ABSTRACT At hatch, the chick skeleton is a miniature of that of the adult bird. The hen deposits calcium, phosphorus, and trace minerals (copper, zinc, and manganese) along with vitamin D into the egg to allow development of the embryonic skeleton. The main source of calcium is the eggshell, whereas phosphorus, trace minerals, and vitamin D are mainly derived from the yolk. Calcium is absorbed from the eggshell and transferred to the embryo and yolk through the chorioallantoic membrane, whereas phosphorus and trace minerals are simultaneously mobilized by the yolk sac membrane. These processes start at day 12 of incubation and peak at around day 17. While the eggshell provides a steady supply of calcium until 19 d of incubation, phosphorus and trace mineral reserves decrease considerably and minimal skeletal development occurs in the last 3 d of incubation. Whether the low levels of phosphorus and trace minerals at late incubation prevent further bone growth, or some other biological control exists preventing further mineralization towards hatching is unknown. Maternal transfer of minerals and the influence of trace mineral form in the hen diet to advance the state of skeletal development at hatch have received increased research attention. Minimal effects on yolk mineral composition and bone growth were observed in the offspring of hens fed different forms of trace minerals. Embryos from young hens had inferior bone development towards the end of incubation and at hatch relative to chicks from older hens. This effect is likely a consequence of limited egg nutrient resources in eggs from young hens. The influence of maternal nutrient transfer on embryonic bone development has been clearly established. However, attempts to increase the state of skeletal development at hatch through increasing egg mineral content have met with limited success. The focus of this paper is the relationship between skeletal mineralization of the chicken embryo throughout incubation and egg mineral supply. INTRODUCTION Modern commercial broiler chicks (Gallus gallus) grow extremely rapidly after hatch. In the grow-out period, the bird's body weight increases from approximately 44 g at hatch to 1.396 kg and 4.202 kg at 28 and 56 d of age, respectively (Zuidhof et al., 2014). Genetic selection for growth and meat yield, however, has come with unintended consequences; in particular, skeletal abnormalities and poor walking ability (Dinev et al., 2012; Shim et al., 2012; Duggan et al., 2015; González-Cerón et al., 2015), which are the most prevalent causes of culling and late mortality in the modern broiler production (Knowles et al., 2008; Grandin, 2010). Bone mineralization and development starts during early embryo development (Pechak et al., 1986a,b), so it is feasible that skeletal disturbances may originate while the chicken is developing within the egg. Although growth-selected and unselected birds have similar periods of rapid bone formation (4 to 18 d) and mineralization (4 to 11 d) in the post-hatch period, the cortical bone tissues of selected birds are less mineralized (Williams et al., 2004). Furthermore, selected embryos and chicks have more porous bones when compared to unselected birds (Rawlinson et al., 2009); this may lead to bone deformities. Therefore, the skeleton of modern chicks might not be adequately calcified in the pre- and post-hatch period to support body growth. In recent years, there has been increased focus to understand factors that affect skeletal mineralization early in life and its influence on leg problems in broiler chickens during the post-hatch period. For example, mineral and vitamin supplementation either via the maternal diet (Favero et al., 2013; Torres, 2013; Saunders-Blades and Korver, 2015) or through in ovo feeding (Yair et al., 2012, 2013, 2015; Bello et al., 2014a,b; Oliveira et al., 2015a,b) increased bone size (Favero et al., 2013), ash (Yair et al., 2013; Oliveira et al., 2015b), and bone mechanical properties of the chicken embryo (Yair et al., 2013, 2015). Chicks with healthier skeletons at hatch may be more able to reach feed and water after placement at the farm, resulting in earlier access to nutrients and thus increased post-hatch growth. Furthermore, newly hatched chicks with a strong, well-formed skeletal frame might be less likely to be reluctant to move and therefore have decreased carcass defects like breast blisters and hock burns later in life. This review will present some important aspects of skeletal mineralization of the chicken embryo in the context of mineral availability to the embryo and to maternal age. OVERVIEW OF CHICKEN EMBRYO BONE DEVELOPMENT Embryonic Bone Physiology During development in ovo, bones grow in 2 different ways. Mesenchymal cells can directly differentiate into bone in a process known as intramembranous ossification, which is the source of most of the craniofacial skeleton (Pechak et al., 1986a,b). Alternatively, these cells can differentiate into cartilage, which provides a template for bone morphogenesis. This process is known as endochondral ossification and results for the formation of the appendicular and axial skeleton of chickens (Pechak et al., 1986a,b; Roach and Shearer, 1989; Roach, 1997). The process of endochondral ossification starts in the first week of incubation and involves a highly coordinated sequence of events that begins with differentiation of mesenchymal cells into chondrocytes and osteoblasts in the diaphysis (Pechak et al., 1986a,b). Chondrocytes lay down an organic matrix of cartilage, which initially forms a miniature bone-shaped structure similar to that of the long bones of the mature skeleton (Osdoby and Caplan, 1981; Pechak et al., 1986b). Osteoblasts lay down layers of collagen type I which becomes mineralized by the deposition of bone mineral, primarily hydroxyapatite (Ca10(PO4)6(OH)2). This model of the limb skeleton will expand to full size over time as blood vessels deliver calcium and phosphorus for upcoming mineralization of the cartilage core region (Kerschnitzki et al., 2016a,b). Bones grow in width by radial growth (Pechak et al., 1986a,b). In this way, cross-sectional area of the medullary cavity increases throughout incubation (Yair et al., 2012) by resorption of existing bone from the inner surface (endosteum), and bones get thicker by synthesis and deposition of new mineral by osteoblasts on the outer surface of the bone (periosteum; Roach and Shearer, 1989; Roach, 1997; Chen et al., 2008; Kerschnitzki et al., 2016a,b). Longitudinal growth of bones occurs through expansion of both the proximal and distal epiphyses, as opposed to the radial growth of the bone diaphysis. In the chicken embryo, the thickness of the growth plate increases considerably as the bone elongates because expansion of the proliferative zone occurs more rapidly than bone resorption at the distal area of the growth plate (Roach, 1997). The end result is that bones grow extremely rapidly during incubation with the tibia increasing its length by about 2.3-fold between 15 d of incubation and hatch (Torres, 2013; Figure 1A). Figure 1. View largeDownload slide Bone growth and mineral metabolism in the chicken embryo. (A) Between day 15 and 18 of incubation, greatest increase in the size and calcium content of tibia of the chicken embryo occurs. (B) Synchronously to increased skeletal mineralization, eggshell calcium and yolk phosphorus are mobilized by the growing embryo. Data are modified from Torres (2013), Kubota et al. (1981) and Li et al. (2014). Figure 1. View largeDownload slide Bone growth and mineral metabolism in the chicken embryo. (A) Between day 15 and 18 of incubation, greatest increase in the size and calcium content of tibia of the chicken embryo occurs. (B) Synchronously to increased skeletal mineralization, eggshell calcium and yolk phosphorus are mobilized by the growing embryo. Data are modified from Torres (2013), Kubota et al. (1981) and Li et al. (2014). Osteons (Haversian canals) are channels within the bone that are the basic structure of bone growth. The osteons generally are parallel to the length of the bone, and form the compact bone. Osteons start off as tubes, which are gradually mineralized on the inner surface by osteoblasts, leaving only a narrow channel in mature bone (Kerschnitzki et al., 2016a). Because embryonic growth rate of fast-growing embryos exceeds the capacity of the broiler to deposit bone mineral (Williams et al., 2004), the osteons do not fill in completely, leaving a porous bone structure that is not fully mineralized at the time of hatching (Rawlinson et al., 2009). Calcium and Phosphorus: the Main Players for Embryonic Mineralization The Role of Calcium and the Chorioallantoic Membrane Throughout incubation, the yolk is the main source of phosphorus, trace minerals (Yair and Uni, 2011), and vitamin D3 (Ono and Tuan, 1991), each of which is required for skeletal growth. For the first 7 to 10 d of incubation, the yolk is the main source of calcium for embryonic growth (Richards and Packard, 1996), but because there is a relative lack of bone calcification at this stage, there are minimal changes in calcium content of the embryo (Johnston and Comar, 1955). Following the complete development of the chorioallantoic membrane (CAM) between 9 and 14 d of incubation (Gabrielli et al., 2004; Gabrielli and Accili, 2010), calcium begins to be absorbed from the shell and incorporated into both yolk and embryonic tissues (Johnston and Comar, 1955). The CAM is a highly vascularized extraembryonic membrane which performs multiple functions during embryonic development, including calcium transport from the shell to the embryo (Gabrielli et al., 2004). This membrane first makes contact with the shell membranes from day 9 to 12 (Bell and Freeman 1971; Makanya et al., 2016) of incubation. However, calcium release from the eggshell and transport across the CAM start a few days later, when villus cavity and capillary-covering cells present in the chorionic epithelium are fully differentiated (Narbaitz and Tolnai, 1978; Tuan, 1979, 1980; Tuan and Ono, 1986; Tuan et al., 1986a; Narbaitz et al., 1987; Soleimani and Narbaitz, 1989; Ono and Tuan, 1991; Akins and Tuan, 1993). Calcium from the eggshell is released by villus cavity cells, which express carbonic anhydrase. This enzyme creates local acidification required for the dissolution of mineral calcite (Kubota et al., 1981; Tuan and Ono, 1986; Ono and Tuan, 1991; Tuan et al., 1986b; Akins and Tuan, 1993; Gabrielli et al., 2004; Matschke et al., 2006), thus making calcium ions available for transport throughout the CAM into embryonic circulation. Free calcium released from the eggshell is then taken up at the apical surface of the capillary covering cells by calcium-binding protein (Tuan et al., 1986b; Ono and Tuan, 1991), calcium ATPase ion transporter (Tuan, 1986), and carbonic anhydrase (Tuan et al., 1986b; Akins and Tuan, 1993; Narbaitz et al., 1995; Gabrielli et al., 2001). The expression of these proteins increases progressively to day 15, prior to the peak of calcium transport activity throughout the CAM (Tuan and Scott, 1977; Narbaitz et al., 1981; Tuan et al., 1986b; Gabrielli et al., 2001; Packard and Lohmiller, 2002). Calcium transport activity through the CAM increases by about 20-fold from day 12 to a peak at day 19, followed by a sharp decrease in activity thereafter (Kubota et al., 1981; Figure 1B). Calcium transport activity correlates with rapid calcium flux through the CAM and mineralization of the embryo skeleton (Tuan and Scott, 1977; Kubota et al., 1981; Figure 1A). Therefore, from day 12 to 19 of incubation, the eggshell becomes the major supply of calcium for skeletal mineralization of the chick embryo (Johnston and Comar, 1955; Kubota et al., 1981; Chien et al., 2009). Phosphorus and Egg Yolk Calcium and phosphorus are the 2 main minerals required during embryonic development. Most research on bone development during embryogenesis has focused on the metabolism of calcium, and much less is known about embryonic phosphorus metabolism. Total yolk phosphorus content at setting is about 0.37 mg/g egg (Li et al., 2014), which represents a fixed resource for embryonic development, given that the albumen and shell contain minimal amounts of phosphorus (Yair and Uni, 2011). About 90% of the yolk phosphorus and 60% of the total yolk phosphoproteins are in the form of phosvitin. The majority of phosphorus needed for skeletal mineralization is provided by this protein (Li et al., 2014). The total phosphorus content of the embryo starts to rise as a result of increased dephosphorylation of yolk phospvitin starting on day 9 of incubation (Li et al., 2014). This process matches well with the increased calcium content in the egg and chick embryo (Johnston and Comar, 1955; Kubota et al., 1981) (Figure 1B). Yolk phosphorus uptake by the chicken embryo is greatest between day 13 and 17; this pattern correlates with skeletal growth of the chick (Li et al., 2014) (Figure 1B). Not much is known, however, about the mechanisms by which the free phosphorus released from phosvitin is transported from the egg yolk matrix into embryonic circulation, and the role of the yolk sac membrane in this process. The NPT2b is the major sodium-phosphate cotransporter in several tissues (Takeda et al., 2004) including the yolk sac membrane (Yadgary et al., 2011). Gene expression of NPT2b in the yolk sac membrane increased on day 11 of incubation, reaching a maximum at day 17, 20, and 21 (Yadgary et al., 2011), indicating an increased mobilization of yolk phosphorus. This finding suggests that free phosphorus is then taken up by the yolk sac NPT2b transporter present in the yolk sac membrane and released to the embryonic circulation. The end result is a constant decrease in yolk phosphorus reserves as incubation (Yair and Uni, 2011) and embryonic bone development proceed (Li et al., 2014). Limited yolk phosphorus reserves towards the end of incubation might explain the reduced concentration of phosphate in the plasma of embryos in the last days of incubation (Taylor et al., 1975), and therefore the reduced rate of mineralization as the embryo approaches hatching (Yair et al., 2012; Li et al., 2014). Behind the Scene: Vitamins and Trace Minerals During Embryonic Mineralization Vitamin D Mineral flux through the CAM during embryonic development is a unidirectional and active process that is highly specific for calcium, and is regulated by vitamin D (Tuan and Ono, 1986; Tuan et al., 1986b; Narbaitz et al., 1987; Narbaitz and Tsang, 1989; Soleimani and Narbaitz, 1989; Ono and Tuan, 1991; Akins and Tuan, 1993; Narbaitz et al., 1995). The concentration of the calcium-binding receptor for 1,25(OH)2D3 and the activity of carbonic anhydrase in the CAM are reduced in vitamin D-deficient embryos (Elaroussi and DeLuca, 1994). The transport of calcium ions released from the eggshell by the CAM is reduced in domestic fowl and Japanese quail embryos from hens deficient in vitamin D (Elaroussi et al., 1993; Elaroussi and DeLuca, 1994; Hart et al., 1985, 1986; Narbaitz and Tsang, 1989; Ono and Tuan, 1991), which in turn decreases calcium accumulation in the embryo (Elaroussi and DeLuca, 1994). As a result of reduced calcium usage from the eggshell, embryos present severe calcium deficiency (Elaroussi et al., 1988, 1993) and fail to achieve the pipping pre-hatching position to start pulmonary respiration (Hart and DeLuca, 1985; Elaroussi et al., 1993) resulting in increased late embryonic mortality (Hart and DeLuca, 1985). Trace Minerals Copper, zinc, and manganese are stored mainly in the egg yolk (Richards, 1997; Yair and Uni, 2011) and are mobilized throughout incubation to support development of the cartilage model and the cartilage growth plate of the bone (Caskey et al., 1939; Kienholz et al., 1961; Simpson et al., 1967). A severe and prolonged maternal trace mineral deficiency increases the incidence of embryonic anomalies (including skeletal disorders) and mortality (Caskey et al., 1939; Kienholz et al., 1961; Simpson et al., 1967). Manganese is a cofactor of polymerase and galactotransferase, enzymes involved in the synthesis of the mucopolysaccharide chondroitin sulfate (Leach et al., 1969), one of the main components of the bone hyaline cartilage model (Eyre, 2004). The most dramatic effect of manganese deficiency in chick embryos is characterized by short, thick legs and shortened wings (Caskey et al., 1944). Copper deficiency blocks the formation of cross-links in bone collagen and arterial elastin (Rucker et al., 1975). Maternal copper deficiency leads to rupture of the aorta of chicken embryos (Simpson et al., 1967; Rucker et al., 1975). Although the effect of copper deficiency on embryonic bone metabolism has not been studied, chicks fed a copper-deficient diet after hatch had brittle and distorted bones (Carlton and Henderson, 1964; Rucker et al., 1975) as a result of reduced activity of the copper-dependant enzyme lysyl oxidase (Rucker and Rogler, 1969; Rucker et al., 1998). Zinc is a cofactor of collagenase and bone alkaline phosphatase (Starcher et al., 1980; Seo et al., 2010). Chicks deficient in zinc have reduced collagenase activity and thus reduced bone collagen synthesis (Starcher et al., 1980), and chicks hatched from zinc-deficient hens have grossly impaired skeletal development (Kienholz et al., 1961). Similar to phosphorus, the yolk sac membrane transfers trace minerals from the yolk to the embryo (Richards, 1997; Yair and Uni, 2011). About 90% of the total copper, zinc, and manganese is mobilized by the embryo during the second week of incubation (Yair and Uni, 2011), which matches with calcium release from the eggshell and its transportation by the CAM, as well as the release of yolk phosphorus by the yolk sac membrane. Towards the end of incubation and after hatch, phosphorus and trace mineral reserves in the residual egg yolk are minimal (Yair and Uni, 2011; Duan et al., 2013; Li et al., 2014), which might represent a limitation to bone growth in the newly hatched chick. Yolk calcium reserves at hatch, however, are similar (Yair and Uni, 2011) or even higher (Richards and Packard, 1996) compared to eggs at setting, as a result of eggshell calcium being transferred to the yolk (Johnston and Comar, 1955). In this case, yolk calcium reserves as hatching approaches would likely be sufficient to support neonatal skeletal requirements, but bone growth may be limited by the low egg phosphorus content. These findings, in addition to observations that embryonic bone growth reaches a plateau after 19 d of incubation (Kubota et al., 1981; Chen et al., 2008; Yair et al., 2012; Li et al., 2014), suggest a nutritional limitation to embryonic bone growth late in the neonatal period and therefore the need for completion of skeletal growth by day 19 of embryonic development. However, it is not known whether there may be other physiological reasons for the cessation of bone growth 2 d before hatch. Enrichment of the embryonic environment with copper, zinc, manganese, and vitamin D in order to promote skeletal growth during the neonatal period and grow-out phase have been tested (Yair and Uni, 2011; Bello et al., 2013, 2014a,b,c, 2015; Yair et al., 2013, 2015; Oliveira et al., 2015a,b). Maternal supplementation of vitamin D3 or 25-hydroxycholecalciferol (25OHD3) increased tibia ash at hatch in broilers relative to chicks from hens fed a vitamin D-deficient diet (Atencio et al., 2005). On the other hand, maternal vitamin D supplementation as 25OHD3 did not affect bone density of the progeny at hatch relative to chicks from hens fed equivalent levels as vitamin D3 (Saunders-Blades and Korver, 2015); the lack of effect indicates the offspring had adequate vitamin D activity in the egg and therefore no effect of vitamin D source was observed. When 17-day-old embryos received an in ovo solution containing a blend of vitamin D3, zinc, copper and manganese, calcium, phosphorus and carbohydrates, yolk content and uptake of zinc, copper and manganese significantly increased from day 18 of incubation to hatch (Yair and Uni, 2011; Yair et al., 2015). However, despite increased size, limited effects on bone ash of the embryos (at day 19 only) and resultant chicks were observed. Similar results were reported when 25OHD3 (Bello et al., 2013, 2014a,c, 2015) or zinc, copper, and manganese solutions at various levels (Oliveira et al., 2015a,b) were injected into the amniotic fluid of the developing embryo at day 17 to 18 of incubation. Taken together, these data suggest that embryonic bone formation has largely ceased by this point and a limited nutrient supply is not necessarily the cause of the plateau on bone development as previously stated. It is important to note, however, that phosphorus content in the in ovo solution was only 2 to 2.2% of the initial yolk phosphorus content of the egg at setting (Yair et al., 2013, 2015). The temporal effects of in ovo feeding on late bone mineralization of the embryo might have been due to an insufficient concentration of phosphorus in the nutrient in ovo nutrient solution. As described previously, phosphorus is essential for bone mineralization, and a positive relationship exists between yolk phosphorus release and bone growth of the chicken embryo (Li et al., 2014). MATERNAL AGE AND TRACE MINERAL NUTRITION AS FACTORS INFLUENCING EMBRYONIC BONE GROWTH The effect of trace mineral bioavailability in the maternal diet on egg shell quality and progeny performance has been extensively investigated; very little is known about the effect on skeletal growth of the progeny (Favero et al., 2013; Torres, 2013; Kidd et al., 1992). Organic trace minerals are minerals chelated to an organic molecule (often an amino acid or organic acid); the chelate is less susceptible to binding by anti-nutritional factors that adversely affect the uptake of trace minerals ions in the gastrointestinal tract (Yi et al., 2007). Organic trace minerals have been reported to be more bioavailable than inorganic forms (Huang et al., 2009; Zhao et al., 2010). Maternal supplementation of copper, zinc, and manganese (as a combination of sulfates and mineral-amino acid chelates) at 10, 100, and 100 mg/kg of diet, respectively, increased whole egg zinc content as well as skeletal size and calcification of the embryos in comparison to eggs from hens fed similar mineral levels as sulfates only (Favero et al., 2013). Egg yolk mineral levels and offspring bone development were similar when 50 mg/kg zinc, 60 mg/kg manganese, and 10 mg/kg copper were fed as 2-hydroxy-4-(methylthio) butanoic acid as when 100 mg/kg zinc, 120 mg/kg manganese, and 10 mg/kg copper were fed as sulfates (Torres, 2013). Similarly, Mabe et al. (2003) reported that relative to a basal diet low in trace minerals, copper, zinc, and manganese at 10, 60, and 60 mg/kg diet, respectively, increased yolk zinc and copper content regardless of mineral form. However, when diets were supplemented at 50% of those levels as organic forms, yolk zinc content was still increased relative to the inorganic mineral diet. These responses indicate that as compared to the levels of supplementation commonly used by the broiler industry, organic forms of trace minerals at 50% were still sufficient to meet requirements for egg mineral content (Mabe et al., 2003; Torres, 2013) and to support embryonic skeletal development (Torres, 2013). Egg trace mineral content also changes as the hen ages (Favero et al., 2013; Torres, 2013, Sun et al., 2012). Sun et al. (2012) reported an increase in yolk copper, zinc, and manganese as hens aged from 43 to 52 wk olds. The yolk of hens at 59 wk of age contained higher levels of copper and zinc in comparison to eggs from hens at either 32 or 45 wk of age (Torres, 2013). Similarly, Favero et al. (2013) reported an increase in copper and manganese in whole eggs from 55-wk-old hens in comparison to the same flock at 35 wk of age. Zinc content in the egg, however, was similar up to 55 wk of age and decreased by about 8% thereafter (Favero et al., 2013). Bone development follows a non-uniform pattern among embryos from hens at different ages. Embryos and chicks from older hens have increased bone size and strength compared to those from younger breeder flocks (Yalcin et al., 2001; Alfonso-Torres et al., 2009; Shaw et al., 2010; Favero et al., 2013; Torres, 2013). As previously described, the chicken embryo utilizes yolk trace minerals reserves throughout incubation (Yair and Uni, 2011), which is used to build the embryonic cartilage model (organic matrix). Older breeder hens produce eggs with increased egg yolk (Nangsuay et al., 2013), fat (Yadgary et al., 2010), energy, and protein (Nangsuay et al., 2013). The concentration of yolk copper, zinc (Torres, 2013), and manganese (Favero et al., 2013) also increased in eggs from old hens. In general, increased trace mineral content in the eggs were associated with enhanced bone size and mineralization of embryos from older flocks in comparison to those from younger hens (Favero et al., 2013; Torres, 2013). Embryos from 32-wk-old hens at 17 d of incubation had greater femur and tibia size compared to embryos from 45- and 59-wk-old hens (Torres, 2013). Bone calcification of embryos from the youngest hens was intermediate at 17 d and lower at 20 d of incubation compared to embryos from the older hens. Ultimately, bones of chicks from hens at 45 and 59 wk of age were stronger at hatch relative to those from chicks from hens at 32 wk of age. Embryos from very young hens might have sufficient yolk mineral to support the greatest rate of embryonic bone development up to day 17, but is limited afterwards, allowing bone development of chicks from older hens to surpass that of the chicks from younger hens. Whether differences exist regarding the pattern of egg yolk trace mineral absorption and its correlation to skeletal growth by embryos from diverse hen ages throughout incubation has yet to be investigated. Further research is also needed to investigate the relationship between maternal flock age on skeletal quality and leg health of the offspring during the grow-out phase. CONCLUSIONS In the developing skeleton, the long bones, such as those of the limbs, arise from the process of endochondral ossification, in which cartilage serves as the initial skeletal model and is later replaced by bone. The multiple cell types present in the CAM, yolk sac membrane, and embryonic tissues must act sequentially and in concert for proper skeletal development of the growing chicken embryo. Chicken embryos rely on the eggshell and to a lesser extent the yolk as the sources of calcium for bone development. However, the yolk is the main source of phosphorus and trace minerals to support skeletal growth of chicken embryos; mineral yolk reserves are low as the embryo prepares to hatch. Further research is needed in order to clarify whether this is due to a lack of phosphorus and trace minerals in the egg near hatching, or some other biological control. Yolk mineral concentration and bone growth in the offspring were not greatly influenced when hens were fed different forms of trace minerals. 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Poultry Science – Oxford University Press
Published: May 21, 2018
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