Circulating thyroid hormones in the chicken before and after hatching

GENERAL

AND

COMPARATIVE

Circulating

ENDOCRINOLOGY

Thyroid

29, 21-27 (1976)

Hormones in the Chicken before and after Hatching T. F. DAVISON

Houghton Poultry Research Station, Houghton,

Huntingdon

PEl7 2DA, Cambs, England

Accepted October 30, 1975 Plasma protein bound iodine (PBI) and total thyroxine (TTJ change markedly during and after hatching. These changes do not follow exactly the same patterns although both show a peak, 6.0 pg I/100 ml and 3.0 kg TJlOO ml, in the l-day-old chick and decline in the week after hatching. Evidence is presented to show that PBI is not a good index of plasma iodohormone concentration in the chicken. T, and T3 are the only identifiable iodoamino acids in plasma from l-day-old chicks and the T,:T3 ratio is 4. Only minute amounts of T, (0.13-0.28%) and T, (O.ll-O.l%) are in free solution in chicken plasma. The absolute concentration of free T, falls in the 3 weeks after hatching from 8.4 to 3.1 ng T.JlOO ml.

Thyroxine (T4) and triiodothyronine (T3) with the albumin fraction (Farer et al., 1%2; Tritsch and Tritsch, 1%5). As much have been identified in the blood of domestic birds but their ratio differs considembly as 40% T, and 74% T, have been reported from that in mammalian blood (Wentworth to be in free solution in chicken plasma and Mellen, 1961). Moreover, they are (Heninger and Newcomer, 1964), although equipotent in chickens and have similar half more recently the proportion of free T, has lives (Heninger and Newcomer, 1964), been shown to be only 0.34% (Refetoff ef whereas in mammals T3 is 5-7 times more al., 1970). In view of these reported differpotent and possesses a shorter half life (see ences in the pattern of binding and disttibuHeninger and Newcomer, 1%4). The con- tion of T, and T, in birds and mammals and centration of circulating iodohormones in the increasing emphasis being placed upon the chicken is of the order of l-2 ~/RIO ml the free moiety of a hormone as the plasma, less than half that found in domes- physiologically active form (see Westphal, tic mammals or man. 1970) it seemed prudent to measure the The markedly low protein bound iodine proportions of free T, and T, in chicken (PBI) and total thyroxine (TTJ concentmplasma. tions have been attributed to differences in The thyroid gland has been implicated in the binding of thyroid hormones by the the initiation of active hatching in the chicken plasma proteins (Ringer, 1%5). The chicken (Freeman, 1974) but little attention plasma of most mammals contains an inter- has been paid to any changes in circulating a-globulin (thyroxine binding globulin, T, and T, which occur during the perinatal TBG) which selectively binds T, and to a period. In mammals the thyroid axis is very lesser extent T, and carries the major labile during the perinatal period with proportion of the circulating iodohormones marked increases in the concentration of (Farer et al., 1%2). However this protein is thyroid stimulating hormone (TSH), T.,, T, not present in avian blood (Tata and Shella- and the dialysable fraction of T, immedibarger, 1959) and T, is transported in ately after parturition (see Nathanielsz, association with the albumin and pre-albu1975). The present paper describes some of min fractions, while T, is only associated the changes in plasma thyroid hormones 21 Copyright All rights

@ 1976 by Academic Press. Inc. of reproduction in any form reserved.

22

T. F. DAVISON

during hatching chicken.

and development

in the

METHODS Birds Eggs and chickens from the Houghton Poultry Research Station’s Light Sussex and Rhode Island Red flocks, reared in conventional conditions, were used.

Blood Sampling Embryos. Fertile eggs were incubated in a Curfew still air incubator and blood samples were obtained from the umbilical vein of embryos after removing the overlying shell membranes from the narrow pole of the egg. Blood samples were pooled to provide 2 ml. After centrifugation the plasma was removed and stored at -20” until required for analysis. Chickens. Samples were obtained by frontal cardiac puncture and where necessary were pooled to provide an adequate volume of blood. With chickens aged 8 weeks or more, blood was collected from the alar vein. The blood was allowed to clot at room temperature and the serum was collected by centrifugation at 2500s at 4”. The serum was stored at -20” until required. When plasma was required, blood was collected in heparinized syringes, centrifuged and the plasma was stored at -20”.

PBI Meakurement The alkaline, wet ash method of Acland (1957) as modified by Foss et al. (1960) was used. Iodine losses during protein precipitation and incineration were estimated with a standard serum (Dade).

TT 4 Measurement Plasma TT, concentration was measured by competitive protein-binding analysis using 1.6% human serum, in 50 m&f barbitone buffer @H 8.6). as a source of thyroxine binding globulin. The ion exchange resin procedure of Murphy and Jachan (1%5) was followed with modifications suggested by Ekins et al. (1%9). In view of the low concentration of thyroxine in fowl serum duplicate I ml samples of ethanol extract were dried and used for analysis. Loss of thyroxine during the ethanol extraction was determined by labeling plasma with 1Z51-Td, determining T, loss and applying the appropriate correction factor to the results. An indication of the precision of the assay was obtained from estimations of T, in 10 identical pooled plasma samples. This gave a value of 1.28 ).g TdIOO ml plasma with a SEM of 0.05.

contained in 0.1 ml sterile isotonic saline, was injected through a small hole in the blunt polar region of the shell onto the vascular bed beneath. The hole was then sealed with a little molten paraffin wax (mp 60”) and the eggs were returned to the incubator. Blood was obtained from the chicks 24 hr after hatching. Separation uf iodoamino acids. The iodoamino acids from 5 ml plasma were separated and concentrated on a column of Dowex 5OW x 8 (200-400 mesh) resin as described by Goldsmith et al. (1969). The amino acids, together with a small amount of iodide, were eluted with 4 ml 2 M triethylamine in acetone:water (l:l) evaporated to dryness and the residue was dissolved in 100-200 ~1 methanol ammonia (1: 1) and streaked onto Whatmann 3 mm filter paper for ascending chromatography in a solvent system consisting of n-butanol:ethanol:0.5 M NH,OH (5:1:2). The area about the origin was treated with a solution of 0.1% bovine serum albumin and dried before the applicaion of the extract to reduce deiodination in the sample during application (Taurog, 1963). The system was run for 24 hr at room temperature. Samples of nonradioactive monoiodotyrosine (MIT), diiodotyrosine (DIT), T,, T, and NaI, each at a concentration of 2 mg/ml, were applied to the paper to determine the RI values. Upon completion the strip of paper containing the nonradioactive standards was removed after drying, sprayed with 0.2% ninhydrin in n-butanol:acetic acid (95:5) to locate the positions of the iodoamino acids and 0.1% PdCI, in 10% HCl to locate the NaI. The Revalues were: T,, 0.56; T,, 0.76; DIT, 0.18; MIT, 0.27 and NaI, 0.39. The strip of paper containing the lZJI labeled sample was divided into I cm sections and each section counted in a y counter (Packard Autogamma).

Measurement of the Dialysable Fraction of T4 and T3

The percentage dialysable fraction of T, was measured by the magnesium precipitation method of Sterling and Brenner (1966). Approximately 0.8 /.Ki losI-T, with a specific activity of 50 PC&g was added to 3.5 ml serum. The percentage dialysable fraction of T, was similarly estimated (Nauman et al., l%7) using 3.5 ml serum labeled with 1.2 &i *2sI-T3, specific activity 50 pCi/l*g. The purity of individual isotopically labeled compounds was confirmed by ascending chromatography on Whatmann 1 filter paper at room temperature in a solvent system consisting of n-butanol:glacial Separation of T 4 and T 3from Plasma acetic:water (75: IO: 15). Standard solutions (2 m&I/ml) Thirty 15-day embryos were each injected with 10 of nonradioactive NaI and the sodium salts of T., or &i carrier-free NaY, specific activity 71 &i/pg T, were used to determine Rf values. The dried (Radiochemical Centre, Amersham). The isotope. papers were stained with ninhydrin to locate the

CHICKEN

THYROID

23

HORMONES

reached a peak in the l-day-old chick. It had declined thereafter to reach a constant level of 2 #g/lo0 ml plasma at 7 days. Changes in plasma TT, concentration did not mimic those in PBI concentration (Table 1). TT, concentration was highest in the 17-day-old embryo but declined until the day of hatching (see 0 day, Table 1). After a transient increase (P = 0.02) in the ldayold chick the TT., concentration remained FIG. I. Changes in the plasma PBI concentration in the chicken from 4 days before hatching (17 days relatively stable during the following 14 incubation) to &week-old. The day hatching took weeks. No sex differences were noted at 3, place is marked by H and the vertical bars represent 4 or 6 weeks. In females (males were not standard errors. examined) the concentration fell during maturation but increased (P < 0.05) later. standards, divided into 1 cm sections and counted in When the T, iodine concentration (Tr a y counter. Contamination of the lo51-Ta and 1251-Ts Q-O.65 x T4 concentration (Table l&is with nonhormonal It51 was never more than 2%. compared with the PBI concentration (Fig. RESULTS 1) the ratio of PBI:TJ can be obtained. PBI concentration (Fig. 1) increased after This ratio increased from 1.4 in the 17day the 19th day of incubation (P = 0.01) and embryo to a maximum of 4.6 in the Zday,

l-11

TABLE CHANGESINTHE

PLASMA

TOTAL

THYROXINE

1

CONCENTRATION

IN EMBRYOSANDCHICKENSOF

VARIOUS

AGES n

T4 oLg/loO ml)

Age

(days)

0 and 8

Embryos 16 17 18 19

3.44 f 4.39 2 2.73 k 2.41 2

Chickens 0 1 2

2.18 k 0.30 (7) 3.00, O.lO(42) 1.86? 0.09 (10)

0.42 0.03 0.15 0.16

d

0 (4) (5) (8) (3)

(weeks) 1 2 3 4 6 14 19 33 55

2.06 4 1.45 + 1.65 2 2.02 2 1.81 2

0.06 (9) 0.11 (8) 0.07 (17) 0.08 (18) O.lO(15)

I .62 + 2.00 -t 1.90 2 2.07 2 I .42 k 1.38? 1.69 ”

’ Mean rt SEM, the number in the sample is in parentheses.

0.07 0.09 0. I3 0.08 0.07 0.09 0.10

(8) (10) (8) (IO) (9) (12) (7)

1.67 2 0.09 (9) 2.05 2 0.15 (8) 1.71 2 0.16 (7)

24

T. F. DAVISON

old chick. It fell to 1.7-l .8, thereafter. In an attempt to account for these large differences in the neonate, the plasma iodoamino acids of l-day-old chicks were endogenously labeled with Na*251 and separated chromatogmphically (see Fig. 2). Only three iodine containing compounds were found, iodide, T, and T,. The approximate T,:T, ratio was 4. It is clear, therefore, that a substantial proportion of PBI in the neonate chick must be nonhormonal iodide. The largest proportion of free T, occurred in the week after hatching (Table 2), but had declined (P = 0.01) at 3 weeks and again (P = 0.02) at 7 weeks. There was a smaIl increase between 14 and 19 weeks. The percentage of free T3 was highest in the l-day-old but was significantly lower by 7 weeks. The proportion of free T, was significantly greater than free T3 in 1- and 7day-old chicks (P < 0.01) but not at 7 and 19 weeks. The absolute concentrations of free T, (FTJ was obtained by multiplying the percentage free T, (Table 2) by the mean T, concentration (Table l+individual results were not used as pooled samples were obtained from younger birds (Fig. 3). The FT, concentration was highest in the l-dayold chick but it fell thereafter to a new constant at 7 weeks. Plasma

DISCUSSION PBI concentrations

in chickens

FIG. 2. A histogram showing the net counts per minute of a chromatogram of an extract of lz51 labeled plasma run in a solvent system consisting of n-butanol:ethanol:O.SN NH,OH (5:1:2). 0 represents the origin and I the solvent front; further details are given in the Methods.

TABLE THE PERCENTAGE TRIIODOTHYRONINE, PERCENTAGE

2

FREE THYROXINE AND MEASURED AS THE

DIALYSABLE FRACTION, OF CHICKENS OF VARIOUS

IN THE SERUM AGES”

% free Age (weeks)

T4

T3

0.28 2’ 0.04 (9) 0.28-c 0.02

0.19+ 0.01

(6)

(6)

4

0.19%‘0.02 (9) -

7

0. I3 2 0.01

0.14~‘0.01 (3 0. I I * 0.02 (4) -

1 day I 3

(6)

I4

0.13 + 0.005

I9

0.19r‘0.03

(6) 0.16~ 0.005 -

(6) (6)

O.l2* 0.03 (5)

” Standard errors are given and the number in the sample is indicated in parentheses.

aged 1 week or older (Fig. 1) were similar to other published values (Bumgardner and Shaffner, 1957; Mellen and Hardy, 1957; Pefetoff cf al 1970). Changes in PBI throughout thk’ perinatal period in the chicken have, hitherto, not been reported; their significance is discussed below. Plasma TT, concentrations in chickens aged 2 days or older (Table 1) are within

1 0 FIG. 3. Changes in the concentration of tree thyroxine in the serum of chickens from l-day-old to lPweek-old.

CHICKEN

THYROID

the range of values obtained by competitive protein-binding (May et al., 1973) and radioimmunoassay (Newcomer, 1974). In the assay system utilizing human TBG as a binding protein, T, possesses one third of the binding activity of T, (May et al., 1973); in view of the concentrations of T, present in chicken plasma (Fig. 2; Newcomer, 1974) the TT, concentrations reported in Table 2 are unlikely to overestimate the true concentration by more than 8%. The large plasma concentrations of T, and T4 reported by Sadovsky and Benasdoun (1971) were not confirmed. The results (Fig. 2) confirm that T, and T, are the only measurable iodoamino acids present in the plasma. The T,:T, ratio of 4 in the neonate chick is higher than that reported by Wentworth and Mellen (l%l) but smaller than reported by Newcomer (1974). However, both of these groups used older birds. The observed T, concentmtion is substantially greater than in euthyroid mammals, including man (see Burke and Eastman, 1974). It is now appreciated that in mammals T, is at least as important as T, (Anon, 1973; Hoffenberg, 1973). Indeed some consider T, is a prohormone, being deiodinated to T, peripherally (Sterling et al., 1970). At present, it is not known if a similar mechanism exists in the fowl. The results in Fig. 1 and Table 1 suggest that PBI measurements overestimate by a quarter or more the concentration of thyroid hormones, particularly during hatching. PBI is not considered, therefore, a good index of thyroid hormone concentration in the chicken. In both fowl and mammal only minute proportions of T, and T3 exist in the unbound form. The values for percentage free T, (Table 2) are similar to those of Refetoff et al. (1970). Thus, although thyroxine binding globulin is absent, chicken plasma albumins and pre-albumins are capable of binding almost all of the circulating T, and T,. The decline in free hormone levels during the first 3 weeks after hatching (Table 2)

HORMONES

25

may be the result of the increase in the concentration of serum binding proteins during this time, particularly albumin (see Van Stone et al., 1955). Similar changes have been noted in the neonate pig (Slebodzinski, 1%5) and in man (Van den Shriek, 1%9). When absolute concentrations of free T, were calculated for birds aged 3 weeks or older, the values were similar to those for euthyroid mammals (Fig. 3: Refetoff et al., 1970). The free T3 fraction (Table 2) was a quarter of that observed in euthyroid man (Burke and Eastman, 1974). However, if the plasma TT, concentration in immature chickens is about 200 ng TdlOO ml (Newcomer, 1974) and 0.11% is in the unbound state (Table 2) then the absolute concentmtions of free T, will be approximately 200 pg/lOO ml, within the range reported for euthyroid man (Burke and Eastman, 1974). Thus while circulating concentrations of total T, and T, may differ considerably from the values reported in mammals, the absolute concentrations of the free hormones may be similar. Changes in thyroid gland activity and increased PBI and TT, concentrations have been observed in several mammalian species during the perinatal period. In the bovine plasma TSH falls several days before parturition (Nathanielsz, 1975) resulting in a decline in TT, concentration. Increases in T, concentration have been observed in the lamb in the 24 hr after birth (Nathanielsz, 1975), in man (Danowski et al., 1950; Fisher and Odell, 1%9) and the pig (Slebodzinski, 1%5). These increases would seem to be due to the rapid release of TSH from the pituitary as a response to parturition augmented by postnatal cold stress (Fisher and Odell, 1%9). There are similarities in the perinatal changes in thyroid hormones in the chicken and in mammals. The large increase in TT, (Table 1) in the neonate chick could be due to the rapid release of TSH at hatching; the maximum response to exogenous TSH is observed within a few hours (T. F. Davi-

26

T. F. D AVISON

son, unpublished data). After hatching the large increase in body surface, exacerbated by the evaporation of water from the down feathers could act as a cold stress of sufficient magnitude to stitiulate pituitary secretion of TSH. The thyroid gland has been implicated in the hatching of the chick (Freeman, 1974) although it is not certain whether the thyroid hormones exert a general effect on tissues or a specific stimulus for hatching. The aspect requires further investigation. ACKNOWLEDGMENTS The author is grateful to J. Rea for expert technical assistance and to the Director of the Institute of Animal Physiology, Babraham and Mr. J. Bounden of the Radioisotope Department of the same institute for the provision of accommodation during part of this work.

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CHICKEN

THYROID

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HORMONES

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