Bone growth is controlled by a precise mechanism and any deviation from normal bone growth will result in bone disorders that have the potential to present a major economic problem to the poultry industry. The enormous losses attributed to skeletal disorders in poultry are caused by increases in mortality and condemnations at the processing plant, and further downgrading caused by the trimming of breasts and legs. From the economic point of view, the major skeletal disorders are associated with the rapid increase in growth rate that has been achieved during the last few decades. Whether the disorders are direct effects of the growth rate or indirect effects that result from increased body weight and improper development of bone, muscles and/or tendons is still to be determined. The economic dilemma facing the industry lies in the balance between continued selection for rapid growth, on the one hand, and the losses caused by bone and other metabolic disorders, on the other. The regulation of bone growth and development appears to be complex, with various levels of interactions among the regulating agents. Multidisciplinary research at various levels, such as the genomic and proteomic approaches, cell culture methodology, genetic selection, nutritional manipulation, and environmental control will provide us with better understanding of the molecular mechanisms underlying bone disorders. Such understanding might enhance our knowledge and perhaps help in the design of rational strategies for the treatment of bone related diseases.
Introduction
The skeleton provides the physical support for and determines the shape of the body, and its mineral fraction serves as a calcium reservoir that is used for the maintenance of the extracellular Ca2+ concentration. Calcium concentration is of the utmost importance in the management of normal cellular function and of intracellular information processing. Bone growth is controlled by a precise mechanism, as can be inferred from the near-symmetry of the limbs and the hereditary element of adult height. Any deviation from normal bone growth will result in bone disorders that have the potential to present a major economic problem to the poultry industry (Sullivan, 1994). Several extensive reviews of various aspects of poultry bone disorders have been written during the last few years (Orth and Cook 1994; Julian 1998; Cook, 2000; Edwards, 2000; Rath et al., 2000; Pines et al., 2004). The aim of this presentation is to provide an introduction to the field, but by no means to cover all aspects of poultry bone disorders.
Bone development
Bone development starts in the early stages of embryo development and continues throughout maturity. Even after adult stature is attained, bone development continues, for repair of fractures and for remodelling to meet various challenges. There are two types of ossification: intramembranous and endochondral.
a) Intramembranous bone formation
Intramembranous ossification involves the replacement of sheet-like connective tissue membranes with bony tissue. Bones formed in this manner are called intramembranous bones; they include certain flat bones of the skull and some of the irregular bones. During intramembranous formation mesenchymal cells migrate to the site of eventual bone formation. These cells condense, align and secrete an organic framework of extracellular matrix (ECM), to form the osteoid. The cells continue to proliferate during the entire osteogenic process, and all cells involved in bone formation retain this ability. Osteoblasts line the osteoid and begin to deposit bone ECM and the inorganic salt components that impart strength, some flexibility and the ability to hold a defined structure. The organic strands – the trabeculae – are mineralized and consecutive growth rings are added to them to increase the thickness of the lamella. The lamella is added in the course of cycles of osteoid secretion and mineralization-appositional growth. When multiple trabeculae within the developing bone contact one another a lattice structure forms. Regions of bones may become completely filled in with mineralized osteoid to form compact bone; if not completely filled in they form primary cancellous bone. Most bones are mixtures, comprising a compact outer layer and a cancellous interior.
b) Endochondral bone formation
Mesenchymal cells migrate to the site of eventual bone formation and become chondrocytes. The chondrocytes proliferate into a very dense mass of cells, devoid of blood vessels, and form cartilage in the shape of the forming bone. They then secrete ECM, which forces them apart, and they become encapsulated within the cartilage, which grows in a process called interstitial growth. The cartilage is surrounded by a layer of mesenchymederived connective tissue cells the perichondrium. These secrete ECM and add to the cartilage by adding more layers, so causing appositional growth. Blood vessels invade the cartilage, which becomes replaced with bone. The outer layer of mesenchyme cells, which support appositional growth over areas where cartilage has been replaced with bone, is now called the periosteum. As the cartilage is degraded, remaining strands of cartilage act as templates for osteoblasts to secrete additional ECM, which is subsequently calcified.
Calcium homeostasis
In contrast to the concentration of cytosolic calcium, that of extracellular calcium remains almost constant and, under normal circumstances, varies by only a few percentage points throughout life. The near constancy of the extracellular calcium concentration is maintained by a complex homeostatic system, which primarily involves the secretion of parathyroid hormone (PTH) from the parathyroid glands (PGs), calcitonin (CT) from the C cells of the ultimobrachial glands, and vitamin D, and their effects on calcium metabolism at renal, skeletal and intestinal sites.
Changes in the set point of the PGs elicit major changes in PTH secretion at any given concentration of extracellular calcium, and these changes, in turn, are closely related to the steady-state concentration of the serum calcium. The G-protein-coupled calcium-sensing receptor (CaR) of the PGs is the major regulator of PTH secretion (Brown, 2000; Yarden et al., 2000).
Vitamin D, either obtained from the diet or derived from sunlight, is a major regulator of calcium homeostasis. The first hydroxylation reaction, which takes place in the liver, produces 25-hydroxyvitamin D3. This reaction is not tightly regulated and so an increase in the amount of dietary intake will immediately result in an increased amount of circulating 25-hydroxyvitamin D3. The second hydroxylation reaction, at the α position of carbon 1, takes place in the proximal convoluted tubules of the kidney, and results in 1,25-dihydroxyvitamin D3, which is the most biologically active form. PTH, thyroid hormones, oestrogens and calcitonin regulate this reaction. The 1,25-dihydroxyvitamin D3 stimulates intestinal calcium absorption and resorption of calcium and phosphates from bone and kidneys in order to ensure the presence of enough minerals for bone mineralization (Norman, 1990). When the level of calcium in the blood decreases, the parathyroid glands are stimulated to secrete the PTH that will activate renal 1α-hydroxylase, which then catalyzes the second hydroxylation reaction of vitamin D3 (Norman and Hurwitz, 1993).
Inherited and rare bone disorders
Nanomelia is a recessively inherited connective tissue disorder of chickens; it affects cartilage development and is characterized by chondrodystrophy and shortening of the long bones. It involves low aggrecan production and diminished aggrecan mRNA levels. Aggrecan mRNA is present in the nucleus of the nanomelic chondrocyte but its level is greatly reduced in the cytoplasmic compartment. A stop codon has been identified at codon 1513, which is located in the eighth repeat of the chondroitin sulfate 2 domain of the large tenth exon (Primorac et al., 1994).
Ametapodia is a mutation associated with abnormal limb development that appeared in a strain of Light Brown Leghorn chickens. The mutants are characterized by the complete absence of the tarsometatarsals, and severely hypoplastic development of the metacarpals is also present. The disease is inherited as an autosomal recessive and the affected chicks do not normally survive beyond 2-4 days of age (Smyth et al., 2000).
Bone disorders of the spine
Scoliosis is characterized by a lateral deviation of the spine, with rotation of the vertebrae. The effect of intense light on melatonin secretion by the pineal gland is probably involved in the incidence of the disease (Nette et al., 2002). A genetic study of an inherited form of scoliosis in chickens revealed that the incidence of expression of a scoliotic parent line implicated three major autosomal, recessive genes (McCarrey et al., 1981).
Spondylolisthesis (kinky-back) is characterized by a ventral dislocation of the anterior end of the articulating fourth thoracic vertebra, with over-riding of the posterior end by the fifth, which causes pinching of the spinal cord. Dislocation may also occur between other cervical and thoracic vertebrae. Damage to the spinal cord causes leg weakness that is usually followed by partial posterior paralysis (Riddell and Howell, 1972; Wise, 1973; McNamee et al., 1998). Incidence was greatest in chickens fed ad libitum and kept in batteries, and it varies between flocks (Khan et al., 1977).
Bone disorders due to mycoplasma infection
Leg weakness characterized by chondrodystrophy of the hock joints, clear fluid in hock joint spaces, valgus deformities and shortening of the tarsometatarsal bones, and curled toes were observed in turkey poults infected with mycoplasma (Trampel and Goll, 1994).
Joint lesions and curved toes were observed in turkey embryos inoculated with mycoplasma, and scanning electron microscopy of the tarsometatarsal joints showed fissures in the cartilage (Lam et al., 2004).
Vitamin and mineral deficiencies
There are numerous studies describing the involvement of vitamins, minerals and their interactions on skeletal disorders in poultry (Baker et al., 1998; Williams et al., 2000b; Jin et al., 2001; Zhang et al., 2003). For example broilers suffering from pyridoxine deficiency had tibias of reduced dry weight and cortical thickness. Histomorphometry revealed a disproportionately large eroded surface, reduction in the amount of osteoid tissue and in the width of mineralized trabeculae and in the number of secondary ossification centers, along with coarse trabeculation. The metaphyseal cartilage showed irregular trabeculae and a markedly reduced amount of collagen and, probably, impaired collagen cross-linking (Masse et al., 1990, 1994). The effect of deficiencies of other minerals and vitamins such as manganese, biotin, and choline have also been studied (Stock and Latshaw, 1981; Liu et al., 1994).
In poultry, the involvement of vitamin D and its metabolites is by far the most studied factor in relation to bone disorder (Edwards, 2000). Decreased extracellular phosphate, which occurs in vitamin D deficiency, may play a key role in rickets. The involvement of vitamin D in longitudinal bone growth and, especially, on the growth plate at the end of the long bones has been studied (Ben-Bassat et al., 1999; Nilsson et al., 2005). In rickets the width of the hypertrophic zone of the growth plate is increased and mineralization is defective. The effects of vitamin D on the growth plate are mediated primarily through the vitamin D receptor, and are expressed in intestinal epithelial cells, which exhibit increased calcium and phosphate absorption from the intestinal lumen. Vitamin D metabolites, however, may also act directly on the growth plate. For example, injection of 24,25-dihydroxyvitamin D3 directly into rachitic chick growth plates resulted in healing (Lidor et al., 1987). In vitro, 24,25-dihydroxyvitamin D3 stimulated differentiation but partly inhibited proliferation in resting zone cells, whereas 1,25-dihydroxyvitamin D3 decreased proliferation in the resting and the proliferative zones (Boyan et al., 2002). The effects of vitamin D (Atencio et al., 2005), 1α-hydroxycholecalciferol (Driver et al., 2005), and 25-hydroxycholecalciferol (Bar et al., 2003) on bone development and disorders have been evaluated. Addition of phytase in combination with 1α-hydroxycholecalciferol or 1,25-dihydroxyvitamin D3 ameliorated leg problems and decreased the incidence of rickets, respectively (Mitchell and Edwards, 1996; Driver et al., 2005).
Bone and rapid growth
From an economic point of view the major concern is the rapid increase in growth rate achieved by genetic selection (Havenstein et al., 2003), and its impact on bone development. Comparison between fast-growing and slower-growing strains revealed less mineralization, more porous cortical bone and increase in the Ca/P ratio in the former (Williams et al., 2000a). The porosity was a result of rapid primary osteon formation at the periosteal surface, and incomplete infilling of the resultant canal with osteoblasts. These reductions in density and mineral content resulted in altered biomechanical properties, which caused high rates of bone breakage during catching, transport and handling at the processing plant. Use of feed restriction led to the suggestion that the rapid growth rate, and not the genetic potential, was responsible for the changes in the biochemical properties of the bones (Williams et al., 2004).
Tibial dyschondroplasia
Tibial dyschondroplasia (TD) is one of the most common skeletal abnormalities that result in deformed bones and lameness (Leach and Gay, 1987; Leach and Lilburn, 1992; Orth and Cook, 1994; Pines et al., 2004). The economic loss caused by TD, as manifested in the high rates of mortality and morbidity, is enormous: up to 30% of birds may be affected during an outbreak. Those with severe lesions are more susceptible to fractures during handling at the processing plant, with consequent economic loss. In addition, lameness associated with TD in broilers is a welfare issue. TD is a disease of rapidly growing birds, especially in broilers (Leach and Lilburn, 1992) and turkeys (Wyers et al., 1991), growing at their maximal genetic potential. Thus, genetic selection for growth rate has actually resulted in increased incidence of this skeletal disease. Various factors have been found to be involved in the aetiology of the disease. They include dietary (Sauveur, 1984; Thorp et al., 1993; Rennie et al., 1993), environmental (Riddell and Classen, 1992); and genetic (Leach and Lilburn, 1992; Wong-Valle et al., 1993) factors.
TD is a disease of the growth plates at the end of the long bones. The growth plate is populated by chondrocytes, arranged in columns parallel to the long axis of the bone, and it comprises several zones arranged in succession from the proximal to the distal border: (a) the resting or reserve zone, containing stem cells; (b) the proliferative zone, with stacks of flattened cells (cell division occurs mostly in the longitudinal direction and leads to cell column formation); (c) the hypertrophic zone, containing hypertrophic chondrocytes; and (d) the degenerative zone, with a partially calcified matrix and invading capillaries. Each chondrocyte, once formed, remains in a spatially fixed location, in which it accomplishes all of its physiological functions, throughout its cellular life cycle.
Chondrocytes located in the various zones differ in their morphology, secretion of extracellular matrix components, activity of various enzymes, and expression of hormone receptors. Synthesis of collagen type II is characteristic of chondrocytes in the proliferative state whereas that of collagen type X is restricted to the hypertrophic state (Knopov et al., 1997). The alkaline phosphatase (AP) activity observed in the hypertrophic zone is of particular interest, since its appearance marks the onset of calcification. The receptor of parathyroid hormone (PTHR), the major hormone responsible for the minute-to-minute regulation of the extracellular calcium concentration, is expressed in the maturation zone (Ben-Bassat et al., 1999). Chondrocyte maturation is accompanied also by changes in the rate of proteoglycan synthesis, and synthesis of non-collagenous phosphorylated matrix proteins such as osteopontin (OPN) and bone sialoprotein (BSP) (Pines et al., 1999).
In comparison with the mammalian growth plate, the avian one contains longer columns of cells, which become randomly oriented. In the hypertrophic and calcified zones, cell columns are no longer apparent. More cells are found in each zone and the metaphyseal blood vessels penetrate more deeply, in the avian than in the mammalian growth plate (Leach and Gay, 1987; Pines and Hurwitz, 1991). The growth plate of a 4- to 7-week-old chicken contains approximately 200 cells per column as compared with 25 cells in that of the rat. The transition between zones was observed to be more orderly in Leghorns than in broilers (Reiland et al., 1978). A wider proliferative zone was found in a heavy than in a light turkey strain (LeBlanc et al., 1986). For many species of birds, the changes in the length of the proliferative zone correlate strongly with variations in growth rate (Kember et al., 1990). In chickens and turkeys, age-dependent growth rates were correlated with the width of the hypertrophic zone rather than with that of the proliferative zone (Hurwitz et al., 1992), suggesting that, as in mammals (Breur et al., 1991), cell hypertrophy is the main determinant of longitudinal growth in these species.
Similarly to TD, rickets is also a disease of the growth plate. In rickets the proliferative zone of the growth plate is enlarged, and there are additional chondrocytes in each column. It is of the utmost importance to distinguish between the two lesions when studying TD and designing strategies to prevent the disease. This is especially important since in some cases TD-afflicted birds have a rickets background. It is possible to distinguish between them visually, but determination of molecular markers such as PTHR gene expression is much more accurate. In TD, normal expression of PTHR is observed, whereas in rickets the receptor is down-regulated because of high parathyroid hormone levels, and no expression of the receptor is observed (Ben-Bassat et al., 1999).
TD is characterized by the appearance of a plug of unvascularized, unmineralized, opaque white cartilage, which dominates the proximal metaphysis of the tibiatarsus and, occasionally, the tarsometatarsus (Hargest et al., 1984). The various morphological and biochemical manifestations of the TD lesion, such as changes in carbonic anhydrase (Gay et al., 1985), AP activity, collagen type II and X production (Chen et al., 1993; Knopov et al., 1995), and OPN and BSP synthesis (Pines et al., 1999) suggest that TD-chondrocytes fail to undergo the complete differentiation that normally leads to cartilage vascularization and mineralization. In addition, OPN is probably involved in vascularization since its expression was demonstrated in the front of new blood vessels in the growth plate (Knopov et al., 1995). There are various tools for the study of TD; each has its advantages as well as limitations that should be considered.
Field TD - Many of the studies of TD were performed on samples taken from the field at the final stage of the disease when lameness was obvious and the lesions were enlarged. The percentage of afflicted birds in each flock varies widely and some of the features observed during a progressive stage of the disease are secondary and do not necessary reflects the early events responsible for the TD lesion. For example, reduction in matrix vesicle formation, which is important for mineralization in the TD-affected growth plate (Nie et al., 1995), may be due to necrosis resulting from energy depletion, impaired oxidative metabolism and a lack in tissue vascularization (Hargest et al., 1984).
Selected lines - The incidence of TD can be changed by genetic selection although to date no specific genetic defect has been identified. An experimental breeding program selecting for TD usually results in only 60-70% of the birds developing the disease. Use of lines selected for high and low incidence of TD makes it possible to study the time-dependent morphological, endocrinological and molecular events associated with chondrocyte differentiation (Ben-Bassat et al., 1999; Pines et al., 1999; Ling et al., 2000; Shen et al., 2004) and environmental and nutritional effects in relation to the development of TD (Mitchell et al., 1997a, b; Punna and Roland, 2001).
Induced TD - TD can be induced by a variety of protocols, all leading to the same phenotype, which suggests a down-stream common pathway. For example, TD can be induced by manipulation of nutritional factors such as the calcium-to-phosphorus ratio (Rennie et al., 1993), cysteine supplement (Bai and Cook, 1994), and by Fusarium-infected feedstuff (Chu et al., 1996). Various dithiocarbamates, such as thiram, are used to induce very high incidence of TD (Ben-Bassat et al., 1999; Rath et al., 2004). This model has the advantage that following thiram removal spontaneous recovery can be studied, and induction of TD by thiram can be prevented by copper supplementation, as demonstrated by AP activity, and expression of collagen type II and X gene (Pines et al., 2004). It remains to be determined whether all the modes of TD induction lead to a result that completely resembles the field TD.
Organ cultures - The regulation of limb growth, and the signals involved in chondrocyte proliferation, maturation, and hypertrophy can be studied in organ cultures (Di Nino et al., 2001). Organ cultures taken from embryos of different ages can be used for evaluating the local effect of various agents simultaneously, in a system where cellular interactions are intact.
Cell cultures - Isolated growth plate chondrocytes in culture form the most widely used system. Chondrocytes are easy to grow and can be manipulated in culture, by means of ascorbic acid, to change their phenotype from proliferative (collagen type II positive) to hypertrophic (collagen type X, OPN, AP positive) cells (Halevy et al., 1994; Barak-Shalom et al., 1995). Cells can be isolated from normal and TD-afflicted chicks (Farquharson et al., 1995) and can be fractionated into distinct populations by means of the Percoll density gradient (Farquharson et al., 1999). Various growth factors (Praul et al., 2002), growth factor receptors (Halevy et al., 1991, 1994) and hormone receptors (Monsonego et al., 1993, 1997; Ben-Bassat et al., 1999) have been studied by using these cells.
TD can be induced by nutritional manipulations or by toxic agents, as well as by selective breeding (Sauveur, 1984; Orth and Cook, 1994; Ben-Bassat et al., 1999). Thus, the various protocols that result in TD may initially act via distinct pathways, but downstream they probably share common pathway(s) that lead to the same phenotype. An association between TD and rickets was demonstrated by the finding that supplementation of high doses of vitamin D or its analogs could ameliorate TD in selected lines (Edwards, 2000; Whitehead et al., 2004; Atencio et al., 2005).
The divergent selection of broilers for low or high TD altered the physiological response to nutritionally inadequate levels of dietary D3 (Shirley et al., 2003). During recent years various strategies were used in attempts to understand the causes of TD. Aspects investigation included: the mechanisms involved in chondrocyte differentiation (Knopov et al., 1997; Pines et al., 1999; Farquharson et al., 2001); chondrocyte apoptosis (Praul et al., 1997; Ohyama et al., 1997; Rath et al., 1998); effects of hormones and their receptors (Ben-Bassat et al., 1999; Webster et al., 2003); and fingerprint techniques to compare gene expression in normal and TD chondrocytes (Jefferies et al., 2000).
Conclusion
The regulation of bone growth and development appears to be complex, with various levels of interactions among the regulating agents. A high degree of precision in the genetic control is essential, and deviation beyond a certain threshold would cause abnormal bone growth. Multidisciplinary research at various levels, such as the genomic and proteomic approaches, cell culture methodology, genetic selection, nutritional manipulation, and environmental approaches will provide us with better understanding of the molecular mechanisms underlying bone disorders. Such understanding might enhance our knowledge and perhaps help in the design of rational strategies for the treatment of bone-related diseases.
The enormous losses attributed to skeletal disorders in poultry are caused by increases in mortality and condemnations at the processing plant, and further downgrading caused by the trimming of breasts and legs. From the economic point of view, the major skeletal disorders are associated with the rapid increase in growth rate that has been achieved during the last few decades. Whether the disorders are direct effects of the rapid growth rate or indirect effects that result from increased body weight and improper development of bone, muscles and/or tendons is still to be determined. The economic dilemma facing the industry lies in the balance between continued selection for rapid growth, on the one hand, and the losses caused by bone and other metabolic disorders, on the other hand.
References are available on request
From Proceedings of the “19th Australian Poultry Science Symposium”, New South Wales, Australia.
M. PINES
Institute of Animal Sciences,
Volcani Center, Israel
