Calcium Homeostasis in the Laying Hen.

PHYSIOLOGY AND REPRODUCTION Calcium Homeostasis in the Laying Hen. 1. Age and Dietary Calcium Effects MAHMOUD A. ELAROUSSI,1-2 LEONARD R. FORTE,3-4 SAMMY L. EBER,3 and HAROLD V. BIELLIER1 Department of Animal Sciences and Department of Pharmacology, University of Missouri, Columbia, Missouri 65212 and Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri 65212

1994 Poultry Science 73:1581-1589

INTRODUCTION Calcium is one of the key elements required for maintenance and production of laying hens. It is the most abundant inorganic component of the skeleton and

Received for publication February 28, 1994. Accepted for publication June 29, 1994. department of Animal Science. 2 Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706. 3 Deparrment of Pharmacology and Harry S. Truman Memorial Veterans Hospital. 4 To whom correspondence should be addressed.

plays a major role in a wide variety of biological functions. The commercial laying hen lays, in a 50-wk period, about 280 eggs, each weighing approximately 60 g. This constitutes a considerable loss of body material, which is estimated to be 12 times that of the hen's body weight (Gilbert, 1983). Of concern is the loss of Ca in the eggshell, which weighs 5 to 6 g and contains about 2 g of Ca. This amount of Ca is equal to 10% of the total body Ca, and thus in 1 yr of production a hen will lose Ca equal to 30 times the hen's total body Ca (see Gilbert, 1983). This implies that the laying

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ABSTRACT An experiment was carried out to investigate the effects of age of laying hens (young = 22 wk vs old = 120 wk) in maintaining Ca homeostasis during periods of Ca depletion then repletion with Ca. Plasma Ca and P, tibia breaking strength and percentage ash, renal 25-hydroxycholecalciferol-lhydroxylase (la-hydroxylase), and parathyroid hormone (PTH)-stimulated adenylate cyclase activities were studied during 28 d of Ca depletion on a .08% Ca diet (LC) and 28 d of Ca repletion on a 3.75% Ca diet (HC). When laying hens on a HC diet were placed on a LC diet, plasma Ca and P, tibia breaking strength and ash percentage, and renal PTH-dependent adenylate cyclase activity were significantly depressed, but renal la-hydroxylase activity was significantly stimulated. These changes were greater in the young hens than in the older hens; therefore an interaction between age and dietary Ca was found. These changes were of a lesser magnitude at 28 d of Ca depletion, probably due to the cessation of egg laying and to the desensitization of hormone-mediated function. la-Hydroxylase activity was significantly less during the repletion period. The age effect was most pronounced for la-hydroxylase, with the younger birds expressing significantly higher activity and ability to respond to hypocalcemia. There was a significant increase in kidney weights in Cadeficient groups at 14 and 28 d of Ca depletion. It is concluded that younger hens have greater adaptive responses to Ca restriction than do older hens. (Key words: age, calcium, cholecalciferol, 25-hydroxycholecalciferol-lhydroxylase, adenylate cyclase)

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known to respond to Ca restriction by increased resorption of mineral, and the kidney by increased tubular Ca reabsorption (DeLuca et al, 1990). Calcium may control l,25(OH)2D3 production indirectly, through increasing PTH secretion by parathyroid glands and then PTH acting through a cyclic adenosine monophosphate (cAMP)-dependent process, or directly through affecting cellular mitochondrial Ca and thus adenosine triphosphate (ATP) production (Favus and Langman, 1986). There is a reciprocal relationship between the circulating concentrations of PTH and Ca2+ (Singh et al, 1986). Horst et al (1978) reported that intestinal Ca transport of rats diminishes with age, and the diminished transport of Ca is accompanied by decreased concentrations of plasma l,25(OH)2D3. They suggested that formation of l,25(OH)2D3 decreases with age because of a decline in the need of Ca for growing bones. Furthermore, Armbrecht et al. (1979) reported that rat intestinal adaptation to low dietary Ca (.014%) decreased with age. They attributed the changes in Ca active transport with age and diet to a parallel change in intestinal content of vitamin D-dependent Ca-binding protein. In addition to the agerelated decrease in intestinal Ca transport, Armbrecht and Forte (1985) reported an age-related decrease in rat renal l,25(OH)2D3 production in response to dietary Ca restriction (.02% Ca). Recently, Chen et al. (1992) suggested that the observed in vivo decrease in rat renal calbindin-D28K with age is primarily due to reduced circulating l,25(OH)2D3 and not to a diminished renal responsiveness. It is known that a severe decrease in eggshell quality associated with age is a major problem in laying hens. The decrease in eggshell quality leads to a higher occurrence of egg breakage during production and processing. It also has been reported that la-hydroxylase activity decreases markedly with age (Abe et al, 1982). In the present study, the effects of age and dietary Ca on some parameters of Ca homeostasis were investigated in young and old laying hens, with an ultimate goal of establishing ways to improve eggshell quality in older hens.

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hen possesses a remarkable Ca homeostatic mechanism. Calcium homeostasis is achieved by balancing the efficiency of intestinal Ca absorption, renal Ca excretion, and bone mineral metabolism to the bird's Ca requirements. The main hormones controlling this balance are parathyroid hormone (PTH), calcitonin, and 1,25-dihydroxycholecalciferol [l,25(OH)2D3] produced by the renal conversion of 25-hydroxycholecalciferol [25(OH)D3] through the activity of the enzyme 25-hydroxycholecalciferol-1-hydroxylase (la-hydroxylase). In laying birds, increased Ca demands during the laying cycle are accommodated by an appropriate increase in intestinal Ca absorption (Bar et al, 1978) and a decrease in renal Ca excretion (DeLuca et al, 1990). Both renal la-hydroxylase activity and plasma l,25(OH)2D3 concentrations are significantly higher during the active stage of eggshell calcification than in other stages (Abe et al, 1979). During reproductive activity in the female chicken, endogenous estrogen mediates changes in the function of the kidney that involve the two major Caregulating hormones: PTH and 1,25 (OH)2D3 (Elaroussi et al, 1993). The number of PTH receptor sites and the activity of PTH-dependent adenylate cyclase are elevated in the kidney of mature egglaying females, relative to either the mature male or immature chicken of either sex (Forte et al, 1983). It has been known since 1920 (Buckner and Martin, 1920) that restricting the quantity of Ca in the diet of laying chickens markedly decreases their egg production and causes the eggshells to become thinner. When no Ca supplement is included in the feed, production ceases. Hypocalcemia stimulates la-hydroxylase, suppresses 25-hydroxycholecalciferol-24hydroxylase (24-hydroxylase), causes accumulation of l,25(OH)2D3 in serum, and increases intestinal levels of the vitamin Ddependent Ca-binding protein, calbindinD28K (Norman, 1990). Nicolaysen et al (1953) showed that intestinal Ca absorption increased in animals placed on a lowCa diet and fed cholecalciferol (vitamin D3). Under such conditions of adaptation to low-Ca intake, more l,25(OH)2D3 was produced by the kidney. The skeleton is

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MATERIALS AND METHODS

TABLE 1. Composition and calculated analysis of the experimental diets Calcium Ingredients and calculated analysis

3.75%

.08% l°'„\ u

Corn, ground yellow Soybean meal (48% CP) Ground limestone Sand Mono-basic potassium phosphate (KH2P04) DL-methionine Salt Vitamin mix1 Trace mineral mix2 Calculated analysis Protein ME, kcal/kg Calcium Total P Available P

64.08 24.09

l )

64.08 24.09 9.66

9.66 1.55 .01 .40 .05 .15

1.55 .01 .40 .05 .15

17.33 2,786 .08 .68 .45

17.33 2,786 3.75 .68 .45

Provided per kilogram of diet: vitamin A (retinyl palmitate), 10,658 IU; cholecalciferol, 3,300 IU; vitamin E, 24.35 IU (dl-a-tocopherol acetate); vitamin B12, .03 mg;riboflavin,6.84 mg; D-calcium pantothenic acid, 21.58 mg; niacin, 75.68 mg; choline, 1,055.32 mg; folic acid, 1.0 mg; thiamin, 2.0 mg; pyridoxine, 5.0; biotin, .2 mg; and menadione sodium bisulfite, 2.0 mg. 2 Provided per kilogram of diet: manganese, 65 mg; iodine, 1 mg; iron, 55 mg; copper, 6 mg; zinc, 55 mg; and cobalt, .2 mg.

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were killed by cervical dislocation, and kidneys and tibias were collected. Sampling time was kept consistent throughout the Birds and Diets experiment, and blood and tissues were White Leghorn laying hens, 45 birds each collected at any time point within a at 22 and 120 wk of age, were maintained in 1-h period. individual cages and were fed diets conLaying hens that were chosen to study taining either .08 or 3.75% Ca (Table 1). The Ca repletion were returned to the HC diet diets were isocaloric (2,786 kcal ME/kg) after 28 d on the Ca-deficient ration. In this and isonitrogenous (17.3% CP). Birds were group, the remaining 12 birds were killed provided with 16 h of light/d (0400 to 2000 after 28 d of HC diet. h). Feed and water were available at all times. All birds were maintained on the Kidney Homogenates high-Ca diet (HC, 3.75%) before the start of the experiment, and six birds were killed on The kidneys from individual chickens Day 0 to serve as a control at this time point. were used for each preparation from euNine birds remained on the HC diet to be thanatized hens. Freshly isolated kidneys killed at other time points for control were placed in ice-cold SET buffer (.25 M groups. All other birds were switched to the sucrose, 1 mM EDTA, and 10 mM Tris-HCl, low-Ca diet (LC, .08%), and six birds for pH 7.4) to effect a 25% (wt/vol) suspension. each age were sampled after remaining on The tissue was homogenized using a motor the LC diet for 7, 14, and 28 d. At all time driven Teflon® pestle and then centrifuged points, birds were chosen for euthanasia at 300 x g for 10 min. The supernatant was following morning oviposition and were removed for the mitochondrial fraction; the those that had laid the same number of pellet was set aside for the plasma memeggs. Blood was collected at this time by brane fraction. The supernatant was cencardiac puncture; and, after the birds were trifuged at 3,000 x g for 10 min, and the sedated with ether under the hood, they resulting pellet was resuspended in STAM

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membrane preparations as previously described (Forte et al, 1982). In brief, the reaction mixture (75 /xL), contained 50 mM Tris-HCl (pH 7.5), 6.7 mM MgCl2, 12 mM creatine phosphate, .1 mM cAMP,7 16 mM caffeine, 1.2 mM [a-32P]ATP (8 x 10* cpm/ jtmol),8 166 ng/mL bovine serum albumin; 13.3 U/mL creatine phosphokinase; and 100 ng plasma membrane protein. Incubations were carried out at 30 C for 15 min in the presence of synthetic bovine PTH-(134) .9 Production of cAMP was linear under these conditions. The reaction was terminated by placement of sample tubes in a 100 C water bath for 3 min. As an internal measure of the recovery of cAMP, [3H]cAMP (5 to 10 x 103 cpm/mL) was added in a volume of 1 mL. Then [32P]cAMP was separated from other 32p-iabeled compounds by the Dowex-50 (H+) alumina6 chromatography by the method of Salomon et al. (1974). Recovery of cAMP ranged from 75 to 85%. Results were expressed as picomoles cAMP/15 min incubation per mg protein. Bone Breaking Strength

Bone breaking strength has been used by nutritionists as a response criterion for Plasma Calcium and Phosphorus. determining the bioavailability of minerals, Plasma concentrations of total Ca were and it is a measure of force. At euthanasia, measured by atomic absorption spec- tibias were collected from all birds for the trophotometry. Plasma P was measured determination of mechanical properties and colorimetricaUy by the Rapid-Stat Kit.5 percentage of ash. The bones were all from Protein Analyses. All protein determi- the right side. Bone breaking strength was nations were made according to the method determined on fresh bones by a Flexure test of Lowry et al. (1951). Bovine serum al- with an Instron Universal Testing Mabumin6 was used as the reference standard. chine10 (Crenshaw et al, 1981). The bone In Vitro Assay of Renal 1-Hydroxylase was treated as a beam supported at each Activity. The determination of 1- end and a force was applied at midshaft. hydroxylase activity was performed by Force was applied at a constant rate (5 mm/ modification of the procedure described by min) to all bones, and a recorder was used Tanaka et al. (1976). The modified procedure was described in detail by Martz et al. to plot the force-deflection curve. The test was stopped when the curve reached a peak (1985). maximum force, and the ultimate stress Assay of Adenylate Cyclase Activity. or of bone was determined and expressed in Enzyme activity was measured in plasma kilograms. Analytical Methods

5

Pierce Chemical Co., Rockford, IL 61105. SSigma Chemical Co., St. Louis, MO 63178-9916. 7 New England Nuclear, Boston, MA 02118. 8 ICN Biomedicals, Inc., Irvine, CA 92713. 'Peninsula Laboratories, San Carlos, CA 94002. 10 Model 1123, Instron Corp., Canton, MA 02021.

Bone Ash Bones were cleaned from adhering tissues and dried in an oven at 105 C for 24 h; after cooling in a desiccator a dry weight was recorded. Then bones were fat ex-

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buffer (.25 M sucrose, 15 mM Tris-acetate, and 1.9 mM-Mg-acetate, pH 7.4). After a second centrifugation at 3,000 x g for 10 min, the pellet was suspended in STAM buffer to yield 6 to 8 mg protein/mL. Assay of 25(OH)D3-l-hydroxylase activity was performed on freshly prepared mitochondrial fractions. The original pellet set aside for the plasma membrane fraction was resuspended in SET buffer and centrifuged at 1,500 x g for 10 min. The supernatant was discarded, and the pellet was resuspended in 2.0 M sucrose and centrifuged at 14,600 x g for 10 min. The supernatant was diluted in 7 vol of ice-cold deionized water and centrifuged at 39,000 x g for 10 min. The pink fluffy layer of the pellet was resuspended in SET buffer to yield 5 mg protein/mL. Aliquots of the plasma membrane preparations were stored at -70 C until assayed for adenylate cyclase activity. The isolation of renal mitochondrial and plasma membrane fractions is based on techniques described by Castillo et al. (1977) for mitochondrial fraction and Fitzpatrick et al. (1969) and Nissenson and Arnaud (1979) for plasma membranes.

CALCRJM HOMEOSTASIS IN THE LAYING HEN

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tracted, weighed again, and ashed in a muffle furnace at 600 C for 24 h to obtain the ash weight. Results were expressed as percentage ash of dry fat-free weight. Statistical

Analysis

Yjj = (i + A ; + Bj + (AB)ij + eijk 40 i

where i = 1,2...5 dietary treatments; and j = young or old age. Significance of difference between means were compared by Fisher's least significant difference test (Snedecor and Cochran, 1967). Statistical significance was taken at P < .05, unless otherwise indicated. RESULTS Plasma Ca and P were determined at the time of euthanasia. The decreases in plasma values were more pronounced in younger birds for both Ca and P after 1 wk of feeding the diets (Figure 1). In general, older birds showed less change than did the young birds, probably due to the fact that younger birds laid more eggs. At Day 28 of Ca repletion, the plasma Ca and P values returned to their normal levels. Figure 2A shows the ash content of tibias. Bones were depleted of their Ca reserves during Ca depletion, especially for the young birds that laid more eggs, such that by 14 d of LC diet, bones contained significantly less ash. Bone mineral concentrations were restored at 28 d of Ca repletion. The results of the bone breaking strength are shown in Figure 2B. There was a significant difference in tibia strength between young and old hens. Calcium depletion resulted in a decrease in bone strength that is greater in young birds compared with the older hens. As the birds were Ca repleted, tibia strength was partially restored, but did not reach the initial level after 28 d of Ca repletion.

33

E

£3 CL

Cont

7 14 28 LC LC LC Days on Dietary Treatments

28 HC

FIGURE 1. Effect of Ca depletion and repletion on plasma P (A) and Ca (B) of young and old laying hens. Data are x ± SE of 15 birds of either age at the control level, 12 birds for the repleted ones and 6 birds for the remaining time points. Columns with no common letter differ significantly (P < .05). There were no differences in either plasma Ca or P due to age. Cont is the control, LC indicates the use of a low-Ca diet (.08%), and HC refers to high-Ca diet (3.75%) for the duration of times during depletion and repletion, respectively.

The levels of la-hydroxylase activity of kidney homogenates (Figure 3) showed a significant difference between young and old birds, with the young birds having higher enzyme activity. As the birds become more Ca depleted, the activity of the la-hydroxylase enzyme increased, especially after 14 d of LC diet, with a threefold increase in enzyme activity. When these birds stopped laying eggs, the dietary Ca needs were less and thus, la-hydroxylase activity decreased by Day

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Results are presented as x ± SE. Data were analyzed by analysis of variance (General Linear Models procedure; SAS Institute, 1986) with age and dietary treatment as main effects and the first order interaction involving them. The statistical model for analysis of variance was:

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ELAROUSSI ET AL. 5,000-,

1

4,000

S

3,000

ra 2 , 0 0 0 Q o

o

1,000

7 14 28 LC LC LC Days on Dietary Treatments

28 HC

FIGURE 3. Effect of Ca depletion and repletion on renal 25-hydroxycholecalciferol-l-hydroxylase [25(OH)D-l-hydroxylase]. Data are expressed as x ± SE. Columns with no common letters are significantly different (P < .05). Older control birds have significantly less (P <, .001) enzymatic activity than do young birds. Cont is the control, LC stands for lowCa diet (.08%), and HC is for high-Ca diet (3.75%) during periods of depletion then repletion.

a c

en c m 0)

m

Cont

7 LC Days

on

14 LC Dietary

28 LC

28 HC

Treatments

FIGURE 2. Effect (x ± SE) of Ca depletion then repletion on tibia percentage ash (A) and bone breaking strength as determined by peak force, kilograms, in the deformation curve (B). Columns with no common letter are significantly different (P < .05). Cont is the control, LC stands for low-Ca diet (.08%), and HC refers to high-Ca diet (3.75%) for the duration of times during depletion and repletion. There was a significant age difference between the Cont group (P <, .025), LC at 14 d (P <, .025), and LC at 28 d (P < .005). Age differences in tibia strength occurred for Cont (P < .01), LC14 (P < .001), LC28 (P < .025), and HC28 (P < .001).

28 of the LC diet. After the birds were Ca repleted, their renal la-hydroxylase activity was below the initial levels, and there was no significant difference in enzyme activity due to age. Studies of the renal adenylate cyclase activity of plasma membranes from these birds revealed that PTH-dependent enzyme activities in membranes from older birds was significantly different from

young birds (Figure 4). Parathyroid hormone-dependent activity was significantly reduced in young birds during Ca depletion. In addition, it was noticed while weighing the kidneys during their preparation to measure the 1-hydroxylase and adenylate cyclase activities, that there was a significant increase in the kidney weights after 14 and 28 d of Ca depletion, and kidney weights returned to their normal weight after Ca repletion (Figure 5, shows only young birds). DISCUSSION The present investigation evaluated the ability of young and old laying hens to maintain Ca homeostasis during periods of Ca depletion and repletion. The results reported here suggest that one of the possible causes of the increased rate of cracked or soft-shelled eggs associated with older laying hens is related to the decrease in the renal 25(OH)D 3 -lhydroxylase activity; thus, an impairment in the biosynthesis of l,25(OH) 2 D 3 may occur in older hens. Reduced production of this hormone is important, because l,25(OH) 2 D 3 is a major Ca homeostasis

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Cont

CALCIUM HOMEOSTASIS IN THE LAYING HEN

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1.2-, CD

1.0 o

•a

FIGURE 4. Renal parathyroid hormone (PTH)dependent adenylate cyclase activity during Ca depletion and repletion. Renal plasma membranes were prepared from each pair of kidneys and used for assay of adenylate cyclase activity (triplicate assays per preparation and condition). Data are expressed as x ± SE of enzyme-specific activity. Basal enzyme activity was subtracted from the PTHsrimulated activity. Columns with no common letter are significantly different (P < .05). The same number of birds were used for all time points. There was greater significant difference between ages at control level (P < .025), and at 14 d of Ca depletion (P < .005). Cont is the control, LC refers to Ca depletion on a low-Ca diet (.08%), and HC is for Ca repletion on a high-Ca diet (3.75%). cAMP = cyclic adenosine monophosphate.

regulating hormone in birds and mammals. Furthermore, the study demonstrated that both age groups do eventually adapt to dietary Ca restriction in terms of increased l,25(OH) 2 D 3 . However, both the rapidity and magnitude of the response is decreased in older hens compared with younger hens. Similar results on the effects of age on la-hydroxylase were reported by Abe et al. (1982) and Tanaka and DeLuca (1984). Hypocalcemia and increased circulating PTH cause an increase in l,25(OH) 2 D 3 production (DeLuca et al, 1990). The young hens showed a continued plasma Ca decrease during the duration of the 28 d duration of LC. In contrast, the plasma Ca of old hens was significantly decreased only during the 1st wk of depletion (Figure 1). Likewise, plasma P showed a similar response. The absence of a significant decrease in plasma Ca in older hens at 2 and 4 wk of Ca

7 14 28 28 LC LC LC HC Days on Dietary Treatment

FIGURE 5. Effect of Ca depletion and repletion on kidney weights (grams per 100 g BW) of young laying hens. Data are x ± SE. Asterisks indicate a significant increase in kidney weight at 14 d (P < .01) and at 28 d (P < .001) of Ca depletion. Cont is the control, LC stands for the use of a low-Ca diet (.08% Ca) for either 7, 14 or 28 d, and HC refers to the use of a high-Ca diet (3.75%) for 28 d of repletion.

depletion could result in a smaller increase in circulating PTH; thus, the modest response of la-hydroxylase activity was observed. Therefore, it may be concluded that plasma Ca levels of laying hens is a strong regulator of this enzymatic system. This conclusion is in agreement with previous reports (Bar et al, 1978; Forte et al, 1983; Armbrecht and Forte, 1985; Martz et al, 1985; Elaroussi et al, 1993). The decrease in bone strength and percentage of dietary ash during dietary Ca depletion was an indication for depletion of the skeletal mineral reserves on the LC diet. The number of eggs laid and the Ca loss when hens were fed a low-Ca diet appear to be dependent upon their Ca reserve. Due to the limited reserves of Ca in young hens, it appeared that younger birds were more sensitive to Ca restriction than were the older hens. Zallone and Mueller (1969) have indicated that histologically, only cortical bone shows signs of osteoclastic activity at bone resorption. This finding agrees with those of Hurwitz and Bar (1969), who found an indication of age-dependent differences in the ability of hens to utilize Ca from cortical bone reserves. During prolonged Ca deficiency,

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Cont Days on Dietary Treatments

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present investigation as a result of hypocalcemia, has been reported also in laying hens as they approach sexual maturity, a time of an increased Ca demand (Forte et al, 1986). The renal hypertrophy may be an adaptive response of the kidney to the Ca deficiency. Such a view is consistent with the finding that a low-Ca diet resulted in an enhancement of the renal hypertrophy that occurs following unilateral nephrectomy in rats (Jobin et al, 1984; Jobin and Bonjour, 1986). The administration of l,25(OH) 2 D also increased the renal hypertrophy, suggesting that l,25(OH) 2 D might cause the increased kidney weights of hens fed the LC diet in the present study. The present report demonstrated that the increased rate of cracked or softshelled eggs seen in older laying birds could be a result of disorders associated with the Ca homeostatic mechanism. It is concluded that younger laying hens have greater adaptive responses to Ca restriction than do older laying hens. ACKNOWLEDGMENTS This research was supported by the Truman Veterans Administration Hospital and by the National Institutes of Health Grant DK-32848. The excellent technical assistance of Richard Poelling is greatly appreciated. Also, the preparation of this manuscript by Kristin Nelson is very much appreciated by the authors.

REFERENCES Abe, E., H. Horikawa, T. Masumura, M. Sugahara, M. Kubota, and T. Suda, 1982. Disorders of cholecalciferol metabolism in old egg-laying hens. J. Nutr. 112:436^46. Abe, E., R. Tanabe, T. Suda, S. Yoshiki, H. Horikawa, T. Masumura, and M. Sugahara, 1979. Circadian rhythm of la, 25-dihydroxy vitamin D3 production in egg-laying hens. Biochem. Biophys. Res. Commun. 88:500-507. Armbrecht, H. J., and L. R. Forte, 1985. Adaptation of middle aged rats to long-term restriction of dietary vitamin D and calcium. Arch. Biochem. Biophys. 242:464-469. Armbrecht, H. J., T. V. Zenser, M.E.H. Brusn, and B. B. Davis, 1979. Effect of age on intestinal calcium absorption and adaptation to dietary calcium. Am. J. Physiol. 236(Endocrinol. Metab. Gastrointest. Physiol. 5):E769-E774. Bar, A., A. Cohen, S. Edelstein, M. Shemesh, G.

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the body responds by increasing the size of the labile medullary reservoir at the expense of cortical bone (Zallone and Mueller, 1969). The less dynamic bone of older hens could be attributed to less synthesis of l,25(OH) 2 D 3 in those hens. Chen et al. (1992) concluded that the decrease in renal calbindin-D28k with age is due primarily to the lowered circulating l,25(OH) 2 D 3 . Singh et al. (1986) suggested that the major endocrine change during Ca deficiency is an increase in parathyroid activity. They showed a reciprocal relationship between the concentrations of PTH and Ca 2+ . Mean concentrations of PTH in the birds given their low-Ca diet were more than twice as high as in the control birds during shell formation, when there is a greater need for Ca. The results reported with the PTH-dependent adenylate cyclase of kidney showed a reduction in enzyme activity during Ca depletion and an increase during Ca repletion. The decrease in renal PTH-dependent adenylate cyclase during depletion could be explained by the induction of a secondary hyperparathyroid state resulting from feeding diets deficient in either vitamin D or Ca, which leads to a marked and progressive loss of renal PTH receptors that correlated with the decreased adenylate cyclase (Forte et al, 1982). Calcium deficiency not only increases PTH levels but also amplifies the expression of the PTH gene, and a fivefold increase in the PTH mRNA was reported after 3 wk of Ca deficiency (Naveh-Many and Silver, 1990). When the laying hens were repleted with Ca, an increase in PTH-stimulated adenylate cyclase was observed, perhaps reflecting restoration of PTH receptors in the kidney. Forte et al. (1983) have reported a similar increase in PTHdependent adenylate cyclase with the increase in estradiol that occurs before hens start laying eggs. Based on the ability of the kidney to adapt to extracellular concentrations of PTH (Carnes et al, 1978), it can be hypothesized that the upregulation of renal PTH-dependent adenylate cyclase activity observed in Ca-repleted hens results, in part, from the low levels of the peptide hormone in the circulation. Hypertrophy of the kidneys, found in the

CALCIUM HOMEOSTASIS IN THE LAYING HEN

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