Social, Reproductive and Contextual Influences on Fecal Glucocorticoid Metabolites in Captive Yangtze Finless Porpoises (Neophocaena asiaeorientalis asiaeorientalis) and Bottlenose Dolphins (Tursiops truncatus)

Round 1

Reviewer 1 Report

• English (sentences format and punctuation) should be reviewed by an english native speaker.
• What is the LSS in the two dolphinaria?
• Table 4: number of fecal samples collected is not clearly reported. 
• Check references reported (reference 23 is not on BD and reference 41 is not on beluga)

Author Response

We thank the reviewers for their comments and constructive criticism.  We have amended the manuscript per their suggestions in the manuscript using the Track Changes function. Our comments to specific criticism are in bold below and any changes within the manuscript are also below with the in text change in blue and underlined.

Reviewer 1:

• English (sentences format and punctuation) should be reviewed by an english native speaker.

Two of the authors are english native speakers and carefully reviewed the manuscript

• What is the LSS in the two dolphinaria?

The information was added

• Table 4: number of fecal samples collected is not clearly reported. 

This has been corrected. Please note that we have re-ordered Tables 3 and 4 from the original manuscript and Table 4 is now Table 3.

• Check references reported (reference 23 is not on BD and reference 41 is not on beluga)

We have corrected this mistake.

Reviewer 2 Report

The manuscript by Serres and colleagues describes the establishment of an enzyme immunoassay to monitor alterations in faecal glucocorticoid metabolite (FGCM) concentrations in Yangtze Finless Porpoises and Bottlenose dolphins. The authors further looked at various potential stressors and how those influence FGCM output in the two species in a captive setting. The outline of the paper is clear and although it could be shortened a bit overall (e.g. section 4.2 in the discussion), it’s well-structured and the results are mostly clearly presented. Thus, there are only a few mostly minor aspects requiring revision or additional information.

Line 3: The study investigates influences on ‘fecal glucocorticoid metabolite concentrations’ not FGCMs per se. Please adjust and also double-check throughout the manuscript when referring to ‘GC’, ‘GCs’, and ‘FGCMs’, as ‘concentration(s)’ is sometimes missing.

Line 101-2: Please specify if faecal collection wasn’t successful or if it wasn’t tried for that individual

Line 119: The unit in the length column is indicated with ‘m/h’, please correct. Please also convert the length for the YFPs into meter (m)

Line 151: According to table1 its 49 samples for the BDs

Line 158: I suggest ‘…of a stressor being perceived.’

Line 169-70: The range of faecal wet weight used for steroid extraction for DBs is rather large and thus an impact of sample weight on fGCM concentration can’t be excluded (see Millspaugh & Washburn, General and Comparative Endocrinology 138 (2004) 189–199). Is there any chance that he authors can still test for that?

Line 180: It is highly unlikely that native hormone concentrations were measured in the respective faecal samples; thus please change ‘Cortisol’ to ‘GCM’ (again please check throughout the manuscript)

Line 181: According to the title, the reference cited (85) refers to a progestagen assay not a GC one, please clarify.

Line 183-4: Please explain the abbreviations ‘FP’ and ‘Tt”

Line 189: Please indicate how many wells were used on the plate to determine the Intra-assay CVs. Please explain further how two Intra-assay CVs were calculated when ‘only a pool of extracts’ were measured.

Line 190-2: Inter-assay CVs were determined twice, why? What were the two quality controls mentioned in line 190?

Line 196: omit ‘using’

Line 201-4: I might have missed it, but shouldn’t be ‘ID’ included as independent variable here?

Line 214: As sex-related differences in GC metabolism can be expected (see Touma et al. 2003 General and Comparative Endocrinology 130: 267-278), a comparison between males and females of a species is already questionable, but an inter-specific comparison is definitively not acceptable (see e.g. Palme 2019). Please revise this part of the analysis and subsequent parts in Results and Discussion.    

Line 249ff & 254ff: Those tables should be switched, as the authors currently referring to table 4 first.

Line 258 (Fig 1): Please correct y-axis label ‘FGCM concentrations (ng/g wet weight)’. Please also correct x-axis label for panel A: Male YFP; Female YFP (non-pregnant), and Female YFP (pregnant). Omit data for Male BD completely

Line 287-99: As pointed out in Palme 2019 and elsewhere, absolute FGCM concentrations could only be compared within a species if the extraction and EIA procedure were identical, thus the authors will have to revise parts of that paragraph and refer to trends and patterns instead of absolute values when comparing their findings with data from the literature.

Line 306: The authors provided a biological validation (comparison of FGCM values from pregnant and non-pregnant animals) for establishing the EIA for YFP. A respective validation is missing for DB and should be pointed out.

Author Response

We thank the reviewers for their comments and constructive criticism.  We have amended the manuscript per their suggestions in the manuscript using the Track Changes function. Our comments to specific criticism are in bold below and any changes within the manuscript are also below with the in text change in blue and underlined.

Reviewer 2:

The manuscript by Serres and colleagues describes the establishment of an enzyme immunoassay to monitor alterations in faecal glucocorticoid metabolite (FGCM) concentrations in Yangtze Finless Porpoises and Bottlenose dolphins. The authors further looked at various potential stressors and how those influence FGCM output in the two species in a captive setting. The outline of the paper is clear and although it could be shortened a bit overall (e.g. section 4.2 in the discussion), it’s well-structured and the results are mostly clearly presented. Thus, there are only a few mostly minor aspects requiring revision or additional information.

• Line 3: The study investigates influences on ‘fecal glucocorticoid metabolite concentrations’ not FGCMs per se. Please adjust and also double-check throughout the manuscript when referring to ‘GC’, ‘GCs’, and ‘FGCMs’, as ‘concentration(s)’ is sometimes missing.

This has been corrected throughout the manuscript.

• Line 101-2: Please specify if faecal collection wasn’t successful or if it wasn’t tried for that individual

We have added the information.

The group included five individuals, but one of the females was excluded from the study due to insufficient training for routine fecal sample collection.

• Line 119: The unit in the length column is indicated with ‘m/h’, please correct. Please also convert the length for the YFPs into meter (m)

We corrected these mistakes.

• Line 151: According to table 1 its 49 samples for the BDs

We corrected this mistake.

A total of 145 samples were collected for YFPs and 49 for bottlenose dolphins (Table 1).

• Line 158: I suggest ‘…of a stressor being perceived.’

This has been changed.

Based on previous research in BDs that has demonstrated acute stress event influences on FGCM concentrations occur within ~4.5 h of a stressor being perceived . . .

• Line 169-70: The range of faecal wet weight used for steroid extraction for DBs is rather large and thus an impact of sample weight on fGCM concentration can’t be excluded (see Millspaugh & Washburn, General and Comparative Endocrinology 138 (2004) 189–199). Is there any chance that he authors can still test for that?

We based our acceptable weight ranges on a publication by Hayward et al., Eliminating the artificial effect of sample mass on avian fecal hormone concentration Gen Comp Endo (2010) 169:117-122 and also cited and used for cetacean fecal GC metabolite analysis by Ayres et al., Distinguishing the impacts of inadequate prey and vessel traffic on an endangered killer whale (Orcinus orca) population. PLoS ONE, 7:e36842. Ranges for fecal samples in the Ayres et al. study ranged from 0.02 to 0.1 g of dried feces. Because we used wet feces, we aimed for 0.5 g of wet weight with a minimum requirement weight of 0.1g (20% of 0.5g), which was the same ratio for Ayres et al. (0.02g minimum requirement weight of 0.1g). However, the mean and medium weights for BD feces analysed were 0.471g and 0.512g, respectively. Eight out of 49 samples (16.3%) had weights < 0.4g and only four out of 49 (8.2%) had weights < 0.25g. Thus, any influence of low sample weight was minimal. We have clarified this in the manuscript.

For BD fecal samples, mean and median samples weights were 0.471g and 0.512g, respectively, and 4/49 samples (8.2% of total) had sample weights that were < 0.25g

• Line 180: It is highly unlikely that native hormone concentrations were measured in the respective faecal samples; thus please change ‘Cortisol’ to ‘GCM’ (again please check throughout the manuscript)

We have corrected this throughout the manuscript.

• Line 181: According to the title, the reference cited (85) refers to a progestagen assay not a GC one, please clarify.

The incorrect citation was included.  This has been corrected in the manuscript.

Munro, C. J., Lasley, B.L., 1988. Non-radiometric methods for immunoassay of steroid hormones. In: Anderson, B.D., Haseltine, F.D. (Eds), Non-radiometric assays: technology and application in polypeptide and steroid hormone detection. A.R. Liss Inc., New York, pp. 289-329.

• Line 183-4: Please explain the abbreviations ‘FP’ and ‘Tt”

We used the incorrect abbreviations for Yangtze Finless Porpoise (YFP) and Bottlenose Dolphin (BD). This has been corrected.

[YFP and BD males]

• Line 189: Please indicate how many wells were used on the plate to determine the Intra-assay CVs. Please explain further how two Intra-assay CVs were calculated when ‘only a pool of extracts’ were measured.

Two fecal extract pools, one female pool and one male pool, were made and tested on randomized wells on one microtiter plate (n = 16 replicates for each pool). To obtain the intra-assay CV of the replicates for each pool, the mean FGCM concentration for each pool was divided by its standard deviation and expressed as a percentage. We have clarified this in the manuscript.

To test for intra-assay variation, two pools of fecal extracts (one female, one male) were run across various, randomized wells (n = 16 for each sex) on the microtiter plate. The mean concentration for each sex was determined from all replicates and the coefficient of variation (CV) was calculated by dividing the mean by the standard deviation. The CV was expressed as a percentage and was 4.7% for females and 5.1% for males.

• Line 190-2: Inter-assay CVs were determined twice, why? What were the two quality controls mentioned in line 190?

In our laboratory, standard operating procedure is to have two sets of controls. The first set of controls is comprised of diluted, analytical standards and bind at approximately 30% and 70%, respectively. These controls are run on every assay in our laboratory to test for intra-assay variation within each specific hormone assay, regardless of species or sample matrix. Thus, we can analyse the performance of the hormone assay over time (currently 10+ years) to assess quality control and assay variation. Secondly, to analyse inter-assay variation within each species and sample matrix, we then have a second set of controls specific for each species and sample matrix type. These values are the second set of CVs that were reported. We are aware that many laboratories only run one set of controls, but they may not be species or matrix specific, and we want to ensure we are thorough in our analytical assay validations and quality control analysis for each study we conduct. We have clarified this in the manuscript.

Species specific biological controls made up of finless porpoise fecal extracts and to test for both species and matrix specific variation had inter-assay CVs of 9.2 and 12.6% (n = 6, 5) for males and females, respectively.

• Line 196: omit ‘using’

This has been corrected.

• Line 201-4: I might have missed it, but shouldn’t be ‘ID’ included as independent variable here?
• Line 214: As sex-related differences in GC metabolism can be expected (see Touma et al. 2003 General and Comparative Endocrinology 130: 267-278), a comparison between males and females of a species is already questionable, but an inter-specific comparison is definitively not acceptable (see e.g. Palme 2019). Please revise this part of the analysis and subsequent parts in Results and Discussion.    

We apologise but we are unsure of the exact meaning of this comment. However, as you are aware, including sex as an independent variable is an appropriate method to account for the variations observed within the dependent variable (hormone) that have been caused differences between sex. In addition, hormone concentrations from both species were determined within the same assay system at the same time, therefore, it is meaningful and we feel interesting to identify interspecies variations in the concentrations of hormones. The actual physiologic reason for these differences is certainly speculative and may or may be due to behavioural or physiological differences between species or many other factors – however we do not attempt to explain the species differences within the manuscript. We therefore feel that we will keep the results and discussion.

• Line 249ff & 254ff: Those tables should be switched, as the authors currently referring to table 4 first.

This has been corrected in the manuscript. Tables 3 and 4 have been re-ordered such that Table 3 is now Table 4 and vice versa. This has been corrected throughout the manuscript.

• Line 258 (Fig 1): Please correct y-axis label ‘FGCM concentrations (ng/g wet weight)’. Please also correct x-axis label for panel A: Male YFP; Female YFP (non-pregnant), and Female YFP (pregnant). Omit data for Male BD completely

We have modified the labels as requested but have chosen to keep the male BD results. We also used NP Female and Pregnant instead of your suggestions due to idiosyncrasies of the graphics software. As mentioned earlier and as described in the methods all samples from both species were determined by the same assay system at the same time, therefore interspecies comparisons of absolute concentrations is relevant and provides and interesting reference point. We do not discuss physiologic significance of these results within the paper.

• Line 287-99: As pointed out in Palme 2019 and elsewhere, absolute FGCM concentrations could only be compared within a species if the extraction and EIA procedure were identical, thus the authors will have to revise parts of that paragraph and refer to trends and patterns instead of absolute values when comparing their findings with data from the literature.

There are no hormone trends or patterns to refer to in this paragraph. Not referring to absolute values would imply to omit them. We mentioned the different assay methods used for each reference and have modified the paragraph to include the statement that comparisons of quantitative data may not be appropriate.

In a previous study in the BD using radioimmunoassay (RIA), FGCM concentrations ranged between 150 to 4450 ng/g with a maximum concentration of 21,000 ng/g after a stress test [6]. A range of 500 to 5000 ng/g for RIA assayed FGCM concentrations in wild killer whales has been reported [28]. In zoo-based killer whales, and using similar sample processing methods and the same assay methodology in the present study, ranges of 38 to 571 ng/g for FGCM concentrations during a biological stress response test have been reported [7], similar to the ranges reported here for both the YFP and BD. However, in another study, RIA assayed FGCMs ranged between 0.2 to 9.5 ng/g for five, healthy BDs [41]. The different ranges given by Champagne et al. [6] and Biancani et al. [41]might be explained by the differences between facilities or the features of studied animals (e.g., sex, age, dominance status). However, the methods used to process and extract cortisol and its metabolites from feces and the analyses (e.g., EIA versus RIA, varying antibody cross reactivity) can lead to differing quantitative results. As a result, quantitative comparisons of FGCM concentrations between studies may not be appropriate, but when a large enough data set is available, comparisons of trends and profiles are possible. Moreover, other factors that we did not include in our analysis may have also played a role and influenced FGCM concentrations (e.g., type of food), making any direct comparisons irrelevant.

• Line 306: The authors provided a biological validation (comparison of FGCM values from pregnant and non-pregnant animals) for establishing the EIA for YFP. A respective validation is missing for DB and should be pointed out.

We have added this.

Biological validation for BDs was not possible in our study, but has been performed previously for this species [6,54].