperiod for the other half. 2. Age at first egg was lower by 4.0 d for birds on the fast lighting regime but there were no overall effects of lighting on bone quality at either 25 or 70 weeks. 3. Additional vitamin K3 resulted in higher proximal tarsometatarsus cancellous bone volumes at 15 weeks and throughout the laying period compared with controls. Plasma osteocalcin concentrations were unaffected by vitamin K3 supplementation during growth. 4. The combination diet resulted in beneficial responses of 12 to 20% in most bone characteristics in hens at 70 weeks. The magnitude of these effects was similar to a previous study involving a particulate calcium source alone (Fleming et al., Poultry Science, 39: 434-440, 1998b). We conclude that the beneficial effects of the combined treatment over the lifetime of the hens were attributable mainly to the presence in the diet of a calcium source in particulate form.

Differences in composition of avian bone collagen following genetic selection for resistance to osteoporosis.

1. Collagen characteristics were compared in the tibiotarsus and humerus from 103 females and 38 males aged 68 to 72 weeks from the G6 generation of lines of laying hen selected for resistance or susceptibility to osteoporosis (high and low bone index (BI) lines). 2. Selection over the latest generation resulted in further divergence in the breaking strengths of humerus (from 12.3 to 21.8%) and tibia (from 22.3 to 37.3%) in hens. Males also showed line differences in bone strengths. 3. Plasma pyridinoline concentration was higher in hens in the low BI line, suggesting a greater rate of bone resorption in this line. 4. There were few differences between the lines in collagen and calcium concentrations in humerus and tibiotarsus cortical bone. 5. There were no differences between the lines in either sex in reduced immature collagen cross-link content of humerus or tibiotarsus. 6. Mature collagen cross-link content was higher in the high BI line in the male humerus but this effect was not apparent in the male tibiotarsus nor in either bone in the females. 7. Pyrrolic cross-link contents were higher in the high BI line in the female humerus and tibiotarsus and in the male tibiotarsus. 8. Over both lines combined, there were positive correlations between humeral and tibiotarsal pyrrole contents and strengths in females and between tibiotarsal pyrrole content and strength in males. 9. It is concluded that an increase in cross-linking, particularly pyrrolic cross-linking, in the collagen matrix contributes in part to the improvement in bone strength in the high BI line.

Prediction of breaking strength in osteoporotic avian bone using digitized fluoroscopy, a low cost radiographic technique.

Bone fragility in caged laying hens is a severe welfare problem, with fracture incidences in commercial flocks of up to 30% of all hens during their life. This fragility has been attributed to osteoporosis, the etiology of which is multifactorial in birds, as in humans, with genetic, environmental, and nutritional components. Greater understanding of the development of the disorder in hens could be obtained from the same kind of in vivo assessments available in human studies of osteoporosis. These high technology techniques for evaluation of bone mineral density (BMD), such as single or dual energy X-ray absorptiometry (SXA or DXA), quantitative computerized tomography (QCT), or attenuation by ultrasound (US), are so far not widely available to nonclinical researchers. We have modified an older X-ray film technique, radiographic absorptiometry (RA) by digitization of the analog video signal from a Philips BV-25 image intensifier, in single pulse fluoroscopy mode, and subsequent computer analysis with the public domain software package, NIH-Image 1.60. Compared with conventional RA, which uses standard X-ray film, our modified technique reduces X-ray exposure and allows the operator to digitize, store, and analyze many more images in a shorter time. We have called this modified technique "digitized fluoroscopy" (DF). In a longitudinal study of humeral radiographic density in a flock of 165 laying hens, significant relationships (P < 0.001) were observed between assessments made as early as 25 weeks, utilizing this DF technique in the humerus, and breaking strengths (and other postmortem indicators of osteoporosis) measured at 70 weeks. We conclude that DF can predict some eventual parameters of bone mass measured at 70 weeks from 25 to 40 weeks onward in bones from the same site in laying hens. The relationship between DF measurements made in the humerus and postmortem measurements of radiographic density and breaking strength made at another site (tibia) are less strong but still significant from 40 weeks onward.

Osteoporosis in cage layers.

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.

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.