effects of environmental temperature on thyroid hormones in the barn ...

Acta Veterinaria Hungarica 54 (3), pp. 321–331 (2006) DOI: 10.1556/AVet.54.2006.3.3

EFFECTS OF ENVIRONMENTAL TEMPERATURE ON THYROID HORMONES IN THE BARN OWL (TYTO ALBA) Á. KLEIN1*, Margit KULCSÁR2, Virág KRÍZSIK3, R. MÁTICS4, P. RUDAS 2, J. TÖRÖK1 and Gy. HUSZENICZA2 1

Behavioural Ecology Group, Department of Systematic Zoology and Ecology, Eötvös Loránd University, H-1117 Budapest, Pázmány P. sétány 2, Hungary; 2 Faculty of Veterinary Science, Szent István University, Budapest, Hungary; 3 Department of Genetics, Eötvös Loránd University, Budapest, Hungary; 4 Faculty of Medicine, Institute of Medical Biology, University of Pécs, Pécs, Hungary (Received 2 November 2005; accepted 13 February 2006)

The basic patterns of thyroid hormones [thyroxine (T4) and 3,3’,5triiodothyronine (T3)] and the T4 and T3 responses induced by thyrotropin releasing hormone (TRH) are reported in captive female barn owls (Tyto alba) during the non-breeding period. The main findings of the study, conducted on a total of 10 owls, are as follow: (1) The thyroid gland of barn owl can be stimulated by the classical TRH stimulation test. (2) T3 response was much more pronounced both under cold (around 10 °C) and warm (around 20 °C) conditions, whereas T4 response ranged so widely that we could not point out any significant change in it. (3) Basal T3 plasma level was significantly (p = 0.036) higher in birds exposed to cold temperature, and they responded to TRH treatment with a lower plasma T3 elevation than the birds kept in a warm chamber. This pattern, however, cannot be explained by increased food intake, but is in agreement with the fact that enhanced T3 level may account for higher avUCP mRNA expression, which results in higher heat production on the cell level. From the results it is concluded that altering T3 plasma level plays a significant role in cold-induced thermoregulation. Key words: Thyroid hormones, thyrotropin releasing hormone, barn owl, temperature, thermoregulation

The effects of thyroid hormones (THs) and the factors influencing the thyroid functions themselves are very diverse. Because THs are important for metabolic adjustment in endothermic organisms (Burger and Denver, 2002), a sub*

Corresponding author: Ákos Klein; E-mail: [email protected]; Phone: 0036 (20) 322-5620; Fax: 0036 (1) 381-2194 0236-6290/$ 20.00 © 2006 Akadémiai Kiadó, Budapest

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stantial proportion of the most recent avian thyroid research is related to thermoregulation, thermal adaptation and conditioning during embryonic and post-hatch growth (Olson et al., 1999; Moraes et al., 2003). Thorough understanding of thermal regulation is significant from the evolutionary, conservational and economic points of view. Thyroid hormones are essential for thermal regulation (Decuypere et al., 2005) and adaptation (Collin et al., 2003). The environment-independent maintenance of high and constant body temperature was a huge evolutionary breakthrough which allowed birds and mammals to colonise several unfavourable habitats. Thus, comparative evolutionary research in thyroid functions on thermoregulation at different taxonomic levels can shed light on ecological patterns and developmental plasticity (Dufty et al., 2002). For example, it helps to understand the physiological background of some aspects of the post-hatch life-history strategies, namely that different TH profiles play a cardinal role in altricial and precocial development (Silverin and Rudas, 1996; Olson et al., 1999). Another important branch of thyroid surveys deals with the decrease in quality or quantity of prey-availability of top predators. Nutritional stress can alter metabolic rate and affect overwintering via reduced body reserves in temperate climates, which can drive the change in survival and population dynamics (Northern cardinal: Burger and Denver, 2002; harbour seal: Oki and Atkinson, 2004). Finally, impacts of endocrine disrupters are very considerable topics of scientific discussion, since agricultural pollution has remained an unsolved phenomenon for decades (Bowerman et al., 2000; Scollon et al., 2004). The third principal question deals with thermal conditioning to improve thermotolerance and productivity in domestic fowl, which is a hot-point of stock-raising especially under tropical conditions (Moraes et al., 2003). To our knowledge there is hitherto no study which endeavours to explore the relationship between thyroid functions and thermoregulation of an obligate predator nocturnal bird species. Former investigations presented data mainly on domestic fowl and common passerine bird species (European starling: Dawson et al., 1985; blue tit: Silverin and Rudas, 1996; red-winged blackbird: Olson et al., 1999; white-crowned sparrow: Scollon et al., 2004; house sparrow: Chastel et al., 2003) because of the relatively large sample size and simple experimental requirements. However, the low diversity of the investigated bird species and orders can be a serious limitation to the exact understanding of thyroid functions and does not allow any evolutionary overview. For these reasons, we chose the barn owl (Tyto alba), which has many remarkable characteristics in connection with thyroid research, such as asynchrony in hatching, being a top predator in agricultural habitat, and showing frequent population fluctuations under temperate climate because of reduced ability to obtain sufficient fat reserve. In this study we investigated the relationship between ambient temperature and thyroid function during the non-breeding period in late winter. The barn owl Acta Veterinaria Hungarica 54, 2006

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evolved mainly in subtropical and tropical areas: its thermoneutral zone is between 25–33 °C, and this species is unable to deposit as much fat as other European owl species (Strigidae) of the same size (Handrich et al., 1993). Under the climatic conditions of the Carpathian Basin the functional characteristics of the adenohypophysis–thyroid gland axis are supposed to be critical factors of metabolic adaptation, particularly in the winter period, before the nesting season. This is the first part of a series of ongoing experiments, which tries to explore the thyroid function of barn owls from some evolutionary, physiological and conservational aspects. The purpose of the current paper is to describe the basic thyroxine (T4) and 3,3’,5-triiodothyronine (T3) patterns, as well as the thyrotropin releasing hormone (TRH) induced T4 and T3 responses of barn owls exposed to different temperatures below the thermoneutral zone. Materials and methods Experimental animals As barn owl is an endangered species in Europe (Hagemeijer and Blair, 1997), we carried out the experiment with injured individuals instead of capturing healthy ones. Several months before this study (between 2001 and 2004) these birds suffered severe traumatic injuries. They recovered completely, but with irreversible consequences (loss of a part of one wing or leg, badly reossified broken bones, etc.), so they were not releasable. Sex was determined by DNAPCR method with P2 and P8 primers according to Rosivall et al. (2004). Because of the low numbers of males we involved only females in the experiment. The females were adults ranging from 1 to 5 years old, held in a community outdoor aviary on natural photoperiod and temperature, and fed with mice and day-old chicks ad libitum. Apart from their former traumatic injury, the owls were healthy and in good condition. The experiments were performed in March, before the breeding season. The holding of birds and the experimental design were in accordance with the Laws of Environmental Protection and Animal Care. Experimental design Before the nesting season – at the beginning of March 2005 – the owls were weighed and transferred indoors from the outdoor aviary. They were kept in wooden boxes (0.7 × 0.5 × 0.8 m), one individual per box. Boxes were similar to those in which the owls were roosting in the outdoor aviary before the experiment, except that these had a hole on the top (diameter 0.1 m) because of the diffuse illumination through the hole. Boxes were housed in two dark rooms illuminated by 15W bulbs. The lighting schedule was 12L:12D imitating the natural light conditions. Owls were fed with laboratory mice ad libitum. Acta Veterinaria Hungarica 54, 2006

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The temperature of the two rooms differed. However, as the rooms were not precisely regulated climatic chambers, we continuously recorded the temperature in each room with two digital thermometers. Cold conditions were maintained in the unheated room (Tmax = 10.0 ± 2.3 °C, Tmin = 4.5 ± 1.6 °C, N = 16) whereas there was a heater in the warm one (T max = 22.0 ± 0.8 °C, Tmin = 17.3 ± 0.5 °C, N = 19). The 10 birds were randomly divided into two groups (Group 1 and Group 2 with 5 individuals in each). The experiments lasted for 8 days. First, owls were acclimated to the respective indoor conditions for two days and then Group 1 was submitted to cold temperature whereas Group 2 to warm conditions for 3 days (Period 1). At the end of day 5, weighing, TRH stimulation test and blood sampling were carried out and we changed the groups between the two rooms (Period 2). Weighing, stimulation and blood sampling were repeated on day 8. After the 8 days of the experiment, the owls were transferred back to the community outdoor aviary. None of them suffered any new injuries during the investigation. Blood collection At the end of both Period 1 and Period 2, birds were weighed and blood samples (500–800 µl) were taken from the brachial vein with a 25-G heparinised needle. Samples were immediately centrifuged and plasma was stored at –20 °C until assessing basal T4 and T3 levels (T4t0, T3t0). After bleeding, each bird received 20 µg of TRH (thyrotropin releasing hormone acetate: P 2161; SigmaAldrich Corporation, St. Louis, USA) by i.v. injection (according to Bartha et al., 1989). Exactly 60 min thereafter another blood sample was drawn from the opposite wing vein. After centrifugation plasma was stored at –20 °C until assessing induced T4 and T3 levels (T4t60, T3t60). Laboratory procedures The thyroid hormone levels were determined with 125I-RIA. The T4 kit ( I-T4 CT-spec. RIA, Institute of Isotopes Co. Ltd., Budapest, Hungary) was originally developed for human use, and was slightly modified for assaying a lower concentration range of T4 in animal samples (Huszenicza et al., 2000). This version of method was validated for assaying T4 in barn owl plasma without any further modification. The inter- and intraassay coefficients of variation (CV) were determined by adding 3–5 parallels of a low (mean: 10.5 nmol/l) and a high (mean: 85.7 nmol/l) control plasma into each run of assay. Depending on the concentrations the intra- and interassay CVs varied between 1.0–9.0% and 4.2–17.2%, respectively. The sensitivity of the assay was 3.8 nmol/l. The human T3 kit (125I-T3 CT RIA, Institute of Isotopes Co. Ltd., Budapest, Hungary) was suitable for assaying animal samples without modification in our earlier work (Huszenicza et al., 2000), and after validation for barn owl plasma it was also 125

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used in the current study (sensitivity: 0.67 nmol/l; the intra- and interassay CVs were 25.3 and 1.6 %, respectively). In both assays the binding pattern of serially diluted barn owl samples was parallel to that of the standard curves, and the recovery of added T4 and T3 to plasma varied between 96 and 105%. Repeated determinations of THs were not possible due to the limited quantity of samples. Statistical analysis All data were analysed using parametric statistics available in STAT 7.0 software. Both T4 and T3 values were log10 transformed prior to statistical analysis to normalise distribution. To evaluate the effects of thermal treatment, TRHinduced release, change in body weight and the two experiments, we used repeated measure ANOVA (rmANOVA), or ANCOVA design in generalised linear model (GLM) procedure. By model selection we performed stepwise backward deletion of non-significant terms. Change of body weight (in percent of the starting body mass) was involved in the models as a covariant, periods (Period 1, Period 2) and temperature (warm, cold) as categorical predictors, and the repeated measure variables were T3t0–T3t60 and T4t0–T4t60. To compare two means we applied Student’s t-test. Statistical significance was accepted at the 0.05 level. Results Controlling body weight Body weight declined steadily during the 8 days of the experiment (rmANOVA: F(2, 18) = 12.369, p = 0.0004) (Fig. 1). However, there were no significant differences between Group 1 and Group 2 during the two periods (Student’s t-test; Period 1: meanwarm = 349.0, meancold = 360.8, t = –1.29, df = 8, NS; Period 2: meanwarm = 344.0, meancold = 336.5, t = 0.621, df = 7, NS). Neither weight nor experiments had any significant effect on T3t0, T3t60, T4t0 and T4t60. Analysing the correlation between body weight loss and the different hormone values separately, we only found significant relationship between basal T4 level and body weight loss. The greater the body weight loss, the lower was the basal T4 (GLM, F = 6.63, df = 1, p = 0.019). Plasma thyroxine (T4) and 3,3’,5-triiodothyronine (T3) level As the patterns during Period 1 and Period 2 were completely consistent regarding both T4 and T3 plasma levels, we pooled the data gained from the two experiments (Table 1). There was no significant difference in mean basal T4 plasma level between the two groups regarding the temperature, while mean basal T3 plasma concentration was significantly lower in warm-exposed animals (Table 2). Acta Veterinaria Hungarica 54, 2006

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375 370

Body weight weight (g) Body (g)

365 360 355 350 345 340 335 330

BeforePeriod Period1 Before 1

After Period11 After Period

Period22 Period

Time of of weighing Time weighing Fig. 1. Change of body weight during the course of experiment. Values show means ± SE Table 1 Student’s t-test for comparing TH plasma levels (mean ± SD, log10 nmol/l) in Period 1 and Period 2. None of them differed significantly between the two experiments log

T4t0 T4t60 T3t0 T3t60

Mean ± SD (Period 1)

Mean ± SD (Period 2)

1.41 ± 0.049 1.43 ± 0.24 0.63 ± 0.09 0.76 ± 0.10

1.39 ± 0.084 1.46 ± 0.14 0.61 ± 0.09 0.75 ± 0.06

t-value

df

p

0.634 –0.343 0.629 0.234

17 17 17 17

0.53 0.74 0.54 0.81

Table 2 Mean (± SD, N) basal T4 and T3 plasma concentrations (nmol/l) under cold and warm conditions. The p values of Student’s t-tests are derived from log-transformed data Cold

Warm

p (t-test)

T4t0

Average (nmol/l) ± SD N

26.2 2.88 10

25.1 4.73 9

0.54

T3t0

Average (nmol/l) ± SD N

4.7 0.79 10

3.9 0.77 9

0.033



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An injection of TRH increased plasma T3 levels irrespective of temperature (rmANOVA: F(1.17) = 25.98, p < 0.01), however the rise of T4 level was not statistically verifiable (rmANOVA: F(1.17) = 1.54, p = 0.23) (Fig. 2). Birds held under cold conditions exhibited a significantly decreased sensitivity to TRH stimulus (Fig 3). The change of T3 plasma level (expressed in percentile of the basal T3 value) due to TRH treatment was higher in warm-exposed birds (Student’s t-test for dependent samples: meanwarm = 131.97%, meancold = 113.88%, t = 3.06, df = 7, p = 0.022). Comparing only the basal values of T3, owls exposed to cold temperature were characterised by significantly higher T3 plasma level than individuals kept in the warm room (Student’s t-test for dependent samples, meanwarm = 0.582, meancold = 0.678, t = –2.596, df = 8, p = 0.036). 1,58 1.58 1.56 1,56 1,54 1.54

T4(log (log10 nmol/l) T4 10 nmol/l)

1,52 1.52

T4 t0 T4 t60

1,50 1.50 1,48 1.48 1,46 1.46 1.44 1,44

1.42 1,42 1,40 1.40 1,38 1.38 1,36 1.36 1,34 1.34 1,32 1.32

warm warm

cold cold

Temperature Temperature Fig. 2. The effect of temperature and TRH provocation test on T4 plasma level. Values are the log10 transformed hormone levels before TRH injection (t0) and 60 min thereafter (t60). Columns show means ± SE

Discussion To our knowledge, this is the first study reporting thyroid functions in response to different ambient temperatures in captive barn owls. Indeed, many reports on avian TH concentration are available regarding changing temperature. Expanding the diversity of study animals can aid the understanding of thyroid function and related adaptive processes in evolutionary context. Acta Veterinaria Hungarica 54, 2006

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0.80 0,80 0.78 0,78

T3(log (log10 nmol/l) T3 10 nmol/l)

0.76 0,76 0.74 0,74

T3 t0 T3 t60

0.72 0,72 0.70 0,70 0.68 0,68 0.66 0,66 0.64 0,64 0.62 0,62

0,60 0.60 0.58 0,58 0,56 0.56 0,54 0.54 0,52 0.52

warm warm

cold cold

Temperature Temperature Fig. 3. The effect of temperature and TRH stimulation test on T3 plasma level. Values are the log10 transformed hormone levels before TRH injection (t0) and 60 min thereafter (t60). Columns show means ± SE

One of the basic results of this survey was that the thyroid gland of adult barn owl can be stimulated by the classical TRH stimulation test. However, T3 response was much more pronounced both under cold and warm conditions, whereas T4 response varied so much that we could not point out any significant change in it. These results are consistent with the statement that TRH increases the plasma concentration of T4 only in chicken embryos and growing chicks, but in adults TRH does not induce the release of thyrotroph hormone, challenging the subsequent increase in T4 and T3 levels (Kühn et al., 1988). In adults, TRH exerts a stimulatory effect on the somatotropic axis, resulting in elevated growth hormone (GH) level which provokes increased hepatic T4 to T3 conversion (De Groef et al., 2005). Nutrition, body condition and fat deposition can all affect thyroxine level. Hence, variation in T4 levels could be brought about by individual variability in loss of body weight, since the extent of weight loss co-varied only with basal T4 level: the greater the body weight loss, the lower the basal T4 plasma level. This finding is inconsistent with the results of Bartha et al. (1989), who concluded that restricted feed intake in adult chickens results in reduced T4 to T3 conversion via lower deiodination activity in the liver. Based on this result we would have expected decreased T3 and increased T4 plasma levels during the course of the experiment, since body weight declined steadily. However, there was no difActa Veterinaria Hungarica 54, 2006


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ference in hormone levels between Period 1 and Period 2. The strong loss of body weight was an unexpected factor in this study, which was caused by the insufficient food intake attributable to the changed environment or perhaps to the quite short habituation time. In the future it would be advisable to test the effect of restricted feed intake on THs under thermoneutral conditions, allowing a longer acclimatisation period to the owls. Surveys in thermal biology are of crucial importance in barn owl research, particularly in the temperate climate. Despite this fact, only few papers were published on this topic in the last decade (e.g. Handrich et al., 1993; McCafferty et al., 2001), and none of them investigated the hormonal background of thermoregulation. In the present survey we exposed owls to two different temperatures, both of them being below the thermoneutral zone of 25–33 °C (according to Handrich et al., 1993), where owls do not need to increase their metabolic rate in order to maintain their constant body temperature around 40 °C. Basal T3 plasma level was significantly higher in birds exposed to cold temperature, and such birds responded to TRH treatment with a lower plasma T3 elevation than birds kept at warm temperature. The lower sensitivity of the pituitary–thyroid axis to TRH stimulation is due to the elevated serum T3 levels. The higher T3 levels at cold temperature and the positive correlation between T4 and temperature seem to be general patterns in birds (Dawson et al., 1992; Burger and Denver, 2002). Although most of the studies explain the seasonal variation in THs by an increased food intake in response to cold temperature, our results fail to support this hypothesis. The owls in the present study went through a continuous weight loss because they were unwilling to feed satisfactorily in spite of the ad libitum availability of food. After all, without any increased food intake, the T3 level elevated under cold conditions. In harmony with the report of Collin et al. (2003), we suppose that the driving force behind the TH variation in response to altering ambient temperature might be not necessarily food intake or the related energy equivalents. Collin et al. (2003) pointed out that the higher T3 plasma level of cold-exposed chicks was associated with enhanced thermogenesis due to the higher avian uncoupling protein (avUCP) mRNA expression. This mitochondrial protein is known to uncouple phosphorylation from oxidation and hence to be involved in heat production in mammals, and presumably in birds as well. In conclusion, the barn owl seems to be an appropriate study organism for investigating the hormonal background of thermal regulation and adaptation in a wild-living predator bird species. Elevated T3 plasma level might be the result of enhanced thermogenesis rather than increased food intake. Further investigations are needed to clarify the role of feed restriction in thyroid functions under different temperatures ranging from frost to thermoneutral or warmer conditions.


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Acknowledgements We highly appreciate the diligent technical assistance and support of Zs. Böddi, A. Vona-Nagy, E. Szöllősi and B. Rosivall. We are also very grateful to The Hungarian Barn Owl Foundation for supporting our work. The study was financed by the National Research Fund of Hungary (project number: OTKA/TS-049 756), and a PhD scholarship at the Eötvös Loránd University (to ÁK), which are gratefully acknowledged. Two anonymous referees provided helpful comments on the manuscript.

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