Thyroid hormone concentrations in harbour seals (Phoca vitulina): No evidence of involvement in the moult

Comp. Biochem. Physiol. Vol. 99A, No. 1/2, pp. 185-194, 1991 Printed in Great Britain

0300-9629/91 $3.00+ 0.00 © 1991 PergamonPress plc

THYROID HORMONE CONCENTRATIONS IN HARBOUR SEALS (PHOCA VITULINA): NO EVIDENCE OF INVOLVEMENT IN THE MOULT DEANE RENOUF*t and GEORGE BROTEA *Department of Psychology and Ocean Sciences Centre and Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland, Canada A1B 3X9 (Received 24 August 1990) Al~tract--1. Weekly measurement of total thyroxine and triiodothyronine, and free thyroxine in the serum of five captive harbour seals over a 12 month period revealed no consistent statistically significant relationship between the moult and thyroid hormone levels. 2. It is proposed that the putative involvement of these hormones in moulting according to the literature is unwarranted. 3. As in other studies of seals, thyroid hormone levels in these seals were substantially lower than in other mammals.

INTRODUCTION Thyroid hormone levels are lower in seals than in terrestrial mammals. Six reports of serum and plasma concentrations of thyroxine ('1"4)and triiodothyronine (T3) revealed substantial variation in levels depending on the age and sex of the animal, however the highest concentrations were still lower than those found in other mammals (Chopra, 1986: Table 1). It is believed that seals' thyroid hormone levels change during the animal's moult each year, in two instances reported as a decline (Ashwell-Erickson and Eisner, 1981; Riviere et al., 1977), in one as an increase (John et al., 1987), and in the most thorough study as falling at the start and increasing at the end of moulting (Ashwell-Erickson et al., 1986). In all but this last study, the seals were sampled relatively infrequently. Ashwell-Erickson and Eisner (1981) measured plasma total T4 six times over a ten week period surrounding the moult in three yearling harbour seals (Phoca vitulina) of the same but unspecified sex. Riviere et al. (1977) did so nine times in ten months using six yearling harbour seals, male and female. The most recent study measured both total T3 and T4 in three adult female harp seals, Phoca groenlandica, sampled four times during a five month period which incorporated moult. Serum T4 and T 3 were significantly lower during the premoult measurement, thereafter T4 remained high, whereas T 3 fell again to the pre-moult level at the final sampling in early June. The authors suggested that a high T3/T4 ratio marked the initiation of moult (John et al., 1987). In an extensive study of moulting in harbour and largha seals (Phoca largha), Ashwell-Erickson et aL (1986) concluded that serum free and total T4 and T3 tPresent address: Bio-Science Laboratory (Ontario) Ltd, Windsor, Ontario, Canada, NSX 4T2, and Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada, H9B 3P4. cnPA99/,/2-M

declined at the onset of moulting and peaked when hair growth was most rapid near moult's end. However, there were notable differences among ages and species. Total T4 declined slightly near the onset and rose towards the end of moulting in the yearling largha and harbour seals and the adult female harbour seal. The two year old and adult largha seals showed a slight increase in this hormone at moulting onset, and a larger increase near its completion. Free T4 was measured only in the yearlings and in the adult largha seals and showed minima not clearly associated with the timing of moulting, and maxima during the period of new hair growth in one yearling and both adults. T 3 declined near the onset of moulting or new hair growth in all but the two adult largha seals, and peaked near the period of maximum hair growth in all largha seals, but sometime after the moulting period in the two harbour seals. Engelhardt and Ferguson (1980) measured total "1"4 and T 3 in harp and grey seal (Halichoerus grypus) pups during the moulting of their white coats near weaning time. They found higher plasma levels of these hormones than those found in adults, but the effect of hair growth could not be assessed in this three week study. Leatherland and Ronald (1979) also found these hormones to be at higher levels in harp seal pups than in adults, however no samples were taken during moulting. Levels of thyroid hormones are known to show diel and seasonal variation in other mammals (Junger, 1979; Leatherland and Ronald, 1981; Nilssen et al., 1984). They are also likely to be suppressed by stress (St Aubin and Geraci, 1988; Sterling et al., 1970), and there are significant variations depending on the sex and age of the animals (Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980; Kieffer et al., 1976; Fisher et al., 1977). Though variation within and among seals was high in all of the studies reviewed above, statistical evaluation of the relationship between moult and thyroid hormone concentrations were made only by John et al. (1987), results

185

186

DEANE RENOUF and GEORGE BROTEA Table 1. Thyroxine (T4)and triiodothyronine (T3) levelsfor pinnipeds. The values shown are means __.standard error of the mean across all ages and sexes investigated. When * is indicated, values were extracted from a graphical presentation and are therefore approximate

T4 Species (Total nmol/l:Free pmol/l) Harp seal (total: serum) 21.14 +_ 18.46 Harp seal (total: plasma) 56.89 + 13.91 Harp seal (total:plasma)* 90.91 + 23.53 Grey seal (total :plasma) 44.07 + 23.96 Harbour seal (total :plasma)* 49.34 + 5.49 Harbour seal (total :serum)* 27.51 +_3.63 free T4 31.77 + 8.91 Largha seal (total :strum)* 24.79 + 1.28 free "I"4 35.28 + 1.86 Harbour seal (total :plasma)* 19.82 + 3.12 Harbour seal (total:serum) 36.92 + 0.73 free T4 20.82 + 0.55

otherwise presented only for empirical interpretation. Since all b u t one instance involved infrequent b l o o d sampling, the variations seen in h o r m o n e concent r a t i o n s near the m o u l t i n g season are difficult to assess in relation to the variations which occur in these h o r m o n e s for o t h e r reasons. T h e present study was u n d e r t a k e n to examine levels of free thyroxine, total thyroxine a n d triiodthyronine in h a r b o u r seals sampled weekly over a twelve m o n t h period f r o m the t e r m i n a t i o n o f m o u l t i n g in 1988 until the end o f the s u b s e q u e n t m o u l t in 1989, so t h a t statistical evalua t i o n o f putative m o u l t related changes could be made. MATERIALS AND METHODS

Five harbour seals (Phoca vitulina concolor) were mainmined in captivity on a diet of herring and vitamins (Renouf et al., 1988; Renouf and Noseworthy 1990). Every Friday the seal pool was drained between 10.30 and 12.30, and each seal was confined inside a plywood box from which the hind flippers extended. Blood (5 cc in untreated or clot vacuminers) was obtained from a hind flipper venous plexus as described by Geraci (1971). This procedure was relatively unstressful insofar as we were occasionally able to acquire the blood without using the box, or restraint of any other kind. The blood was kept in a cool room for approximately two hours, and was then centrifuged to collect the serum which was frozen at -70°C. The animals had not eaten for at least 20 hours prior to blood sampling. Serum samples were thawed at room temperature before assays were performed. Free T 4, and total T4 and T 3 were measured using the following techniques: Free "1"4levels for specimens collected before May 1989 were determined using an analogue based radioimmunoassay (Wilkins et al., 1985) Amerlex-M Free T, RIA kit obtained from Amersham Canada Ltd., Oakville, Ontario. The rest of the serum specimens were assayed for free "1"4levels using a DELFIA (Dissociation Enhanced Lanthanide Fluoroimmunoassay) free thyroxine kit obained from Pharmacia (Canada) Ltd., Bale d'Urfe, Quebec. Total T3 and T4 were determined using DELFIA thyroxine and DELFIA triiodothyronine kits obtained from Pharmacia. Amerlex-M Free T4 RIA and DELFIA Free Thyroxine kits are both solid phase immunoassay, but their principles of analysis are different. Amerlex-M uses as radioactive tracer a labelled thyroxine derivative which has been chemically modified to inhibit the binding to the endogenous T4 binding proteins in human serum, but which binds normally to antibodies to T4. The tracer competes with the free T4 in the specimen for a limited number of binding sites on the T4 antibody, and the proportion of bound tracer is inversely related to the concentration of free T 4 present in the serum.

T3 (nmol/1) 1.41 + 0.23 2.63 __.0.43 2.50 + 0.46 2.59 + 0.88 0.62 + 0.05

Reference Leatherland and Ronald (1979) John et aL (1987) Engelhardt and Ferguson (1980) Engelhardt and Ferguson (1980) Ashwell-Erieksonand Eisner ( 1981) Ashwell-Erickson et aL (1986)

0.61 __.0.06

Ashwell-Erickson et al. (1986)

0.71 + 0.02

Riviere et al. (1977) Renouf and Brotea (present report)

A very small amount of T 4 antibody is used in order to prevent the disturbance of the normal equilibrium between free T 4 and the T 4 bound to serum protein. DELFIA free thyroxine assay is based on the back titration principle. Sample free T 4 is first reacted with a small amount of antibody T4 monoclonal antibody derived from mice, which in turn binds to the solid phase anti-mouse IgG. The binding sites on the anti "1"4antibody sites which remain empty after this first step are back-titrated with europium labelled T 4 (Hemmila, 1985; Ekins, 1985). The fluorescence of bound europium (measured after being enhanced by chelating reagents) is inversely proportional to the amount of free 1"4 in the sample. The results obtained with the Amerlex-M method were multiplied by a correction factor (obtained by doing correlation studies between the two methods using human sera: Brotea et al., 1990). The similarity of the results obtained with each method suggests that the characteristics of the thyroxine binding proteins of humans and seals are alike. Human serum controls were run with each batch of specimens in order to determine the precision of the methods. The between assay co-efficient of variation for free T4 and total T4 and T 3 was less than 5% for controls having similar concentrations as in the reported results. RESULTS The levels of total a n d free T4, a n d T3 are s h o w n for each a n i m a l in Fig. l a - e . T h e dates w h e n m o u l t i n g was first observed as a hair t h i n n i n g a r o u n d the eye orbits, a n d w h e n m o u l t i n g was finished in t h a t n o old hair r e m a i n e d are s h o w n as horizontal black bars o n these graphs. It is clear t h a t the weekly v a r i a t i o n in all three h o r m o n e levels is as high as any shifts occurring before, d u r i n g a n d after moulting. T h e year was p a r t i t i o n e d into four periods for each seal: (1) from the end o f the m o u l t in 1988 until 30 days before the s u b s e q u e n t m o u l t (2) within 30 days of the onset o f m o u l t i n g in 1989 (3) d u r i n g a n d (4) after this moult. Analyses o f variance c o m p a r i n g the m e a n total T4, m e a n free T4, a n d m e a n T3 a n d the ratios o f T3 to each T4 for each seal across these four periods revealed n o consistent significant differences (Tables 2a a n d b). T h e youngest a n i m a l showed the m o s t variation with significantly higher levels o f total T4 a n d free T4 d u r i n g his moult, while T3 was elevated 30 days before, d u r i n g a n d after moulting. Free "1"4 also was higher in one adult male d u r i n g moulting, a n d showed a significant decline in the s u b a d u l t after moulting. The ratio o f T3 to either T4 showed

Hormone concentrations in harbour seals

187

ADULT MALE 1 60,

50,

40,

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188

DEAN~RENoUFandGEoRGE BROTEA

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Fig. lb

Hormone concentrations in harbour seals

189

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190

DEANE RENOUFand GEORGEBROTEA

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H o r m o n e concentrations in harbour seals

191

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Fig. le Fig. 1. (a-e) Levels of total thyroxine and triiodothyronine and free thyroxine in two adult males, one adult female, one subadult male and one juvenile male harbour seal over twelve months. The moulting period from the first signs of hair loss around the orbits to the completion of the new pelage is indicated as the solid bar above the graphs.

Table 2. (a) Thyroxine (total T 4 and free T4) and triiodothyronine (T3) and ratios for five harbour seals during four periods: (1) prior to 30 days before moult was visible (2) during the 30 days before visible moult (3) between the start and end of visible moulting (4) after the end of visible moulting. Values shown are means (number of blood samples)

Seal Adult Male 1 Adult Male 2 Female Subadult Juvenile*

(l) Prior to 30 Days pre-moult 37.5385 (26) 36.4839 (31) 36.2692 (26) 35.5238 (21) 38.0476 (21)

Total T4 (nmol/l) Period (2) 30 Days (3) pre-moult Moulting 37.0000 (4) 28.0000 (1) 39.0000 (2) 39.0000 (2) 41.3333 (3)

41.5000 (2) 39.3333 (3) 29.3333 (3) 54.0000 (l) 70.0000 (1)

(4) Post-moulting 34.3333 (3) 29.7500 (4) 28.2000 (5) 35.1111 (9) 37.6250 (8)

Table 2. continued overleaf

192

DEANE RENOUF and GEOROE BROTEA Table 2. (continued)

(I) Seal Adult Male 1" Adult Male 2 Female Subadult* Juvenile*

Prior to 30 Days pre-moult 18.9018 (28) 21.6344 (31) 19.6641 (32) 18.0729 (24) 19.5977 (29)

(I) Seal Adult Male 1 Adult Male 2 Female Subadult Juvenile*

Seal Adult Male 1 Adult Male 2 Female Subadult Juvenile*

Seal Adult Male 1' Adult Male 2* Female Subadult Juvenile*

Prior to 30 Days pre-moult 0.6468 (26) 0.8120 (30) 0.7215 (33) . 0.6146 (24) 0.6579 (24)

(1) Prior to 30 Days pre-moult 0.0176 (26) 0.0221 (30) 0.0194 (26) 0.0187 (22) 0.0173 (21)

(1) Prior to 30 Days pre-moult 35.6 (23) 38.5 (27) 38.2 (30) 35.3 (21) 33.0 (21)

Free T4 (pmol/l) Period (2) 30 Days (3) pre-moult Moulting 25.1042 (4) 17.8333 (2) 22.4167 (2) 13.5833 (2) 16.1944 (3)

27.7083 (2) 29.8056 (3) 21.8611 (3) 26.1667 (1) 46.1667 (1)

T3 (nmol/l) Period (2) 30 Days (3) pre-moult Moulting 0.6333 (3) 0.8900 (1) 0.7350 (2) 0.6450 (2) 0.8400 (3)

0.8000 (1) 0.7667 (3) 0.6667 (3) 1.0800 (1) 2.1000 (1)

Ratio T3/T4 Period (2) 30 Days (3) "pre-moult Moulting 0.0160 (3) 0.0179 (2) 0.0165 (2) 0.0202 (3)

0.0163 (1) 0.0193 (3) 0.0228 (3) 0.0200 (1) 0.0300 (l)

Ratio T3/Free "1"4 Period (2) 30 Days (3) pre-moult Moulting 23.1 (3) 68.5 (1) 32.8 (2) 48.3 (2) 52.2 (3)

25.1 (1) 25.3 (3) 30.5 (3) 41.3 (1) 45.5 (1)

(4) Post-moulting 25.3333 (3) 24.6250 (4) 20.1333 (5) 24.2222 (9) 27.1354 (8)

(4) Post-moulting 0.4333 (3) 0.7000 (4) 0.5400 (5) 0.6875 (9) 0.9625 (8)

(4) Post-moulting 0.0135 (3) 0.0248 (4) 0.0192 (5) 0.0 t 86 (8) 0.0256 (8)

(4) Post-moulting 17.4 (3) 29.0 (4) 27.0 (5) 26.9 (8) 35.5 (8)

*Indicates some values are significantly different (P < 0.05) from others for that particular hormone and seal, using Seheff6 ranges after oneway analyses of variance across periods. Table 2(b) on next page.

significant variation for three seals, but in a different temporal pattern in each animal. The correlation between total and free T4 was insignificant (r = 0.1989; P = 0.1030), however there was a significant though small positive correlation between total T 4 and T 3 (r = 0.5635: P = 0.0000) and free T4 and T3 (r = 0.3685: P = 0.0080).

DISCUSSION

The present results reveal no clear relationship among thyroid hormone levels and moulting. In the few instances where significant differences were found during and near moulting, they were opposite to the reports of lowered levels by Riviere et al.

Hormone concentrations in harbour seals

193

Table 2. (b) F ratios for the values shown in (a) Seal/hormone F ratio df Probability Scheff6 Adult Male 1 T4 FT4 T3 T3/T 4 T3/FT4

0.5628 9.0493 2.9401 0.9322 6.5781

3,31 3,33 3,29 3,29 3,26

0.6436 0.0002 0.0497 0.4377 0.0019

Adult Male 2 I"4 FT4 T3 T3/T4

2.2414 3.6462 0.6759 1.6196

3,35 3,36 3,34 2,34

0.1007 0.0215 0.5728 0.2129

NS NS NS NS

T3/FT 4

7.4538

3,31

0.0007

2 ~ 1,3,4

Female T4 FT4 T3 T3/T4 TJFT4

2.6238 0.9042 1.4769 0.3947 1.8878

3,32 3,38 3,29 3,32 3,36

0.0674 0.4481 0.2358 0.7576 0.1491

NS NS NS NS NS

Subadult T4 FT4 T3 T3/T4 T3/FT4

1.2868 6.5420 2.4724 0.0710 2.0359

3,29 3,32 3,31 3,29 3,28

0.2975 0.0014 0.0802 0.9750 0.1316

NS 4 ~-2,1 NS NS NS

Juvenile 1"4 FT4

5.9191 28.6937

3,29 3,37

0.0028 0.0000

T3

16.1687

3,32

0.0000

3 ~ 1,2,4 4 ~ 1,2: 3 ¢: 1,2,4 3 ~ 1,2,4: 4~1

7.5635 4.9366

3,29 3,29

0.0007 0.0069

T3/T 4 T3/FT 4

NS 1 # 2,3,4 NS NS I~ 4

4 g- I 2 -~ I

NS indicates no significantcomparisons. Significantcomparisons are indicated by differencesin periods number 1-4 as shown. (1977) and Ashwell-Erickson and Eisner (1981), but similar to some of the results described by AshwellErickson et al. (1986). There is no indication from the present data that T3/T 4 ratios m a r k the initiation of moult as suggested by John et al. (1987). Though the descent of the hair bulb occurs well before there are visible signs of the moult (Ashwell-Erickson et al., 1986), there is no consistent evidence in the present study of any period before moulting when thyroid h o r m o n e levels are elevated or reduced (see Fig. l). It is possible that previous results suggesting that thyroid hormones are related to moulting are a product of infrequent blood sampling of yearlings (Riviere et al., 1977; Ashwell-Erickson and Elsner, 1981). The youngest male in our study showed the largest fluctuations in hormone concentrations. John et al. 0987) used adult females, but sampled them only four times: we found no significant hormone changes in the female in the present study. Ashwell-Erickson and Elsner (1981) found a lowering of plasma thyroxine in yearling harbour seals at the onset of moulting. These authors measured oxygen consumption using indirect calorimetry immediately after blood sampling. They found the decrease in thyroxine was correlated with a reduction of basal metabolic rate to 83% of pre-moult levels. AshwellErickson et al. (1986) reported resting metabolic rate declined early in the moult in association with lowered thyroid hormone levels. If thyroid hormones are unrelated to moulting, it is possible that they are a

reflection of metabolic changes occurring in relation to their feeding cycles (Renouf and Noseworthy, 1990). Acknowledgements--We would like to thank Grant Brown, Linda Gaborko, David Rosen, Mary Scott, Sheldon Stone, Sean Todd, and Damian Whitten. We appreciate the review and tutelage of Dr David Idler. We thank Pharmacia (Canada) Ltd. for providing the DELFIA reagents. This research was funded by NSERC grant number A6364 to DR and is OSC contribution number 100. REFERENCES

Ashwell-Erickson S. and Elsner R. (1981) The energy cost of free existence for Bering sea harbor and spotted seals. In The Eastern Bering Sea Shelf: Oceanography and Resources. (Edited by Hood D. and Calder J.) Vol. 2, pp. 869-899. Ashwell-Erickson S., Fay F., Eisner R. and Wartzok D. (1986) Metabolic and hormonal correlates of molting and regeneration of pelage in Alaskan harbor and spotted seals (Phoca vitulina and Phoca largha). Can. ,I, Zool. 64, 1086-1094. Brotea G., Mill A., Prabhakaran V,, Walsh R. and Whelan V. (1990) Evaluation of the DELFIA system. Clin. Chem. 36, 1204. Chopra I. (1986) Nature, sources and relative biological significance of thyroid circulating hormones. In The Thyroid (Edited by Ingbar S. and Braverman L.) pp. 128-153. Lippincott, Philadelphia. Ekins R. (1985) Principles of measuring free thyroid hormone concentrations in serum. NucCompact 16, 305-313.

194

DEA~a~RENOUFand GEORGEBgOTF~

Engelhardt F. R. and Ferguson J. (1980) Adaptive hormone changes in harp seals, Phoca groenlandica, and gray seals, Halichoerus grypus, during the postnatal moult. Gen. Comp. Endocrin. 40, 434---445. Fisher D., Sack J., Oddie T., Pekary A., Hershman J., Lam R. and Parslow M. (1977) Serum T4, TBG, T a uptake, T3, Reverse T 3 and TSH concentrations in children l to 15 years of age. J. Clin. Endocrinol. Metab. 45, 191-198. Geraci J. (1971) Functional hematology of the harp seal, Pagophilus groenlandicus. Physiol. Zool. 44, 162-170. Hemmila T. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem. 31, 359-370. John T., Ronald K. and George J. (1987) Blood levels of thyroid hormones and certain metabolites in relation to moult in the harp seal (Phoca groenlandica). Comp. Biochem. Physiol. 88A, 655457. Junger W. (1979) A longitudinal study of serum concentrations of thyroxine and triiodothyronine in the BUF rat. M.Sc. thesis, Memorial University of Newfoundland, St John's, Newfoundland, Canada. Kieffer J., Mover H., Federico P. and Maloof F. (1976) Pituitary-thyroid axis in neonatal and adult rats: comparison of the sexes. Endocrinology 98, 295-304. Leatherland J. and Ronald K. (1979) Thyroid activity in adult and neonate Harp seals Pagophilus groenlandicus. J. Zool. Lond. 189, 399-405. Lcatherland J. and Ronald K. (198 l) Plasma concentrations of thyroid hormones in a captive and feral polar

bear (Ursus maritimus). Comp. Biochem. Physiol. 70A, 575-577. Nilsson K., Sundsfjord J. and Blix A. (1984) Regulation of metabolic rate in Svalbard and Norwegian reindeer.Am. J. Physiol. 247, R837-R841. Renouf D., Almon M. and Noseworthy E. (1988) Variations in feeding and social behaviour in a captive breeding group of harbour seals (Phoca vitulina). Mar. Behav. Physiol. 6, 287-299. Renouf D. and Noseworthy E. (1990) Feeding cycles in harbour seals: weight gain in spite of reduced food intake and increased thermal stress. Mar. Behav. Physiol. 17, 203-212. Riviere J. E., Engelhardt F. R. and Solomon J. (1977) The relationship of thyroxine and cortisol to the moult of the harbor seal Phoca vitulina. Gen. Comp. Endocrin. 31, 398-401. St. Aubin D. and Geraci J. (1988) Capture and handling stress suppresses circulating levels of thyroxine (T4) and triiodothyronine (T3) in beluga whales. Delphinapterus leucas. Physiol. Zool. 6, 170-175. Sterling K., Milch P. and Ridgway S. (1970) The day of the dolphin: thyroid hormone metabolism in marine mammals. In Thyroid Hormone Metabolism (Edited by Harland W. and Orr J.) pp. 241-248. Academic Press, London. Wilkins T., Midgiey J. and Barron N. (1985) Comprehensive study of a thyroxine-analog based assay for free thyroxine (Amerlex FT4). Clin. Chem. 31, 1644-1653.