The role of calcitonin in controlling hypercalcaemia in the domestic fowl (Gallus domesticus)

THE

IN CONTROLLING ROLE OF CALCITONIN HYPERCALCAEMIA IN THE DOMESTIC FOWL (GALLUS DOMESTICUS) K. G. BAIMBRIDCE* and T. G. TAYLOR

Department of Nutrition, School University of Southampton.

of Biochemical Southampton

(Received 4 Auyust

and Physiological Sciences. SO9 5NH, England

1980)

Abstract-l. Calcitonin (CT) was assayed in the plasma of 10 laying hens by a sensitive radioimmunoassay at the time of oviposition and at 4-hr intervals thereafter. 2. The hormone was not detectable in any of the birds when active shell calcification was in progress but it was present in 6 birds at the time of oviposition at a mean concentration of 1.54ng synthetic salmon CT/ml (SEM f 0.23, range 0.95-2.48 ngjml). 3. These results are consistent with the view that CT is secreted in laying birds when the plasma level of ionic calcium exceeds 1.5 mmolil and that one of the physiological roles of the hormone is to limit the extent of hypercalcaemia after the completion of shell calcification. 4. The effect of different levels of dietary calcium (11, 30 and 60 g/kg) on plasma CT levels in chicks were studied from 6-10 wk of age. The level of CT in the control birds (given 1I g calcium/kg diet) varied from 1.05-1.64 ng synthetic salmon CT equivalents/ml and this level rose to a maximum of 2.5 ng/ml at the highest level of dietary calcium. 6. Concentrations of plasma calcium increased slighly and those of plasma phosphorus decreased markedly as the level of dietary calcium increased. 7. These results confirm that CT secretion in chicks is stimulated by hypercalcaemia but indicate that the hormone is unable to restore the plasma calcium to normal when diets high in calcium are fed.

INTRODUCTION

Until recently, no experimental work has been published that provides convincing evidence for a hypocalcaemic action of calcitonin (CT) in normal birds with a normal circulating levels of calcium and this has raised doubts as to the significance of CT in calcium homoeostasis in birds. However, there is ample evidence that hypercalcaemia stimulates CT secretion by the ultimobranchial glands (Ziegler et al., 1969; Copp rt al., 1970) and Luck et al. (1980) reported that salmon CT significantly reduced the concentration of ionic calcium, but not that of total calcium, in the plasma of laying hens that were not actively engaged in egg shell calcification. Furthermore, Baimbridge & Taylor (1980) observed a significant hypocalcaemia in chick embryos in which endogenous release of CT was stimulated by isoprenaline. Two reasons may be suggested to explain the failure of other workers (e.g. Gonnerman et a/., 1972) to demonstrate a hypocalcaemic action for CT in normal birds viz. (1) only total calcium was measured rather than ionic calcium; (2) due to the speed with which parathyroid hormone acts in birds (Candlish & Taylor, 1970) the hypocalcaemia induced by CT was rapidly overcome. The normal level of plasma calcium in laying birds is approximately twice that of immature birds of both sexes and mature male birds but the level of ionic calcium is the same, except during periods of active * Present address: Department of Physiology, Faculty of Medicine, University of British Columbia, 2075 Wesbrook Mall, Vancouver, B.C., Canada. 647

egg shell formation, when it is substantially reduced (Luck & Scanes, 1979). The extra calcium in the blood of laying birds is bound to the phospholipoglycoprotein, vitellogenin, which is synthesised in the liver and transported thence to the egg yolk (Urist er al., 1960). The ultimobranchial glands respond to the level of ionic calcium in the blood and the only time during the egg cycle when an elevated level of ionic calcium is found is immediately after the calcification of an egg shell has been completed and before the calcification of the next shell begins (Luck & Scanes, 1979). The rate at which calcium is removed from the blood for shell formation is extremely high and this drain is balanced by the absorption of calcium from the gut and the mobilisation of calcium from the medullary bone (Taylor, 1965). When shell calcification ceases, therefore, the rate at which calcium enters the blood exceeds the rate of its removal, thus inducing a hypercalcaemia. The levels of CT in the plasma of birds during the egg cycle have been studied by Dacke et al. (I 972). In Japanese quail the mean plasma CT level was significantly lower during active shell calcification than when no calcification was occurring but CT could not be detected in the blood of laying hens either during shell formation or immediately after oviposition. Dacke er nl. (1972) used a rat bioassay for their CT assays but in the present work a more sensitive radioimmunoassay was used to investigate the CT levels of hens during the laying cycle. Relatively little work has been carried out on the normal circulating level of CT in young chickens but Kenny (1971) found values of 178 and 267~ MRC units/ml in single samples of pooled plasma from, re-

K. G. BAIMBRIDGE and T. G. TAYLOR

648

spectively, male and female chicks using a rat bioassay. In the present CT levels were determined in chicks taining differing amounts of calcium mine the effect on CT secretion of induced by diet.

MATERIALS

Laying

2 months of age work, the plasma fed on diets conin order to detera hypercalcaemia

AND METHODS

hens

A hybrid stram of laying fowl of medium body weight (Ross I) was used in this work. The birds were laying regularly and they had been in lay for about 3 months. They were housed singly in battery cages and fed on a commercial layers’ mash containing 32 g calcium/kg. The lighting regime used was 14 hr light-10 hr dark and 10 birds were studied. Blood samples were taken from the claws (Hertelendy & Taylor, 1961) within 15 min of oviposition and five further samples were collected at intervals of 4 hr to cover the main period of calcification of the next egg to be laid. After the final bleeding, 20 hr after oviposition, the hens were observed every 30 min for a further 16 hr and the time of the second oviposition recorded. Any bird that did not lay within this period was checked every 6 hr for a further 12 hr. Chicks Day-old chicks (Ross I) were reared to 5 wk of age in electrically heated tier brooders on a conventional maizesoya diet containing 11.4 g calcium and 9.4 g total phosphorus/kg (by analysis). At this time three groups of 14-16 birds of each sex were transferred to floor pens and given one of three diets (1) the original rearing diet (control): (2) the original diet supplemented with 19g calcium/kg (as calcium carbonate) to provide a total of 30g calcium/kg: (3) the control diet supplemented with 49g calcium/kg to provide a total of 60g calcium/kg. After 1, 3 and 5 wk on the experimental diet, blood samples (2 ml) were taken from the wing vein of each bird. The birds were weighed at

the end of the experiment. Blood analysis Heparin was used as anti-coagulant for all samples and blood was kept on ice until the plasma could be separated by centrifugation. The plasma was stored in liquid nitrogen until assayed for CT by a sensitive radioimmunoassay using an antibody raised to synthetic salmon CT in rabbits with synthetic salmon CT itself as the standard (Baimbridge & Taylor, 1980). Calcium was determined in the plasma by atomic absorption spectrophotometry (Pybus et ul.. 1970) and inorganic phosphorus with a Technicon

Series II autoanalyser.

RESULTS The full results of the hen experiment are shown in Table 1. Only 4 of the 10 birds laid within 48 hr of the original oviposition, 2 after 24 hr (Bird Nos 2 and 3) and 2 after approx 3 hr (Bird Nos. 1 and 7). It is clear that ovulation was delayed in the 2 latter birds, as it is most unusual for birds to lay at night, and it seems likely that they were upset by the handling and bleeding. In general, the decreases in diffusible plasma calcium values associated with shell calcification are accompanied by decreases in total calcium and, after oviposition, increases in total calcium reflect increases in

diffusible calcium (Taylor & Hertelendy, 1961) and the levels of total calcium observed in this experiment were consistent with this earlier work. Thus, in all 10 birds the plasma calcium values were higher 4 hr after oviposition than at the time of oviposition itself, when the birds were still suffering from the effects of shell calcification. Otherwise, the total calcium figures were somewhat variable. In most instances, the plasma concentrations of inorganic phosphate were higher during active shell calcification than in the absence of shell formation, confirming the observations of Luck & Scanes (1979). The CT results may be summarized as follows: 1. In 6 of the 10 birds CT was detectable shortly after oviposition at a mean concentration of 1.54 ng synthetic salmon calcitonin/ml (SEM 0.23. range 0.95-2.48 ng/ml). 2. CT was not detectable during active shell calcification. 3. In birds having a laying pause of more than 48 hr, i.e. in birds laying the last egg of a sequence, CT was not detectable between 4 and 20 hr after oviposition. 4. In the 4 birds that laid a second egg within 48 hr of the first, CT was detectable until about the time of the next ovulation. The weights of the chicks at the end of the experiment, when they were 10 wk of age, are given in Table 2. The cock birds fed on the diet with the highest amount of calcium were significantly lighter than the controls (P < 0.01, r-test) but none of the treatment differences was significant in the pullets. Food spillage was too great for food intake to be measured but it seems likely that food intake was depressed in the cocks given the diet containing 60 g calcium/kg. The mean plasma calcium, phosphorus and CT values observed in the chick experiment after the birds had been on the experimental diets for 3 wk are shown in Fig. 1. The results after 1 and 5 wk were similar and there were no consistent differences between the sexes. The general picture was one of a progressive increase in the concentrations of calcium and CT in the plasma as the level of calcium in the diet increased, coupled with a progressive decline in plasma inorganic phosphorus levels. The mean control value for plasma calcium was 2.55 mmol/l and the maximum concentration reached with the highest level of dietary calcium was 3 mmol/l, an increase of about 17%. Mean plasma inorganic phosphorus concentrations fell from 1.7-2.0 mmol/l in the controls to a minimum of 1.2 mmol/l for the diet containing 60 g calcium/kg. The control birds all had mean circulating levels of CT within the range 1.05-l 64 ng synthetic salmon CT equivalents/ml and the maximum levels observed (with the birds fed on the diets containing the most calcium) were 2.2-2.5 ng/ml), slight variations occurring with age and sex. DISCUSSION

The results pothesis first major role for restriction of the time when

of the hen experiment support the hyput forward by Taylor (1970) that a CT in laying birds is the prevention or hypercalcaemia in the period between calcification of one shell ends and that

Plasma calcitonin in laying hens and chicks

649

Table 1. Concentrations of calcitonin (CT, ng synthetic salmon calcitonin/ml~. tota calcium (mmol~l) and inorganic phosphorus (mmol/I) in the plasma of individual hens at oviposition and at 4-hourly intervals thereafter Hen No.

Interval between eggs (hr)

Time of first

Time of second

oviposition

oviposition

0930

20.30

35

0945

1035

25

1135

1225

25

I200

-

>48

1205

-

>48

1230

-

>48

1240

0230

38

I425

-

>48

1510

-

>48

1515

-

>48

ND

Time after first oviposition (hr) Plasma constituent

FT Ca P CT Ca P CT Ca RCa P

4

8

12

16

20

1.47 4.58 1.15 1.22 5.25 0.73 I .90 5.53 0.72 ND 6.05 1.37 0.95 5.00 1.28 ND 5.63 1.15 2.48 4.80 1.11 ND 5.78 1.31 1.21 5.75 1.40 ND 7.83 1.66

1.80 4.70 1.21 0.74 5.58 0.93 0.66 6.25 1.30 ND 8.23 1.78 ND 5.50 1.75 ND 5.88 1.18 2.31 5.05 0.95 ND 6.35 2.32 ND 5.95 2.33 ND 7.93 2.60

0.75 4.70 1.14 ND 5.32 1.12 ND 5.95 1.32 ND 6.30 2.12 ND 5.73 1.96 ND 5.63 1.12 2.45 5.00 0.78 ND 5.20 1.47 ND 5.50 1.48 ND 8.25 1.95

1.00 4.65 2.04 ND 5. I3 1.14 ND 5.80 I .39 ND 6.13 1.80 ND 6.53 1.65 ND 5.90 0.93 I.12 5.35 2.84 ND 5.48 1.51 ND 4.65 1.27 ND 6.70 1.44

ND 4.65 2.32 ND 5.45 1.68 ND 5.68 1.12 ND 5.23 I.44 ND 5.35 2.12 ND 4.78 0.88 ND 4.78 1.43 ND 4.63 I .33 ND 4.20 1.19 ND 6.88 1.32

ND 4.10 2.42 ND 5.50 1.13 ND 5.63 0.51 ND s.t3 1.38 ND 4.7s 0.97 ND 5.50 0.94 ND 4.30 I.1 1 ND 4.63 0.98 ND 5.00 0.74 ND 5.50 IS8

not detectable (co.5 ngiml).

of the next one begins, the period during which the highest concentration of ionic calcium occurs (Luck & Scanes, 1979). In the 4 hens sampled in the middle of a laying sequence, CT was detectable until at least the time of the next ovulation and in the 2 birds that laid a second egg 25 hr after the first the hormone was detectable until the latter egg entered the shell gland (about 4 hr after ovulation). CT could not be detected during active shell formation in any of the birds and using the values reported by Luck & Scanes (1979) it would appear that CI is secreted when the plasma level of ionic calcium exceeds 1.5 mmol/l. Six of the hens in this experiment were studied after the oviposition of the last egg of a laying sequence Table 2. Live weights of the experimental birds at the age of 10 wk after 5 wk on diets differing in their concentration of calcium (Mean values for 16 birds/group + SEM) Dietary calcium (g&g)

Cocks Pullets

CT Ca P CT Ca P CT Ca P CT Ca P CT Ca P CT Ca

0

(CoZrol) I558 k 35 1049 f 25

30

60

1497* + 16 1078 + 38

1394t f 16 1023 f 18

* 14 birds only. t Significantly less than controls (P <:0.01, r-test) (all other comparisons between treatments non-significant).

and in only 2 of these birds was CT detectable. No information is available on the plasma concentration of ionic calcium at this time but the low CT values observed suggest that the levels of ionic calcium were below the threshold value for CT release and that the absorption of calcium from. the gut and/or the release of calcium from the skeleton of these birds was less than in hens that were in mid-sequence. This, in turn, suggests that the production of the major kidney hormone controlling calcium metabolism, 1.25-dihydroxycholecalciferol (I ,25-DHCC) may have been reduced in the birds laying the terminal egg of a sequence. In birds that are laying regularly, high concentrations of oestrogen (Peterson & Common, 1972), androgens (Peterson et al., 1973) and progesterone (Peterson & Common, 1971) are present in the plasma 4-6 hr before oviposition but the pre-ovulatory peaks of these reproductive steroids would not have occurred in the 6 birds that laid the final egg of a sequence. Tanaka et al. (1978) have shown that oestrogen, androgen and progesterone, given together, exert a major stimulatory action on the renal 25-hydroxycholecalciferol-1-hydroxylase in castrated male chickens but it is doubtful if changes in the plasma concentrations of these steroids could themselves induce the short-term changes in 1,25-DHCC production that we are discussing here. However, both Tanaka et al. (1976) and Sedrani & Taylor (1980) have demonstrated a marked synergism between para-

K. CLBAIMBRIDGE and T. G. TAYLOR

650

MALES

3.2

FEMALES

r (a)

2.6 2.4

1.6 (b)

Fig. I. The effects of diets differing in their concentration of calcium on the plasma levels of (a) total calcium, (b) inorganic phosphorus and (c) calcitonin in male and female chicks after 3 wk on the experimental diets when the birds were 8 wk of age. Mean values for 16 chicks per group except for the group of males given 30g calcium/kg in which N = 14. Error bars represent SEM. Means were compared with control means (I 1 g calcium/kg) by t-test and significant differences indicated by *(P < 0.05), **(P< 0.01). ***(P< 0.001). n 11 g calcium/kg 0 30 g calcium/kg, LSI60 g calcium/kg. thyroid hormone (PTH) and reproductive steroids in activating the renal I-hydroxylase and this synergism may provide an explanation for the differences in CT secretion between ovulating and non-ovulating birds

observed in this study, i.e. PTH may have stimulated a greater production of 1,25-DHCC in the presence of the elevated levels of reproductive steroids occurring in the birds that went on to lay again. The results of the chick experiment demonstrate conclusively that CT is secreted in the growing chick in response to hypercalcaemia. However, the hypercalcaemic stimulus of the high-calcium diets was too powerful for the hypercalcaemia to be overcome by CT and the relatively high concentration of CT in the control birds suggests that they too were under a certain hypercalcaemic stress. (The recommended intake of calcium for birds of this age is 7.5 g/kg (Agricultural Research Council, 1975). whereas the control diet used in this experiment supplied 11 g calcium/kg.) On the control diet the concentration of CT in the plasma was 1.05-1.64 ng synthetic salmon CT equivalents/ml, i.e. 4800-7850 p MRC units/ml, 20-30 times higher than the values observed by Kenny (1961) for 2 month-old chicks.

In the hen experiment the concentration of CT in the plasma at the time of oviposition varied between 1 and 2.5 ng synthetic salmon CT equivalents/ml, a range essentially the same as that observed in these chicks, the minimum corresponding to the mean level observed in the control cocks after 3 wk and the maximum to that in the pullets given the very high calcium diet at the same stage of the experiment. The mean level of CT in chicks at hatching is 2.7 ng synthetic salmon CT/ml and in l-day old chicks 1.8 ng/ml but in 2-day old chicks CT is not detectable (co.50 q/m!) (Baimbridge & Taylor, 1980). It would be of interest to determine at what age between 2 days and 6 weeks the level of CT in the plasma starts to rise. The relationship between dietary calcium and circulating CT when less than the recommended intake of calcium is fed should also be investigated and we would expect that plasma CT would fall as the level of dietary calcium is reduced. We conclude from these experiments that hypercalcaemia is a major stimulus to the secretion of CT in birds and that there is no reason to doubt that one of the physiological roles of CT in birds, as in mammals, is to cc ribat hypercalcaemia, just as one of the

Plasma calcitonin in laying hens and chicks physiological roles of PTH is to overcome h_vpucalcaemia rather than to produce hJ,percalcaemia. In our view, altogether too much attention has been given to the difficulty of inducing a hypocalcaemia in birds by exogenous CT. Acknowledgement-We wish to thank Dr J. W. Bastian (Armour Pharmaceuticals Company. Kankakee, IL U.S.A.) for a generous gift of salmon calcitonin.

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