Improving Egg Production and Hen Health with Calcium

Chapter 30

Improving Egg Production and Hen Health with Calcium Patricia Y. Hester Department of Animal Sciences, Purdue University, West Lafayette, IN, United States

Calcium metabolism and turnover in egg-laying hens is extraordinary when compared to mammals because of its role in the reproductively active female avian in forming the eggshell. However, this ion is also critical for other biological functions. Similar to other vertebrates, calcium is a secondary messenger in signal transduction pathways, and is an enzymatic cofactor for biochemical reactions such as clotting of the blood. Calcium ions are needed for muscular contraction, the release of synaptic neurotransmitters, and bone integrity. It is the bone that serves as a major reserve for calcium during the formation of the eggshell (Scanes, 2015). Circulating extracellular calcium is found in unbound form as an ionized salt; it can be bound to proteins (e.g., albumin, phosvitin, or vitellogenin) or complexed to anions (e.g., lactate, citrate, phosphate, or bicarbonate). The protein-bound calcium is a storage reservoir for ionized calcium. Due to its inability to cross capillary membranes, extracellular protein-bound calcium, which represents about 40% of the total calcium, is physiologically inactive. It is the ionized pool of calcium that is retrieved from the blood by the uterus of the oviduct to be used for shell formation. Circulating concentrations of ionized calcium remain stable because some of the protein-bound calcium is released to replace the calcium delivered to the shell. In addition, ionized calcium is tightly regulated by the classic calcium-regulating hormones:1,25-dihydroxycholecalciferol, parathyroid hormone, and calcitonin. A laying hen has the capability to respond to hypocalcemic conditions within minutes, whereas in mammals, the response time is more typically 24 h or more (Taylor, 1970; Stanford, 2006; de Matos, 2008). Domesticated chickens used to produce table eggs for human consumption have been bred to efficiently lay a large number of eggs. A pullet reaches sexual maturity at 19 weeks of age and for the next 61 weeks or 427 days of her egglaying cycle, she has the genetic potential to produce 378 eggs, which is not quite an egg a day. Unlike the much larger broiler breeder hen that lays fertile hatching eggs for propagation of the commercial meat-type broiler, the egg-laying strain of chickens has been genetically selected for a small body size and skeletal frame mainly to improve egg production and feed efficiency as body weight and egg laying are negatively correlated traits. For instance, a White Leghorn pullet, fed ad libitum that lays white eggs, will typically weigh 1.25 kg at 17 weeks of age. Brown hybrid hens, responsible for producing brown eggs, are slightly larger and weigh 1.44 kg at 17 weeks of age. In contrast, a broiler breeder female of similar age that has been on a feed restriction program to prevent obesity weighs 1.7 kg. Leghorns consistently produce eggs with as little as 95 g of feed daily. An average conversion rate for Leghorns from 20 to 80 weeks of age is 1.86 kg of feed/kg of egg. In other words, a single 63-g large egg requires about 117 g of feed. As feed represents about 60–70% of the cost of producing eggs, it is no wonder that table eggs are an economical source of high-quality protein, essential fatty acids, vitamins, and minerals (Bell and Weaver, 2002).

EGG FORMATION It takes about 24–25 h for a hen to form an egg, which means a hen could lay one egg daily. Chickens, similar to many other species of birds, lay eggs in clutches, i.e., they lay an egg a day without interruption, then pause for one or several days before resuming egg production. Unlike their ancestor of the wild junglefowl, the domesticated hen of today typically has long clutches before taking a break from egg laying. It is not uncommon for a White Leghorn hen to lay a clutch of 200 eggs or 1 egg per day for 200 days without interruption. More typically, an average clutch for a laying hen in the first half of her egg-laying cycle is 50 eggs laid in succession without pause. These extended clutches have not only been accomplished Egg Innovations and Strategies for Improvements. http://dx.doi.org/10.1016/B978-0-12-800879-9.00030-5 Copyright © 2017 Elsevier Inc. All rights reserved.

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through breeding for increased output of high-quality eggs, but also by providing hens with an appropriate environment, managerial care, and a highly nutritious diet to maximize their genetic potential (Bell and Weaver, 2002). For a hen to create an egg, she requires an extraordinary reservoir of nutrients. If the egg is fertilized, these nutrients will be utilized by the developing embryo during incubation. The yolk is packed full of lipoproteins that are assembled in the hen’s liver before transport via the blood to the developing ova of the left ovary. A follicular hierarchy of multiple-sized growing ova or yolks exists in the left ovary with the largest ovum being the next in sequence to ovulate (Fig. 30.1). Once ovulation occurs, the second ova in the hierarchy (Fig. 30.1) now succeeds to the first position and has an additional 24 h to grow, accumulating additional lipoprotein, before it too ovulates for the next day’s egg. The ovulated yolk is now free floating in the abdominal cavity while the follicular sac, which originally contained the ova, remains attached to the ovary and is referred to as the postovulatory follicle (Fig. 30.1). The ovum or yolk is engulfed by the funnel-shaped infundibulum of the oviduct (Fig. 30.2) where fertilization occurs if sperm are present. With the exception of backyard enterprises where roosters may be present, table eggs for human consumption are generally not fertile, as the hens are never exposed to roosters. Twenty minutes following ovulation, the yolk, which may or may not be fertilized, travels to the magnum through contraction of smooth muscles lining the oviduct, a process referred to as peristalsis. In just 3 h, the magnum of the oviduct has deposited the albumen or egg white. Peristalsis continues to forward the developing egg to the narrowed section of the oviduct called the isthmus where the inner and outer shell membranes are laid down in 1.5 h. The forming egg spends the longest amount of time (20 h) in the uterus or shell gland of the oviduct. It is here where the egg is plumped with water and salts, and the shell, its pigment, and protecting cuticle are laid down before oviposition or egg laying occurs. Of the total 20 h that the egg remains in the uterus, it is the last 16 h that most of the calcium carbonate is deposited on the shell at a rate of 125 mg of calcium/h. Feed is the major source of calcium for forming the eggshell. It is absorbed from

FIGURE 30.1  The left ovary from a laying hen (chicken) shows the follicular hierarchy of multiple sized growing ova or yolks. Typically in the female chicken, there are four to six large yolk-filled or yellow follicles with diameters that range from 2 to 4 cm (1, 2, and 3 are F1, F2, and F3, respectively). There are numerous small yellow follicles with diameters of 6–12 mm (4) and many small white follicles less than 6 mm in diameter (5). Note the postovulatory follicle (6) from which the last ovum ovulated. The postovulatory follicle is metabolically active for several days following ovulation. Resorption of the postovulatory follicle in chickens occurs gradually over a 6–10 day period following ovulation. The follicular sac ruptures at the stigma line (7), which lacks vascularization, to release the ovum for capture by the infundibulum of the oviduct. (Johnson, A.L., 2015. Reproduction in the female. In: Scanes, C.G. (Ed.), Sturkie’s Avian Physiology, sixth ed. Academic Press, London, United Kingdom, pp. 635–665 (Chapter 28))

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FIGURE 30.2  The left ovary (1) and oviduct of the reproductive tract of a White Leghorn depicts the infundibulum (2, the funnel that engulfs the ovum and the site of fertilization), magnum (3, egg white deposition), isthmus (4, shell membranes are formed), uterus (5, shell is deposited), vagina (6, passageway for the egg), and the exterior vent (7). Note that in the uterus (5), an egg is undergoing shell calcification.

the intestines and delivered via the blood to the uterus; however, because of the high demand for calcium during eggshell formation, the intestines cannot absorb the calcium fast enough and this is when the hen turns to her skeletal reservoir as a secondary source (Taylor, 1970; Johnson, 2015).

CALCIUM A shell, once formed, contains 2–2.5 g of calcium, which corresponds to 10% of the hen’s total body calcium (Taylor, 1970). If a hen lays 280 eggs in a year, she will secrete a phenomenal quantity of calcium into her eggshells that is equal to about 30 times of her total body calcium reserve (Johnson, 2015). With this much calcium placed on the shell of an egg on an almost daily basis, the laying hen has been used as an intriguing research model to further elucidate the mechanisms of calcium metabolism. Without replenishment, the hen’s body reserve of calcium would be quickly diminished, leading to a precarious condition known as cage layer fatigue. As the name of this noninfectious disease implies, it is caged hens rather than noncaged layers that typically succumb to this condition. As calcium supplies are depleted from the hen’s body, eggs are laid with thin shells, or they are shell-less (Fig. 30.3). Eventually, the fatigued hens stop laying eggs. They can no longer stand on their own two feet and collapse on the cage floor. Providing supplemental calcium revives some, but not all, of the hens with cage layer fatigue bringing them back into lay (Couch, 1955; Bell and Siller, 1962). Cage layer fatigue is not common in today’s modern flocks because nutritionists fortify the diet of laying hens so that they consume up to 4.5 g of calcium daily (Hy-Line W-36 Performance Standards Manual, 2012) (Table 30.1). Feed ingredients, such as oyster shell, limestone, and steamed bonemeal are the major sources of calcium that are added to the diet. However, too much calcium in the hen’s diet has to be avoided as it depresses appetite. Any calcium fed in excess is excreted through the urine (Coon, 2002). Excessive dietary calcium fed to pullets can also result in urate deposits in the kidney (Crespo, 2014).

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FIGURE 30.3  A shell-less egg (1) laid prematurely as compared to a normal white hard-shelled egg (2) that resided in the uterus for the normal amount of time (20–21 h) before oviposition.

TABLE 30.1 Typical Recommendations for the Inclusion of Calcium, Phosphorus, and Vitamin D3 in the Diets of Reproductively Active Avian Females Species

Calcium (%)

Available Phosphorus (%)

Vitamin D3 (IU/kg of diet)

White egg layera

4.00–4.50

0.40–0.50

3300

Brown egg layera

4.00–4.90

0.35–0.44

3300

Broiler breederb

3.00–3.20

0.32–0.35

3500

Turkey

c

2.85

0.38

5000

Quaild

3.10

0.45

—

Duck

3.30e

0.42e

2500d

Goose

2.80

0.38

2500

Pheasantd

2.60

0.42

2500

Guinea fowl

3.00

0.40

—

Ostrichd

1.80

0.45

—

d

d

a

Hy-Line International Online Management Guide: Management Guide for all Hy-Line Varieties of Laying Hens, 2010. Available from: http://www.hylinena. com/redbook/Nutrition_recommendations/Nutrition_recommendations.html b Ross 300 Parent Stock: Nutrition Specifications. 2013. Available from: http://en.aviagen.com/assets/Tech_Center/Ross_PS/Ross308PS-NutritionSpecs2013FINAL.pdf c Aviagen Turkeys: Breeder Nutrition Recommendations. 2010. Available from: https://www.aviagenturkeys.com/media/183522/nicholas_breeder_feed_ recommendations_usa_2010.pdf d Lesson, S., Summers, J.D., 2009. Commercial Poultry Nutrition, third ed. Nottingham University Press, Nottingham, England. e Davis, G.S., Anderson, K. E., 2014. Management programme for raising breeder duck flocks. The Poultry Site. Available from: http://www.thepoultrysite. com/articles/3337/management-programme-for-raising-breeder-duck-flocks

VITAMIN D Similar to calcium, deficiencies of vitamin D can also contribute to cage layer fatigue and osteomalacia, a nutritional deficiency disease that weakens bone and can cause the keel to become deviated with an S-shaped curve (Fig. 30.4) (Wilson and Duff, 1991). Without vitamin D, calcium cannot be absorbed from the lumen of the hen’s small intestines to be delivered to the blood, and the calcium is, therefore, excreted in the feces. Vitamin D’s role in facilitating intestinal calcium absorption is to stimulate the synthesis of the carrier protein of calbindin-D28k, which transports calcium across the intestinal wall to be deposited into the blood. Sex steroid–induced calbindin-D28k is also involved in the transfer of calcium from the blood into the uterus of the oviduct for shell deposition in sexually mature avian females. With respect to bone, the function of vitamin D is dependent on circulating levels of calcium and phosphorus. When blood calcium levels are normal, vitamin

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FIGURE 30.4  A keel bone with an S-shaped curvature noted during the necropsy of a White Leghorn hen. Feathers and skin have been removed to expose the breast muscle.

D stimulates osteoblasts (bone cells responsible for making bone) to produce osteocalcin, a biomarker of bone turnover (Skjødt et al., 1985). Under conditions of hypocalcemia or hypophosphatemia, vitamin D’s role changes, stimulating osteoclasts (cells that resorb bone) to mobilize calcium from bone. Vitamin D also retrieves calcium from the renal filtrate and returns this valuable ion to the circulation so that it is not excreted in the urine when an animal is hypocalcemic. The most biologically active form of vitamin D involved in calcium regulation is the steroid hormone 1, 25-dihydroxycholecalciferol. The hen synthesizes vitamin D in her kidney through hydroxylation of 25-hydroxycholecalciferol. In fact, if sunlight is available, the featherless skin (e.g., tarsometatarus or shank) of a hen can synthesize vitamin D using the ultraviolet radiation of sunlight to convert 7-dehydrocholesterol into cholecalciferol (vitamin D3). Cholecalciferol is then transported via carrier proteins, such as albumin, to the liver for hydroxylation to form 25-hydroxycholecalciferol before being transported to the kidney for its final hydroxylation. Many commercial hens are not exposed to natural daylight because their enclosed housing is light tight. To prevent a vitamin D deficiency with any housing systems, hens are given synthetic vitamin D3 or 25-hydroxycholecalciferol in the diet through the trace mineral premix (Table 30.1). Vitamin D2 or ergocalciferol from plants has low-biological potency in birds because it does not bind tightly to circulating transport proteins and is excreted conjugated to bile (Norman and Hurwitz, 1993; de Matos, 2008).

PHOSPHORUS There is very little phosphorus in the eggshell, yet this mineral, along with calcium and vitamin D3, needs to be added to the feed to maintain egg production and bone integrity (Table 30.1) (Sohail and Roland, 2002). A deficiency of phosphorus in the diet leads to a drop in egg production (Klingensmith and Hester, 1983) and bone becoming osteoporotic

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(Simpson et al., 1964; Wilson and Duff, 1991). Similar to calcium, too much phosphorus (0.6% available phosphorus) in the laying hen diet can be deleterious to hen health, reducing bone strength (Sohail and Roland, 2002).

AVIAN BONE Bone is a reservoir for calcium and phosphorus. Approximately 99% of total body calcium is located in the skeleton with the remaining 1% found either intracellularly or extracellularly (de Matos, 2008). Mineralization of bone is successful if there are adequate local concentrations of skeletal calcium and phosphorus and if nucleators are present in bone to promote hydroxyapatite [Ca5(PO4)3(OH)] crystallization. Vitamin D3 is key to providing the skeleton with sufficient quantities of calcium to promote mineralization of osteoid, the unmineralized organic portion of bone (Norman and Hurwitz, 1993). The avian, as well as the mammalian adult skeleton consist of trabecular and cortical bone. Due to the reticular appearance of trabecular bone and its foam-like structure, it is also called spongy bone. The vertebrae and joints of long bones, such as the tibia and femur of the leg and the humerus in the wing contain trabecular bone. The sponge-like trabecular bone absorbs mechanical loads and impacts, similar to the Styrofoam packaging material used during shipping. The dense, tough, and strong outer most layer is the cortical or compact bone, which is more prevalent than the trabecular bone. However, as there is more surface area in the trabecular as compared to the cortical bone, it is more metabolically active with much greater turnover (Whitehead, 2004). Besides trabecular and cortical bone, the reproductively active hen has developed an additional type of bone to avoid a negative calcium balance. The ovarian estrogen surge at sexual maturity causes the formation of medullary bone in the marrow of some bones. Medullary bone has also been discovered in the bone fossils of egg-laying female dinosaurs (Schweitzer et al., 2005) and in crocodiles. Examples of medullary avian bones include the tibia, femur, and keel bone. During active shell formation, the thin spicules of loosely woven bone found in the marrow provide a quick, labile source of calcium. Under the influence of parathyroid hormone from the parathyroid gland, calcium is retrieved from the hydroxyapatite crystals of medullary bone and transported in the blood to such sites as the shell gland or uterus (Whitehead, 2004; de Matos, 2008). Some but not all bones of the avian skeleton are hollow or pneumatic and are connected to the air sacs of the respiratory system, allowing for air to move in and out of the bones. These pneumatic bones are generally, but not always, nonmedullary. Examples of pneumatic bones include the humerus, coracoid, parts of the sternum (keel), some ribs, pelvic girdle, thoracic and cervical vertebrae, and skull bones (King, 1957). Some bones, such as the humerus and keel can be pneumatic, but also contain medullary bone (Whitehead, 2004). At the cellular level, there are three major cells involved in bone metabolism, which includes the formation (osteoblasts), break down or resorption (osteoclasts), and maintenance (osteocytes) of the skeleton. The functional activities of the osteoblasts, osteoclasts, and osteocytes create a dynamic environment of constant remodeling. Osteoclastic degradation is immediately followed by osteoblastic regeneration (Dacke et al., 2015). The bone-forming uninucleated osteoblasts synthesize and release the osteoid, which is composed of type I collagen and noncollagenous protein, into the extracellular matrix. Eventually, the osteoid is mineralized with hydroxyapatite. Osteoblasts express receptors for many bone-active mediators such as vitamin D metabolites, parathyroid hormone, parathyroid hormone–related protein (PTHrP), adrenal and gonadal steroids, as well as specific growth factors and cytokines (Dacke et al., 2015). The large, multinucleated, bone-resorbing osteoclasts are derived from hematopoietic marrow precursors. The ruffled border of osteoclasts use adhesion molecules to attach to the bone to initiate bone catabolism. Hydrogen and chloride ions are secreted by the osteoclasts to aid in the dissolution of mineral. Cysteine proteinases, metalloproteinases, and lysosomal proteases, active at low pH or acidic conditions, dissolve the bone matrix. Mature osteoclasts have few receptors unlike the receptor-rich osteoblasts, but they do have calcitonin receptors. Avian calcitonin (32 amino acids) is derived from the ultimobranchial gland, whereas mammalian calcitonin originates from the C-cells of the thyroid gland. Circulating levels of calcitonin in birds is relatively high. Its function is to oppose parathyroid hormone, serving as a check and balance. Although not everything is known about the role of calcitonin in avian bone metabolism, it has a limited response to hypercalcemia by lowering blood calcium through inhibition of skeletal calcium entry into the circulation. Osteoclasts succumb to apoptosis once bone resorption is completed (Stanford, 2006; Dacke et al., 2015). Once an osteoblast becomes surrounded by mineralized bone, it differentiates into an osteocyte, which resides in a small space of bone called a lacuna. Compared to the osteoblast, the osteocyte is not as metabolically active. The osteocyte has developed cytoplasmic processes that communicate through narrowed channels called canaliculi with other osteoblasts, osteocytes, and the vasculature of the bone in general. This vast network of interconnecting canaliculi allows for bone mineral exchange during periods of high-metabolic activity. Osteocytes are involved in mechanical loading by responding to movement and exercise stimulating the remodeling of bone (Dacke et al., 2015).

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FIGURE 30.5  Bone cells communicate with one another through cytokines to prevent overzealous bone formation or resorption. A cytokine called receptor activator of nuclear factor kappa-B ligand (RANKL) is expressed on the membranes of bone-forming cells called osteoblasts. Receptors present on the membranes of osteoclast precursor cells called RANK recognize RANKL. The binding of RANK with RANKL facilitates communication between the osteoblast and the osteoclast precursor cell, ultimately stimulating the osteoclast precursor cell to differentiate into the activated osteoclast whose function is to resorb bone. As a counter balance to RANK is the homolog, osteoprotegerin (OPG), an estrogen-stimulated cytokine produced by bone marrow cells called megakaryocytes. The OPG competes with RANK for RANKL. In the presence of increasing concentrations of OPG, the interaction between osteoclast and osteoblast is diminished, thus preventing excessive bone resorption. (Nakashima, T., Takayanagi, H., 2012. New regulation mechanisms of osteoclast differentiation. Ann. NY Acad. Sci. 1240, E13–E18).

To maintain a balance between bone formation and resorption, it is germane that these bone cells communicate with one another. Cytokines serve as the mediators of communication. The mechanism of communication between bone cells has been defined in mammals and increasing evidence suggests that it is similar in avian bone. One cytokine called receptor activator of nuclear factor kappa-B ligand (RANKL), located on the membrane of the osteoblast, is a member of the tumor necrosis factor family. The RANKL ligand binds to a receptor found on the osteoclast precursor cell that is called RANK. Binding of RANK with the ligand of RANKL culminates in osteoclastic differentiation leading to bone resorption. As a check and balance system, osteoprotegerin (OPG) is a cytokine that is also a member of the tumor necrosis factor family. The OPG is a RANK homolog that can compete for the RANKL ligand. In the presence of increasing concentrations of OPG, the interaction between osteoclast and osteoblast is diminished, thus preventing excessive bone resorption. Stimulated by estrogen, OPG is being considered as a possible treatment for human osteoporosis (Oursler, 2003; Nakashima and Takayanagi, 2012; Dacke et al., 2015) (Fig. 30.5).

OSTEOPOROSIS Though cage layer fatigue is uncommon is today’s egg-laying flocks, aging laying hens responsible for producing table eggs experience a decrease in mineralized structural bone causing osteoporosis. Whereas structural bone declines in aging osteoporotic hens, medullary bone deposition actually increases with age. However, the increase in medullary bone does not compensate for the decline in structural bone, leading to bone breakage in aging hens. Osteoporosis is systemic, affecting all bones of the skeleton. The prevalence is widespread in today’s commercial caged flocks contributing to about 20–35% of all mortalities during the egg production cycle (McCoy et al., 1996; Anderson, 2002). Caged hens suffer up to a 30% incidence of broken bones at end of lay when hens are caught and transported to processing plants (Gregory and Wilkins, 1989). A hen at the end of her egg-laying cycle is referred to as spent. In the past, meat processed from the spent hen was placed in soup; however, today, most soup-based companies use broiler meat instead of the meat from spent hens because bone splinters render the soup unsafe for human consumption. Most spent hens today are rendered or euthanized on the farm and composted (Webster, 2004). It is well known that genetics affects skeletal integrity of egg-laying strains of hens. High- and low-bone strength lines of chickens were selected for over nine generations with marked differences in bone quantity and quality traits, as well as susceptibility to osteoporosis (Bishop et al., 2000; Fleming et al., 2006). The tibia cortical width and medullary bone content of the tarsometatarsus were greater in the high-bone strength line (Whitehead, 2004). Qualitatively, there were distinct differences, including improvements in the organic matrix (collagen pyrrolic cross-linking) (Sparke et al., 2002) and fewer osteoclasts resulting in lower bone resorption of the endosteal surface of cortical bone in the high-bone strength line (Whitehead, 2004).

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After genetics, exercise has the next biggest impact on skeletal quality (Fleming et al., 2006). Hens housed in conventional cages have poorer bone strength as compared to hens in noncage systems, such as littered floor systems and aviaries, though hens in aviary systems experience more broken bones due to crash landings (Lay et al., 2011). Adding perches to conventional cages stimulates pullet and hen activity (Enneking et al., 2012a,b; Hester, 2014) improving bone mineralization in laying hens through mechanical loading (Hester et al., 2013). Birds that exercise have fewer osteoclasts resorbing the endosteal bone surface than nonactive birds. However, this suppression of osteoclastic activity does not persist throughout life. As hens age, the differences in the number of osteoclasts between active and nonactive hens dissipate. Ultimately, at some point in the aging process, exercise fails to maintain the suppression of osteoclastic resorption leading to bone loss. Another possibility for bone loss with aging hens is the downregulation of the osteoblastic estrogen receptor, which may contribute to exercise having less of an effect in maintaining skeletal quality (Fleming et al., 2006).

RELATIONSHIP BETWEEN EGG PRODUCTION TRAITS AND OSTEOPOROSIS Hens resistant to osteoporosis lay fewer eggs and place less calcium into their eggshells (Whitehead, 2004). There is a negative correlation between numbers of eggs laid and skeletal integrity in laying hens (r = −0.36, P  < 0.001) (Whitehead, 2004). Specifically, lower-producing hens that lay fewer than 250 eggs during a typical egg-laying cycle up to 68 weeks of age had higher skeletal integrity (bone index value) than highly productive hens. The recurrent interruptions in egg laying and the poor persistency of lay with short clutches most likely provided more opportunities for these lower-producing hens to rebuild structural bone. Even hens laying 250–332 eggs to 68 weeks of age still had a negative correlation between their bone index values and the number of eggs laid, but the relationship was weaker with lower correlation values (r = −0.055, P < 0.001), suggesting that these hens laid more persistently with longer clutches, but also had opportunities for bone rebuilding between clutches or during a natural molt. Whenever hens stop laying eggs, e.g., during a molt, estrogen levels plummet causing medullary bone to become depleted. However, during ovarian arrest when little to no eggs are laid, structural bone replenishment occurs, restoring structural bone integrity. The eggs laid by the hens with improved skeletal integrity (rate of lay was 86.9% as compared to 87.3% for the low-bone index line) had poorer shell quality traits, such as lower shell weight (mg/cm2) and more cracked eggs. As males in the high- and low-bone index lines responded similar to the females with respect to bone traits, Whitehead (2004) suggested that hens resistant to osteoporosis have decreased bone osteoclastic resorption resulting in reduced calcium available for eggshell formation.

STRATEGIES TO IMPROVE BONE STRENGTH AND EGG PRODUCTION IN LAYING HENS Nutritional manipulation is already being used as a tool to improve skeletal health and shell quality through the incorporation of large-size particles of limestone or oyster shell into the diets of laying hens. Through strong muscular contractions of the gizzard, feed is slowly ground to a smaller size before the ingesta proceeds to the small intestines. Large-particle sizes as compared to fine particles of calcium are retained in the gizzard of the hen for a longer period of time allowing for a slow, sustained release of calcium into the small intestines for eventual absorption into the blood. Making a dietary source of calcium available throughout shell formation, especially at night when hens are not eating, replenishes the skeletal calcium source (Guinotte and Nys, 1991; Fleming et al., 1998). In addition, prelay diets are typically given to pullets for consumption from 16 to 18 weeks of age before switching to the calcium-enriched laying hen diet (Table 30.1). Consumption of prelay diets prevents the depletion of medullary bone that is rapidly forming as the pullet approaches sexual maturity and egg laying. A prelay diets typically consists of 2.25% calcium and 0.42% available phosphorus, which is higher than the preceding grower diet the pullets consume (Lesson and Summers, 2009). Breeding companies have a repertoire of approximately 30 genetic traits that they use in selecting outstanding candidates for their primary breeding stock (Lay et al., 2011). Traits of economic importance, such as egg production, shell quality, livability, and feed efficiency receive heavy emphasis in the genetic selection program. The more traits used in a genetic selection program, the less progress is made in the improvement of a particular trait of interest. Skeletal health is one trait that the breeding companies have not placed emphasis on, as there is no easy way to assess bone health in breeder candidates. If a biomarker correlated to bone health can be easily measured or genes involved in osteoporosis can be identified, this would aid in improving skeletal health. The negative correlation between egg production traits and good bone integrity makes the genetic selection process more difficult as no breeding company wants their genetic lines to lay fewer eggs with thinner shells as a result of a genetic selection for stronger bones. A balanced bird with good health, as well as excellent egg-laying characteristics is the goal of all breeding companies. As the negative correlation between egg production and high-bone integrity is low for high-producing hens (Whitehead, 2004), it is hopeful that genetics will be a future tool used by breeding companies to improve skeletal health.

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ANALYTICAL METHODS Plasma or serum calcium concentrations: Circulating levels of total calcium (30 mg/100 mL of plasma) in sexually mature laying female avians are about 3 times higher than in mammalian counterparts (Taylor, 1970). Both total and ionized calcium decrease in the blood as the shell is being formed in the uterus (Dacke et al., 1973). Measurement of ionized calcium as compared to total calcium in the blood is a more precise assessment of calcium status especially when an animal is diseased or unhealthy. It is the ionized calcium that is physiologically active. Ion-specific electrodes are used to measure ionized calcium in plasma (Stanford, 2006), whereas total calcium is measured using a colorimetric assay (serum only) (Baron and Bell, 1957; Connerty and Briggs, 1966) or by atomic absorption spectrophotometry (Zettner and Seligson, 1964). When ionized calcium is being measured, blood samples should be chilled and the collecting vesicle receiving the blood should be filled to minimize exposure to air. Carbon dioxide from the air or generated from glycolysis occurring in erythrocytes can lower pH and affect the accuracy of the measurements. Some laboratories recommend measurement of ionized calcium as soon as possible following blood collection (Stanford, 2006). Plasma vitamin D concentrations: As the half-life is longer for 25-hydroxycholecalciferol and it is more abundant, it is this vitamin D metabolite that is usually measured in plasma to assess if levels are normal. Heparin is used as the anticoagulant, and samples are frozen immediately following collection for subsequent analysis. Exposure of samples to light is avoided. Measurements are done using a radioimmunoassay or more recently, an enzyme-linked immunosorbent assay (ELISA) (Hollis, 1996; Stanford, 2006). Plasma phosphorus concentrations: A colorimetric assay using a spectrophotometer can be used to determine plasma levels of phosphorus (Goldenberg and Fernandez, 1966). Plasma parathyroid hormone concentrations: A human radioimmunoassay kit for the 1–34N section of parathyroid hormone has been used successfully to measure plasma levels in African gray parrots. This section of the parathyroid hormone has a short half-life. As parathyroid hormone is labile, the assay, using the anticoagulant of ethylenediaminetetraacetic acid for blood collection, should be conducted within 1 h of collection or the plasma should be immediately frozen at −70° C for further analysis (Stanford, 2006). Skeletal integrity: Bone quantity and quality can be assessed by using invasive techniques, such as ashing, mineral analysis of ash, breaking force, histomorphometry (Hudson et al., 1993), or histological examination of bone. Radiographic density is an additional invasive technique in which the midshafts of excised long bones can be radiographed using a Faxitron 405 X-ray apparatus (Fleming et al., 1994). The humerus in most sexually mature avian female skeleton contains little to no medullary bone; therefore, a decrease in radiographic density of this bone can serve as an indicator of a decline in structural mineralized bone and diagnosis of osteoporosis (Whitehead and Fleming, 2000). Radiographic densities were highly correlated (P < 0.0001) with the breaking strengths of the tibia (r = 0.64) and humerus (r = 0.76; Fleming et al., 1994). Invasive techniques require euthanasia of the animal for measurements and the use of more animals over time. To measure skeletal mineralization sequentially, noninvasive techniques have been developed. Past research with poultry has shown that photon absorptiometry is an effective tool in detecting differences in bone mass in poultry in vivo (Meyer et al., 1968; Miller and Sunde, 1975; Akpe et al., 1987), as well as ex vivo with excised bones (Orban et al., 1993). In addition, dual-energy X-ray absorptiometry (DEXA) has been successfully applied both in vivo and ex vivo for assessing bone mineralization in chickens (Schreiweis et al., 2003, 2005; Hester et al., 2004). High correlations (r = 0.82–0.94, P < 0.001) were reported between DEXA bone scans conducted in live birds as compared to respective excised bones (Schreiweis et al., 2005). In addition, in vivo bone mineral density scans were positively correlated with the bone breaking force and the bone ash weight (Schreiweis et al., 2005). As bone mineral density (measured using DEXA) decreased in Leghorns, the incidence of bone breakage increased (r = −0.54, P < 0.05) (Mazzuco and Hester, 2005). Single- or dual-energy X-ray absorptiometry is commonly used in humans for the diagnosis and monitoring of osteoporosis (Wahner, 1989; Faulkner et al., 1991; Johnston et al., 1991). Quantitative computed tomography (QCT) can also be used in poultry for assessing bone mineral density both in vivo and ex vivo. The bone mineralization values of avian bones as measured by QCT were correlated with bone ash, breaking strength, and mineral analysis. An advantage of QCT over DEXA is its ability to distinguish trabecular from cortical bone. The ability to compartmentalize and measure different bone types allows investigators to better understand skeletal dynamics during bone formation and resorption, as well as during eggshell formation (Korver et al., 2004). Digitized fluoroscopy and ultrasound have also been used in vivo to identify osteoporotic hens (Fleming et al., 2004).

CONCLUSIONS l

Calcium metabolism and turnover in egg-laying hens is extraordinary when compared to mammals because of its use in eggshell formation. A shell contains 2 g of calcium, which corresponds to 10% of the hen’s total body calcium. If a hen lays 280 eggs in a year, she will secrete a phenomenal quantity of calcium into her eggshells that is equal to about 30 times of her total body calcium reserve.

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l

A hen uses calcium from feed and bone to maintain calcium homeostasis. Repercussions of a negative calcium imbalance include cage layer fatigue and osteoporosis, conditions whereby the skeleton becomes weak due to loss in mineralization of the structural bone. l Genetic selection can be used as a tool to improve skeletal health in egg-laying hens, but genes involved in skeletal integrity and a practical, easily measurable biomarker need to be identified for use in genetic selection programs. l Providing opportunities for exercise and activity, such as perches, improves skeletal strength. l Larger particle sizes of limestone or oyster shell consumed by laying hens are retained in the gizzard for a longer period of time as compared to powdered calcium sources. The slow, sustained intestinal absorption of dietary calcium prevents the hen from relying heavily on her bone reserves as a calcium source, especially at night when she is not eating and her shell is being formed. l

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