Effect of Thyroidectomy of Immature Male Chickens on Circulating Thyroid Hormones and on Response to Thyroid-Stimulating Hormone and Chronic Cold Exposure1

PHYSIOLOGY AND REPRODUCTION Effect of Thyroidectomy of Immature Male Chickens on Circulating Thyroid Hormones and on Response to Thyroid-Stimulating Hormone and Chronic Cold Exposure 1 EID E. HADDAD AND MAGDI M. MASHALY2 Department of Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT Previous studies in the author's laboratory established that thyroidectomized (Tx) immature male chickens had significant levels of circulating thyroid hormones, and it was proposed that extrathyroidal tissue might be present. Three experiments were conducted to further investigate this possibility. In all experiments, thyroid glands were removed surgically at 3 wk of age. In the first experiment, birds were kept until 20 wk of age. It was found that only triiodothyronine levels were reduced significantly in the Tx birds. In the second experiment, Tx as well as sham-operated control groups received a single iv injection of bovine thyroid-stimulating hormone (TSH) to determine if extrathyroidal tissue in Tx birds would respond to exogenous TSH. It was found that circulating thyroxine (T4) concentrations in sham-operated control birds, but not Tx birds, were increased following TSH injection. In the third experiment, Tx and sham-operated birds were exposed chronically to cold (7 C), and only circulating T 4 was found to be elevated in both groups. It was concluded that extrathyroidal tissue in Tx birds does not respond to TSH. (Key words: thyroidectomy, thyroid hormones, thyroid-stimulating hormone, cold exposure) 1989 Poultry Science 6 8 : 1 6 9 - 1 7 6 INTRODUCTION

It is well established that the mammalian as well as avian thyroid gland is the unique site for production of the thyroid hormone, thyroxine (T4), and the site of part of the production of triiodothyronine (T3). Yet there have been several reports indicating that measurable levels of thyroid hormones exist in the circulation of the surgically thyroidectomized (Tx) dog (Kallfelz, 1973; Li et al., 1986), rat (Taurog and Evans, 1967; Obregon et al., 1981), Japanese quail (Peczely et al., 1980), chicken (Harvey et al., 1983; Mashaly et al., 1983; Bachman and Mashaly, 1986; 1987), and radiothyroidectomized chicken (Mellen and Wentworth, 1962). The half-life of T 4 and T 3 in chickens lies in the range of a few hours in length (Singh etal., 1967; May et al., 1974; Davison, 1978), which is shorter than the half-life of the hormones' counterparts in mammalian species. In mammals, the oldest age to which a Tx animal was kept under experimental conditions was 6 mo (Obregon et al., 1981). Nevertheless, it has

1 Paper Number 7746 in the journal series of the Pennsylvania Agricultural Experiment Station. 2 To whom correspondence should be addressed.

been proposed that the source of thyroid hormones in the circulation of Tx animals is extrathyroidal tissue. However, little is known about its nature. In Tx rats, a daily injection of 5 mg of iodide for up to 4 wk produced a significant circulating concentration of T 4 (Taurog and Evans, 1967). No changes in serum T 4 and T 3 levels were found in Tx dogs injected with thyroid stimulating hormone (TSH) or thyrotrophin releasing hormone (TRH) (Kallfelz, 1973; Li et al., 1986). Similar results were reported in Tx chickens injected with TRH (Harvey et al., 1983) or exposed to a longer period of light (Peczely et al., 1980). The effect of other thyroidal stimulators or inhibitors on Tx chickens has not been investigated. Exposure to cold has been reported to be associated with an increase in several aspects of thyroid function in the euthyroid rat, including synthesis (Cottle and Carlson, 1956), secretion (Heroux and Brauer, 1965; Straw, 1969), and fecal loss (Straw and Fregly, 1967) of thyroid hormones. Rats exposed chronically to temperatures of 4 C exhibited no changes in serum T 4 concentration, whereas T3 concentrations were increased (Hardeveld et al., 1979). In the domestic fowl, cold exposure stimulated thyroid function in a manner similar to that in mammals (Kuhn and Nouwen, 1978). Exposure of

169

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

(Received for publication August 20, 1987)

HADDAD AND MASHALY

170

MATERIALS AND METHODS

Single Comb White Leghorn male chicks were used in the present study. Chicks were maintained in a starter battery for 1 mo, then moved to a growing battery, with feed and water available ad libitum. Birds were exposed to a 14 h light: 10 h dark photoperiod. In the first experiment, at 3 wk of age, one group of 15 chicks was surgically thyroidectomized under Allobarbitol general (3.5 mL/kg BW) and lidocaine local anesthesia. A second group of 10 chicks served as sham-operated controls. Blood samples (1 mL) were taken from the brachial vein of the same birds 1 day prior to surgery and at 4, 6, 7, 10, 15, and 20 wk of age. Body weights were recorded at 10, 15, and 20 wk of age. In a second experiment, six groups of chicks were used. Three groups of 30 each were surgically thyroidectomized as described above, and the remaining birds (10 chicks/group) were sham operated and served as controls. At 7 wk of age, birds were cannulated (through the brachial vein) under lidocaine local anesthesia. One group of each surgical treatment (Tx birds or sham-operated controls) was injected intravenously with .2 mL saline, .25 IU/kg BW of bovine thyroid-stimulating hormone (TSH; Sigma, St. Louis, MO) or 1IU TSH/kg BW. Blood samples (1 mL) were taken immediately prior to and at 30, 60, 120, and 180 min after either TSH or saline injection. The same birds were bled over the different time periods. In a third experiment, at 3 wk of age a group of chicks (20 chicks) was surgically thyroidectomized as described above. Another group of 15 chicks served as sham-operated controls. Chicks were allowed a 1-wk postoperative re-

covery period, then moved to a temperature-controlled room (7 C) where they were kept for 2 wk. Food and water were available ad libitum, and the same light-dark period established prior to cold exposure was used. Blood samples (1 mL) were drawn from the brachial vein of the same birds immediately prior to and at 1 and 2 wk after cold exposure. All blood samples were taken at the same time of day for each experiment in order to avoid potential diurnal variations in thyroid hormone concentrations (Newcomer, 1974; Klandorf et al., 1978). The success of a complete thyroidectomy was subsequently confirmed at autopsy by gross visual observation and histological examination of the surgical site covering the region from the carotid body to the last lobe of the thymus. Data from chicks that showed any residual thyroid tissue were not included in the results. Blood samples were maintained for 4 h at room temperature to allow clot formation and retraction and then refrigerated (4 C) overnight. Samples were centrifuged, and sera were removed and stored frozen (-20 C) for subsequent radioimmunoassay (RIA). The RIA kits for T 3 and T 4 were purchased from Cambridge Medical Diagnostics (Boston, MA). Radioimmunoassay of serum T 3 and T 4 were validated using three procedures. First, a doseresponse curve was established with various volumes of pooled chicken serum from both Tx and sham chicks. The correlation coefficient between the serum volume and observed hormone concentration was greater than .99 for both surgical treatments. Second, stripped chicken serum was prepared by stirring serum with activated charcoal (10%) overnight under cold (4 C), and centrifuged at 1,100 X g for 15 min. The supernatant was filtered using .22-|xm paper filter purchased from Millipore Corp. (Bedford, MA). Percentage recovery was established by measuring the concentration of thyroid hormones in constant volumes (10 fiL each) of stripped chicken serum to which known quantities of thyroid hormone standards had been added (Tables 1 and 2). Third, T 4 and T 3 levels in stripped serum from both Tx and sham-operated birds were measured and established to be less than the lowest dose of the standard solutions that were provided in the kit (10 and .30 ng/mL for T4 and T 3 , respectively). Most importantly, the fact that high and low concentrations of both hormones were measurable further validates the RIA.

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

Japanese quail to acute cold (-1 to-6 C) resulted in elevation of serum T 3 and T 4 , although serum T 4 levels during the 1st h were decreased (Bobek et al., 1980). Acute exposure of T x birds to cold (10 C) stimulates T 4 to T 3 conversion (Rudas and Pethes, 1986). However, the effect on the profile of thyroid hormones of chronic cold exposure of Tx birds has not been investigated. Under such a condition, it would be expected that serum concentration of both T 4 and T3 would be essentially nondetectable. The profiles of thyroid hormones in Tx chickens and the birds' response to TSH as well as to a chronic cold exposure were investigated.

THYROIDECTOMY AND THYROID HORMONES TABLE 1. Percentage recovery of thyroxine added to stripped serum T 4 Observed

T4 Added

T 4 Recovery

<%)

eig.1 12.5 16.5 35.5 64.0 117.0 197.5

125 83 89 128 117 99

Thin-layer chromatography (TLC; Fisher Scientific, Pittsburgh, PA) was used to further ascertain that substances detected by the assays were, in fact, thyroid hormones. Techniques employed were as described by Taurog et al. (1956) and modified by Wentworth and Mellen (1961) with the exception that 5 and 10 (iL of standard and extracted samples, respectively, were applied. Standards were prepared for both T 4 and T 3 (Free acid, Sigma Chemical Co., St. Louis, MO) using .2 u.g of each dissolved in 1 mL of butanol-ethanol-2 N NH4OH, 5:1:2 and applied directly to the silica gel. An ultraviolet light was used to locate the position of both hormones on the silica gel. Both T 4 and T3 standards were located. Extracted serum from both sham-operated and Tx birds was found to contain compounds corresponding to the position of T 4 but not the T 3 standard. Statistical Analyses. Analyses were conducted for all experiments using the general linear model procedures for repeated measurement described in the SAS (1982) User's Guide. Main effects in the first and third experiments were surgical treatment (Tx or sham-operated controls) and either age (first experiment) or time (third experiment). Interactions between

the two main effects were also analyzed. Main effects for the second experiment were surgical treatment (Tx or sham-operated controls), saline, or TSH injection and time. Interactions were also analyzed statistically. In all experiments, means were compared using Duncan's multiple range test. RESULTS

Experiment 1. Average body weights of Tx birds were significantly (P<.05) lower than those of sham-operated controls at all ages (Figure 1). There were no significant differences in serum T 4 concentrations due to either surgical treatment or interaction between treatment and age (Figure 2). However, there was a significant difference in serum T 4 concentrations due to age. The removal of the thyroid gland significantly (P< .05) reduced serum T 3 concentrations to 1.76 ± .29 ng/mL (x ± SE) below those of sham-operated controls (2.81 ± .21 ng/mL) (Figure 3). There was also a significant (P<.05) difference in serum T3 concentrations due to age or the interaction between surgical treatment and age. Experiment 2. Thyroidectomy significantly (P<.05) reduced overall serum T 4 concentrations (11.14 ± .78 ng/mL, x ± SE) below those of sham-operated birds (19.85 ± 1.96 ng/mL) (Figure 4). Changes in serum concentrations of T 4 and T 3 in response to a single iv injection of TSH are depicted in Figures 4 and 5, respectively. The changes are expressed as percentage responses relative to basal levels (zero time). The TSH, but not saline, injections

TABLE 2. Percentage recovery of triiodothyronine (T3) added to stripped serum T 3 Observed

T3 Added

(%)

l'ig; .35 .70 2.60 5.20 10.40

T 3 Recovery

.43 .67 2.68 5.62 13.11

123 96 103 108 126

Age(Wk)

FIGURE 1. Body weights of both thyroidectomized (Tx) and sham (Sh) birds at different ages (n = 4 and 7 for Tx and Sh, respectively). Means with no common superscripts are significantly different (P<.05).

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

10 20 40 50 100 200

(Tt)

171

172

HADDAD AND MASHALY

60 ..Tx

50-

40

•— 201 10

-i

0

1

1

2

1

4

1

1

1

6

1

8

1

1

1——i

1

1

1

1

1

1

1 0 1 2 1 4 1 6 1 8

1

r

20

Age(Wk) FIGURE 2. Serum concentrations of thyroxine (T4) in both thyroidectomized (Tx) and sham (Sh) birds at different ages (n = 4). Values are x ± SEM. Means with no common superscripts are significantly different (P<.05).

7

-Sh -TX

6 ^5 4

c

'32 11 0

-1

2

4

6

1

1—i

8

1

10

1

1

1

1-

1 2 1 4 1 6 1 8

20

Age(Wk) FIGURE 3. Serum concentrations of triiodothyronine (T3) in both thyroidectomized (Tx) and sham (Sh) birds at different ages (n = 4). Values are x ± SEM. Means with no common superscripts are significantly different (P<.05).

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

CT>30" C

THYROIDECTOMY AND THYROID HORMONES

TABLE 3. Overall serum concentration (x ± SEM) of thyroxine (TA) and triiodothyronine (T3J in thyroidectomized (Tx) and sham-operated (Sh) birds exposed for 2 weeks to cold (7 C)

in the sham-operated birds significantly (P< .05) elevated serum concentrations of T 4 but not T 3 approximately two-fold. The TSH injections of the sham birds caused a progressive increase in circulating T 4 level, which reached a plateau between 60 and 180 min after the injections. In the Tx birds, however, neither TSH nor saline injections changed serum T 4 or T3 concentrations. There was a significant interaction between surgical treatments, TSH, and time for serum concentration of T4 but not T 3 . Experiment 3. A significant difference (P< .05) in overall serum T 4 and T3 concentrations

Surgical treatment

T4

T3

Sh Tx

34.1 ± 2.1 a 26.4 ± l.l fc

(ng/mL) 3.79 ± .21 a .90 ± .15fc

a' b Means with no common superscripts within each hormone are significantly different (P<.05).

-saline -.25IU TSH -1IUTSH

Sh 80 60

—Saline —.25IU TSH —-1IUTSH

40 20 0 -20 -40

-i

1

1

v o> c o o

1-

o

-60 -80

80

Tx

60

40

40

20-I

20 0

0-20-

-20

-40-

-40-I

-60-80

1

0

30 60 120 Time(min)

180

FIGURE 4. Percentage changes of thyroxine (T4) concentrations in serum of both thyroidectomized (Tx) and sham (Sh) birds following injection of saline or different doses of bovine thyroid stimulating hormone (TSH) (n = 4 or 5). Values are x ± SEM.

1

-60

0

30 60 120 Time(min)

180

FIGURE 5. Percentage change of triiodothyronine (T3) concentrations in serum of both thyroidectomized (Tx) and sham (Sh) birds following injection of saline or different doses of bovine thyroid stimulating hormone (TSH) (n = 4 or 5). Values are x ± SEM.

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

480 - Sh 420 360 300 240 180 120 60 0 » c o -60

173

174

HADDAD AND MASHALY DISCUSSION

601 50

140 CT>30

c "20-

Time(Wk) FIGURE 6. Serum concentrations of thyroxine (T4) in both thyroidectomized (Tx) and sham (Sh) birds at different times following exposure to cold (7 C; n = 9). Values are x ± SEM. Means with no common superscripts are significantly different (P<.05).

was found due to the surgical treatment (Table 3). Data for serum concentrations of T 4 and T 3 , throughout the experiment, are presented in Figures 6 and 7, respectively. Overall serum concentration of T4 but not T3 was significantly (P<.05) increased with age. There was no interaction effect between surgical treatment and time on serum concentration for either T4 or T3.

>!J en c

f0

1

2

Time(Wk) FIGURE 7. Serum concentrations of triiodothyronine (T3) in both thyroidectomized (Tx) and sham (Sh) birds at different times following exposure to cold (7 C; n = 9). Values are x ± SEM. Means with no common superscripts are significantly different (P<.05).

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

10

Although histological examinations of the thyroid region at autopsy revealed no thyroidal tissue in the thyroidectomized birds, thyroid hormones, especially T4, had been measured in notable quantity in the circulation of the Tx birds for as long as 17 wk postsurgery. There have been several explanations for the possible source(s) of thyroid hormones in the circulation of hypothyroid T x animals. Sil va (1986) reported that in mammals there are two types of 5'monodeiodinase enzymes (5'D)-5'DI and 5'DII. The former is found mainly in the liver and kidney, whereas the latter is responsible for local conversion of T 4 to T 3 in pituitary, brain, and brown adipose tissue (BAT). In euthyroid animals, both are active, whereas in athyroid animals, only 5'DII is active. The activity of the 5'D enzymes have been also investigated in euthyroid chicken embryos (Hughes and McNabb, 1986). It is possible that measured T 3 in the circulation of Tx birds may have been a product of the activity of 5'D enzymes; however, presence and activity of 5'D systems after thyroidectomy in avian species have not been well established. In addition, Griminger (1986) reported that avian species do not have BAT. Measured T 4 in the circulation of Tx birds is of unknown source. However, the T 4 may actually be immunoreactive protein (Klandorf etal., 1981; Harvey et al., 1983). It may also be synthesized from a precursor other than thyroglobulin, such as thyroalbumin (Lissitsky etal., 1968; Desai et al., 1974). This possibility could be true, as Taurog and Evans (1967) reported that administration of iodide, in the milligram range, to severely hypothyroid rats resulted in a thyroid hormone-like effect. This finding (Taurog and Evans, 1967) suggests that a thyroid hormonelike material, to some extent, is being synthesized in hypothyroid Tx animals. Obregon et al. (1981) and Rudas and Pethes (1986) reported that tissues from T x animals have a notable quantity of T 4 and T3 even several weeks after thyroidectomy. Evidence supporting the possibility that thyroid hormones may be synthesized extrathyroidally has been reviewed (Taurog, 1974). The existence of extrathyroidal tissue has been suggested by many investigators. Approximately 50% of adult dogs have extrathyroidal tissue embedded in fat on the intrapericardial aorta (Martin and Capen, 1979). Nevertheless, other glands concentrate iodide in a manner that is unaffected by thyroid hormones or TSH, as

THYROIDECTOMY AND THYROID HORMONES

REFERENCES Ahren, B., 1984. Influence of VIP on thyroid hormone secretion. Peptides 5:305-308. Ahren, B., 1986. Thyroid neuroendocrinology: Neural regulation of thyroid hormone secretion. Endocrine Rev. 7:149-155. Ahren, B., J. Alumets, M. Ericsson, J. Fahrenkrug, L. Fahrenkrug, R. Hakanson, P. Hedner, I. Loren, A. Melander, C. Rerup, and F. Sundler, 1980. VIP occurs in intrathyroidal nerves and stimulated thyroid hor-

mone secretion. Nature 287:343-345. Ahren, B., R. Hakanson, and C. Rerup, 1982. VlP-stimulated thyroid hormone secretion: Effect of other neuropeptides and a- or ^-adrenoceptor blockade. Acta Physiol. Scand. 114:471^173. Bachman, S. E., and M. M. Mashaly, 1986. Relationship between circulating thyroid hormones and humoral immunity in immature male chickens. Dev. Comp. Immunol. 10:395-403. Bachman, S. E., and M. M. Mashaly, 1987. Relationship between circulating thyroid hormones and cellmediated immunity in immature male chickens. Dev. Comp. Immunol. 11:203-213. Banerjee, R. K., and A. G. Datta, 1981. Gastric peroxidase localization, catalytic properties and possible role in extrathyroidal thyroid hormone formation. Acta Endocrinol. 96:208-214. Banerjee, R. K., and A. G. Datta, 1982. Iodide transport and organification in extrathyroidal tissues. Indian J. Biochem. Biophys. 19:171-174. Bobek, S., J. Niezgoda, M. Pietras, M. Kacinska, and Z. Ewy, 1980. The effect of acute cold and warm ambient temperatures on the thyroid hormone concentration in blood plasma, blood supply, and oxygen consumption in Japanese Quail. Gen. Comp. Endocrinol. 40:201210. Cottle, M., and L. D. Carlson, 1956. Turnover of thyroid hormones in cold-exposed rats determined by radioactive iodine studies. Endocrinology 59:1-11. Davison, T. F., 1978. The turnover of thyroxine in the plasma of the domestic fowl (Gallus domesticus). Gen. Comp. Endocrinol. 36:380-387. De, S. K., C. K. Ganguly, T. K. Chakraborty, A. K. Bose, and R. K. Banerjee, 1985. Endocrine control of extrathyroidal peroxidases and iodide metabolism. Acta Endocrinol. 110:383-387. Deme, D., J. Pommier, and J. Nunez, 1978. Specificity of thyroid hormone synthesis: The role of thyroid peroxidase. Biochem. Biophys. Acta 540:73-82. Desai, K. B., M. N. Mehta, M. C. Patel, S. M. Sharma, L. Ramanna, and R. D. Ganatra, 1974. Familial goitre with absence of thyroglobulin and synthesis of thyroidal hormones from thyroidal albumin. J. Endocrinol. 60:389-397. Griminger, P., 1986. Lipid metabolism. Page 355 in: "AvianPhysiology," P. D. Sturkie, ed. Springer-Verlag, New York, NY. Hardeveld, C. Van., M. J. Zuidwijk, and A.A.H. Kassenaar, 1979. Studies on the origin of altered thyroid hormone levels in the blood of rats during cold exposure. Acta Endocrinol. 91:473-483. Harvey, S., R. J. Sterling, and H. Klandorf, 1983. Concentrations of triiodothyronine, growth hormone, and luteinizing hormone in the plasma of thy roidectomized fowl (Gallus domesticus). Gen. Comp. Endocrinol. 50:275-281. Heroux, O., and R. Brauer, 1965. Critical studies on determination of thyroid secretion rate in cold-adapted animals. J. Appl. Physiol. 20:597-606. Hughes, T. E., and F. M. Anne McNabb, 1986. Avian hepatic T-3 production by two pathways of 5'monodeiodination: Effects of fasting and patterns during development. J. Exp. Zoo. 238:393-399. Kallfelz, F. A., 1973. Observations on thyroid gland function in dogs: Response to thyrotropin and thyroidectomy and determination of thyroxine secretion rate. Am. J. Vet. Res. 34:535-538.

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

is the thyroid. However, these glands, in contrast to the thyroid, cannot store appreciable quantities of iodine (Banerjee and Datta, 1982). Furthermore, other peroxidases (lactoperoxidase and gastric peroxidase) have the characteristic of thyroid peroxidase in catalyzing iodination of amino acid in thyroglobulin (Taurog et al, 1974; Deme et al, 1978; Banerjee and Datta, 1981; 1982). This suggests that some of these glands may be stimulated, in the absence of the thyroid gland, to synthesize thyroid hormones. This possibility is supported by the finding of De et al. (1985) that in Tx rats, iodide concentration in the stomach is stimulated. However, the mechanism of synthesis and secretion of thyroid hormone-like material by these glands is unknown. In the present study, Tx birds did not respond to TSH. These results are in agreement with findings of Kallfelz (1973), Harvey et al. (1983), and Li et al. (1986), who reported that Tx animals did not respond to either TSH or TRH administration. It may be possible that endogenous TSH is maximal or that the proposed extrathyroidal tissue requires a higher level of TSH to respond. It may also be possible that this extrathyroidal tissue is controlled by a system other than the TRH-TSH system. For example, vasoactive intestinal polypeptide (VIP) has been reported in mammals to act as an intrathyroidal regulator (Ahren et al., 1980; 1982; Ahren, 1986). It is secreted by VIPergic nerves that innervate the thyroid follicular cells. It is also found in endocrine cells throughout the gastrointestinal tract. It has been suggested that VIP activates thyroid hormone secretion by a mechanism mat is distinct from that of TSH, though adenosine 5'-monophosphate may mediate both effects (Ahren, 1984). Based on conditions and results of the present study, the data suggest that there may be another tissue that has the ability to synthesize thyroid hormone-like material in the absence of the thyroid gland.

175

176

HADDAD AND MASHALY tomized rats. Endocrinology 100:908-918. Peczely, P., G. Pethes, and P. Rudas, 1980. Interrelationship between thyroid and gonadal function in female Japanese quail kept under short and long photoperiods. J. Endocrinol. 87:55-63. Rudas, P., and G. Pethes, 1986. Acute changes of the conversion of thyroxine to triiodothyronine in hypophysectomized and thyroidectomized chickens exposed to mild cold (10°). Gen. Comp. Endocrinol. 63:408-413. SAS, 1982. SAS User's Guide: Statistics. SAS Inst. Inc., Cary, NC. Silva, J. E., 1986. Brown adipose tissue: An extrathyroidal source of triiodothyronine. New Physiol. Sci. 1:119122. Singh, A., E. P. Reineke, and R. K. Ringer, 1967. Thyroxine and triiodothyronine turnover in the chicken and the Bobwhite and Japanese Quail. Gen. Comp. Endocrinol. 9:353-361. Straw, J. A., 1969. Effects of fecal weight on thyroid function in cold exposed rats. J. Appl. Physiol. 27:630633. Straw, J. A., and M. J. Fregly, 1967. Evaluation of thyroid and adrenal pituitary function during cold acclimation. J. Appl. Physiol. 23:825-830. Taurog, A., 1974. Biosynthesis of iodoamino acids. Page 101 in: Handbook of Physiology: Endocrinology, Vol. 3. Sec. 7. R. O. Greep and E. B. Astwood, ed. American Physiological Society, Washington, DC. Taurog, A., M. L. Dorris, andL. Lamas, 1974. Comparison of lactoperoxidase- and thyroid peroxidase-catalyzed iodination and coupling. Endocrinology 94:12861294. Taurog, A., and E. S. Evans, 1967. Extrathyroidal thyroxine formation in completely thyroidectomized rats. Endocrinology 80:915-925. Taurog, A., J. D. Wheat, and I. L. Chaikoff, 1956. Nature of the I 13 ' compounds appearing in the thyroid vein after injection of iodide I 13 '. Endocrinology 58:121— 131. Wentworth, B. C , and W. J. Mellen, 1961. Circulating thyroid hormones in domestic birds. Poultry Sci. 40:1275-1276.

Downloaded from http://ps.oxfordjournals.org/ at H.E.C. Bibliotheque Myriam & J. Robert Ouimet on June 28, 2015

Klandorf, H., P. J. Sharp, and I.J.H. Duncan, 1978. Variations in levels of plasma thyroxine and triiodothyronine in juvenile female chickens during 24- and 16-hr light cycles. Gen. Comp. Endocrinol. 36:238-243. Klandorf, H., P. J. Sharp, and M. G. Macleod, 1981. The relationship between heat production and concentrations of plasma thyroid hormones in the domestic hen. Gen. Comp. Endocrinol. 45:513-520. Kuhn, E. R., and E. J. Nouwen, 1978. Serum levels of triiodothyronine and thyroxine in the domestic fowl following mild cold exposure and injection of synthetic thyrotropin-releasing hormone. Gen. Comp. Endocrinol. 34:336-342. Li, W. I., C. L. Chen, A. A. Tiller, and G. A. Kunkle, 1986. Effect of thyrotropin-releasing hormone on serum concentrations of thyroxine and triiodothyronine in healthy, thyroidectomized, thyroxine-treated, and propylthiouracil-treated dogs. Am. J. Vet. Res. 47:163-169. Lissitzky, S., J. Bismuth, J. L. Codoccioni, and G. Cartouzou. 1968. Congenital goitre with iodoalbumin replacing thyroglobulin. Serum origin of thyroid iodoalbumin. J. Clin. Endocrinol. Metabol. 22:1797-1806. Martin, S. L., and C. C. Capen, 1979. The endocrine system. Pages 1124-1136 in: Canine Medicine. E. J. Catcott, ed. American Veterinary Publication, Inc., Santa Barbara, CA. Mashaly, M. M., S. L. Youtz, and R. F. Wideman, Jr., 1983. Hypothyroidism and antibody production in immature male chickens. Immunol. Commun. 12:551563. May, J. D., L. F. Kubena, and J. W. Deaton, 1974. Thyroid metabolism of chickens: 2. Estimation of thyroxine half-life in plasma. Poultry Sci. 53:687-691. Mellen, W. J., and B. C. Wentworth, 1962. Observations on radiothyroidectomized chickens. Poultry Sci. 41:134-141. Newcomer, W. S., 1974. Diurnal rhythms of thyroid function in chicks. Gen. Comp. Endocrinol. 24:65-73. Obregon, M. J., J. Mallol, F. Escobar Del Rey, and G. Morreale De, 1981. Presence of L-thyroxine and 3,5,3'-trisodo-L-thyronine in tissues from thyroidec-