General and Comparative Endocrinology 197 (2014) 73–81

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Thyroid hormone concentrations in relation to age, sex, pregnancy, and perinatal loss in bottlenose dolphins (Tursiops truncatus) Kristi L. West a,⇑, Jan Ramer b, Janine L. Brown c, Jay Sweeney d, Erin M. Hanahoe e, Tom Reidarson f,1, Jeffry Proudfoot b, Don R. Bergfelt g a

Hawaii Pacific University, College of Natural and Computational Sciences, Kaneohe, HI 96744, United States Indianapolis Zoo, 1200 W. Washington St., PO Box 22309, Indianapolis, IN 46222, United States Department of Reproductive Sciences, Conservation and Research Center, National Zoological Park, Smithsonian Institution, Front Royal, VA 22630, United States d Quest Global Management/Dolphin Quest, 4467 Saratoga Ave., San Diego, CA 92107, United States e Dolphin Quest Hawaii, Waikoloa Beach Drive, Waikoloa, HI 96738, United States f Sea World of California, San Diego, CA 92109, United States g US Environmental Protection Agency, Washington, DC 20460, United States b c

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Article history: Received 23 August 2013 Revised 13 November 2013 Accepted 15 November 2013 Available online 7 December 2013 Keywords: Bottlenose dolphin Thyroid hormones Age Sex Pregnancy Perinatal loss

a b s t r a c t This study evaluated circulating concentrations of thyroid hormones in relation to age, sex, pregnancy status, and perinatal loss in bottlenose dolphins (Tursiops truncatus) under human care. A total of 373 blood samples were collected from 60 individual dolphins housed at nine aquariums/oceanariums. Serum concentrations of total and free thyroxine (T4) and triiodothyronine (T3) were analyzed with commercial RIA kits validated for use with dolphins. While the effect of age was indicated by higher (P < 0.0001) concentrations of total and free T4 and T3 in juveniles than adults, the effect of sex on thyroid hormones was inconclusive. The effect of pregnancy was indicated by higher (P < 0.035) total and free T4 and T3 during early pregnancy compared to non-pregnancy. For both successful and unsuccessful pregnancy outcomes, maternal concentrations of thyroid hormones were highest during early, intermediate during mid, and lowest during late pregnancy (P < 0.07 to P < 0.0001). Compared to live and thriving births, concentrations of total and free T4 and total T3 were lower (P < 0.08 to P < 0.001) in dolphins with perinatal loss. Lower concentrations ranged from 10% to 14% during early, 11% to 18% during mid, and 23% to 37% during late pregnancy. In conclusion, the effects of age, reproductive status and stage of pregnancy on thyroid hormone concentrations are necessary factors to take into account when assessing thyroid gland function. Since perinatal loss may be associated with hypothyroidism in dolphins, analysis of serum T4 and T3 should be considered for those dolphins that have a history of pregnancy loss. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction More than 40 years ago, reports of thyroid hormone insufficiency in bottlenose dolphins (Tursiops truncatus), common dolphins (Delphinus delphis), and beluga whales (Delphinapterus leucas) led to the suggestion that captivity may be responsible for thyroid abnormalities observed in these species (Harrison, 1969). More recently, congenital goiter was recognized in neonatal bottlenose dolphins under human care as a possible cause of substantial calf mortality (Garner et al., 2002). Thyroid pathologies have also

⇑ Corresponding author. E-mail addresses: [email protected] (K.L. West), [email protected] (J. Ramer), [email protected] (J.L. Brown), [email protected] (J. Sweeney), [email protected] (E.M. Hanahoe), [email protected] (Tom Reidarson), [email protected] (J. Proudfoot), [email protected] (D.R. Bergfelt). 1 CEO for the Reidarson Group, Marine Animal Specialists, NV reidarsongroup@me. com. 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.11.021

been observed in stranded bottlenose dolphins and appear similar to those observed in humans (Baloch and LiVolsi, 2002; Slovik, 2009) and terrestrial mammals (Dalefield and Palmer, 1994; Wakeling et al., 2007). Pathologies reported in dolphins (Cowan and Tajima, 2006) include hyperplastic nodules (8% of thyroid glands examined), adenomas (3%), colloid cysts (8%), and squamous cysts (15%). Despite the occurrence of these thyroid gland perturbations, clinical assessment of thyroid function is not typically performed in cetaceans under human care. The lack of data on circulating concentrations of thyroid hormones and factors that may influence changes in concentrations in cetaceans under human care precludes clinical examination and interpretation of results in these species. The hypothalamic-pituitary-thyroid axis involves a negative feedback loop to regulate the thyroid hormonal pathway. In the circulation, thyroxine (T4) and triiodothyronine (T3) are considered biologically inactive when bound to carrier proteins; smaller amounts of both hormones circulate as the free or bioactive forms.

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Generally, the less abundant but more potent T3 is derived from the more abundant but less potent T4 primarily within the liver and kidneys and to a lesser extent in peripheral target tissues where both hormones exert their role in metabolism, growth, and development (Ganong, 1999). In humans, thyroid hormones are transported in the circulatory system bound to three blood proteins, thyroxine binding globulin (TBG), transthyretin (TTR; formerly known as thyroxine-binding pre-albumin, TBPA), and albumin (Glinoer, 1997). Theoretically, free T4 and T3 provide a more reliable measure of thyroid function than protein bound forms since total T4 and T3 (bound plus free) can also be altered by changes in the abundance of binding proteins, which may not be representative of thyroid gland dysfunction. In dogs and cats, circulating concentrations of TSH and total and free T4 and T3 are used diagnostically (Feldman and Nelson, 2004). In humans, free T4 is often used diagnostically because it is less variable (i.e., not affected by binding proteins) and a more direct measure of hormone produced by the thyroid gland (Fitzgerald, 2000). In cetaceans, seasonality influences circulating concentrations of thyroid hormones in wild beluga whales, but annual fluctuations were not observed in wild bowhead whales (Rosa et al., 2007) or bottlenose dolphins either in the wild or under human care (St. Aubin et al., 1996). More recently, ocean temperature was shown to impact circulating concentrations of thyroid hormone secretion in two geographically disparate populations of free-ranging bottlenose dolphins along the Atlantic coast of the United States (Fair et al., 2011). Although knowledge of the influence of environmental factors on thyroid hormone concentrations is fundamentally important, the influence of innate factors such as age, sex, and reproductive status on thyroid hormones is required for developing a clinical approach to assess thyroid function in dolphins and other cetaceans under human care. Innate factors such as these have been examined in a limited number of studies that include bottlenose dolphins under managed care (St. Aubin et al., 1996), wild bottlenose dolphins (St. Aubin et al., 1996; Fair et al., 2011) and wild bowhead whales (Rosa et al., 2007). However, the relationships between these factors and thyroid hormones was not always consistent within or between species, perhaps because of the different environmental conditions, relatively small numbers of animals, unknown pregnancy status, and lack of longitudinal samples within studies. In comparative studies of humans and domestic animal species, thyroid hormones appear to be influenced by age, sex, and pregnancy status (Colodel et al., 2010; Fantz et al., 1999; Feldman and Nelson, 2004; Glinoer et al., 1990; Harada et al., 1979; Hübner et al., 2005; Kapelari et al., 2008; Refsal et al., 1984) and, are therefore taken into consideration during clinical examination and diagnosis of potential thyroid disorders. Bottlenose dolphins are the most common cetacean species housed in aquariums/oceanariums under a relatively controlled environment. In general, dolphins under human care are highly amenable to conditioning, thereby allowing voluntary (unrestrained) and repeated collection of blood samples. Hence, they can serve as a model species to characterize and evaluate temporal patterns of thyroid hormones during different life stages, reproductive statuses, and pregnancy outcomes. The objectives of the present study were to: (1) determine the influence of age (juvenile vs adult), sex (male vs female), and pregnancy status (non-pregnancy vs early, mid and late pregnancy) on thyroid hormone concentrations; (2) compare thyroid hormone concentrations during different pregnancy outcomes (successful vs unsuccessful); (3) evaluate thyroid hormone concentrations following TSH treatment; and (4) develop initial reference ranges of thyroid hormones in bottlenose dolphins under human care.

2. Materials and methods 2.1. Study dolphins The study involved 60 healthy bottlenose dolphins, representing 33 females and 27 males ranging in age from 1 to 38 y at the time blood samples were collected between 1990 and 2003. Study dolphins were under managed care at nine different facilities: (1) Dolphin Quest Hawaii (Waikoloa, HI, USA) (2) Dolphin Quest Oahu (Kahala, HI, USA) (3) Dolphin Quest Bermuda (Dockyard, Bermuda) (4) Dolphin Quest French Polynesia (Moorea, French Polynesia) (5) Indianapolis Zoo (Indianapolis, IN, USA) (6) Sea Life Park Hawaii (Waimanalo, HI, USA) (7) SeaWorld of California (San Diego, CA, USA) (8) Minnesota Zoo (Apple Valley, MN, USA) and (9) West Edmonton Mall Dolphin Lagoon (Edmonton, AB, Canada). Each of the Dolphin Quest oceanariums contained natural seawater in a lagoon setting ranging in size from 1.8 to 7.5 million liters. The Indianapolis Zoo, West Edmonton Mall Dolphin Lagoon, Minnesota Zoo, SeaWorld of California, and Sea Life Park Hawaii contained up to 8.7 million liters of filtered, chlorinated, or ozonized seawater or saltwater. While there may have been slight differences in environmental and management conditions among facilities, study dolphins were generally housed with other male and female adult and juvenile dolphins and fed and managed in compliance with the US Animal Welfare Act and by the Standards and Guidelines of the Alliance of Marine Mammal Parks and Aquariums.

2.2. Age classifications In wild male bottlenose dolphins, circulating concentrations of testosterone and testis length and diameter have been used to define the age at sexual maturity, which reportedly ranges from 8 to 12 y (Wells, 2000). In dolphins under human care, the youngest male known to have sired a calf was 7 y (Duffield et al., 2000). In the present study, actual ages were available for all juvenile dolphins but not adults since most were born in the wild. Males 66 y of age were classified as juveniles and those P13 y were classified as adults. Dolphins ranging between 7 and 12 y were classified as adult when their serum testosterone concentrations were >2 ng/mL (Kirby, 1990) or ultrasound examination of testis size diameter was >2.5 cm (Holley Muraco, personal communication). Hence, blood samples were available from 13 juveniles and 14 adult males. In a demographic study of dolphins under human care, the youngest female known to give birth was 5 y; most females first give birth between 7 and 10 y (Duffield et al., 2000). For the present study, females 64 y were classified as juveniles and those P11 y were classified as adults. Female dolphins between 5 and 10 y were included as adults only if there was evidence of ovulation or a current or previous pregnancy. When ovulation was unknown, females in this age range were excluded from the study. Hence, blood samples were available from seven juvenile and 30 adult non-pregnant females. Blood samples were collected from 19 pregnant dolphins during 28 separate pregnancies. Fifteen pregnancies resulted in live and thriving calves (successful pregnancy) and 12 in perinatal loss (unsuccessful pregnancy). One pregnancy apparently ended in early embryo/fetal abortion and was excluded from the study. Perinatal loss was defined in accord with that used in domestic animal production as those pregnancies in which the fetus or neonate died before, during or within 24–48 h after parturition (Philipsson et al., 1979). The stage of pregnancy was based on the date of parturition (Month 0) and a 12-mo gestation period (Cornell et al., 1987). Retrospective from Month 0, there were 103 blood samples taken during early (months -12 to -9, n = 36 samples), mid (months -8

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to -5, n = 29 samples), and late (months -4 to 0, n = 38 samples) pregnancy. For non-pregnancy, there were 137 blood samples taken at various stages of the estrous cycle; time of estrus or ovulation was not known. 2.3. Blood sample collection and storage Collection and archiving of blood samples were done during routine animal husbandry practices as part of a proactive health and breeding program at all facilities in compliance with the US Department of Agriculture Approved Program of Veterinary Care. Although blood samples were collected voluntarily from dolphins at each of the participating facilities using preconditioned behavior, there were some instances where mild, physical restraint was necessary to stabilize the animals during collections. Preconditioned behaviors consisted of signaling the dolphins to move into a dorsal or ventral-up position with tail flukes placed on the lap of a trainer or on a foam mat while sitting poolside or dockside. A 21-ga winged infusion set or butterfly needle attached to a syringe was then used to draw blood from the arterial venous network of the tail. Blood from each dolphin was placed in a 10-mL serum separator tube or thrombin containing tube and centrifuged within 45 min of collection. Serum was transferred to vials, labeled with dolphin identification and date, and stored at 20 to 70 °C at respective facilities. 2.4. Thyroid Gland Stimulation Test A test of thyroid function was conducted on a healthy adult non-pregnant female approximately 19 y of age that was housed at the Indianapolis Zoo and trained for sequential blood sampling over a 24-h period. A 1.0 mL solution of human TSH (0.9 mg/mL, ThyrogenÒ, Genzyme Corporation, Cambridge, MA, USA) was administered via an intramuscular injection. Blood samples were collected immediately before TSH at hour 0 and, thereafter, at hours 3, 6, 12, and 24. Blood samples were collected, processed, and stored similar to that described above except that heparin was used as an anticoagulant in the collection tubes to harvest plasma. Reportedly, heparin does not influence thyroid hormone measurements (Hightower et al., 1971). Serum and plasma were later shipped from all facilities on dry ice to the University of Hawaii at Manoa where samples were stored at 20 °C until hormone analysis. 2.5. Thyroid hormone assays Thyroid hormones (total T4 and T3, and free T4 and T3) were measured in serum or plasma samples using commercial solid phase radioimmunoassay (RIA) kits (Siemens Medical Solutions Diagnostics, Los Angeles, CA; total T4 catalog number TKT45, total T3 catalog number TKT35, free T4 catalog number TKF45, free T3 catalog number TKF35). All assays were conducted in accord with the manufacturer’s instructions and validated for use in dolphins. Validation was based on observed parallelism between serial dilutions of pooled serum samples and respective standard curves and >90% recovery of respective hormone standards from pooled serum samples. Assay sensitivities were 2.5 ng/mL for total T4, 0.1 pg/mL for free T4, 0.07 ng/mL for total T3, and 0.02 pg/mL for free T3. For all assays, intra- and inter-assay coefficients of variation were 0.05 and P 6 0.1 indicated that a difference approached significance. The data are presented as the actual means (±SEM) unless otherwise indicated.

3. Results 3.1. Characteristics of total and free T4 and T3 Combining the data for age, sex, reproductive status, and pregnancy outcomes (n = 290 serum samples) across all facilities, circulating concentrations of total thyroid hormones (protein bound plus free) were mostly in the form of T4 (99.3%) compared to T3 (0.71%). Correspondingly, the combined forms of thyroid hormones were mostly protein bound (99.9%) compared to free (0.01%). The effect of facility on thyroid hormone concentrations was not examined.

3.2. Relationship of age and sex on thyroid hormones Circulating concentrations of thyroid hormones were evaluated in juvenile and adult, female and male dolphins and depicted with results of statistical analysis as shown in Fig. 1. Juveniles were born under human care so their actual ages were known. Mean age for females was 2.9 ± 0.2 y and for males 4.8 ± 0.4 y. For pregnant and non-pregnant adults, actual ages were not known but were estimated to range from 6 to 38 y. Significant main effects of age and sex were indicated by higher (P < 0.05) mean concentrations of total and free T4 and T3 in juveniles than adults, and higher (P < 0.05) mean concentrations of total and free T4 and total T3 but not free T3 in females than males. There were no significant interactions between age and sex for total and free T4 concentrations (panels a, b), but there were for total (P < 0.02) and free (P < 0.007) T3 (panels c, d). For total T3 (panel c), mean concentrations were higher (P < 0.05) in juvenile females than corresponding males. For free T3 (panel d), mean concentrations were higher

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(a)

Sex: P