C. C. WHITEHEAD
Roslin Institute, Roslin, UK
Structural weakness and/or distortion lead to bone fracture in live birds and during processing, especially with automated systems for deboning or mechanical meat recovery. Genetic, nutritional and environmental factors influence both bone strength and deformity. Bone strength in end-of-lay hens has been found to be highly heritable and divergent selection on this basis has resulted in lines with markedly different bone strengths. A quantitative trait locus (QTL) for bone strength has been identified and it is possible that genetic markers for bone strength can be developed for use in breeding programmes. Calcium, phosphorus and vitamin D are important nutrients for bone strength, but recent studies have also highlighted the beneficial effects of n-3 fatty acids. Bone deformities, particularly broiler leg bone deformities also have a genetic component and the importance of including criteria of leg quality in broiler breeding programmes is paramount.
Tibial dyschondroplasia (TD) can be selected against but it also has a strong nutritional component, with vitamin D especially important. Dietary supplementation with different vitamin D metabolites, as well as endogenous production of vitamin D by UV irradiation, has been shown to minimise TD. Exercise is also plays a part in minimising leg bone deformities and broiler house layout, lighting pattern and stocking density are all important factors affecting birds’ locomotion.
The problem of bone fractures in poultry is important for two reasons. Firstly, the pain and discomfort caused by fractures is a cause of poor welfare for the birds. Secondly, the presence in meat products of bone splinters from the fractures impairs the quality of poultry meat products. This is especially likely with automated systems for deboning or mechanical meat recovery. The problem is especially severe with spent laying hens where a survey by Gregory and Wilkins (1989) showed that 29% of hens had one or more bone fractures during life and that 98% of carcasses had fractures after processing. There is more recent information to suggest that the problem is getting worse, with over 50% of hens showing some form of skeletal damage during life (Sandilands et al., 2005). This skeletal fragility of hens is making them largely worthless from the point of view of meat recovery. The problem of bone fractures also occurs with broilers. In the case of these birds, the problem is often associated with leg bone deformities which can contribute to fractures, particularly during processing, in two ways. Firstly, deformities may result in excessive loads being placed on bones when legs are forced into shackles. Secondly, deformed leg bones may impact with machinery during automatic deboning. Any weakness in bones will increase the chances of fracture during either process. This review discusses the various factors that can influence bone strength and deformity in poultry.
II. Bone strength
Bone is a living tissue and its strength can be influenced by a number of variables. Genetic factors can determine the way a bone grows and subsequently remodels. Nutrition supplies the components from which bone is constructed. But bone is also a dynamic tissue and its growth and remodelling can respond to mechanical forces placed upon the bone. All of these factors come into play in relation to the major problem of bone strength in laying hens. In these birds, osteoporosis brings about a progressive loss of structural bone and bone strength over the reproductive period with resultant increased susceptibility to fracture.
Genetic effects on bone strength have been studied most extensively in relation to laying hen osteoporosis. Heritabilities of a range of bone traits have been measured and have been found to be particularly high for the 3-point breaking strengths of tibia (0.45) and humerus (0.30). A bone index (BI) was devised as a basis for selection for increased bone strength and resistance to osteoporosis: it contained functions for tibia and humerus strength, keel bone density and a negative function for bodyweight to prevent bone strength increasing in response to greater bodyweight. The heritability of the BI was 0.40
BI = 0.61 x tibia strength (N) + 0.37 x humerus strength (N) + 0.27 x keel radiographic density (mm Al eq.) – 0.35 x bodyweight (kg)
Divergent selection in a strain of White Leghorn hens using this BI resulted in the production of two lines, H (high BI) and L (low BI) lines. Divergence in bone strength, particularly tibia strength, was rapid and after 3 generations the lines differed by 25% for tibia strength, 13% for humerus strength and 19% for keel radiographic density at end of lay (Bishop et al., 2000). The divergence has continued and after 9 generations of selection there is now a 83% difference in tibia strength and 35% difference in humerus strength. The selection has been carried out on hens only with males in the breeding programme chosen on the basis of family values. However, males have also shown considerable effects of selection on bone traits. Interestingly, these changes have occurred without any corresponding changes in egg output or shell quality. Biological comparisons have shed some light on the mechanisms involved. The improvement in bone strength was attributable to a small increase in bone formation during the growing period and a large decrease in cortical bone resorption during the laying period in the H line. The latter observation could be explained by the decrease in numbers of osteoclast (bone resorbing) cells in the H line. There was also a small increase in the amount of medullary bone in long bones. Medullary bone is a labile source of calcium for shell formation and is not as strong as structural bone, but it does make a contribution to overall bone strength.
These results show that it is possible to select birds for improved bone strength. The BI selection procedure has involved retrospective selection on the basis of post mortem values. It is quite labour intensive and not particularly suited to application in commercial breeding programmes. A simpler, more predictive system based on genetic markers would be preferable. In an effort to understand the genetic basis of bone strength, an F2 reciprocal cross was established between the H and L lines. The F2 was used to detect and map quantitative trait loci (QTL) affecting the BI and the component traits of the index (Dunn et al., 2007). Phenotypic data from 372 hens in 32 families were analysed by within-family regression analyses using 136 microsatellite markers in 27 linkage groups. A significant QTL was found on chromosome 1, centred on 370 cM, with an F ratio of 9.29 which exceeded the P=0.05 F-statistic threshold. The region also contained QTL for two of the components of the bone index, tibia and humerus breaking strength. The F-ratio for tibia breaking strength QTL was
13.2 which exceeded the P=0.01 F-statistic threshold and the humerus breaking strength QTL was just below the P=0.05 F-statistic threshold. After full genotyping there were no other locations detected that contained significant QTL at the P<0.05 level for any of the traits analysed. Putative QTL on chromosomes 3, 6 and 9 were not confirmed after genotyping of the whole population and the inclusion of more markers. The additive effects for tibia breaking strength represented 34% of the trait standard deviation or 7.6% of the phenotypic variance of the trait. This is the first significant QTL related to bone quality in poultry and is particularly important as it is directly relevant to commercial populations. The next steps in the project will be to identify single nucleotide polymorphic (SNP) marker(s) within the region of the QTL that would have predictive value across different genotypes. If suitable markers can be established for laying hens, it will be of considerable interest to determine whether they also have predictive applicability to other types of poultry such as broilers and turkeys.
Influences of genetic selection have also been seen in broilers. Continued selection for faster growth rate has been shown to result in greater cortical porosity and also an increased ratio of Ca:P in bone mineral (Williams et al., 2000a; 2000b). The implications of this for bone strength are as yet unclear but it will be advisable for broiler breeders to pay attention to possible changes in bone strength in the newer genotypes. These genetic changes are already having an implication for nutrition, as discussed next.
Bone is a composite material of 30% organic matrix and 70% hydroxyapatite mineral. Hydroxyapatite is composed mainly of calcium and phosphorus in the weight ratio of 2.15:1. Bone is therefore highly dependent on the dietary supply of constituent nutrients, particularly Ca and P. The retentions of Ca and P from the diet are linked, but can be independently regulated to a small extent. Thus nutritionists endeavour to supply these nutrients in the diet in the approximate proportions of 2.15:1, cf NRC (1994) broiler starter requirement of 10 g Ca, 4.5 g available P/kg. Ca and P absorption and homeostasis are strongly regulated by vitamin D and requirements for this nutrient increase substantially as dietary Ca/P deviates from the ideal. Vitamin D is discussed more fully in a later section. The vitamin can help the bird adjust its absorption and retention of Ca and P within limits, but any substantial supply of Ca and/or P below the requirement or outwith the ideal ratio will result in rickets, characterised by soft bones and growth plate abnormalities.
The genetic changes in bone composition referred to above involved increases in the bone Ca:P ratio to up to 2.5:1 at about 10 days of age. Evidence that this may be changing the requirements of young broilers has come from a factorial study involving different dietary Ca and available P concentrations (Williams et al., 2000b). Optimum growth plate morphology was observed when the diet contained 11 to 12g Ca and 4.5g available P/kg. It may thus be advisable to increase the Ca content of broiler starter diets above the amount recommended by NRC (1994).
Other vitamins and minerals have effects on bone growth and composition. However, under current conditions of commercial nutrition, these nutrients do not seem to be associated with any particular practical problems.
Bone development is influenced by a wide range of metabolic regulatory factors. Prostaglandins are important regulatory factors that are derived transiently from polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series. They influence many metabolic pathways, including bone formation and development. Prostaglandins derived from n-6 fatty acids have been shown to have some inhibitory effects on bone development, while prostaglandins derived from the n-3 series can have more beneficial effects of stimulating osteoblast function and bone formation, as demonstrated in cell culture studies (Chang et al., 1998). Broiler diets, especially starter diets, are usually rich in n-6 fatty acids and providing a better dietary n-3/n-6 fatty acid balance in the diet may thus benefit bone development in birds. Studies have shown that feeding diets containing PUFA from fish oil can improve tibial strength in quail (Lui et al., 2003a; b).
A recent study has confirmed an effect of dietary n-3/n-6 ration on bone strength in broilers (McCormack et al., 2007). Experimental diets comprised a basal diet based mainly on wheat and soybean meal with natural oil content of 28g/kg and containing normal concentrations of other nutrients from which isoenergetic and isonitrogenous experimental diets of different n-3/n-6 balances were obtained by adding different combinations of supplemental maize oil (MO) and salmon oil (SO). These oils were chosen because MO is rich in n-6 fatty acids, mainly linoleic acid, and has a low content of n-3 fatty acids whilst SO is a rich source of n-3 fatty acids, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The results are shown in Table 1. There were significant improvements in liveweight in the diets containing SO in experiments 2 (P<0.05) and 3 (P<0.01). Feed-to-gain ratios were improved by SO addition in all experiments though the differences did not attain statistical significance (P>0.05). Three point breaking strengths of the tibia showed significant (P<0.001) increases with SO inclusion in all experiments. These treatment differences were also significant (P<0.01) after covariate analysis for liveweight. Tibia ash % was increased by SO inclusion in experiments 2 (P<0.001) and 3 (P<0.01).
The morphometric analyses in experiment 2 did not reveal any dietary effects on cortical structure or bending stress. The results indicated that diets with a low ratio of n-3/n-6 fatty acids resulted in sub-optimal performance and also gave poorer bone strength. The improvement in bone strength was partly a response to greater liveweight but was also directly attributable to effects of dietary fatty acids as confirmed by covariate analysis for liveweight. Increasing the n-3/n-6 ratio above 0.04 by provision of long chain PUFAs present in SO resulted in progressive improvements in performance and bone strength. It is concluded that addition of SO to diets of low n-3 fatty acid content has a beneficial effect on bone strength and performance in young broilers. The results do not give any indication of the relative merits of other n-3 fatty acids such as linolenic acid. However, it seems probable that diets lacking a source of n-3 fatty acids will give suboptimal bone strength.
Environment, particularly the opportunity it gives a bird for exercise, can have a strong effect on bone strength. Thus housing systems that allow birds the opportunity to fly are particularly effective in increasing bone strength. However, this may not reduce fracture incidence in the husbandry system (Gregory et al., 1990), because such birds may experience more violent accidents, but might improve processing quality. Genetic and housing effects on bone strength are additive (Whitehead, 2006) so selecting birds for better bone strength will benefit birds irrespective of housing system.
III. Bone deformity
Leg weakness is a widespread and longstanding problem in broiler production. The term covers a wide range of pathologies and causes can include genetic predisposition, nutritional and management inadequacies and disease. Almost all forms of leg abnormality, with the exception of femoral head necrosis, involve angular or rotational deformities of leg bones and/or joints and give rise to the type of problems that are likely to lead to bone fracture or splintering during processing or meat recovery.
Genetic predisposition, as a result of concentration on selection for growth rate, is a common underlying cause of leg problems and can be exacerbated by nutritional practices, either those that maximise growth or are deficient in some nutrients, and management. Some abnormalities, e.g. TD, can directly involve both genetic and nutritional factors. There is now an increasing awareness of the importance of including leg health characteristics among broiler selection criteria. Procedures can include assessing bird gait characteristics and using procedures to identify specific lesions.
TD is a specific abnormality that can be directly selected against using a ‘Lixiscope’ to identify lesions in real time in live birds. With experience, it is possible to identify large lesions, but small lesions are more problematical. Nevertheless experimental and commercial use of this approach has led to lines and strains of broilers whose susceptibility to TD is decreased, though not eliminated.
Nutrition has an important role in combating leg deformities. Where the primary cause of the problem is genetic predisposition linked to fast growth, nutritional procedures aimed at slowing growth can be effective. This can be achieved by feeding diets of lower nutrient density. This is best done at an early stage of growth, because deformities can be initiated quite early in the chick’s life and is often most effective when applied during the second week of life. The optimum procedure can be to give the chicks a fast start during the first week, as this has been shown to stimulate development of the digestive system and give them greater long-term growth potential, then slow growth during the 2nd and 3rd weeks before returning birds to a high density nutritional regime that will encourage compensatory growth.
TD is a common cause of leg bone deformity, with moderate to severe lesions causing a marked bowing of the tibia. A range of nutritional factors have been shown to affect the occurrence of TD, including Ca/P ratio and electrolyte balance, but the most effective means of preventing TD is via vitamin D metabolism. Dietary supplementation with 1,25-dihydroxyvitamin D3 (1,25D) has been found to reliably prevent TD (Rennie et al., 1993). 1,25D in its crystalline form has a low margin of safety between effective (~3.5μg/kg) and toxic threshold (~5μg/kg), depending upon dietary Ca concentrations (Rennie et al., 1995). However, herbal extracts containing 1,25D derivatives are becoming available and may have lower toxicity. Another metabolite that can be effective against TD is 25-hydroxyvitamin D (25D). This is available commercially (Hy-D) and effective doses are from 70μg/kg upwards. Earlier studies did not suggest that vitamin D itself was effective against TD, but a more recent study (Whitehead et al., 2004) has shown that very high dietary concentrations (up to 10,000IU/kg) can have a marked effect in countering TD. This concentration is above the legal limit of 5000IU/kg in the EU so alternative strategies are needed in Europe. These involve use of 25D and/or water-soluble vitamin D supplements over the first few days of the chicks’ lives. It seems to be particularly important to maximise the vitamin D status of the chick during the early stages of growth when TD lesions can first be initiated. The need to provide vitamin D in such high doses raises the question of whether it is perhaps operating by a pharmacological rather than nutritional route. Regulation of gene expression is an important metabolic function of vitamin D. If the genetic component in TD involves dysfunction of a vitamin D-dependent gene, perhaps a gene regulating chondrocyte differentiation, it is possible that a greater signal from vitamin D (or its main metabolite, 1,25D) can overcome the gene defect and prevent TD. An alternative means of providing this signal is explained in the next section.
Exercise is well known for its effects in strengthening bones and has also been found to diminish the incidence of leg bone deformities. The latter effect may be related to better muscular development rather than solely to increases in bone strength. Broilers can be encouraged to exercise by such management procedures as increasing to distance between feeders and waterers. Stocking density can also affect a bird’s opportunity for exercise. There has been a heated debate in Europe over optimal stocking densities. Using bodyweight as a criterion, quite high stocking densities can be achieved, provided adequate environmental conditions are maintained. However, it is noticeable that the incidence of leg problems increases at the higher stocking densities, presumably as a result of difficulty in movement (Sorensen et al., 2000; Dawkins et al., 2004).
Lighting has also been used as a means of improving leg quality, and this effect may also be related to changed patterns of behaviour and movement. Thus intermittent lighting and ‘normal’ dark periods of 7 to 8 hours have been found to be beneficial.
More specific effects of UV lighting have been reported. Edwards et al. (2003) found that exposing birds soon after hatching to UV light from below directly on to the skin of the legs decreased the incidence of TD. It was suggested that this effect was related to endogenous photosynthetic production of vitamin D. Exposure of chicks to UV light in a hatchery is impractical so we have been studying effects of different durations and intensities of UV light in situations that could be applied in a broiler house. In our most recent experiment, day-old chicks were exposed to UV light for 12 h from bulbs suspended 1.5m above them and fed a diet imbalanced in Ca/P to magnify the occurrence of TD.
The results (Table 2) show significant improvements in bone strength and mineralisation and a marked decrease in growth plate abnormalities, mainly TD and rickets. Measurements of 25D showed that plasma concentrations remained elevated in the treated group for several days post irradiation. These results show that it is not essential to irradiate from below; irradiation from above is also effective and did not result in ocular damage. We are now progressing to trials in a broiler house where the normal light bulbs are replaced by UV bulbs for 12h immediately after the chicks are placed. These findings confirm the central role of vitamin D in preventing TD. Whether the effect of UV irradiation is associated solely with the formation of vitamin D (cholecalciferol) or also promotes the formation of other highly-effective hydroxylated metabolites is uncertain at the moment.
Genetic selection for bone strength and freedom from leg bone deformities should have a high priority in breeding programmes. Nutritionally, careful attention should be paid to Ca, P and vitamin D supply. Fatty acids of the n-3 series are also important. Vitamin D has a central role in prevention of TD and use of vitamin D metabolites, high concentrations of vitamin D itself or induction by UV irradiation can all be effective. Environmental factors such as exercise, lighting pattern and stocking density also affect bone deformities. If, despite attention to these factors, bone splinters still occur in meat products, X-ray detection is a last ditch strategy.
Bishop, S.C., Fleming, R.H., McCormack, H.A., Flock, D.K. and Whitehead, C.C. (2000). British Poultry Science 41: 33-40.
Chang, D.J., Ji, C., Kim, K.K., et al. (1998). Journal of Biological Chemistry, 273: 48924896.
Dawkins, M.S., Donnelly, C.A. and Jones, T.A. (2004). Nature, 427 (6972): 342-324
Dunn, I.C., Fleming, R.H., McCormack, H.A., Morrice, D., Burt, D.W., Preisinger, R. and Whitehead C.C. (2007). Animal Genetics (in press).
Edwards, H.M. (2003). British Journal of Nutrition, 90: 151-160.
Gregory, N.G. and Wilkins, L.J. (1989). British Poultry Science, 30: 555-562.
Gregory, N.G., Wilkins, L.J., Eleperuma, S.D., Ballantyne, A.J. and Overfield, N.D. (1990). British Poultry Science, 31: 59-69.
Liu, D, Veit, H.P., Wilson, J.H. & Denbow, D.M. (2003a). Poultry Science, 82: 831-839.
Liu, D, Veit, H.P., Wilson, J.H. & Denbow, D.M. (2003b). Poultry Science, 82: 463-473.
McCormack, H.A., Fleming, R.H., McTeir, L. and Whitehead, C.C. (2007). British Poultry Abstracts (in press).
NRC (1994). Nutrient Requirements of Poultry, ninth revised edition. National Academy Press, Washington,D.C.
Rennie, J.S., Whitehead, C.C. and Thorp, B.H. (1993). British Journal of Nutrition, 69: 809-816.
Rennie, J.S., McCormack, H.A., Farquharson, C., Berry, J.L., Mawer, E.B. & Whitehead,
C.C. (1995). British Poultry Science, 36: 465-477. Sandilands, V., Sparks, N., Wilson, S. and Nevison, I. (2005). British Poultry Abstracts, 1: 23-24.
Sorensen, P., Su.G. and Kestin, S.C. (2000). Poultry Science, 79: 864-870.
Whitehead, C.C. (2006). Australian Poultry Science Symposium,
Whitehead, C.C., McCormack, H.A., McTeir L. & Fleming R.H. (2004). British Poultry
Science, 45: 425-436. Williams, B., Solomon, S, Waddington, D, Thorp, D and Farquharson, C. (2000a) British Poultry Science, 41: 141-149. Williams, B., Waddington, D., Solomon, S., and Farquharson, C. (2000b) Research in Veterinary Science, 69: 81-87.
From Proceedings of the “19th Australian Poultry Science Symposium”