Comparative Biochemistry and Physiology, Part A 272 (2022) 111268 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa Blubber and serum cortisol concentrations as indicators of the stress response and overall health status in striped dolphins Clara Agusti a, *, Annaïs Carbajal b, Sergi Olvera-Maneu b, Mariano Domingo b, c, Manel Lopez-Bejar b, d a Zoo Animal Welfare Education Centre (ZAWEC), Veterinary Faculty, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain c CRESA (Centre de Recerca en Sanitat Animal), Campus Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain d College of Veterinary Medicine, Western University of Health Sciences, Pomona 91766, CA, USA A R T I C L E I N F O A B S T R A C T Editor: Michael Hedrick The impacts of environmental changes and anthropogenic threats in marine mammals are a growing concern for their conservation. In recent years, efforts have been directed to understand how marine mammals cope with Keywords: stressors and to assess and validate stress biomarkers, mainly levels of glucocorticoid hormones (e.g. cortisol) in Stress certain body tissues. The aims of this study were to assess the impact of different causes of stranding (chronically Blubber affected and bycaught striped dolphins) on cortisol concentrations in serum and in blubber; and to evaluate the Cortisol association between cortisol levels in these tissues. Blubber and blood samples were collected from striped Striped dolphin (Stenella coeruleoalba) Stranding dolphins (n = 42) stranded on the Mediterranean coast between 2012 and 2018. Cortisol concentrations were Bycatch measured by using enzyme immunoassay. A high correlation was found between circulating and blubber cortisol concentrations (R2 = 0.85, p < 0.01). Necropsies and pathological studies concluded that a third of the dolphins were bycaught in fishing nets and released by fishermen (Bycaught animals group), while the other two thirds were euthanized, or died, due to a disease or chronic condition (e.g. calves separated from the mother or animals infected with dolphin morbillivirus or Brucella ceti) that impeded survival (Chronically affected animals group). Cortisol concentrations (mean ± SD) were six times higher in chronically affected animals (35.3 ± 23 ng cortisol/g blubber and 6.63 ± 3.22 μg cortisol/dl serum) compared to those bycaught in fishing nets (6.2 ± 4.3 ng cortisol/g blubber and 1.15 ± 1.51 μg cortisol/dl serum). Results suggests that serum and blubber cortisol concentrations can contribute in inferring the overall health and welfare of free-ranging cetaceans. However, further research is required to understand better the kinetics of blubber cortisol incorporation and removal, the factors involved in these processes, and the local conversion of cortisol in the blubber. 1. Introduction cope with stress and maintain populations' sustainability (Fair and Becker, 2000). Marine mammal communities have been described to be As inhabitants of a changing environment, wild animals are exposed at high-risk in 47% of coastal waters worldwide (Avila et al., 2018). In to physical or psychological events that can disrupt their homeostasis. this context, understanding the interactions between stressors and ma­ The stress response occurs when the central nervous system perceives rine mammal welfare, fitness and survival is necessary to guide effective these events as a threat, developing a variety of behavioural and phys­ conservation efforts (Papastavrou et al., 2017). iological responses that allow the animal to cope with a dynamic envi­ The hypothalamic-pituitary-adrenal (HPA) axis is a crucial element ronment (Moberg and Mench, 2000). Anthropogenic stressors in marine of the stress response, releasing glucocorticoids into the bloodstream ecosystems, such as boat traffic, fisheries interactions, noise or pollution, when activated (Sapolsky et al., 2000). When released after an exposure are increasing and could be affecting the ability of marine mammals to to short-term stressors they are considered beneficial and indicative of Abbreviations: HPA, hypothalamic-pituitary-adrenal; BCC, blubber cortisol concentrations; SCC, serum cortisol concentrations; EIA, enzyme immunoassay; CBG, cortisol binding globulin. * Corresponding author. E-mail address: clara.agusti@awec.es (C. Agusti). https://doi.org/10.1016/j.cbpa.2022.111268 Received 14 February 2022; Received in revised form 4 July 2022; Accepted 5 July 2022 Available online 8 July 2022 1095-6433/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). C. Agusti et al. Comparative Biochemistry and Physiology, Part A 272 (2022) 111268 resilience (Mostl and Palme, 2002), but the continued releasing due to 2. Materials and methods persistent stressors can have a direct negative effect on fitness (Romero et al., 2009). Glucocorticoids are also mediators of essential metabolic 2.1. Ethics statement processes such as fat metabolism or energy regulation (Sapolsky et al., 2000). Cortisol is one of the main glucocorticoid hormones secreted in All samples for this study were collected post-mortem after carcasses marine mammals and there is an increasing interest in its assessment as a were recovered by the Marine Fauna Stranding Network of the Catalan biomarker for stress, health and physiological state (Atkinson et al., Government. No animals were directly targeted for this or any other 2015; Kellar et al., 2015; Kershaw et al., 2017; St. Aubin et al., 2001; associated study. Handling of carcasses was done with the official Trana et al., 2016). governmental permits AG-2015-474, PTOP-2016-663 and PTOP-2021- Cortisol concentrations in cetaceans can be measured in multiple 14, matrices such as serum or plasma, saliva, respiratory vapour (‘blow’), urine, faeces, blubber or skin (Bechshoft et al., 2015; Champagne et al., 2.2. Study area and animals 2018; Champagne et al., 2017; Fair et al., 2014; Hart et al., 2015; Mingramm et al., 2019; Pedernera-Romano et al., 2006; Suzuki et al., Samples were collected from 42 stranded striped dolphins along the 1998). Alternatives to the use of blood present advantages as most are North-western Mediterranean Sea, between June 2012 and February less invasive and facilitate hormonal assessments in free-ranging in­ 2018. All carcasses were necropsied at the Veterinary School of the dividuals. Moreover, alternative matrices can offer different insights of Universitat Autònoma de Barcelona (UAB) by ECVP-certified patholo­ cortisol levels over time, and are less affected by the time of the day, the gists, according to established procedures (Kuiken, 1991). Details about individual differences in circadian rhythms, and the transient exposure the subsequent pathological investigation can be found at (Cuvertor­ to acute daily stressors or capture stress (Lightman et al., 2008). et-Sanz et al., 2020). The preservation status of the carcasses was clas­ Blubber is the specialized hypodermic adipose tissue found in marine sified on a scale from 1 (very fresh) to 5 (very autolytic), according to mammals and circulating cortisol, as a lipophilic compound, can diffuse (Jauniaux et al., 2005). The percentages of carcasses in the preservation to fatty tissues (Hunt et al., 2013). Blubber cortisol concentrations (BCC) states 1, 2, 3 and 4 were 14%, 86%, 10% and 7%, respectively. have been measured in a variety of marine mammals in recent years Individuals included were 19 females and 23 males and were clas­ (Atkinson et al., 2019; Galligan et al., 2019; Kellar et al., 2015; Kershaw sified into four life history categories following guidelines of previous et al., 2017). authors (Calzada et al., 1997; Gómez-Campos et al., 2011): (1) calves, For instance, beach-stranded short-beaked common dolphins including neonates and individuals up to 1 year of age; (2) juvenile (Delphinus Delphis) showed sixfold higher BCC than bycaught individuals animals, (considering juvenile animals as those which measure <187 cm (Kellar et al., 2015) while ice-entrapped beluga whales (Delphinapterus for females and <190 cm for males); (3) mature females, including leucas) presented a sevenfold increase of BCC when compared with resting, lactating and pregnant females; and (4) mature males. harvested ones (Trana et al., 2016; Trana et al., 2015). Moreover, The nutritional status was classified as ‘non-emaciated’ or ‘emaci­ elevated BCC have been related with higher calf mortality and disease in ated’ with reference to anatomical parameters such as the evidence of indo-pacific humpback dolphins (Sousa chinensis) (Guo et al., 2022). certain prominent bones, the dorso-axial muscular mass, and the Overall, these studies revealed blubber as an alternative matrix to blood absence or limited presence of fat, taking account the age of the animal. useful for assessing the stress response in cetaceans. Moreover, blubber Blubber thickness was assessed during necropsy from the medium cortisol concentrations increased during fasting season in wild harbour lateral region behind the flipper. Lastly, bycaught animals were diag­ seals (Phoca vitulina) (Kershaw and Hall, 2016) and during substantial nosed following the criteria described by (Vázquez et al., 2016). nutritional deficits in short-beaked common dolphins (Kellar et al., Quetelet's index (mass/length2; Kershaw et al., 2017) was used for 2015) and harbour porpoises (Kershaw et al., 2017), evidencing the role assessing the correlation of BCC and SCC with body condition index in of cortisol during highly energetically demanding periods in these striped dolphins. Prior to relate Quetelet's index with BCC and following species. the recommendations from Kershaw et al. (2017) to validate a body Interestingly, elevations in BCC in association with serum cortisol condition index, we checked if Quetelet's index was able to differentiate concentrations (SCC) have been observed in samples collected simulta­ between emaciated and non-emaciated individuals (classified by visual neously (Champagne et al., 2018; Champagne et al., 2017). This rela­ inspection), death causes, age classes, and life-history stages. tionship was detected after performing hormonal challenges with hydrocortisone (Champagne et al., 2017) and out-of-water stress tests 2.3. Causes of death (Champagne et al., 2018) in captive bottlenose dolphins (Tursiops trun­ catus). Recently, Mingramm et al. (2019) and Sherman et al. (2021) also Thirteen individuals were included in the Bycaught group (6 females found that patterns in blubber and serum steroid hormones levels of and 7 males). General findings for these animals were absence of dis­ bottlenose dolphins were positively correlated. Overall, previous work ease, good body condition, full stomach, net marks, visceral congestion highlights that BCC determination can be a good tool to assess cortisol and marked muscular exertion. levels in free-ranging animals. Twenty-nine individuals were included in the Chronically affected The striped dolphin (Stenella coeruleoalba) is the most abundant group (13 females and 16 males). The cause of chronic stranding was cetacean in the Mediterranean Sea. It is considered ‘Vulnerable’ by the attributed to six contributing factors characterized as: calves separated IUCN (Aguilar and Gaspari, 2012), with incidental captures in fishing from the mother (n = 6); animals infected with dolphin morbillivirus nets listed as one of the main threats. In the Catalan coast, the most (DMV) (n = 6); animals infected with Brucella ceti (n = 5); animals with frequent cause of death in stranded striped dolphins between 2012 and septicaemia of unknown bacteria (n = 3); extensive parasitism and 2019 was bycatch (Cuvertoret-Sanz et al., 2020), which emphasizes an emaciation (n = 1); and unknown (n = 8). existent need to study and address better this threat in the western Mediterranean Sea (Aguilar and Gaspari, 2012; Bearzi, 2002). 2.4. Sample collection The aims of this study were to assess the impact of different causes of stranding on dolphins. We assessed serum and blubber cortisol con­ Full depth integument samples (N = 42) were collected from the left centrations in two groups: dolphins that died during bycatch events and lateral flank and stored at − 20C◦ . Cardiac blood (N = 28) was collected dolphins that stranded as a result of chronic effects (bycatch and through a cardiac puncture at the central point of a coronal line from chronically affected, respectively). Furthermore, we evaluated the as­ sternum to axillar fold, using a 20 ml syringe and a 16 G, 1.7 × 133 mm sociation between cortisol levels in serum and blubber. catheter needle (AngiocathTM). Samples were placed in sterile serum 2 C. Agusti et al. Comparative Biochemistry and Physiology, Part A 272 (2022) 111268 collector tubes and centrifuged (1372 g 6 min). Then, serum was ali­ cause of death: (1) Bycaught, that included stranded animals suspected quoted in 1 ml cryotubes and frozen at − 80 ◦ C analysis. to have been captured in fishing nets and released after incidental capture; and (2) Chronically affected animals, that included alive and 2.5. Sample preparation and hormone extraction dead stranded animals which were euthanized or died due to a disease or chronic condition that impeded survival. Epidermis and outer layer of the dermis, mostly composed of con­ Data were processed and analysed using SPSS software package nective tissue, were removed from all samples leaving only fat-filled version 17.0 (SPSS, Chicago, IL). All the values are presented as mean ± panniculus adiposus, which is going to be referred to as blubber. Each SD. A P-value <0.05 was considered for significance. Prior to analyses, a sample was then divided in two equal subsamples: the first one was kept Shapiro–Wilk test was performed to check normality of all the data. Data frozen at − 20C◦ until hormone extraction, whereas the second one was with non-normality distribution were square root and logarithmic (log freeze-dried (Kinetic Thermal System: condenser Dura-Dry Model 10) transformed and normality was evaluated again. Data in which an FD2055D0TOO, US) for 96 h as suggested by (Dunkin, 2005) before improvement in normality was not achieved were analysed by non- cortisol extraction. Water content was determined by weighing sub­ parametric tests. samples before and after freeze-drying. One-way analyses of variances (ANOVA) (p = 0.05) were performed Blubber hormone extractions were based on the methods described to determine if there were significant differences between striped dol­ by (Kellar et al., 2015) with some modifications. Outer edges of the phins life history categories in blubber thickness, amount of lipid subsamples were removed to avoid potential contamination or changes extracted, and water content (blubber composition parameters). Inde­ in the tissue by direct contact with air. Prior to the extraction, 0.1 ± pendent sample t-tests were used to determine significant differences 0.002 g of all blubber subsamples (fresh and freeze-dried) were trans­ between non-emaciated and emaciated dolphins in blubber thickness, ferred to a 2 ml eppendorf tube (T1). The contents of T1 were pre- amount of lipid extracted and water content. Spearman's correlation test grinded using small scissors for one minute and fixed inside a 10 ml was used to examine the association between cortisol values in fresh and stainless-steel grinding jar to homogenize the contents using a ball mill freeze-dried blubber samples, in serum and blubber and between BCC for 15 min at 25 Hz (MM200, Retsch, Haan, Germany). Grinding media using grams of blubber and grams of lipid as denominator units. Pear­ was separated and transferred to a new eppendorf tube (T2) that was son's correlation test was used to determine the co-linearity between the rinsed with 0.5 ml of ethanol, and the washed contents were transferred amount of lipid extracted from wet blubber and the water content. into a falcon tube (T3). The homogenization tube (T1) was then rinsed Correlations of blubber thickness and amount of lipid extracted were with 0.5 ml of ethanol and all content was transferred into T3. This step performed with a simple linear regression. was repeated once more, resulting in a total of 1.5 ml of ethanol with the Ten factors were assessed as potential correlates with BCC and SCC: homogenate inside T3. Two ml of a 4:1 ethanol:acetone solution were 1) cause of death (Bycaught or Chronically affected animals), 2) added to T3 and the resulting solution was vortexed and centrifuged at stranded death or alive, 3) sex, 4) emaciation 5) life history category, 6) 2800 g for 15 min. The supernatant was transferred into a new weighted stranding season, 7) carcass state of preservation, 8) blubber thickness, falcon tube (T4) and evaporated at 37 ◦ C. After evaporation all T4 were 9) amount of lipid extracted in fresh samples, and 10) Quetelet's weighted to obtain the amount of lipid extracted by subtraction of the morphometric index (mass/length2). The factors 1–4 were assessed pre-weighed T4 from the T4 after evaporation (Mello et al., 2017). Two through an independent sample t-test, the factors 5–7 using ANOVA and ml of diethyl ether were added to T4, vortexed and centrifuged. The the factors 8–10 using Pearson's correlation test. Tests including BCC as supernatant collected was transferred into a 10 ml-glass vial and intro­ the dependent variable were run twice using grams of wet blubber and duced into the oven (Heraeus model T6; Kendro Laboratory Products, grams of lipid extracted as denominator units. Langenselbold, Germany) with continuous venting at 30 ◦ C. The boiling A generalised linear model (GLM) using a normal distribution and point of diethyl ether is at 34.6 ◦ C, therefore, this step should always be identity link function was conducted for Quetelet's body condition index performed with caution. The residue was resuspended in 1.5 ml of (N = 42) to test the significance of the explanatory variables death acetonitrile, vortexed and 1.5 ml of hexane were added to the cause, age class and life-history stage. Backwards model selection using acetonitrile-lipid mixture. After vortexing and centrifuging, 1.5 ml of the the stepwise function was used to identify the variables that best explain acetonitrile lower layer were transferred into a new glass vial (T6) and the variation in Quetelet's body condition index and include it in the the process was repeated once more. The final portion of acetonitrile final model based on the smallest AICc. The model fit was assessed using was transferred into a falcon tube (T7) and evaporated. The evaporated the adjusted R2. contents were resuspended in 0.25 ml of 1 M phosphate buffered saline, vortexed and stored at − 20 ◦ C until analysis. 3. Results 2.6. Hormone detection and assay validation tests 3.1. Assay and method validation Cortisol concentrations and validation tests were determined by Mean intra- and inter-assay coefficients of variation were 3.56% and using competitive EIA kits (Neogen® Corporation Europe, Ayr, UK). 4.92% respectively, indicating high repeatability of the test. The line­ Following the essential criteria for immunological validation (Reimers arity of dilution presented a R2 = 0.99% and a mean percentage error of and Lamb, 1991), the accuracy, precision, specificity and sensitivity of − 0.92 showing a high correlation between the obtained and the theo­ the assays were determined. Extracts from fresh blubber, freeze-dried retical cortisol values. The average recovery percentage from the spike blubber and blood serum were separately pooled for assay validation. recovery test was 107.5 ± 7.65%. The sensitivity of the assay was 0.071 Specificity was given by the dilution test, determined by using 1:1, 1:2, μg of cortisol/dl of serum (μg/dl), 2.28 ng cortisol/g of blubber and 5.98 1:5 and 1:10 dilutions of pools with EIA buffer. Accuracy was assessed ng cortisol/g of freeze-dried blubber. through the spike-and-recovery test (N = 7). Precision was evaluated by A significant correlation was detected between cortisol values (mean calculating intra-assay (N = 6 in each assay) and inter-assay (N = 12) ± SD) of fresh (26.26 ± 23.55 ng/g of wet blubber, range 2.28 to coefficients of variation (CV). Finally, sensitivity was given by the 105.79) and freeze-dried (31.42 ± 25.39 ng/g of dry blubber, range to smallest amount of hormone concentration detected. 5.98 to 96.3) blubber samples (Pearson: r(31) = 0.92, p < 0.01). When cortisol concentration was measured as cortisol per gram of lipid 2.7. Statistical analyses extracted (ng cortisol/g lipid), a high correlation was also found be­ tween fresh (mean ± SD) (48.68 ± 47.81 ng/g of lipid extracted from Striped dolphins were divided into two main groups according to the wet blubber, range 4.18 to 165.87) and freeze-dried (36.8 ± 30.83 ng/g 3 C. Agusti et al. Comparative Biochemistry and Physiology, Part A 272 (2022) 111268 of lipid extracted from dry blubber, range to 7.26 to 122.22) blubber samples (Pearson: r(31) = 0.94, p < 0.01). The percentages of extracted lipids in 0.1 g of blubber were (mean ± SD) 86.93 ± 5.65% (range 71 to 95) in freeze-dried samples and 59.17 ± 13.08% (range 33 to 81) in fresh samples. From these results, values of cortisol in blubber were assessed only in fresh tissue (referred as ng/g of blubber) without the freeze-drying procedure throughout the study due to the higher simplicity of the method (Palme et al., 2013). Cortisol concentrations in fresh blubber were expressed using two denominators: ng of cortisol/g of blubber (wet) and ng of cortisol/g of lipid extracted (from wet blubber). As we suspected that the denominator unit used (mass of blubber vs mass of lipid) is a potential confounding factor for interpreting hormone levels, and research lacks for determining which one is the preferred, we decided to use both throughout the study to enable future in­ Fig. 1. Blubber cortisol concentrations were associated with serum cortisol terpretations and comparisons. concentrations. There was a positive correlation between blubber and serum cortisol concentrations (Pearson: r(26) = 0.89, p < 0.01). Yellow dots represent individuals of the Bycaugh group and blue dots individuals of the Chronically 3.2. Blubber thickness and composition affected group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Significant differences in blubber thickness were found among the life history categories considered (ANOVA: F(3, 37) = 2.81, p = 0.05; 3.4. Cortisol levels in relation to the type of death Table 1) as calves had significantly thinner blubber than other groups (juveniles, mature males and females). No significant differences were Serum cortisol concentrations ranged between 0.071 and 11.8 μg/dl. detected across life history categories in the amount of lipid extracted Blubber cortisol concentrations in fresh samples ranged from 2.28 to from wet blubber (ANOVA: F(3, 37) = 2.19, p = 0.11; Table 1), neither 105.79 ng/g of blubber and from 1.03 to 74.05 ng/g of lipid extracted. in the percentage of water (ANOVA: F(3, 27) = 2.27, p = 0.10; Table 1). Blubber cortisol concentrations (mean ± SD) were up to 6 orders of Mean blubber thickness of emaciated mature dolphins was not magnitude significantly higher in individuals of the Chronically affected significantly different from the average of those non-emaciated (t-test: t group (35.3 ± 23 ng/g of blubber and 66.24 ± 47.93 ng/g of lipid (16) = − 0.99, p = 0.119; Table 1). Conversely, there were significant extracted) compared to those from the Bycaught group (6.18 ± 4.35 ng/ differences in the amount of lipid extracted from wet blubber and the g of blubber and 6.18 ± 4.35 ng/g of lipid extracted; t-test: t(32.15) = percentage of water between emaciated and non-emaciated mature − 6.53, p < 0.01 and t(30.13) = − 6.51, p < 0.01 respectively; Fig. 2A).. dolphins (t-test: t(17) = 3.51, p < 0.01 and t(11) = − 5.8, p < 0.01 Mean SCC values of the Chronically affected group (6.63 ± 3.22 μg/dl) respectively; Table 1). were also 6-fold higher compared to values of the Bycaught group (1.15 A significant negative relationship was detected between amount of ± 1.51 μg/dl; t-test: t(22.44) = − 6.06, p < 0.01; Fig. 2B). lipid extracted from wet blubber (59.17 ± 13.08% lipid extracted mass/ wet mass, range from 33 to 81) and percentage of water (22.82 ± 13.05% water mass/wet mass, range 7.56% to 59.91%; Pearson: r(31) = 3.5. Cortisol levels as a function of biological features 0.92, p < 0.01). No significant differences were detected between males and females in SCC (t-test: t(26) = 1.613, p = 0.119), neither in BCC measured as ng/ 3.3. Correlation between cortisol levels in blubber and serum g of blubber (t-test: t(40) = − 0.177, p = 0.86) nor measured as ng/g of lipid extracted (t-test: t(40) = − 0.99, p = 0.922). Similarly, no signifi­ Blubber cortisol concentrations significantly correlated with SCC cant differences were found across life history categories in SCC when BBC were measured as a function of blubber tissue (Pearson: r(26) (ANOVA: F(3, 24) = 1.489, p = 0.243), neither in BCC measured as ng/g = 0.89, p < 0.01) and when BCC were measured as cortisol per gram of of blubber (ANOVA: F(3, 38) = 0.558, p = 0.646) nor measured as ng/g lipid extracted (Pearson: r(26) = 0.8, p < 0.01; Fig. 1). When the of lipid extracted (ANOVA: F(3, 38) = 0.635, p = 0.597). Bycaugh group was analysed separately a high correlation was found Significant difference in BCC was found between stranded alive (36.2 between BCC and SCC for both BCC measured as ng/g of blubber ± 21.9 ng/g of blubber and 72.2 ± 49.9 ng/g of lipid extracted) and (Pearson: r(5) = 0.86, p = 0.01) and measured as ng/g of lipid extracted stranded dead animals (20.16 ± 22.8 ng/g of blubber and 34.2 ± 41 ng/ (Pearson: r(5) = 0.91, p < 0.01). Similarly, a high correlation was found g of lipid extracted; t-test: t(40) = − 3.361, p < 0.01 and t(40) = − 2.687, between BCC and SCC in the Chronically affected group for both BCC p = 0.01 respectively). This difference was not found for SCC concen­ measured as ng/g of blubber (Pearson: r(19) = 0.79, p ≤ 0.01) and trations (stranded alive: 7.21 ± 3.02 μg/dl; stranded dead: 5.7 ± 3.5 μg/ measured as ng/g of lipid extracted (Pearson: r(19) 0.72, p < 0.01). dl). Table 1 Striped dolphins (Stenella coeruleoalba) blubber composition data for each life history category and body condition. Calves Juveniles Mature Mature p value Non-emaciated mature Emaciated p value females males individuals mature individuals Blubber thickness (mm) 8.9 ± 3.1a 12.5 ± 3.1 b 10.6 ± 3.3a,b 10.4 ± 2.2a,b 0.05 10.9 ± 2.6 8.3 ± 1.5 0.119 Lipid extracted mass/wet mass 66.5 ± 61.2 ± 13.2 59.8 ± 14.6 51.9 ± 13.4 0.10 59.8. ± 12 38 ± 4.5 < 0.01 (%) 5.7 14.8 ± Water mass/wet mass (%) 22.6 ± 13.5 17.9 ± 7.2 30.6 ± 15.0 0.10 20.3 ± 8.1 48.2 ± 3.3 < 0.01 4.5 Values are expressed by mean ± S.D. For all measurements, categories with the same letter are not significantly different (p > 0.05) 4 C. Agusti et al. Comparative Biochemistry and Physiology, Part A 272 (2022) 111268 Fig. 2. (A) Blubber cortisol concentrations in bycaught and stranded striped dolphins (Stenella coeruleoalba). Horizontal box lines represent the lower quartile, median and upper quartile values. Whiskers lines indicate range of concentrations. Chronically affected individuals (35.3 ± 23 ng/g of blubber and 66.24 ± 47.93 ng/g of lipid extracted) had on average 6 times more blubber cortisol concentrations than bycaught individuals (6.18 ± 4.35 ng/g of blubber and 6.18 ± 4.35 ng/g of lipid extracted; t-test: t(32.15) = − 6.53, p < 0.01 and t(30.13) = − 6.51, p < 0.01 respectively). (B) Serum cortisol concentrations in bycaught and stranded striped dolphins (Stenella coeruleoalba). Chronically affected individuals (6.63 ± 3.22 μg/dl) had on average 6 times more serum cortisol than bycaught individuals (1.15 ± 1.51 μg/dl; t-test: t(22.44) = − 6.06, p < 0.01). No significant differences were detected across stranding seasons diagnosed as bycaught in fishing nets. This study used a modified (autumn, spring, summer and winter) in SCC (ANOVA: F(3, 24) = 1.138, method for steroid hormone extraction based on that from (Kellar et al., p = 0.354), neither in BCC measured as ng/g of blubber (ANOVA: F(4, 2015) and an enzyme immunoassay (EIA) to measure BCC and SCC in 37) = 1.24, p = 0.311) nor measured as ng/g of lipid extracted (ANOVA: striped dolphin (Stenella coeruleoalba). Serum cortisol concentrations F(4, 37) = 1.039, p = 0.4). No significant differences were detected and BCC were associated and were influenced by the cause of death and across carcass states of preservation (1 to 4) in SCC (ANOVA: F(2, 25) = by some biological features like the body condition. 0.572, p = 0.572), neither in BCC measured as ng/g of blubber (ANOVA: F(3, 38) = 0.253, p = 0.859) nor measured as ng/g of lipid extracted 4.1. Assay and method validation (ANOVA: F(3, 38) = 0.651, p = 0.587).Emaciated individuals presented significantly higher BCC (mean ± SD) (45.8 ± 26.8 ng/g of blubber and Assay results indicate that blubber cortisol detection by EIA with the 95.5 ± 48.6 ng/g of lipid extracted) than those non-emaciated (20.15 ± methodology presented here is successful, even using an EIA kit not 19 ng/g of blubber and 34 ± 37.5 ng/g of lipid extracted; t-test: t(40) = designed specifically for blubber. − 3.361, p < 0.01 and t(40) = − 4.207, p < 0.01 respectively). No sig­ The excellent correlation obtained in BCC between fresh and freeze- nificant differences were detected between emaciated and non- dried blubber samples suggests that both preparation techniques present emaciated individuals in SCC (t-test: t(19.93) = − 1.796, p = 0.088). A similar extraction efficiencies. In addition, these results suggest that the negative correlation was found between BCC and blubber thickness for elimination of water by freeze-drying the samples is increasing the both BCC expressed as ng/g of blubber and ng/g of lipid extracted amount of cortisol detected but in a proportional way. (Pearson: r(40) = − 0.41, p = 0.08 and r(40) = − 0.39, p = 0.01 respectively). 4.2. Blubber thickness and composition There was a significant effect of death cause on Quetelet's body condition index, which showed that the Chronically affected animals Calves presented the thinner blubber thickness compared to the were in poorer condition than Bycaught ones (p < 0.01). The index also other life history categories evaluated (Table 1). This result was ex­ suggested that mature animals were in better condition than calves and pected given that blubber thickness has been related to body mor­ juveniles (p < 0.01), but age was not retained in the best-fitting model phometries (Gómez-Campos et al., 2011). Blubber thickness of (p = 0.74). The model for mass/length2 fitted well to the data with an emaciated individuals did not vary compared to those animals not adjusted R2 value of 0.49. Moreover, mean mass/length2 of the animals emaciated (Table 1) and a negative relationship was found between classified as non-emaciated by visual inspection (0.0019 ± 0.0005) was blubber lipid and water content of all individuals. Blubber thickness may higher than in emaciated animals (0.0014 ± 0.0004; p < 0.05). not significantly diminish during catabolism of fat, as blubber contains Quetelet's index (mass / length2) significantly correlated with BCC many collagen bundles and elastic fibbers in which adipocytes are when measured as a function of blubber tissue (r = − 0.34, p < 0.05) but interspersed (Rommel and Lowenstine, 2001). In emaciated adults, not when measured as cortisol per gram of lipid extracted (p = 0.19). No where lipid content was clearly reduced, water content increased. These correlation was found between Quetelet's index (mass / length2) and results may indicate a replacement of lipid with water, which is thought SCC (p = 0.548). to be a mechanism for maintaining the structural integrity of the tissue No significant differences or correlations were found when analysing to preserve blubber's other functions (Dunkin, 2005). Therefore, blubber BCC and SCC with stranding season, carcass state of preservation and thickness seems an inadequate tool for assessing nutritional condition of amount of lipid extracted in fresh samples (p = 0.311, p = 0.4, p = 0.354 dolphins, coinciding with other authors (Caon et al., 2007; Gómez- respectively). Campos et al., 2011; Joblon et al., 2014). Conversely, blubber lipid content seems to be a better indicator of nutritional status (Gómez- 4. Discussion Campos et al., 2011). The lack of differences between life history categories in the blubber Here, blubber cortisol concentrations were examined as a potential lipid and water contents (Table 1) may be due to the limited number of supplement to serum cortisol concentrations for assessing the physio­ individuals since significant differences have been shown with a larger logical stress response of cetaceans. Concentrations in both matrices sample size (Gómez-Campos et al., 2011). were markedly higher in individuals stranded with a disease or chronic condition that impeded survival than in stranded animals that were 5 C. Agusti et al. Comparative Biochemistry and Physiology, Part A 272 (2022) 111268 4.3. Correlation between blubber and serum cortisol concentrations dolphins (Delphinus capensis and Delphinus delphis) and spotted dolphins (Stenella attenuata): 5 and 4.7 min respectively (Evans, 1971; Heyning There is very little published data about corticosteroid levels in and Perrin, 1994; Scott et al., 1993). marine mammals in simultaneously collected tissue samples. Cham­ The mammalian stress response is characterized by two temporal pagne et al. (2018 and 2017) results indicate an increase in BCC waves of stress mediated actions. The first one occurs in seconds and concomitant with increased SCC within 2 h of the onset of stress. Time includes noradrenaline and corticotropin-releasing hormone (CRH). elapsed since the capture of free-ranging bottlenose dolphins was a Then, with the HPA pathway activated, adrenocorticotropin hormone significant predictor of BCC, thus occurring an increase in BCC due to (ACTH) is released from the anterior pituitary into the bloodstream capture stress between 53 min and 215 min post capture (Galligan et al., stimulating glucocorticoid production in the adrenal cortex. Mammalian 2020). Meanwhile, a high positive relationship of testosterone concen­ endocrinological research shows that generally an increase in circu­ trations has been described in bottlenose dolphin serum and blubber lating glucocorticoids above baseline levels can be measurable within paired samples (r2 = 0.932; Sherman et al., 2021). As it is thought that 3–5 min. Glucocorticoid concentrations continue to rise to peak levels free cortisol diffuses into adipose tissue (Breuner et al., 2013), BCC can after 15–30 min and then decline to baseline levels within 60–90 min somehow reflect circulating cortisol concentrations that are integrated (Ron de Kloet et al., 2005; Sapolsky et al., 2000). A death within a few over a short time period (less than ~2 h). Nevertheless, Galligan et al. minutes of entrapment is likely and could explain the low levels of SCC (2020) did not found this correlation between the two matrices probably of the Bycaught group. If bycaught dolphins had died in a hyper-acute because samples were not collected simultaneously and the intervals way, i.e., in <3–5 min by asphyxia, this would explain both SCC and between blood and blubber collection were different across individuals. BCC low levels. Further, variability in times between entrapment and Overall, our results support previous findings and suggest that this death may have had influence on SCC. association occurs during endogenous cortisol release in free-ranging Mean BCC values obtained in relation to the type of death (Bycaught animals. However, in the case of acute stress responses, the reflection group: 6.18 ± 4.35 ng/g of blubber and 6.18 ± 4.35 ng/g of lipid in blubber would depend on the delay between adrenal cortisol secretion extracted; Chronically affected group: 35.3 ± 23 ng/g of blubber and and its integration in blubber, as well as the clearance rate and the 66.24 ± 47.93 ng/g of lipid extracted) are close to those obtained by sampling moment (discussed in 4.4). Moreover, blubber has been Kellar et al. (2015), who reported mean values of 3.99 ng/g of blubber recognized as a site of active steroid metabolism and it should be and 24.3 ng/g of blubber in bycaught and stranded short beaked com­ considered when quantitatively linking circulating cortisol concentra­ mon dolphins, respectively. Trana et al. (2016) reported mean BCC tions with BCC (Galligan et al., 2018). values of 0.25 and 1.79 ng/g of blubber in blubber from subsistence harvest and entrapped beluga whales respectively. It is noteworthy that 4.4. Cortisol levels in relation to the cause of death although the magnitude of the values differs, the differences observed between groups are very similar in the three species: a sevenfold in­ Blubber cortisol concentrations and SCC values from the stranded crease in entrapped beluga whales (Trana et al., 2016) and a sixfold animals were higher than those detected among bycaught animals. This increase in stranded short beaked common dolphins (Kellar et al., 2015) result was indeed not unexpected as cortisol concentrations can be and the striped dolphins of this study. Moreover, Champagne et al. influenced by many factors such as duration of stress, changes in cortisol (2017) reported baseline BCC of 1.4 ng/g of lipid in captive bottlenose binding globulin (CBG) concentrations and degree of perfusion in the dolphins and in the same animals, after the onset of a stress test, mean case of blubber. blubber cortisol concentrations raised from 3.4 to 16.9 ng/g of lipid at The increased serum cortisol concentrations in the Chronically sample times of 0 and 120 min respectively. Therefore, blubber cortisol affected group (6.63 ± 3.22 μg/dl) are comparable and slightly higher to seems to increase in a similar way during chronic stress and shortly after what has been reported during stressful events in other species: ~4.54 acute stress scenarios. μg/dl (Fair et al., 2014) and ~ 4.27 μg/dl (Hart et al., 2015) after cap­ The time course of cortisol aggregation into blubber may vary as a ture and handling in free-ranging bottlenose dolphins; ~5.04 μg/dl after function of environmental conditions. Ambient temperature and diving- chase and encirclement by a tuna purse seiner in free-ranging pantrop­ surfacing periods are factors that are thought to affect blubber cortisol ical spotted dolphins (Stenella attenuate; St. Aubin et al., 2013); and 3.62 integration. Exposure of blubber to circulating cortisol (and therefore μg/dl (Champagne et al., 2018). the subsequent hormonal quantification) could increase due to heart Meanwhile, serum cortisol concentrations in the Bycaught group rate increase and peripheral vasodilation. This happens during surface (1.15 ± 1.51 μg/dl) are marginally higher than some baseline concen­ intervals, temperature increases and after exercise (Champagne et al., trations reported from captive animals: 0.87 μg/dl (Champagne et al., 2018; Noren et al., 1999, 2012; Williams, 2015). Therefore, stranded 2018) and 0.725 μg/dl (Champagne et al., 2017) in bottlenose dolphins animals that are unconditioned to being out of the water might accu­ and 0.50 μg/dl in Indo-Pacific bottlenose dolphins (Tursiops aduncus). mulate cortisol faster than bycaught ones. Further, illness could lead to Although data is difficult to compare and cortisol levels may vary across foraging decline and therefore to a nutritional deficit that causes pro­ species and due to captivity condition, low levels in comparison to the longed elevated cortisol levels (Kellar et al., 2015). Prolonged percep­ Chronically affected animals and similarity to basal levels in other tion of threat, persistent pain and group separation could also elevate species suggest that SCC in bycaught animals are unexpectedly around glucocorticoid levels in a permanent way. baseline levels of striped dolphins. Cortisol synthesis occurs in pulsatile events in the adrenal gland Pathological studies have concluded that hypoxia and subsequent followed by infusion into the bloodstream (Chrousos, 1995). From asphyxiation is the main cause of mortality of bycaught cetaceans in blood, free cortisol reaches target tissues through capillaries. Blubber bottom-set nets (Ijsseldijk et al., 2021; Soulsbury et al., 2008). Animals accumulates contaminants and hormones by passively diffusing from may not be able to rise to the surface and thus the period between the capillaries found throughout the lipid (Deslypere et al., 1985; Mead capture and death may be in the order of minutes, leaving limited time et al., 1986). To our knowledge, cortisol uptake into mammalian adipose for animals to overstrain (Ijsseldijk et al., 2021; Soulsbury et al., 2008). tissue cells occurs by passive diffusion through the phospholipid bilayer Marked muscular exertion findings in some bycaught dolphins could be because of its lipophilic character (Deslypere et al., 1985; Mead et al., related to a certain degree of physical struggle (Moore et al., 2013). If 1986). Nevertheless, it is not clear whether active transport through the animal struggled to free itself, this would require a greater delivery carriers also occurs in this process (Lee et al., 2016; Rao, 1981). In of oxygen, which, added to the physiological acute stress-response, human adipose intracellular compartment, individual cortisol pulses can could very quickly lead to asphyxiation. The maximum dive duration be distinguished, and cortisol peaks seems to occur with a delay of ~3 of striped dolphins may be similar to that described for common min in adipose capillary bead and of ~35 min. in interstitial and 6 C. Agusti et al. Comparative Biochemistry and Physiology, Part A 272 (2022) 111268 intracellular space (Lee et al., 2016). In addition, intraadipose cortisol calves. Nevertheless, we did not assess other potential indices that might also comes from the local conversion of cortisone into cortisol by the fit better to de data and our sample was not large enough to represent all enzyme 11βhydroxysteroid dehydrogenase type 1 (Stimson et al., 2009). reproductive classes. Moreover, we were not able to assess blubber trunk Thus, BCC measured here may reflect a combination of both circulating lipid mass (BTLM), which was signalled as the best nutritional condition concentrations of cortisol and the local transformation of the hormone in index for striped dolphins (Gómez-Campos et al., 2011). the tissue itself (Kershaw et al., 2017). Analyses showed that BCC when measured as a function of blubber The uptake of steroid hormones by the total adipose mass has been tissue were weakly negatively correlated with body condition index, as described as dependent of fat content, being proportionally higher in described on harbour porpoises in Kershaw et al. (2017). Importantly, obese rats (Zomzely et al., 1959) and humans (Bleau et al., 1974). Bleau this relationship was not found when BCC was measured as cortisol per et al. (1974) suggested that the uptake of cortisol by adipose tissue is gram of lipid extracted, highlighting the significance of the choice of one much less than that of other steroid hormones and that cortisol is not or other unit when interpreting data. Long-term periods of reduced food stored in this tissue for prolonged periods as occur with progesterone. intake or fasting are associated with an increase in the circulating con­ Thus, we suggest that blubber cortisol concentrations are not useful centrations of cortisol in pinnipeds (Champagne et al., 2012). During to distinguish between short (acute) and long-term (chronic) stress. these periods cortisol increases gluconeogenesis (Exton et al., 1972) and However, BCC have been signalled as an indicator of chronic activation is involved in lipolysis to provide energy (Bergendahl et al., 1996). of the HPA axis in different species of cetaceans (Loseto et al., 2017; Therefore, increased cortisol concentrations in the blubber of striped Mingramm et al., 2020; Teerlink et al., 2018; Trana et al., 2016). dolphins in poorer body condition may also be related with increased Blubber cortisol concentrations units vary among reports making in­ mobilization of fat reserves (Kershaw et al., 2017). terpretations and direct comparisons difficult. Here we propose that the Blubber cortisol concentrations should therefore not only be inter­ unit ‘ng of cortisol/g of lipid extracted’ may be more suitable that ‘ng of preted based on the psychological stress but considering the other roles cortisol/g of blubber’ as a large part of the measured cortisol is probably of cortisol in the regulation of lipolysis and overall energy balance in dependent of fat content as discussed before. Either way, it is necessary mammals. In turn, the analysis of cortisol concentrations in remotely to reach a consensus or until then authors should present the results in obtained blubber biopsies may be also informative of the body condition both units or justify the use of either. However, further research of free-ranging cetaceans for which morphometric measurements comparing BCC in both scenarios (acute vs. chronic stress), revealing cannot be obtained. However, further research is needed to differentiate hormone integration and clearance rates in blubber and determining the effects of emaciation, acute and chronic stress on blubber cortisol how much of the cortisol pool in cetacean blubber is derived from local concentrations. production is necessary to confirm these affirmations. Baseline BCC of healthy populations can be obtained through remote 5. Conclusions biopsy sampling while baseline SCC are unlikely to obtain from samples of alive free-ranging marine mammals due to capture stress alteration. Cortisol concentrations in blubber and blood collected from dead So far, circulating hormone concentrations from free-ranging cetaceans stranded animals were successfully measured by using EIA. Blubber have been measured from blood obtained by capturing live animals, a cortisol concentrations and SCC were on average of six to seven times process that is not easy and implies an invasive procedure (Atkinson higher in stranded dolphins compared to those bycaught. Moreover, the et al., 2015; Fair et al., 2014; Hart et al., 2015). We suggest that the use two matrices showed a high correlation, suggesting that in this case BCC of blood collected from dead stranded animals for hormone analysis is reflected circulating cortisol concentrations. The results obtained show possible, but the effect of haemolysis and post-mortem changes in car­ that both BCC and SCC can help to infer in the physiological state and casses on different preservation states should be assessed accurately. overall health status in wild cetaceans. Additionally, mass/length2 ratio appeared to be an appropriate morphometric index for striped dolphins 4.5. Cortisol as a function of biological features that correlated negatively with BCC, highlighting the role of cortisol in the regulation of overall energy balance in mammals. Further, BCC No differences in BCC nor in SCC among sexes, ages or seasons were added to other indicators could provide valuable information about the found coinciding with St. Aubin et al. (1996) and Kellar et al. (2015). physiological state before death in stranded cetaceans and therefore Conversely, other authors detected seasonal variations in SCC (Funasaka about the potential causes of death. However, further research is et al., 2011; Houser et al., 2021; Mingramm et al., 2019; Suzuki et al., required to understand better the kinetics of blubber cortisol incorpo­ 2003) and BCC (Mingramm et al., 2019) as well as sex-related variations ration and removal, the factors involved in these processes, and the in SCC (Houser et al., 2021; Mingramm et al., 2019). To date, these potential local conversion of cortisol in the blubber. relationships are not clear since research on cetacean stress physiology has been carried out from animals under pathological processes, using Funding low sample sizes or isolated samples. This also occurs in the present study, where the specific differences in age, sex, or seasonality, if any, This research did not receive any specific grant from funding would be masked by type of death. Considering that many studies across agencies in the public, commercial, or not-for-profit sectors. vertebrate taxa have found a variation in cortisol levels with respect to sex, age, reproductive state, and season, more studies are needed to Declaration of Competing Interest address if this also occurs in cetaceans. Healthy animals together with larger sample sizes and long-term serial sampling would help to solve The authors declare that they have no known competing financial these issues. interests or personal relationships that could have appeared to influence Mass/length2 appears to be an appropriate morphometric index for the work reported in this paper. inferencing about the body condition of striped dolphins. The index was consistent with the hypothesized relationships between type of death, Acknowledgements life history classes and emaciation classified by visual inspection. Bycaught animals were in better body condition as they died in a sudden We thank Direcció General de Medi Natural i Biodiversitat Permit for event in comparison with the Chronically affected animals where a the authorization to collect and keep samples from stranded striped general debilitation and deterioration in health occurred due to disease dolphins. 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