Blubber steroid hormone profiles as indicators of physiological state in free-ranging common bottlenose dolphins (Tursiops truncatus)

https://doi.org/10.1016/j.cbpa.2019.110583Get rights and content

Highlights

  • Long-term changes in systemic endocrine function are reflected in blubber.

  • Blubber hormone profiles indicate physiological state (maturity, pregnancy, stress).

  • Reproductive hormone profiles in blood and blubber are comparable.

  • Corticosteroid profiles in blood and blubber differ.

Abstract

Blubber has been proposed as a possible alternative to blood in the assessment of endocrine physiology in marine mammals because it can be collected via remote biopsy, which removes some of the confounding variables and logistical constraints associated with blood collection. To date, few studies have directly assessed the relationships between circulating versus blubber steroid hormone profiles in marine mammals, and these studies have been limited to a small subset of steroid hormones, which collectively limit the current utility of blubber steroid hormone measurements. In this study, we used liquid-chromatography tandem-mass spectrometry (LC-MS/MS) to screen for 16 steroid hormones in matched blood and blubber samples from free-ranging common bottlenose dolphins (Tursiops truncatus). Seven steroid hormones were detected and quantified, including two progestogens, two androgens, and three corticosteroids. Using principal components analysis (PCA), we explored relationships between hormones in both matrices and three physiological states: sexual maturity, pregnancy, and acute stress response. Plasma and blubber testosterone and its precursors, 17-hydroxyprogesterone and androstenedione, loaded to the first principal component (PC1), and PC1 scores were higher in mature males. Plasma and blubber progesterone loaded to PC2, and pregnant/probable pregnant females had significantly higher PC2 scores. Pregnant females also had higher PC1 scores than other females, suggesting differences in androgen profiles between these groups. There was disagreement between plasma and blubber corticosteroid profiles, as indicated by their loading to different PCs; plasma corticosteroids loaded to PC3 and blubber corticosteroids to PC4. PC3 scores were significantly predicted by elapsed time to blood collection (i.e., time between initiating the capture process and blood collection), while elapsed time to blubber collection significantly predicted PC4 scores, indicating that corticosteroid profiles shift in both tissues during acute stress. Corticosteroid profiles were not related to demographic group, site-month, body mass index, water temperature, or time spent outside of the water on the processing boat. Overall, these results demonstrate that blubber steroid hormone profiles reflect changes in endocrine function that occur over broad temporal scales.

Introduction

Blood is the most common matrix used for endocrine assessments in vertebrates, but collecting blood from free-ranging wildlife typically requires capture and restraint, which is a stressful event and inherently induces shifts in circulating hormone measurements, particularly of stress hormones. As such, it is difficult to measure endocrinological baselines in wildlife, including marine mammals, using blood and current sampling techniques. Furthermore, in free-ranging cetaceans, collection of blood is a labor-intensive and expensive process (Balmer et al., 2014). Using remotely collected sample matrices for marine mammal endocrine assessments could minimize stress to the animals, allow for the measurement of endocrinological baselines, reduce sampling costs for researchers, and increase the number of animals that can be feasibly sampled. Several such matrices have been used, including: feces (Champagne et al., 2018; Wasser et al., 2017), respiratory vapor (Hunt et al., 2014a), baleen (Hunt et al., 2014b), earwax (Trumble et al., 2013), and blubber. Blubber, a form of subcutaneous adipose tissue in marine mammals, can be collected via remote biopsy (Noren and Mocklin, 2012), and contains measurable concentrations of numerous steroid hormones (Boggs et al., 2017; Champagne et al., 2017; Kellar et al., 2015; Kellar et al., 2009; Kellar et al., 2006; Kershaw and Hall, 2016; Kershaw et al., 2017; Mansour et al., 2002; Pallin et al., 2018a; Pallin et al., 2018b; Pérez et al., 2011; Trego et al., 2013; Vu et al., 2015). Thus, blubber could potentially serve as an alternative to blood in marine mammal endocrine assessments involving lipophilic hormones, such as steroids.

Blubber steroid hormone measurements vary with physiological states in cetaceans, including stress (Kellar et al., 2015), sexual maturity (Inoue et al., 2018; Kellar et al., 2009), and pregnancy (Kellar et al., 2006; Mansour et al., 2002; Pérez et al., 2011; Trego et al., 2013). In general, blubber steroid hormone profiles qualitatively reflect circulating profiles. In adult male cetaceans, both circulating and blubber testosterone (T) values increase during the breeding season (Harrison and Ridgway, 1971; Kellar et al., 2009; Schroeder and Keller, 1989; Vu et al., 2015). Similarly, progesterone (P4) is elevated in the blubber and blood of pregnant cetaceans (Kellar et al., 2006; Kirby and Ridgway, 1984; Mansour et al., 2002; Pallin et al., 2018a; Pallin et al., 2018b; Pérez et al., 2011; Sawyer-Steffan et al., 1983; Trego et al., 2013). Stressor-induced activation of the hypothalamo-pituitary-adrenal axis leads to elevated cortisol (F) concentrations in both blood and blubber in cetaceans (Champagne et al., 2018; Houser et al., 2011; Kellar et al., 2015; Kershaw and Hall, 2016; Schroeder and Keller, 1989; St. Aubin et al., 1996; Thomson and Geraci, 1986). Blubber cortisol levels were also linked to body condition in harbor porpoises (Phocoena phocoena) (Kershaw et al., 2017). However, few studies have explicitly characterized the relationships between circulating and blubber hormone concentrations (Champagne et al., 2017; Champagne et al., 2018; Kellar et al., 2013).

Endocrine glands secrete hormones into blood, which then delivers hormones to peripheral tissues, including hormone target tissues, sites of peripheral hormone metabolism, and blubber. Thus, when changes in central endocrine function occur, circulating hormone values will change rapidly and these changes can be detected nearly instantaneously, while changes in hormone concentrations in peripheral tissue must lag behind changes in circulating concentrations and central endocrine function. Such a relationship was observed in domesticated pigs, in which peak adipose P4 concentrations exhibited a 1–2 day lag behind peak plasma P4 concentrations and returned to baseline concentrations more gradually than plasma concentrations (Hillbrand and Elsaesser, 1983). Thus, it is likely that blubber integrates circulating hormones over some period of time, and blubber hormone concentrations reflect an average circulating value over that period. Blubber hormone profiles are likely also influenced by in situ metabolism of steroid hormones—as demonstrated by Galligan et al. (2018b)—blubber perfusion rates, and perhaps other factors (e.g., lipid composition, concentration of steroid binding proteins in blood, etc.), which are also dynamic.

We currently have a poor understanding of the temporal relationships between circulating and blubber steroid hormone concentrations. In common bottlenose dolphins under human care, circulating F was elevated 15 min following exposure to acute stress, remained high during the 120 min stress exposure, and then had returned to baseline within 1 h post exposure (Champagne et al., 2018). Blubber was sampled at 0 min, 60 min, and 120 min into the stress exposure, and blubber F had significantly increased at both 60 min and 120 min, though the magnitude of increase was lower compared to blood (Champagne et al., 2018). Notably, blubber was not sampled prior to 60 min, thus it is unclear how soon after the initiation of exposure that a change in blubber F could be detected. Furthermore, Champagne et al. (2018) did not sample blubber after cessation of exposure, meaning we do not know how long blubber F is elevated after an acute change in circulating F concentration. Therefore, while an increase in circulating F would indicate stressor exposure within the past several minutes-to-hours, elevated blubber F would indicate stressor exposure at some currently undetermined point(s) in the past, meaning that blubber cannot necessarily be used interchangeably with blood for assessment of acute stress. Conversely, blubber may be interchangeable with blood when measuring pregnancy-related shifts in P4 physiology. In bowhead whales (Balaena mysticetus), during pregnancy when P4 secretion increases and remains high for a prolonged period, circulating and blubber P4 values are strongly correlated with one another (Kellar et al., 2013). This is likely because the persistent increase in P4 secretion that occurs during pregnancy results in relatively stable circulating P4 concentrations over a long period allowing sufficient time for blubber P4 values to equilibrate with blood P4. Taken together, this evidence suggests that blubber steroid measurements likely reflect changes in systemic endocrine function over a broader temporal scale, which has implications for how we interpret blubber hormone values in relation to physiological states.

Importantly, the blubber steroid hormone literature has primarily focused on F, T, and P4 (Champagne et al., 2017; Kellar et al., 2015; Kellar et al., 2013; Kellar et al., 2009; Kellar et al., 2006; Mansour et al., 2002; Pérez et al., 2011; Trego et al., 2013), and no studies to date have directly studied the relationship between T concentrations in blood and blubber. Furthermore, several additional steroid hormones—17-hydroxyprogesterone (17OHP4), 11-deoxycorticosterone (DOC), corticosterone (B), 11-deoxycortisol (S), cortisone (E), and androstenedione (AE)—have recently been measured in free-ranging common bottlenose dolphin blubber and blood (Boggs et al., 2019; Boggs et al., 2017; Galligan et al., 2019; Galligan et al., 2018a). Galligan et al. (2018a) and Boggs et al. (2019) explored relationships between these hormones and various physiological states in blood and blubber, respectively. Both noted positive correlations between several of the hormones in the Δ4 androgen biosynthesis pathway (specifically, 17OHP4, AE, and T) (Fig. 1), and increases in AE during pregnancy. Herein, we build from these studies and conduct a comprehensive assessment of the relationships between physiological state and steroid hormone profiles in dolphin blood and blubber.

In this study we used individual-matched blood and blubber samples to assess the relationships between hormones in both matrices in the context of various physiological states, including sexual maturity, pregnancy, and acute stress response. We hypothesized that generally hormone profiles would be comparable across the two sample matrices. Furthermore, we predicted that the hormones in the Δ4-androgen biosynthesis pathway (Fig. 1) would be elevated in adult males because sexual maturity is marked by an increase in T secretion and is detectable in both matrices (Harrison and Ridgway, 1971; Kellar et al., 2009; Schroeder and Keller, 1989). Progestogens should be elevated in pregnant females, as has been observed previously (Kellar et al., 2006; Kirby and Ridgway, 1984; Mansour et al., 2002; Pérez et al., 2011; Sawyer-Steffan et al., 1983; Trego et al., 2013), and in sexually mature females (Inoue et al., 2018). We suspected that androgens would also be elevated in both tissues in pregnant females as reported in bottlenose dolphins and killer whales (Boggs et al., 2019; Galligan et al., 2018a; Robeck et al., 2017; Steinman et al., 2016). Additionally, corticosteroids should be elevated during pregnancy (Valenzuela-Molina et al., 2018). Finally, the corticosteroids should be positively correlated with elapsed time to sample collection because capture and handling stress induces cortisol secretion (Kellar et al., 2015; Kellar et al., 2013; St. Aubin et al., 1996; Thomson and Geraci, 1986) and be impacted by body condition (Kershaw et al., 2017). This comprehensive comparison of blood-blubber hormone profiles will improve our ability to use remotely collected blubber biopsies to study endocrine physiology in free-ranging marine mammals.

Section snippets

Animals, field data collection, and sample collection

Matched blubber and blood samples were collected from free-ranging common bottlenose dolphins (n = 77) from three locations in the southeastern United States during late spring and late summer/early fall (Barataria Bay, LA [June 2013, 2014; n = 34]; Brunswick, GA [September 2015; n = 16]; and Sarasota Bay, FL [May 2013–2016; n = 27]). Methods for the temporary capture, restraint, sampling, and release of common bottlenose dolphins have been previously described (Schwacke et al., 2014; Smith et

Results

Six hormones were detected and quantified in both blubber and plasma: P4, 17OHP4, AE, T, F, and E (Table 1, Table 2). S was detected and quantified in blubber only (Table 1, Table 2). We did not detect pregnenolone, 17-hydroxypregnenolone, DOC, B, dehydroepiandrosterone, dihydrotestosterone, estradiol, estrone, or estriol in any samples. Hormone concentrations for each individual are reported in Supplemental Table 6.

In the PCA, four components with eigenvalues >1 were extracted explaining

Discussion

The goal of this study was to explore the relationships between blood and blubber steroid hormone profiles in common bottlenose dolphins, and thereby, provide information to subsequently improve our ability to use remotely collected blubber biopsies to assess endocrine status in marine mammals. We accomplished this using LC-MS/MS to measure a broad suite of steroid hormones in matched plasma and blubber samples from free-ranging common bottlenose dolphins. We performed a PCA to explore

Acknowledgements

Funding for this work was primarily provided by the NMFS Marine Mammal Health and Stranding Program. Additional support was provided by the Medical University of South Carolina, the National Institute of Standards and Technology, and the National Marine Mammal Foundation. This research was also made possible in part by a grant from the Gulf of Mexico Research Initiative; those data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at //data.gulfresearchinitiative.org

Declaration of competing interest

The authors declare that they have no conflict of interest in the publication of this manuscript. Commercial equipment, instruments, or materials are identified to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology nor the National Oceanographic and Atmospheric Administration, nor does it imply that the materials or equipment identified are necessarily the best available for the

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