Open Veterinary Journal, (2025), Vol. 15(2): 911-922

Research Article

10.5455/OVJ.2025.v15.i2.40

Fecal glucocorticoid metabolites can be used as a stress indicator in bottlenose dolphins (Tursiops truncatus)

Estelle Ferenczi^1*, Birgitta Mercera², Karl Mercera¹, Caroline Gilbert^3,4, Fabienne Delfour^5,6 and Fanny Pilot-Storck^3,7,8,9

¹ADVETIA Centre Hospitalier Vétérinaire, Vélizy-Villacoublay, France

²Parc Astérix, Delphinarium, Plailly, France

³École nationale vétérinaire d’Alfort, Maisons-Alfort, France

⁴Laboratoire MECADEV, UMR 7179, CNRS/MNHN, Brunoy, France

⁵École Nationale Vétérinaire de Toulouse, Toulouse, France

⁶Animaux et Compagnies, Toulouse, France

⁷Université Paris-Est Créteil, INSERM, EFS, EnvA, IMRB Team Relaix, Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, France

⁸Université de Lyon, VetAgro Sup, Marcy-l’Etoile, France

⁹Université de Lyon, UCBL1 CNRS UMR5261, INSERM U1315, Institut NeuroMyoGene INMG-PNMG Team MNCA, Lyon, France

*Corresponding Author: Estelle Ferenczi. ADVETIA Centre Hospitalier Vétérinaire, Vélizy-Villacoublay, France. Email: estelleferenczi [at] hotmail.fr

Submitted: 22/10/2024 Accepted: 23/01/2025 Published: 28/02/2025

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Abstract

Background: Glucocorticoids increase in response to the hypothalamic–pituitary–adrenal axis stimulation, and their metabolites can be measured in dolphins’ feces.

Aim: This study aimed to assess the welfare of bottlenose dolphins under human care by measuring fecal glucocorticoid metabolites (FGM).

Methods: Our study consisted of measuring glucocorticoid metabolites concentration by enzyme immunoassay in fecal samples from five bottlenose dolphins housed in a dolphinarium. Dolphins were sampled once a month over a year, and 1 day before and 2 days after the three stressful events.

Results: We confirmed the validation of an extraction technique and an enzyme immunoassay to measure FGM and we observed an increase in their concentration after the stressful events, which provides a biological validation of this method. In parallel, we confirmed that males had a higher concentration of FGM than females, with a basal concentration of around 80 and 50 ng/g of dried feces, respectively.

Conclusion: Our study confirms that FGM measurement is a relevant indicator of stress response in bottlenose dolphins under human care, although it needs to take into account the sex and reproductive status of the animals.

Keywords: Bottlenose dolphin, Cortisol, Enzyme immunoassay, Feces, Stress.

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Introduction

Cetaceans face many stressors, both in the wild and in captivity (Lott and Williamson, 2017). Stressful events (or stressors) correspond to internal or external stimuli that threaten homeostasis balance and trigger a set of biological reactions under the control of the hypothalamic-pituitary-adrenal axis (Moberg and Mench, 2000). This results in an increased secretion of glucocorticoids, which are considered as potential indicators of stress. Glucocorticoids activate the cardiovascular system to concentrate blood flow to the muscles, stimulate water secretion, promote immunosuppression, increase anti-inflammatory action, stimulate neoglucogenesis in the liver and inhibit glucose utilisation, mobilise fatty acids from adipose tissue, degrade skeletal muscle proteins to provide substrates for gluconeogenesis, inhibit the release of Gonadotropin releasing hormone and Luteinizing hormone, and reduce the concentration of Luteinizing hormone receptors in the gonads, inhibiting the reproductive function (Sapolsky et al., 2000; Romero and Butler, 2007). In chronic stress, the plasma concentration of glucocorticoids is elevated for a long period, which can alter many functions of the organism, which have been described in cetaceans (Kyrou and Tsigos, 2008, 2009; Atkinson et al., 2015). Glucocorticoids have long-term inhibitory effects on growth and reproduction. Their immunosuppressive effects predispose them to infections. The degradation of skeletal muscle proteins leads to long-term muscle mass loss. A chronically high concentration of glucocorticoids inhibits osteoblastic activity, leading to the loss of bone mass, which can lead to osteoporosis. Visceral adiposity increases as a result of fatty acid mobilization from adipose tissue and the redistribution of fat (Sapolsky et al., 2000; Tsigos and Chrousos, 2002; Charmandari et al., 2005; Romero and Butler, 2007; Kyrou and Tsigos, 2008, 2009; Atkinson et al., 2015).

Assessing welfare should provide a better understanding of stressors and help reduce their impact on animals’ ability to adapt to their environment. Webster’s principle of triangulation (Webster, 2005) states that three categories of parameters (i.e., physiological, behavioral, and cognitive parameters) form a triangle whose center represents the welfare state of the animal. Reliably assessing animal welfare requires identifying and validating relevant indicators of welfare for these three categories.

This study is part of a European project aimed at assessing the welfare of captive bottlenose dolphins (Tursiops truncatus). Using a physiological approach, our study focused on a potential indicator of stress, i.e., a negative welfare indicator: glucocorticoid metabolites. Cortisol is the main glucocorticoid secreted by bottlenose dolphins (Atkinson et al., 2015). Glucocorticoids can be measured in blood, saliva, adipose tissue, expired air, urine, and feces (Möstl and Palme, 2002; Pedernera et al., 2006; Romano et al., 2010; Sheriff et al., 2011; Cook, 2012; Ugaz et al., 2013; Atkinson et al., 2015; Biancani et al., 2017; Monreal-Pawlowsky et al., 2017; Champagne et al., 2018; Mercera et al., 2021), but feces collection is most frequently used in terrestrial mammals since it is non-invasive and does not require to capture the animals (Atkinson et al., 2015). In bottlenose dolphins under human care, retrieval is minimally invasive and is well tolerated (Mercera et al., 2021). The two main available techniques to measure fecal glucocorticoid metabolites (FGM) are radioimmunoassays and enzyme immunoassays (EIAs), which both provide good sensitivity and specificity but differ in how they reveal antibody-antigen binding, i.e., by radioactivity for radioimmunoassays and by colorimetry for EIAs (Sheriff et al., 2011). EIAs thus present the advantage of being easily conducted with few equipment and in a standard laboratory (Skrzipczyk and Verdier, 2006). EIAs have already been technically validated for glucocorticoid metabolites measurement in bottlenose dolphins in saliva (Monreal-Pawlowsky et al., 2017), adipose tissue (Champagne et al., 2016, 2018), and feces (Mercera et al., 2021), yet a biological validation of their capacity to report a stress response in this species is still lacking.

The aim of our study was to determine whether the concentration of FGM, measured by EIA, can be used as an indicator of the stress response in captive bottlenose dolphins. The main objectives were (1) to validate a method for detecting the stress response in captive bottlenose dolphins and (2) to evaluate the influence of season and sex on FGM concentration.

First, we aimed to validate the extraction process by ensuring a satisfactory extraction yield, repeatability, and reproducibility of a method of FGM measurement that was first used by Mercera et al. (2021). Then, we tested whether a stressful event led to a significant increase in FGM concentrations. For this purpose, we measured FGM from fecal samples collected before and after imposed, non-experimentally, stressful events due to facility renovation that occurred at this time. Finally, we collected fecal samples from the same group of dolphins over a year to study the variation in FGM concentration according to season and sex.

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Materials and Methods

Animals

The study focused on five bottlenose dolphins at Parc Astérix dolphinarium (Plailly, France): two adult males (Cecil and Guama) and three females (Aya, Baïly, and Beauty) (Table 1). No dolphin was treated with corticosteroids (e.g., prednisolone, cortisone, or prednisone) during the study. The females Aya, Beauty, and Baïly were treated during the entire study with altrenogest (i.e., REGUMATE^®), a synthetic progestin used to prevent pregnancy.

Housing

The dolphins lived in three pools, permanently open and communicating with each other. The total surface area of the tanks was 1,045 m², with a total water volume of 3,790 m³. The water was heated to 14°C in winter to avoid exposing the dolphins to low temperatures. The outdoor pool was the largest, where presentations to the public were given. It had a surface area of 825 m² for a total volume of water of 3,240 m³ and allowed the evolution of animals in open air under natural lighting. Around the main pool, a large underwater viewing area was used for visitors to observe the animals. To reduce noise pollution, this underground passage was coated with rubber. The other two indoor tanks were not accessible to the public and had an area of 220 m² and a total water volume of 550 m³. They were occasionally used as isolation and care pools. Enrichment of the dolphin environment consisted of the provision of play objects, training sessions, contact with caretakers, and public performances. The facility was accredited by the European Association for Aquatic Mammals and its housing conditions complied with the welfare guidelines published by this association (European Association for Aquatic Mammals, 2019).

Sample collection

The dolphin feces collection technique and the equipment used in this study were identical to those described by Mercera et al. (2021). Before the start of the study, the dolphins were trained in the feces collection technique as part of a medical training program using positive reinforcement. They always participated in a voluntary way by putting themselves in dorsal decubitus. The study period extended from May 2019 to 2020.

Table 1. Status of the bottlenose dolphins of the study.

For the longitudinal study over seasons, caretakers collected fecal samples from the five dolphins once a month, with a total of 65 samples. Four sampling periods were grouped into seasons: from May 20, 2019 to June 20, 2019 and from March 20, 2020 to May 18, 2020 (called “Spring”), from June 21, 2019 to September 22, 2019 (called “Summer”), from September 23, 2019 to December 21, 2019 (called “Autumn”), and from December 22, 2019 to March 19, 2020 (called “Winter”). In case of insufficient samples or lack of cooperation from the dolphins, the caretakers renewed their attempts (with a maximum of four) during the same day and up to four days later. To minimize bias, only four caretakers took the samples.

For the stress response study, caretakers collected three fecal samples for each dolphin when a stressful event was scheduled (i.e., 1 day before the event (D-1), 1 day after (D+1), and 2 days after (D+2)), for a total of 45 samples. During the study, three episodes of construction work occurred in the large outdoor tank (14 August 2019 called “Event 1”; 07 September 2019 called “Event 2” and 22 September 2019 called “Event 3”) and were considered stressful events for the dolphins by the caretakers despite all the precautions they could take. During these construction works, the dolphins were locked into the two indoor tanks for about 10 hours because professional divers had to drill the bottom of the large outdoor tank. Several potential stress factors for the dolphins were thus combined: unfamiliar sounds, confinement, constrained social groups, and the presence of unfamiliar humans in their tanks.

Glucocorticoid extraction

Samples with insufficient fecal amount (i.e., less than one milliliter) or consisting predominantly of mucus, were excluded. The remaining samples underwent the extraction protocol described by Mercera et al. (2021).

Glucocorticoid metabolites measurement assay

Because bottlenose dolphins excrete a significant amount of glucocorticoid metabolites in feces (Biancani et al., 2017), measurement of FGM was performed by competitive EIA with a specific cortisol assay kit (Cortisol EIA kit #402710, Neogen^® Corporation Europe, Ayr, United Kingdom), as by Mercera et al. (2021).

Data and statistical analysis

Calculation of FGM concentrations in fecal samples was performed on myassays.com website from the obtained absorbances, compared to the standard curve. Each sample was assayed in duplicate and sample concentration was determined as the mean of the two values. When the coefficient of variability between duplicates was greater than 10%, a triplicate was performed. The mean of the two closest values was used as sample concentration. When the concentration obtained was above the analysis threshold (i.e., >10 ng/ml), the sample was diluted and then reassayed. FGM concentrations were expressed in nanograms per gram of dried feces (ng/g of dried feces). Kolmogorov-Smirnov test was performed on the Social Science Statistics website (Social Science Statistics, 2021), and statistical tests were performed on the BiostaTGV website (BiostaTGV, 2021) using the statistical software R. A difference was considered significant when the p-value was less than 0.05.

Extraction validation

Validation of the extraction technique consists of verifying that it meets the intended requirements for its use in terms of repeatability and yield (Keay et al., 2006; Palme et al., 2013; Andreasson et al., 2015). The repeatability was determined by dividing five samples into two after homogenization. The extraction yield was calculated by assessing six samples separated into two batches after homogenization, in which a known amount of cortisol (Hydrocortisone, 3867-1GM, Sigma-Aldrich) diluted in 100% ethanol (Ethanol absolute, 1.07017, Sigma-Aldrich) was added to one batch, whereas the same volume of ethanol only was added to the other batch.

Assay validation

Validation of an assay method consists of objective evidence that the technique meets the intended requirements for its use by assessing different parameters, such as precision (i.e., repeatability and reproducibility), parallelism, dilution linearity, and recovery (European Medicines Agency, 2011; Andreasson et al., 2015). In our study, we used the same cortisol detection kit as Mercera et al. (2021), who validated the method with satisfactory results. These measures were reproduced for robustness. The repeatability was determined by assaying two different samples eight times on the same microplate with the same operator. The reproducibility was calculated by assaying four different samples on four different microplates and at different dates with the same operator.

Ethical approval

This study adhered to the ASAB/ABS Guidelines for the Use of Animals in Research and was reviewed and accepted by the scientific and animal welfare committee of Parc Asterix.

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Results

Measurement of FGM concentration

We recovered 109 fecal samples over the 110 expected ones (65 monthly samples and 45 related to stressful events). After excluding inappropriate samples (<1 ml of volume, mucus excess, or <50 mg of dried feces), 102 samples (93%) were usable and underwent extraction and assay. The statistical description of FGM concentrations is presented in Table 2. A Kolmogorov-Smirnov test gave the following values: D=0.12256; p =0.08573, indicating that FGM concentrations followed a normal distribution, thus allowing the use of histograms for result presentation.

Table 2. Statistical description of the measured FGM concentration (n=102).

Extraction and assay validation

The repeatability and yield of FGM extraction were 9.98% and 80.6%, respectively. The repeatability and reproducibility of FGM measurements were 8.5% and 9.2%, respectively. We thus confirmed the validity of this extraction technique and EIA assay for FGM concentration measurement.

Effect of sex on FGM concentration

To confirm the effect of sex on FGM concentration, which was previously reported (Mercera et al., 2021), we addressed it in our study and observed significantly higher FGM concentrations for males than for females (80.52 and 51.08 ng/g, respectively; Student’s t test; p =0.002) (Fig. 1).

Effect of seasons on FGM concentration

When grouping male and female FGM concentrations by season (Fig. 2), the mean FGM concentration was around 65 ng/g of dried feces over the year, and the difference between seasons was not significant (ANOVA test; p =0.93).

When separating FGM concentrations by season between females and males (Fig. 3), the mean FGM concentration of females was stable at around 50 ng/g of dried feces throughout the year, and the difference between seasons was not significant (ANOVA test; p =1.0). The mean FGM concentration of males ranged around 80 ng/g of dried feces over the year, but it was higher in autumn and winter (around 90 ng/g of dried feces) than in spring and summer (around 75 ng/g of dried feces); however, this difference was not significant (ANOVA test; p =0.78).

Fig. 1. Mean (± SD) FGM concentration of female and male dolphins over the entire study (n=60). The numerical value above each histogram represents the mean of FGM concentrations for each group. *: p < 0.05.

Fig. 2. Mean (± SD) FGM concentration of all dolphins by season (n=60). The numerical value above each histogram is the mean FGM concentration for each group.

Fig. 3. Mean (± SD) FGM concentration of female and male dolphins in the different seasons (n=60). The numerical value above each histogram is the mean FGM concentration for each group.

Effect of opening the park to the public on FGM concentration

In this study, the park was open to the public, with dolphin’s performances, from May 2019 to September 2019. When separating the FGM concentrations of dolphins between the periods when the park was open or closed to the public, no significant difference was observed between the two periods (Fig. 4; Student’s t test for paired series; p =0.94).

We then separated the FGM concentrations of females and males when the park was open or closed to the public (Fig. 5). No significant difference was observed between the two periods for females (Student’s t test for paired series; p=0.98) and males (Student’s t test for paired series; p=0.88).

Effect of stressful events on FGM concentration

When looking at the whole group of dolphins, the FGM concentrations after the stressful events, corresponding to the mean of D+1 and D+2 samples, were significantly higher than those at D-1 before the stressful events (102.83 and 66.83 ng/g, respectively; Student’s t test for paired series; p =0.01) (Fig. 6).

Fig. 4. Mean (± SD) FGM concentration of all dolphins according to the opening of the park to the public (n=60). The numerical value above each histogram is the mean FGM concentration for each group.

Fig. 5. Mean (± SD) FGM concentration of female and male dolphins according to the opening of the park to the public (n=60). The numerical value above each histogram is the mean FGM concentration for each group.

We then separated the FGM concentrations in females and males (Fig. 7). For females, the mean FGM concentration after the stressful events was significantly higher than before (75.09 ng/g and 44.88 ng/g, respectively; Student’s t test for paired series; p =0.03). For males, the mean FGM concentration after the stressful events was also higher than before (135.20 ng/g and 92.43 ng/g), yet this difference did not reach a significant level (Student’s t test for paired series; p =0.11).

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Discussion

The aim of our study was to determine whether the FGM concentration, as obtained by EIA, could be used as a reliable indicator of the stress response in captive bottlenose dolphins. For this purpose, we first validated the method to ensure the reliability of the results through three distinct steps: (1) validation of the extraction process, (2) validation of the assay kit, and (3) biological validation.

Validation of the method

Regarding the validation of the extraction process, the reference method that has already been used in several species other than bottlenose dolphins (Mateo and Cavigelli, 2005) relies on radiolabelled cortisol, which requires accredited laboratories. In this study, we assessed the validity of an FGM extraction method and a measurement assay based on EIA.

Fig. 6. Mean (± SD) FGM concentration of all dolphins before and after the stressful events (n=42). “Before” corresponds to the day before the stressful events. “After” corresponds to the 2 days after the stressful events. The numerical value above each histogram is the mean FGM concentration for each group. *: p < 0.05.

Fig. 7. Mean (± SD) FGM concentration of female and male dolphins before and after the stressful events (n=42). “Before” corresponds to the day before the stressful events (D-1). “After” corresponds to the two days after the stressful events. The numerical value above each histogram represents the mean FGM concentration for each group. *: p < 0.05.

Regarding FGM extraction, we obtained satisfactory values for both repeatability and extraction yield (9.98% and 80.6%, respectively), confirming the relevance of this protocol for the measurement of FGM in bottlenose dolphins.

Validation of the assay kit was previously performed by Mercera et al. (2021), who achieved good parallelism as well as satisfactory dilution linearity, recovery, repeatability, and reproducibility. We also achieved satisfactory repeatability and reproducibility (8.5% and 9.2%, respectively), thus confirming the reliability of FGM concentrations measured with this kit.

Physiological validation consists of assessing whether a change in blood glucocorticoid metabolites concentration is reflected in FGM concentration. For this purpose, a change in glucocorticoid metabolite concentration is pharmacologically induced, for example, by an injection of adrenocorticotropic hormone (ACTH), which stimulates cortisol synthesis, or dexamethasone, which, on the contrary, inhibits it. Physiological validation of FGM concentration measurement following an ACTH injection has been performed on several species other than bottlenose dolphins (Mateo and Cavigelli, 2005; Stevenson et al., 2018). However, such injections, as well as dexamethasone injection are prohibited in Europe in captive bottlenose dolphins for ethical and medical reasons (Thomson and Geraci, 1986; Proie, 2013). Biological validation is an alternative to physiological validation and corresponds to the detection of a stress response induced by a natural stressful event (Cook, 2012; Atkinson et al., 2015). During the course of this study, three episodes of construction work occurred in the large outdoor tank and were considered stressful events for the dolphins by caretakers because they combined several stressors for the dolphins: sound, confinement, constrained social groups, and the presence of non-familiar persons in their tanks. We studied the change in FGM concentration between the day before and 2 days following the stressful events. For ethical and practical reasons, fecal sampling was not performed on the day of construction work but on the following two days. This timing was consistent with a previous report that the time lag between glucocorticoid secretion in blood and the fecal excretion of their metabolites was approximately 24 hours (Biancani et al., 2017).

Our results showed that the mean FGM concentration after stressful events was significantly higher than before. This means that the physiological stress response induced by a natural stress event can be detected by FGM concentration measurement. To our knowledge, this is the first biological validation of FGM concentration measurement as an indicator of stress response in captive bottlenose dolphins. This is in accordance with previous observations of an increase in glucocorticoid metabolites concentration after construction work in the saliva of bottlenose dolphins (Monreal-Pawlowsky et al., 2017). Thus, our data highlight that the measurement of FGM concentration is reliable in the two days after a potentially stressful event, compared to baseline, to detect a stress response.

FGM concentration ranges

In this study, FGM concentrations ranged from 13 and 254 ng/g of dried feces with a mean of 72.8 ng/g. In the study of Mercera et al. (2021), using the same extraction and assay protocols, concentrations ranging from 7 to 389 ng/g, with a mean of 80 ng/g, were recorded, which is close to those reported here. Previous studies on FGM concentration in bottlenose dolphins reported consistent values, such as 25 to 338 ng/g (Serres et al., 2020), 4 to 539 ng/g (Miller et al., 2021a), and 17 to 535 ng/g (Miller et al., 2021b), or more different ones such as 0.2 to 10 ng/g (Biancani et al., 2017), 150 to 4450 ng/g (Champagne et al., 2018), and 1,123 to 2,391 ng/g (Houser et al., 2021). The more different ones might be explained by differences between the facilities, dolphin features, and protocols used for extraction or assay. Comparison of the absolute values of FGM concentration between studies should thus be taken cautiously and comparisons of changes between groups or periods should be preferred.

Effect of sex on FGM concentration

Samples collected over a year showed an association between FGM concentration and sex, with males exhibiting a significantly higher FGM concentration than females. This confirms previous results observed in the blood (Houser et al., 2021) and feces (Mercera et al., 2021) of bottlenose dolphins, with the exception of pregnant females who had a significantly higher concentration than males (Mercera et al., 2021). This sex-related difference in glucocorticoid metabolite concentration has also been reported in other marine mammals (Hunt et al., 2004, 2006). This result suggests that gonadal steroids influence glucocorticoid metabolism and may be related to the increased energetic requirements of male dolphins due to sexual behaviors (St. Aubin, 2001). This key result confirms the need to consider dolphin sex and reproductive status when interpreting FGM concentration.

Effect of seasons on FGM concentration

Our study also showed the absence of significant seasonal variation in bottlenose dolphins’ FGM concentration. This lack of seasonal variation in glucocorticoid metabolites concentration was previously observed in blood (St. Aubin et al., 1996) and in feces (Biancani et al., 2017; Serres et al., 2020; Mercera et al., 2021) [although only in females in the study by Mercera et al. (2021)]. On the contrary, a recent study showed that serum glucocorticoid metabolites concentration was significantly higher in winter than in spring (Houser et al., 2021), while another study reported a significant increase in FGM concentration in males in spring compared with autumn and winter (Mercera et al., 2021). In this study, female FGM concentration was stable over the year, whereas male FGM concentration showed a variation, with a higher, but not significant, FGM concentration in autumn and winter. Given these discrepancies and the low statistical power of our study on this point, further experiments are warranted to conclude on the influence of season on FGM concentration in bottlenose dolphins.

Effect of opening the park to the public on FGM concentration

During the study period, Parc Astérix was open from May to September 2019 and closed from October 2019 to May 2020. The closure was accompanied by the cessation of dolphin performances for the public, and the number of caretakers was limited. However, the enrichment of the environment was maintained through training sessions, contact with caretakers, and provision of play objects. FGM concentration presented no significant variation between the periods when the park was open or closed. Some studies suggested that the presence of the public has a positive influence on captive animal welfare (Farrand et al., 2013; Bloomfield et al., 2015; Hosey and Melfi, 2015). On the contrary, other authors have shown that visitors can have a negative influence (Morgan and Tromborg, 2007; Hosey, 2017) or no effect (Margulis et al., 2003; Sherwen et al., 2014) on captive animal welfare. Serres et al. (2020) observed lower FGM concentrations when visitors were not present or rare and higher FGM concentrations when few or many visitors were present. The influence of visitors seemed to depend on the number of visitors, their behavior, the noise level, the enclosure conformation (including the presence of a retreat area), the presence of enrichment and training with caretakers, the species involved, and the personality of the animal ( Hosey, 2017; Sherwen and Hemsworth, 2019; Hashmi and Sullivan, 2020). In this study, the absence of FGM concentration variation suggests that exposure to the public was not a source of stress for the dolphins in the context of this study.

Effect of storage samples on FGM concentration

During our study, fecal samples were stored directly after collection in a freezer at −20°C until extraction. We performed extraction and assays of the samples several months after collection. However, some studies have shown that the storage time of samples influences FGM concentration in baboons (Papio cynocephalus) (Khan et al., 2002) and gorillas (Gorilla gorilla gorilla) (Shutt et al., 2012). Khan et al. (2002) advise that fecal samples should not be stored for more than 120 days at −18°C. Additional studies involving extractions with different time frames after the first sample is collected may provide interesting results on the effectiveness of our storage technique in bottlenose dolphins.

Effect of circadian rhythm on FGM concentration

The fecal samples were collected in the morning or afternoon depending on the schedule of the caretakers to avoid a constraint on their work day. Cortisol secretion follows a circadian rhythm in dolphins. It decreases in the evening and increases in the morning in the blood of Tursiops truncatus (Houser et al., 2021) and Tursiops aduncus (Suzuki et al., 2003) and in the saliva of Tursiops truncatus (Monreal-Pawlowsky et al., 2017). According to Palme (2005), the influence of circadian rhythm is lower in feces because it provides the mean cortisol concentration over several hours. However, circadian rhythms have been observed in feces in some species, such as the mouse (Mus musculus f. domesticus) (Touma et al., 2004). Additional studies involving fecal sampling at different times of the day from the same dolphins will provide insights into the influence of circadian rhythms on FGM concentration in bottlenose dolphins.

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Conclusion

This study aimed to assess a physiological indicator to evaluate the welfare of bottlenose dolphins under human care. It focused on the fecal concentration of glucocorticoid metabolites as a potential indicator of stress, i.e., negative welfare, in these species. In line with a previous report (Mercera et al., 2021), we confirmed the validation of an extraction technique as well as an EIA assay for FGM measurement. Our study further showed that FGM concentration was significantly higher in the 2 days after a stressful event than the day before, providing the first biological validation for this approach. This study thus confirms the interest of FGM measurement to evaluate a stress response in bottlenose dolphins. In parallel, fecal sampling over a year confirmed the influence of sex on FGM concentration, but not season.

This study reveals the interest of FGM measurement as an indicator of the stress response in bottlenose dolphins. However, since other factors such as sex and pregnancy influence FGM concentration, this parameter should be taken cautiously when comparing different animals and should be associated with other indicators of stress in the fields of physiology, behavior, and cognition to reliably conclude that there is a stress response (Webster, 2005; Boissy et al., 2007; Clegg et al., 2019; Delfour et al., 2021).

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Acknowledgments

We are grateful to all the dolphin trainers of Parc Astérix dolphinarium who provided fecal samples. We thank the whole group of comparative medical genetics at the National Veterinary School of Alfort (Maisons-Alfort, France), where we performed the hormone extraction and assay.

Conflict of interest

The authors declare no conflict of interest.

Funding

The authors received specific funding from Parc Astérix dolphinarium for cortisol EIA kits.

Authors’ contributions

Estelle Ferenczi: Conceptualization, Data curation, Methodology, Investigation, Formal analysis, Writing-original draft. Birgitta Mercera: Resources, Data curation, Funding acquisition. Karl Mercera: Conceptualization, Methodology. Caroline Gilbert: Writing-review and editing. Fabienne Delfour: Conceptualization, Methodology, Investigation, Supervision, Writing-review and editing. Fanny Pilot-Storck: Supervision, Methodology, Investigation, Formal analysis, Writing-review and editing.

Data availability

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

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