Birds that lay long clutches (series of eggs laid sequentially before a "pause day"), among them the high-producing, strongly-calcifying Gallus gallus domesticus (domestic hen) and Coturnix coturnix japonica (Japanese quail), transfer about 10% of their total body calcium daily. They appear, therefore, to be the most efficient calcium-transporters among vertebrates. Such intensive transport imposes severe demands on ionic calcium (Ca2+) homeostasis, and activates at least two extremely effective mechanisms for Ca2+ transfer from food and bone to the eggshell. This review focuses on the development, action and regulation of the mechanisms associated with paracellular and transcellular Ca2+ transport in the intestine and the eggshell gland (ESG); it also considers some of the proteins (calbindin, Ca(2+)ATPase, Na+/Ca2+ exchange, epithelial calcium channels (TRPVs), osteopontin and carbonic anhydrase (CA) associated with this phenomenon. Calbindins are discussed in some detail, as they appear to be a major component of the transcellular transport system, and as only they have been studied extensively in birds. The review aims to gather old and new knowledge, which could form a conceptual basis, albeit not a completely accepted one, for our understanding of the mechanisms associated with this phenomenon. In the intestine, the transcellular pathway appears to compensate for low Ca2+ intake, but in birds fed adequate calcium the major drive for calcium absorption remains the electrochemical potential difference (ECPD) that facilitates paracellular transport. However, the mechanisms involved in Ca2+ transport into the ESG lumen are not yet established. In the ESG, the presence of Ca2+-ATPase and calbindin-two components of the transcellular transport pathway-and the apparently uphill transport of Ca2+ support the idea that Ca2+ is transported via the transcellular pathway. However, the positive (plasma with respect to mucosa) electrical potential difference (EPD) in the ESG, among other findings, indicates that there may be major alternative or complementary paracellular passive transport pathways. The available evidence hints that the flow from the gut to the ESG, which occurs during a relatively short period (11 to 14 h out the 24- to 25.5-h egg cycle), is primarily driven by carbonic anhydrase (CA) activity in the ESG, which results in high HCO3-content that, in turn, "sucks out" Ca2+ from the intestinal lumen via the blood and ESG cells, and deposits it in the shell crystals. The increased CA activity appears to be dependent on energy input, whereas it seems most likely that the Ca2+ movement is secondary, that it utilizes passive paracellular routes that fluctuate in accordance with the appearance of the energy-dependent CA activity, and that the level of Ca2+ movement mimics that of the CA activity. The on-off signals for the overall phenomenon have not yet been identified. They appear to be associated with the circadian cycle of gonadal hormones, coupled with the egg cycle: it is most likely that progesterone acts as the "off" signal, and that the "on" signal is provided by the combined effect of an as-yet undefined endocrine factor associated with ovulation and with the mechanical strain that results from "egg white" formation and "plumping".This strain may initially trigger the formation of the mammillae and the seeding of shell calcium crystals in the isthmus, and thereater initiate the formation of the shell in the ESG. (C) 2008 Elsevier Inc. All rights reserved.

Egg laying and shell calcification impose severe extra demands on ionic calcium (Ca2+) homeostasis; especially in birds characterized by their long clutches (series of eggs laid sequentially before a "pause day"). These demands induce vitamin D metabolism and expression. The metabolism of vitamin D is also altered indirectly, by other processes associated with increased demands for calcium, such as growth, bone formation and egg production. A series of intestinal, renal or bone proteins are consequently expressed in the target organs via mechanisms involving a vitamin D receptor. Some of these proteins (carbonic anhydrase, calbindin and calcium-ATPase) are also found in the uterus (eggshell gland) or are believed to be involved in calcium transport in the intestine or kidney (calcium channels). The present review deals with vitamin D metabolism and the expression of the above-mentioned proteins in birds, with special attention to the strongly calcifying laying bird.

1. The effect of age on ovarian function was studied in 245-, 350-, 500-, 700- and 800-d-old Lohmann hens. The effect of three different methods for moult induction on ovarian function and corticosterone concentration was studied in 500-d-old hens. 2. No significant reductions in ovarian weight or in number of follicles before the age of 700 d were found. The ability to produce progesterone and oestradiol-17beta was unchanged up to the age of 700 d and the circadian secretion of these two steroids was identical in young (225 d) and old hens (600 d). 3. The effects of induced moulting by feed withdrawal (FW) and a high Zn (HZn) diet on body weight and ovarian function were very similar; those of a moderate Zn with low Ca (MZn/LCa) diet were smaller. 4. The first significant effect of moulting was a decrease in oestradiol-17beta plasma concentration (d 2). Plasma progesterone decreased more gradually than oestradiol-17beta, and reached a nadir on d 6 in FW- and HZn-treated hens and on d 9 in MZn/LCa-treated ones. 5. Hens treated with either FW or the MZn/LCa, but not those with the HZn diet, showed a very sharp rise in corticosterone concentration on d 2 of treatment. Thus the MZn/LCa diet was less efficient than the other treatments in induction of ovarian involution, but had a similar effect on stress induction, as indicated by increases in plasma corticosterone.

1. A series of 5 trials was conducted with Cobb chickens in order to determine the effect of 25-hydroxycholecalciferol (25OHD3) on their performance and bone development under adequate Calcium (Ca) and Phosphorus (P) supplementation, and under moderate dietary restriction of Ca and P. Formulated beadlets of 25OHD3, trade name HY-D (IsoGen, Naperville, IL, USA) were used as the 25OHD3 source. 2. Five to 10 microg of cholecalciferol (vitamin D3) or 25OHD3/kg diet were sufficient to ensure normal body weight (BW) and bone ash in chickens under continuous lighting. The two materials had similar effects on BW and bone ash. 3. In one out of the three experiments, 25OHD3 increased BW and BW gain, while in the others it had a similar effect to that of vitamin D3, or even a slight negative effect in a trial conducted on the floor, in which the diets were supplemented with the D sources at 75 microg/kg. The effects of both D sources on bone ash and on the severity or frequency of tibial dischondroplasia were similar. 4. 25OHD3 restrained the effect of moderate dietary P restriction, but not of Ca restriction, on BW gain and bone ash in 22-d-old chickens. This effect could not be explained by an higher P bioavailability in the 25OHD3-fed chickens.

A series of trials was conducted in order to study the effects of age and molt on intestinal and eggshell gland (ESG) calbindin, and on bone ash. For this purpose an ELISA for chicken calbindin was developed. Age did not significantly affect duodenal or ESG calbindin. Bone ash increased (but not significantly in this study) from 8 to 16 months of age. During molt induction, egg laying was arrested, duodenal and ESG calbindin almost completely disappeared and ovary mass, plasma estradiol and total calcium (Ca) decreased markedly, whereas bone ash and body mass (BW) decreased moderately. During the non-laying period that followed the feed withdrawal period, duodenal and ESG calbindin remained low, whereas plasma estradiol and other estrogen-dependent variables, such as plasma total Ca and bone ash, increased slightly. At the onset of egg production following molting, duodenal and ESG calbindin levels were similar to pre molt level. Bone ash was higher than at the pre molt period. Body mass, small yellow follicles, ovary and oviduct mass and plasma estradiol were lower than their values prior to molt induction. Bone ash contents in the molted hens at the ages of 583 and 820 days were similar to or even slightly higher than those in the non-molted hens, whereas duodenal and ESG calbindin were not significantly different. These results suggest that the improvement of shell quality in the molted birds does not involve mechanisms associated with calbindin synthesis.

AbstractThree series of experiments were conducted with fast-growing chickens in order: to evaluate the effects of dietary Ca and P on cholecalciferol metabolism and expression; to determine dietary Ca requirements; to determine dietary P requirements. The results of the first series confirmed previous results on the effects of dietary Ca and P on some variables of vitamin D metabolism and expression, Ca homeostasis and P metabolism in the young chicken (1- to 21-d-old), and extended them to older birds (22- to 43-d-old). The bone formation rate and the duodenal calbindin content were maintained at high levels until the age of 43 d. Dietary Ca or P restriction increased duodenal calbindin and decreased bone ash in both 22- and 43-d-old chickens, but the effect on bone ash was less pronounced in the 43-d-old birds than in the younger ones. These results suggest that: (a) the capabilities for adaptation to dietary Ca and P restriction remain high during the whole growing period; (b) the growing broilers express a high adaptive capability even when the diet contains the recommended Ca and P contents. The results of the second and third series of experiments suggest that: (c) unlike the Ca requirements of the 1- to 22-d-old chick, P requirements for growth and bone ash are similar, and are as high in the older chicks as in the younger ones (7·4–8·3 g P/kg or 4·8–5·7 g non-phytate P/kg diet); (d) although growth and bone ash in the 29- to 43-d-old chickens appear to be less sensitive to dietary Ca content, within a range close to the calculated P requirement, 10 g Ca/kg diet appears to be required for best tibia mineralization, and to a lesser extent for better growth at this age.

Shortening daylight (to 10.5 to 11 h/d) slightly reduced the rest period (the interval between last egg and first clutch), whereas feeding a low-protein diet during the 22-d period following 8 d of feed withdrawal (FW) (recovery period) markedly extended it. Feed withdrawal accompanied by a short-daylight regime and a long recovery period led to the best postmolt production. However, production per hen housed during the whole experimental period was only slightly increased. Thus, a long rest period may mask the positive effects of short daylight and recovery diet. At least 140 to 170 d were needed to enable the molted hens to compensate for the loss of eggs during the rest period. Therefore, under certain economic conditions, rearing of nonmolting hens for 640 to 700 d should be an economic consideration. Ten days of feeding of a diet containing 0.06% nicarbazin (NICR) arrested egg production and caused a 22-d rest period but not a typical molt. Few variables of production or shell quality were improved by NICR but to a lesser extent than by FW or Zn feeding. Five days of feeding on a high-Zn diet (20 g Zn/kg; HZn) improved postmolt performances similarly to FW. Ten days of feeding on a modest-Zn (2.8 g Zn/kg), low-Ca, and low-P diet (Zn-CaP) affected postmolt performances inconsistently. In one out of two trials (trial 2), the effects of Zn-CaP were similar to those of FW or HZn; in the other (trial 3), the effects were less pronounced, more time was required for egg arrest, and more eggs were laid occasionally during the rest period. In trial 2, only the Zn-CaP diet was accompanied by short daylight. In both trials, feed intake during the induction period was only slightly reduced. Zn feeding increased the yolk Zn content slightly in eggs laid during the induction period and at the onset of production. In trial 2, only Zn-CaP markedly increased yolk Zn of eggs laid during the first 5 d of production.

1. A series of 5 trials was conducted with 401- to 650-d-old Lohmann, Yafa (local breed with brown eggshells) and Yarkon (local breed with white eggshells) hens fed for 56 to 84 d with diets containing 25 to 50 g/kg calcium (Ca) and 4.5 to 7.6 g/kg phosphorus (P). 2. Increasing dietary Ca from 24-25 to 36-40 g/kg improved egg production, shell weight (SW) and shell thickness (ST), and decreased mortality. 3. Increasing dietary Ca to 48 to 50 g/kg did not affect egg production but increased SW and/or ST. In one out of the 4 trials, this effect was not significant, most likely because of the high shell quality of the eggs from the Yafa hens used for this trial. 4. Dietary P content of 4.5 g/kg (1.0 g/kg added inorganic P) appears to be sufficient for maintaining egg production and shell quality in aged laying hen given 36 to 40 g/kg Ca. 5. Increasing dietary Ca above 40 g/kg may require a higher dietary P content.

Molt was induced at the 431, 501, or 571 d, in Lohmann (L) and Hy-Line W-77 (H) hens, by 8 or 14 d, respectively, of feed withdrawal followed by a rest period of 16 d. Induced molt resulted in increases in egg production, numbers of intact eggs, egg mass per housed or surviving hen, and shell quality and in decreases in egg breakage (not significant), mortality, and culling. Egg weight was only slightly affected by molt, and the EW of hens induced to molt at 431 or 501 d of age were slightly lower than those of the unmolted hens or of those induced to molt at 571 d. Both strains reacted similarly to molt, although the L hens responded better, and expressed their responses more intensively when induced to molt earlier (431 d). This finding suggests that although different breeds have some effects of molt in common, molt protocols should be finely tuned for each breed. Total intact egg production and egg mass of the molted hens became higher than those of the unmolted hens at 650 to 728 d, which suggests that no benefit would be achieved by rearing molted hens for less than 700 to 730 d.

1. The effects of relative humidity (rh=40% to 70%) at high ambient temperature (T-a) on the performance of laying hens at different ages (8 to 10 months, Trial 1; and 16 to 18 months, Trial 2) was evaluated. Laying hens were exposed to 25 degreesC (control) for 3 weeks and thereafter acclimated for 1 week to 35 degreesC and 4 different rh.2. Body weight declined significantly in young and older hens exposed to 60% or 70% and 70% rh, respectively. Food intake declined with increasing T-a, except in the case of older hens exposed to 60% rh, for which it remained relatively constant. Water consumption, however, increased with increasing T-a but the increase was significant in young hens exposed to 70% rh only.3. Egg production was not affected by the changes in T-a. However, a decrease in egg production was observed in older hens exposed to 60% rh.4. Egg weight (EW), shell weight (SW) and shell thickness (ST) were significantly reduced by exposure to elevated T-a, whereas % breakage significantly increased. In young hens, a response to rh was exhibited in ST which was significantly higher in hens exposed to the low rh (40% to 45%) than in those exposed to the highest rh (70% to 75%).5. It can be concluded that T-a is the main environmental factor affecting young and older laying hens while the effect of rh is minor.

Hens forming uncalcified shells synthesized less 1,25-hydroxycholecalciferol (1,25(OH)2D3) and less duodenal and eggshell gland (ESG) calbindin than normal laying hens. Hens forming thin shells had lower intestinal and ESG calbindin and its mRNA. Reducing ESG calcium (Ca2+) transport by the carbonic anhydrase inhibitor acetazolamide, but not by dietary Ca2+ restriction, reduced ESG calbindin and its mRNA. Two sub-populations of hens characterized by shell thickness (ST) maintained this characteristic throughout the whole production period. The differences between the two sub-populations increased with age. In old laying hens, the two sub-populations responded differently to dietary Ca2+ restriction and to exogenous 1,25(OH)2D3. Those forming a thin shell responded to 1,25(OH)2D3 by a significant improvement in ST. The results suggest that: (a) the mechanism responsible for Ca2+ transport to the egg shell consists of a vitamin D-dependent absorption of Ca2+ and a multi-factor-dependent transfer of Ca2+ to the shell; (b) both steps are, most likely, calbindin-mediated; however, the induction of calbindin gene expression in the ESG is predominantly calcium-dependent; and (c) the apparent defect in vitamin D metabolism or its expression in old hens is typical of, or even exclusive, to thin-shell-forming hens.

The aim of this study is to evaluate the regulation of the osteopontin (OPN) gene expression by non-hormonal stimuli, such as calcium flux and mechanical strain during the daily egg cycle in the oviduct of the laying hen. After the egg enters the eggshell gland (ESG), the OPN gene is expressed by the epithelium cells in two waves: first by the basal cells and only then by the apical cells of the epithelium. A reduction in OPN gene expression was observed 1 h prior to laying. The calbindin gene, which marks the onset of calcification, was found to be expressed in the glandular epithelium starting 2 h after OPN gene expression. In addition, the formation of soft shells was accompanied by a reduction in calbindin, but not in OPN, gene expression. The application of a mechanical strain comparable to that induced by an egg led to induction of OPN gene expression at a normally quiescent phase in the cyclical expression of this gene. The induction of the gene was time- and strain-dependent and temporally similar to that induced by the entry of the egg into the ESG. In contrast, the calbindin gene was not affected by mechanical strain. The ESG of the laying hen provides a system to study the effect of a mechanical strain on matrix protein production in vivo, in a relevant physiological setting. The finding suggests that, in contrast to calbindin, OPN gene expression is not regulated by calcium flux but rather by the mechanical strain imposed by the resident egg.

1. Rate of production and shell thickness (ST) decreased, while body weight (BW), egg weight (EW) and percentage breakage increased progressively with age. Shell weight (SW) increased until 8 to 13 months of age and then decreased. 2. Early onset of production resulted in lower BW and EW at the onset of production, and lower pooled averages of BW, EW, SW and ST, as compared with late or medial onset of production. In 4 out of 5 trials, early onset did not result in the production of more eggs during the laying period. 3. Early onset of production is associated with physiological Ca deficiency as indicated by increases in kidney-1-hydroxylase and duodenal calbindin in early layers as compared with late layers. Early layers exhibited a more severe reduction in shell quality as the result of Ca deficiency as compared with late layers. 4. Feeding pullets with a prelaying diet containing 3.9% Ca did not affect unequivocally the performance or shell quality during the whole productive period, whether the birds started to lay early or late. The dietary treatment did not cause renal damage, as indicated by morphological examination and by plasma calcium and uric acid concentration.

Acute and chronic changes in calbindin (Mr 28,000) mRNA and calbindin concentrations were determined to assess the roles of steroid hormones in calbindin mRNA and calbindin synthesis in the eggshell gland (ESG). The results support an earlier suggestion that calbindin gene expression in the ESG is associated with Ca2+ flux through the ESG. The evidence includes wide oscillation of the mRNA during the diurnal egg cycle, in close temporal association with egg shell calcification. Progesterone (single im injection of 1 mg/kg body weight, BW) prolonged the period of egg formation and reduced the rate of Ca2+ transport and the concentration of calbindin mRNA in the ESG. Dexamethasone (single im injection of 5 mg/kg BW) prolonged the period of egg formation, increased shell Ca2+, and reduced calbindin mRNA in the ESG and intestine. Testosterone (single im injection of 2 mg/kg BW) did not affect calbindin mRNA synthesis in the ESG. The effects of estradiol on the synthesis of calbindin mRNA in the ESG of sexually immature or laying birds were minor, while it affected plasma Ca in the same birds. The antiestrogen Tamoxifen (60 mg/kg BW, given orally) reduced plasma Ca, but did not affect the synthesis of calbindin mRNA in the ESG. The antiprogesterone RU-38486 (20 mg/kg BW, orally) increased shell Ca2+ but had no effect on plasma Ca or the synthesis of calbindin mRNA. It appears that estrogens alone cannot account for the markedly elevated synthesis of calbindin mRNA in the ESG of the laying bird. The hypothesis that the regulatory mechanism for the synthesis of calbindin mRNA in the ESG may involve a stimulator(s), associated with the onset of production, and an oscillating depressor(s) is supported and both appear to be closely linked to the reproductive cycle. The specific in vivo effect of progesterone on calbindin mRNA in the ESG, together with its already known changes during the ovulatory cycle in birds, supports the idea that it is a depressor.

Expression of the osteopontin (OPN) gene in the oviduct of the laying hen was studied. It was detected only in the egg shell gland (ESG), where massive calcification occurs. No OPN gene expression was detected in any other part of the oviduct, such as the magnum and isthmus. The OPN gene was expressed in a circadian fashion during the daily egg cycle only during the period of egg shell calcification. No OPN gene expression was detected in the ESG of a pre-laying hen before the onset of reproduction, or after forced removal of the egg close to its entrance into the ESG. OPN was found to be synthesized by the epithelial cells of the ESG lining the lumen. Upon synthesis, OPN is immediately secreted out of cells and accumulates in the egg shell. These findings demonstrate for the first time temporal and spatial association of OPN with egg shell calcification. OPN, which was found to be part of the organic matrix of the egg shell, may play an important role in egg shell calcification.

Regulation of prepro-PTH and vitamin D receptor (VDR) mRNAs in the parathyroid glands was studied in chickens in vivo. The birds were raised to 21 days of age on a vitamin D-deficient diet with 1% calcium and 0.65% phosphorous. At the end of this period, the chicks exhibited marked hypocalcemia and enlarged parathyroid glands. In three separate trials, the birds were repleted for 6 days with vitamin D and different dietary calcium and phosphate concentrations, with 2 micrograms/kg 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and different dietary calcium concentrations (0.5%, 1.0%, or 1.8%), or with 2 or 10 micrograms/kg 1,25-(OH)2D3 and 0.6% or 1.9% calcium or were kept vitamin D3 deficient and fed 0.5%, 1.0%, or 1.8% dietary calcium. Vitamin D treatment when combined with a high level of dietary calcium resulted in an increase in plasma calcium from 6 mg/dl to greater than 10 mg/dl, a decrease in PTH mRNA of 65%, and a 6- to 8-fold increase in VDR mRNA. In another experiment in which no vitamin D source was given and the diets contained increasing levels of dietary calcium, plasma calcium increased significantly (5.5 vs. 7 mg/dl), while PTH mRNA decreased by 40% and VDR mRNA increased by 60%. Neither parathyroid gland weight nor total RNA was significantly affected. When chicks were repleted with 1,25-(OH)2D3, the increase in plasma calcium and VDR mRNA and the decrease in PTH mRNA were considerably more pronounced than those in the absence of the vitamin D source. Furthermore, in the presence of the hormone, parathyroid weight and total RNA decreased significantly with increasing concentrations of dietary calcium. When the chicks were repleted, respectively, with the two levels of 1,25-(OH)2D3, a marked positive interaction was evident between the hormone and dietary calcium in affecting levels of PTH and VDR mRNA. These results suggest that both 1,25-(OH)2D3 and calcium participate in the regulation of PTH and VDR gene transcription in the avian parathyroid gland. Whereas the action of 1,25-(OH)2D3 requires a minimal level of dietary calcium, calcium affects PTH and VDR gene transcription even in the absence of any vitamin D source.

1. Eggshell density (mg/cm2) and eggshell gland calbindin decreased in the aged hens.2. Aged hens which laid eggs with a low shell weight and shell density had significantly lower intestinal and eggshell gland calbindin as compared with those which laid eggs with a high shell weight and shell density.3. Significant correlations were found in aged hens between duodenal or eggshell calbindin and shell weight or shell density.4. The results suggest that: (a) aged hens forming light shells absorbed calcium with a lower efficiency than those forming heavy shells or than young hens; (b) the decline in shell density in the aged hens is caused by a physiological calcium deficiency or by a defect in the hens' ability to alter calbindin synthesis in response to calcium needs; (c) in the aged hens, the deposition of calcium into the eggshell is dependent on, or at least associated with, eggshell gland calbindin.