Non-invasive monitoring of adrenocortical function in female domestic pigs using saliva and faeces as sample matrices

Tanja E Wolf; Norbert Mangwiro; Folorunso O Fasina; Andre Ganswindt

doi:10.1371/journal.pone.0234971

. 2020 Jun 25;15(6):e0234971. doi: 10.1371/journal.pone.0234971

Non-invasive monitoring of adrenocortical function in female domestic pigs using saliva and faeces as sample matrices

Tanja E Wolf ^1,^‡,^*, Norbert Mangwiro ^2,^‡,^*, Folorunso O Fasina ^2,³, Andre Ganswindt ^1,⁴

Editor: Henrik Oster⁵

PMCID: PMC7316308 PMID: 32584863

Abstract

Intensive pig management involves in a commercial setting the housing and implementation of certain procedures, such as castration and tail docking, which may be stressful for the animal. Good farming practices include the reduction of stress due to management processes, but assessing the level of stress perceived entirely through behavioural observations can be challenging. The monitoring stress-related physiological markers, like glucocorticoids (GC), can be an accurate alternative that would presumably be more objective. In order to avoid an additional stressor by taking blood, a non-invasive approach is advisable. We used an adrenocorticotropic hormone (ACTH) stimulation test and the effect of transport to examine the suitability of different enzyme immunoassays (EIAs) for monitoring adrenocortical function in domestic pigs using saliva and faeces as sample matrices. An assay measuring faecal glucocorticoid metabolites (fGCMs) with a 3ß,11ß-diol group has proven suited to determine adrenocortical activity, showing an overall increase of 180% in fGCM concentrations related to ACTH administration and of 70% related to transport, respectively. A cortisol EIA was used to detect salivary glucocorticoid (sGC) concentrations, revealing a 1100% increase in sGC concentrations after ACTH administration. The stability of fGCM concentrations post-defecation was determined to assess possible changes in measured fGCM concentrations in unpreserved faecal material over time, with fGCM concentrations being relatively stable (maximal 12% change) under natural conditions for approximately two days after defecation. This implicates that untreated faecal material from pigs can be analysed for up to two days after collection without appreciable level of depreciation in fGCM concentrations. Being able to assess the physiological stress response of domestic pigs non-invasively can help to improve the well-being of commercially reared pigs.

Introduction

Over 1 billion pigs (Sus scrofa domesticus) can be found worldwide [1], and a large proportion of these animals are intensively managed in confined pens and sow crates, and these housing conditions have raised questions and concerns on animal welfare [2–4]. In this regard, animal welfare issues are often evaluated by monitoring proxies for perceived intrinsic and extrinsic stress [5]. Potential stressors could be periods of weaning, transportation, mixing, and confinement, as well as events like castration and tail docking among others [6]. As pigs are very susceptible to stressors, those can potentially influence the quality of the meat [7]. Due to animal welfare concerns and rising consumer interests, the management and housing conditions of pigs and its potential stress-inducing effects are of utmost interest to the industry [4,8]. So far, it is common practice within the pig farming industry to evaluate stress caused by management practices using indicators like vocalization or stereotypic behaviours, which can be susceptible to observer bias [8]. An alternative approach would be the monitoring of stress-related physiological markers, like glucocorticoids, which would presumably be more objective [3].

In response to a perceived stressor, corticotropin-releasing hormone (CRH), released from the hypothalamus will stimulate the pituitary to release adrenocorticotropic hormone (ACTH), which in turn stimulates the adrenal cortex to release glucocorticoids [9,10]. Glucocorticoids (GC) can therefore be seen as key elements in the neuroendocrine stress axis, and their measurement can give insight into an animal’s well-being [11]. So far, blood, saliva, and to a quite lesser extent urine have been used as hormone matrix for monitoring glucocorticoid alterations in pigs [12–15]. Although blood is still wildly used for determining glucocorticoid concentrations in a wide range of species including pigs, respective results are often of limited value as determined steroid levels are usually affected by episodic fluctuations and a diurnal pattern of hormone secretion [16]. In addition, blood collection often requires restraining of the animal, which is usually perceived as a stressful event in itself [17]. Consequently, minimal- or even non-invasive monitoring of steroid hormone metabolites, using alternative matrices like saliva, urine, and especially faeces, have become an increasingly popular technique [18,19]. However, the collection of saliva still requires some manipulation of the animal, and thus cannot be regarded as entirely feedback free. Furthermore, it can only be applied to a limited extent in free-moving animals [16,20]. Finally, salivary steroid concentrations are also susceptible to circadian rhythm and episodic fluctuations of hormone secretion, and are therefore not ideal, as sampling needs to take place at a standardized time of the day when investigating chronic stress. Using faeces as basic sample material for determining glucocorticoid output in pigs would be an ideal approach, as it does not involve handling and therefore can avoid alteration of the results due to handling stress. Furthermore, faecal samples are less affected by daily variation in hormone secretion [21], and can be comparatively easily collected even under free-roaming conditions. So far, however, attempts to reliably determine adrenocortical activity as a measure of stress in domestic pigs using faeces as sample matrix have led to inconclusive results, leading to doubts whether glucocorticoid alterations can actually be measured reliably using pig faeces [22].

Therefore, the overall aim of this study was to examine the suitability of four enzyme immunoassays (EIAs) for monitoring adrenocortical function in domestic pigs using saliva and faeces as sample matrices. More specifically, the objectives of this study were to: a) determine stress-related physiological responses in domestic pig’s saliva and faeces by performing an adrenocorticotropic hormone stimulation test (ACTH challenge test), b) determine the effect of circadian rhythm on salivary glucocorticoid (sGC) and faecal glucocorticoid metabolite (fGCM) levels, c) verify the effect of transport on fGCM levels, and d) investigate the stability of fGCM levels post-defecation.

Material and methods

Study animals and housing

Six Large White-Landrace F1 gilts (weight between 43.7 and 47.7 kg), approximately 14 weeks of age, were housed at the Biomedical Research Centre (UPBRC), University of Pretoria, South Africa, for the study. After the initial transport to the UPBRC, the pigs were allocated to a straw-bedded pen for acclimatisation to the new environment for 3 days. Thereafter, the animals were individually-housed in metabolic crates and had ad libitum access to food and water. The housing conditions (e.g. temperature, humidity) were monitored and the crates were cleaned on a daily basis.

After the end of the experiment, the animals were moved to standard grow-out pens and not used any further. The entire study was performed under the approval and in accordance with the guidelines of the Animal Ethics Committee of the University of Pretoria (V094-13).

Monitoring of transport and ACTH challenge test

The duration of the entire study was 17 days. To investigate the effect of relocation to biologically validate the most suited EIA, one faecal sample per day was collected from each animal from the day of arrival at UPBRC for three consecutive days (n = 18). No sampling took place for the following four days of acclimatisation. For determining individual baseline values faeces were again collected for 6 days prior ACTH injection. On the day of ACTH administration (early morning), the pigs were minimally restrained by blocking each animal off to a side of the metabolic crate without the use of force. Four of the six animals (T1 –T4) were each injected with approximately 50 IU (10 μg/kg) of ACTH analogue (Synacthen^© Depot, Norvartis Switzerland) in the neck muscle. The remaining two animals served as controls (C1 + C2) and received 0.5 ml physiologic saline (0.9% Sodium chloride) each through the same route. Sample collection continued on day of injection and for another 3 days post injection. In addition, saliva samples were collected frequently from three days prior to three days post ACTH injection.

Sample collection

Throughout the study, saliva was collected, at least twice a day, once between 08:00–08:30 h in the morning and again between 15:00 and 15:30 h in the afternoon. On the day of ACTH/Saline injection, salivary samples were collected approximately an hour prior administration and subsequently 20, 40, 60, 90, and 120 min post injection. Thereafter, saliva was collected at hourly intervals until 8 hours post-administration, and further twice daily until the end of the experiment. Saliva was collected using Salivette® tubes (Sarstedt, Germany). The sponges were either held using forceps or hanged into the crate with a transparent string in order to allow the pigs to chew on it for 1 to 2 min.

To investigate the effect of transport on fGCM levels, individual faecal samples were collected once per day between 12:00 and 18:00h. After the four days of full acclimatisation, faecal samples were collected daily between 08:00 and 18:00h starting six days before the ACTH injection until three days post- injection. During this period, samples from all voided faeces were collected. Faecal samples were collected as soon as they were voided using the outlets on the base and the sides of the metabolic crates. Using gloves, about 10 to 15 g of freshly voided material was taken from the core of each faecal sample, mixed and placed in a sterile plastic container.

Immediately after collection, faecal and salivary samples were stored at -20°C and kept frozen until further processing. A total of 161 faecal samples (n = 72 prior and n = 89 post injection) and 170 saliva samples (n = 42 prior and n = 128 post injection) were collected throughout the experiment.

Effect of collection time and degradation study

In order to investigate a possible influence of collection time on sGC as well as fGCM concentrations, steroid concentrations determined in salivary (six days of collection) and faecal (six days of collection) samples collected in the morning were compared to respective steroid values from samples collected in the afternoon.

To determine the effect of increasing time intervals between sample defecation and freezing on the stability of fGCM concentrations, freshly voided faeces from three pigs were collected additionally, prior to injection. The faecal material was homogenized, and divided into 24 equal sub-samples, which were then stored at ambient room temperature (22°C to 28°C). At different time points (0, 1, 2, 4, 8, 16, 24, and 48 hours post-defecation) three sub-samples were taken and stored frozen at -20°C until analysis [23].

Steroid extraction

The saliva was obtained from the sponge following the protocol by Ruis and colleagues [6]. In brief, the thawed sponge was centrifuged for 10 min at 1500g. The retrieved saliva was then stored at -20°C until further analysis.

The frozen faecal material was lyophilized, pulverized, and sieved through a mesh to remove remaining fibrous material [24]. For steroid extraction, 0.100 to 0.110 g of the faecal powder was vortexed for 15 minutes with 3 ml of 80% ethanol. After centrifugation at 1500g the supernatants were transferred into microcentrifuge tubes and stored at -20°C until analysis [23].

Steroid analysis

Retrieved saliva was measured for salivary glucocorticoid (sGC) concentrations via a cortisol enzyme immunoassay (EIA) using an antibody against cortisol-3-CMO:BSA [25]. Further details on the assay characteristics including a full description of the assay components and antibody cross-reactivities are described in Palme & Möstl 1997 [25]. The sensitivity of the EIA was 0.6 ng/ml, and the intra- and inter-assay coefficients of variation (CVs), determined by repeated measurements of high and low value quality controls, were 9.5% and 11.0% (for Intra-CV) as well as 15.0% and 15.2% (for Inter-CV), respectively.

To identify a suitable EIA for effective assessment of adrenocortical function using faeces as sample matrix, a selected set of faecal extracts (n = 31) from three pigs were measured for faecal glucocorticoid metabolite (fGCM) concentrations. Four different enzyme immunoassays were tested, namely (i) a 11-oxoaetiocholanolone I EIA (detecting 11,17 dioxoandrostanes; 11,17-DOA), (ii) 11-oxoaetiocholanalone II EIA (detecting fGCMs with a 5β-3α-ol-11-one structure; 3α,11-oxo-CM), (iii) a 5α-pregnane-3β,11β,21-triol-20-one EIA (measuring 3β,11β-diol-CM), and (iv) the above mentioned cortisol EIA. Detailed assay characteristics, including full descriptions of the assay components and cross-reactivities were previously described by Palme and Möstl [25] for the 11,17-DOA and cortisol, by Möstl et al. [26] for the 3α,11-oxo-CM, and by Touma et al. [27] for the 3β,11β-diol-CM EIA. After identifying the most suitable EIA with regards to fGCM elevation post-ACTH administration, the entire sample set, including the material from the stability experiment, was assessed using only the 5α-pregnane-3β,11β,21-triol-20-one EIA.

The sensitivity of the assays were 0.6 ng/g faecal dry weight (DW) for 11, 17-DOA, 3α, 11-oxo-CM and cortisol, and 1.5 ng/g DW for the 5α-pregnane-3β, 11β,21-triol-20-one EIA. Intra-assay coefficient of variations, determined by repeated measurements of high and low value quality controls, were 9.5% and 11.0% for the cortisol EIA, 4.9% and 5.9% for the 11, 17-DOA EIA, 6.1% and 8.0% for 3α, 11-oxo-CM EIA, and 4.9% and 6.3% for the 5α-pregnane-3β, 11β, 21-triol-20-one EIA. Inter-assay coefficient of variation, only determined for the 5α-pregnane-3β, 11β, 21-triol-20-one EIA by repeated measurements of high value quality controls, was 9.5%. All assays were performed on microtiter plates following established protocols [28] at the Endocrine Research Laboratory at the Faculty of Veterinary Science, University of Pretoria.

Data analysis

To determine reliability of an EIA by showing an adequate increase in glucocorticoid output post ACTH-administration samples from three individuals from 48 hours pre-injection until 36 hours post injection were used. To establish a baseline individual median fGCM concentrations were calculated using pre- ACTH administration samples. Peak fGCM concentrations following ACTH administration were expressed as percentage increase above baseline concentrations.

Subsequently, determined sGC and fGCM concentrations from all individuals were compared to respective individual baseline levels using the chosen assays. Descriptive statistics were used to describe peak increase after ACTH/saline injection in percentage for sGC and fGCM concentrations.

To biologically validate the chosen EIA the initial transport of the animals was used and the fGCM concentrations in percentage were compared to the subsequent individual baseline levels during the acclimatisation period.

To evaluate the effect of time of day on GC concentrations, samples collected in the morning (saliva: 8:00–8:30h, faeces: 8:00–10:00h) were compared to samples collected in the afternoon (saliva: 15:00–15:30h, faeces: 12:45–18:00h). On six different days, a minimum of one sample per matrix per individual for all six study animals was collected in the morning and afternoon, respectively (total n = 2x 36 samples per matrix) In case that more than one sample was collected from an animal within a respective time window on a given day the mean steroid concentration of those samples was used for analysis. Individual sGC, as well as fGCM concentrations from morning samples were compared to individual afternoon samples using a Wilcoxon signed rank test.

The relative fGCM metabolism rate post defaecation (%) was calculated on pooled samples using the mean value determined at t = 0 as 100%. Differences in fGCM concentrations between sampling subsets were subsequently described as change in percentage. All statistical analyses were performed using R, version 3.0.2 [29].

Results

ACTH challenge test

Administration of synthetic ACTH resulted in an overall median increase of 1100% above baseline in sGC levels (Fig 1), with peak concentrations occurring 40 to 90 minutes after injection. Administration of saline (controls) was reflected in a median increase of 400% above baseline sGC concentrations with peak concentration also present 40 to 90 minutes after injection. Overall sGC concentrations returned to baseline 5 to 24 h post injection for ACTH, and 1 to 4 h for the controls, respectively.

All four assays tested for fGCM concentrations revealed an overall increase between 110% and 180%, and peak values occurred within 12 to 24 h after injection (Table 1). However, the 5α-pregnane-3β, 11β,21-triol-20-one EIA showed the most comparable baseline and peak values for all three animals and was chosen for the analysis of the remaining faecal samples.

Table 1. Baseline and peak fGCM concentrations, and overall median increase (%) for samples from the three animals tested after ACTH injection with four enzyme immunoassays (EIAs).

EIA	11-oxoaetio-cholanolone I	11-oxoaetio-cholanolone II	5α-pregnane-3β,11β,21-triol-20-one	Cortisol
Individual median fGCM baseline (ng/g DW)	31.0	127.4	234.3	35.4
	57.8	273.9	224.7	51.1
	97.9	272.1	205.7	25.3
Individual peak fGCM levels post ACTH injection (ng/g DW)	44.9	289.3	419.5	61.8
	162.0	765.1	549.0	105.8
	372.5	1373.7	559.8	68.6
Overall median increase	180%	180%	140%	110%

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DW = dry weight.

Overall baseline fGCM level, using the 5α-pregnane-3β,11β,21-triol-20-one EIA, for all 6 pigs was 226.4 ng/g dry weight (DW) (range 215.7 to 255.1 ng/g DW). Administration of ACTH resulted in an overall 180% (range: 60–220%) increase above baseline in fGCM levels, with peak concentrations present 24 hours after injection (Fig 2). Overall fGCM concentrations returned to baseline 36 to 48 hours post injection.

Fig 2 — Longitudinal profiles of faecal glucocorticoid metabolite (fGCM) concentrations of six pigs monitored directly after arrival, adaptation period, prior- and post injection of synthetic ACTH (a) or saline (b) using a 5α-pregnane-3β,11β,21-triol-20-one EIA. Arrows indicate transport (1) and injection (2).

Effect of transport on fGCM

The initial transport of the pigs to the experimental facility was used as a biological validation of the chosen EIA. The transport was reflected in overall increased alterations of fGCM levels of 70% against subsequent baseline values between 24 and 48 h after the event (Fig 2). The fGCM levels decreased during the acclimatisation period and reached baseline levels seven days after arrival at the experimental facility.

Effect of collection time

A significant difference was found in sGC concentrations when comparing samples collected in the morning compared to samples collected in the afternoon (V = 571, p < 0.001), with sGC concentrations collected between 08:00–08:30h being higher than sGC concentrations collected between 15:00 to 15:30h. In contrast, no significant difference was found, when comparing fGCM concentrations of samples collected either in the morning (08:00–10:00h) or the afternoon (12:45–18:00h, V = 315, p = 0.786).

Stability test for fGCM concentrations

Within the first hour at ambient temperature, fGCM concentrations showed an initial decrease of 12%. The fGCM concentration than stabilized over the next 24 hours at approximately 90% of the initial concentration (Fig 3). Variation in fGCM concentration between sample subsets ranged from 2.3 to 15.2% for respective measuring points in time.

Discussion

Reliable monitoring of adrenocortical activity in a particular species using fGCMs requires a solid validation of the test system in question as different animal’s gastrointestinal transit (GIT) systems and microbiome activities may differentially produce different fGCM [9,15]. The main route of excretion for glucocorticoids in pigs is urine and so far no assay system has been proven suited for measuring fGCM in pigs [20]. Previous studies have used an ACTH challenge, as well as a dexamethasone suppression test [22], or a biological validation [30] to identify a suitable test system using faeces as sample matrix, however with mixed results. After the injection of ACTH in only three out of six animals an increase in glucocorticoid metabolites could be measured using an 11-oxoaetiocholanolone I EIA [22], while the biological validation comparing pre- and postweaning samples confirmed the validity of the used corticosterone EIA [30]. In this regard, our study demonstrates a non-invasive technique to monitor adrenocortical function based on measuring the concentrations of glucocorticoid metabolites in faeces, using an ACTH challenge test for physiological validation and using the initial transport event as an additional biological validation.

For the saliva samples, a cortisol assay (using an antibody against cortisol-3-CMO:BSA) has been shown to reliably detect salivary GC concentrations. An increase in sGC levels of over 1000% occurred within the first 90 minutes post injection, which is consistent with the findings of Bushong et al. 2000 [31], who also observed a four to six fold peak response of sGC in barrows. The results found in pigs are comparable with other species. In horses for example a significant increase in sGC was found 30 minutes post injection, with a peak after approximately 2 hours [32]. In ewes a similar timeframe was found with a peak in salivary cortisol release approximately 60 minutes after ACTH administration [33].

After the initial screening of 4 available enzyme immunoassays (EIAs) for the measurement of faecal glucocorticoid metabolites an assay using an antibody against 3β,11β-diol-CM has been identified to suitably determine fGCM concentrations in female domestic pigs. Peak fGCM concentrations post injection showed an increase of approximately 180% compared with the baseline values about 24 hours post injection before returning back to baseline values 48 hours post injection. Although individual baseline levels varied between 215.7 and 255.10 ng/g DW, all individuals reacted to the ACTH/saline injection with increased fGCM levels. In horses, a similar peak between 200 and 600% in fGCM levels could be observed between 16 and 34 hours post ACTH injection. In contrast to our study the values in the horses decreased back to basal concentrations after already half a day [22]. In goats a similar long period of elevated fGCM could be found, here the levels did not reach baseline after 24 hours [34].

Whereas sGC concentrations rapidly returned to baseline following administration of ACTH, a delay in fGCM concentrations could be seen and this is most likely attributed to the intestinal passage, which causes a time delay between the circulation of steroids in the plasma and the appearance of their metabolites in the faeces [35].

Not only the actual transport can be a stressful event for animals, but also being in a transporter itself can lead to an increased glucocorticoid release. In pigs it has been shown, that blood cortisol levels increased significantly after being loaded onto a transport vehicle [36]. When the vehicle was moving the plasma cortisol levels remained elevated for a longer time period compared to the vehicle staying stationary. Although the transport in our study caused an increase in fGCM levels, this increase is distinctively lower than the increases found in other studies, for example in sheep an increase of approximately 300% was observed [37]. The observed differences may be due to the durations and conditions of transportations, as well as due to species and measurement differences. The pigs in this experiment were transported for about 20 minutes on a smooth road whereas in the experiment of Lexen et al. 2008 [37], the animals were transported for approximately 60 minutes.

As with many other hormones, plasma glucocorticoids have a circadian rhythm in most vertebrate species [16,20], and diurnal variations of fGCMs were already demonstrated for at least some mammals [16]. In pigs, the basal concentrations of cortisol in blood are generally higher in the morning than in the afternoon and evening [6]. Our findings showed that there was a significant difference in sGC concentrations in samples collected in the morning compared to those collected in the afternoon, with higher concentrations in morning samples. This has also been observed in other studies comparing morning and evening samples [6,38,39]. The results confirm that circadian rhythmicity of cortisol should be taken into consideration when evaluating stressors in pigs using saliva as a sample matrix. However, fGCM concentrations did not show a significant difference between morning and afternoon samples and thus might be a more suited approach to monitor adrenocortical activity if frequent sampling at a specific time cannot be guaranteed.

The stability of fGCMs post-defecation has been determined to assess possible changes in measured hormone concentrations in unpreserved faecal material. Steroid concentrations varied by 13% over a period of 48h. These changes agree with other studies, that showed an increase in fGCM levels within a few hours (4–24 h) post defecation (cattle & horses [22]; sheep [37]). However, a rather negligible change in fGCM levels over several days has been reported in other species [40–42]. While results can differ distinctively even when using the same assay but in different species and vice versa [9,37], to date, there is no empirically conclusive explanation for this observation other than bacterial enzymes and microbiome activities. In different animal species different bacteria may be present in the faeces which further metabolize fGCMs differently [16,43]. Therefore, it is necessary to test storage methods if immediate freezing of samples is not possible [9].

From the findings in this study, fGCM in pig faeces was relatively stable for approximately two days. The implication of this finding is that faecal materials from pigs can be collected from distant locations and transported to the laboratory for up to two days without appreciable level of depreciation in fGCM concentrations.

Potential limitations of the current study is the sex and the age of the animals with only immature females. Further studies with mature individuals of both sexes would be helpful to elucidate the suitability of the described assay.

Conclusion

Animal well-being can be defined as the animal living in harmony within the environment, meaning that it is able to adapt to it physically as well as psychologically [44]. Measuring psychological well-being of an animal is difficult, and therefore evaluating health and behaviour are an alternative [45]. Being able to non-invasively assess the physiological stress response of domestic pigs by using faeces as a sample matrix can help to improve the well-being of commercially reared pigs. Potential applications could for instance be the possibility of measuring the potential effect of different rearing practices on stress in pig, or more specifically the ability to measure potential variability of fGCM levels in different pig housing set ups, such as group housing or confined single stands.

Therefore, the generated information regarding pigs should help to facilitate further studies which examine endocrine responses to putative stressful circumstances in different pig environments and set ups.

Acknowledgments

The authors would like to thank S. Ganswindt and H. Roussow for their expert help with laboratory techniques.

Data Availability

All relevant data files are available from the University of Pretoria Research Data Repository. https://researchdata.up.ac.za/articles/Non-invasive_monitoring_of_adrenocortical_function_in_female_domestic_pigs_using_saliva_and_faeces_as_hormone_sample_matrices/11763420 (DOI: https://doi.org/10.25403/UPresearchdata.11763420.v1).

Funding Statement

Funding for this work was provided partly by the National Research Foundation Incentive funding for Rated Researchers (2016) provided to Fasina FO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0234971.r001

Decision Letter 0

Govindhaswamy Umapathy

23 Dec 2019

PONE-D-19-24903

Non-invasive monitoring of adrenocortical function in domestic pigs using saliva and faeces as hormone matrices

PLOS ONE

Dear Dr Wolf,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The paper of Wolf et al (PONE-D-19-24903) aims at validating a method for evaluating adrenocortical activity via measuring faecal glucocorticoid metabolites (fGCMs) in pigs. The authors performed an ACTH challenge test (physiological validation) and a biological validation (transportation) in 6 female, immature pigs. They tested 4 different EIAs on a subset of sample (3 pigs) and found a 5α-pregnane-3β,11β,21-triol-20-one EIA best suited. Clear increases after ACTH injection and transportation in all 6 animals successfully validated the chosen EIA. An additional storage experiment demonstrated that FGCMs were stable up to 48 h. I think the experimental setup is well designed and the study adds knowledge about fGCMs analysis in an important species, where such methods have not been successfully validated so far. However, I miss a clear statement about the limitations/weaknesses of the study. The authors only used female, immature pigs. Because reproductive status (age) and sex are serious confounders in fGCM analyses, their influence needs to be elucidated (and the method validated in mature animals of both sexes) before a broad application of this method in pigs can be recommended.

Detailed comments (ordered by appearance in the ms).

Title: I suggest indicating that only females (in female domestic pigs, or sows) were used (important as sex differences might be present). Better use “sample matrices” instead of “hormone matrices” (only metabolites are present in faeces)

Abstract: line 24: Replace “In order of not adding” with “To avoid”.

Line28: “sample matrices” (also line 80, 150)

I suggest to reword: “An assay measuring faecal glucocorticoid metabolites (fGCM) with a 3ß,11ß-diol group has proven suited to determine adrenocortical activity , showing…” (the antibody is not against those metabolites).

Line 31: Better: A cortisol EIA was used… (the immunogen is cortisol-3-CMO:BSA)

Lines 33-38: That could be described shorter (especially as collection and transport from distant locations might be a problem in wildlife, but unlikely in domestic pigs, or?)

Line 52: stereotypic

Line 67: Your focus is domestic livestock – so would not be another review better suited here?

Somewhere in the introduction, it might be worth mentioning that pigs are an especially stress susceptible species.

Line 85: ..on the stability of….

Line 90: I wonder when females of this breed get sexually mature, but 14 weeks old animals are too young.

Line 94: normally written in italics and with 2 words: ad libitum

Line 103: 50 IU (10 µg) there is a space between a number and its dimension (many other cases too – e.g. line 134; 138, etc..).

Line 104: Add country! Switzerland?

Line 106: Were samples from all defecations collected? Here and elsewhere: I suggest to replace the hyphen with a word space.

Line 114: Salivette, or?

Line 117-118: I guess the portion was taken from the core of each samples, and then mixed? Or why is it important to take it from the core of a well-mixed sample?

Line 125: .. between defecation and freezing, or? For stability it is less important when the sample was collected!

Line 128-129: I can imagine what was performed, but wording is unclear.

Line 142: via a cortisol enzyme immunoassay…. against cortisol-3-CMO:BSA.

Line 149: “Suited” is better than “reliable”. How would you define the latter? Most important is an EIA, which can monitor expected increases/decreases in adrenocortical activity well.

Line 151 onwards: Four different EIAs were tested – please add EIA to each of the descriptions (e.g. 11-oxoaetiocholanolone I EIA) - I guess GB-spelling is chosen, so aetiocholanolone is correct.

Lines 156-157: not possible to write by [number] – need to write the authors and then [].

Line 159: delete “fGCM”

Line 175-176: Peak fGCM concentrations following ACTH administration were expressed as percentage increase above baseline concentrations.

Line 183-186: How many samples per animal were present (I guess only baseline samples were used)?

Line 187: post defecation.

Line 188: mean value … (no hormones in the faeces).

Line 188/189: Unclear what the authors mean here? Why distribution?

Line 195: ..occuring

Line 209: enzyme immunoassays (EIAs)

Table 1: The two 11-oxoaetiocholanolone have a higher increase – so why was the 5α-pregnane-3β,11β,21-triol-20-one EIA chosen? What means a “more stable” baseline? How is this made objective?

Looking at Möstl et al (1999) seems to show that the 11-oxoaetiocholaonolone I had even higher increases – but it proved unsuited, as not all animals showed the increase, and dexamethasone injection failed to clearly suppress levels. Most important: fGCM were not stable after defecation (a 175% increase after 4 h, 375% after 24 h). The latter may be an argument for choosing the 5α-pregnane-3β,11β,21-triol-20-one EIA.

Line 213: (range: 215.7…) – but the 205.7 given in Table 1 is lower, why that?

Fig 2 (and the same for Fig. 1): How were those 4 chosen (of course this makes someone curious about the other 2 ones (especially as Möstl et al., 1999 found even higher increases in some pigs, but no expressed ones in others). Please show all 6 pigs.

Line 232 (235) – what does “V” mean?

Line 248: first time of “GIT” – spell it out!

Line 249: add [8] to [14]

Line 250: “suited” is better than “sufficient”

Line 251: [20] they also used a dexamethasone suppression test, and the assay was found unsuited, based (at least partly) on the fact that the suppression was not well reflected in fGCM levels.

Line 250/251: Those studies did not perform ACTH test or biological validations “to measure fGCM concentrations” – please reword.

Line 256: .. a cortisol assay (using an antibody against cortisol-3-CMO:BSA) …

Line 259: barrows? Did you mean “farrows”?

Line 263: no hyphen in case of enzyme immunoassay. “glucocorticoid metabolites” – and also reword the rest (see comments above).

Line 265-267: Please reword. I wonder, but cannot find a sustained (up to 48 h) peak in Fig 2?

Line 268: baseline levels between 221.5 and 246.3 ng/g: I’m confused, as this is the third interval given for the baseline levels (Tab 1; line 213), and all are different?

Line 269 – repetition of line 266 (180% increase)?

Line 270: delete one full stop.

Line 272: .. and the levels did not reach baseline after 24 hours [32].

Line 275: “fGCM recovery time”?

Line 276: .. and the appearance of their metabolites in the faeces [33].

Lines 283-286: I suggest adding “other species” and “different measurement method” as the most likely reasons for the observed differences.

LINE 288: “were” instead of “was”

Line 294: .. when evaluating stressors in pigs using saliva as sample matrix.

Line 296: ..might be a more suited..

Line 299 “steroid concentrations”

Lines 298-315: this is a rather long passage dealing with the stability experiment. I suggest focusing on domestic livestock and shortening it (e.g. the sentences lines 307-310 can be easily deleted without losing much). As mentioned before, I doubt there is much application in pigs for collection in remote locations and long sample transport.

Line 322 onwards: I would prefer more straight forward applications in immature pigs (too many potential things/possibilities) – but see also further comments below!

Lines 326-327: Don’t think that this statement is true. Your aim was validating such a non-invasive tool, but the importance must be beyond the tool.

A clear statement about the limitations/weaknesses of the study needs to be added here. Only female, immature pigs were used. Thus results don’t guarantee a broad application of the method in pigs, because reproductive status (age) and sex are serious confounders in fGCM analyses, and thus their influence needs to be elucidated. That clearly limits the application of the method!

References: Not all words should be capitalized in the title [correct for 6, 9, 12,15, 17, 22, 28, 33, 36, 46].

Line 337: last accessed?

Line 346: delete the 1 after cattle.

Line 379: Domestic Animal Endocrinology

Line 388: the journal is missing.

Line 393: “Suppl 2” is missing.

Line 395: Veterinary Research Communications

Line 452: “ethological”

Line 453: better: “last accessed” than “updated”.

Fig 1 and 2: Smaller symbols for the 4 pigs would make the picture clearer, also allowing adding the missing two.

Fig. 2. What about the animal which showed a peak in fGCM concentrations at the time of ACTH injection? Was there a known stressor occurring the day before? There seemed to be a smaller peak (ACTH related) present on Day 1 (based on the broader shoulder).

Fig. 3: The y-axis should be scaled from 0 to 100. This is more correct and also better representing the message of only insignificant changes in FGCM levels over time. Myjor x-axis ticks every 8 hours would fit the intervals better and also help the readers who usually think in days (i.e. 24 h not in 10/20/30 h etc.). Legend y-axis: % of initial fGCM concentration.

Reviewer #2: This is a very well written article. It is clearly explained and well executed. The methods for validation are sound and support the findings. The authors have effectively identified a new method that can used to noninvasively measure adrenal activity in pigs. I have no edits on the manuscript and would accept it in its current format. Great work.

**********

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Reviewer #1: Yes: Rupert Palme

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PLoS One. 2020 Jun 25;15(6):e0234971. doi: 10.1371/journal.pone.0234971.r002

Author response to Decision Letter 0

3 Feb 2020

Reviewers' comments:

We thank the reviewer for the valuable input and we hope that our revised version sufficiently addresses the respective points raised.

Detailed comments (ordered by appearance in the ms).

The title has been adjusted as suggested

Abstract: line 24: Replace “In order of not adding” with “To avoid”.

The sentence has been corrected (line 24)

Line28: “sample matrices” (also line 80, 150)

The sentence has been adjusted as suggested (lines 28, 77, 151)

The sentence has been adjusted as suggested (line 28ff)

Line 31: Better: A cortisol EIA was used… (the immunogen is cortisol-3-CMO:BSA)

The sentence has been corrected (line 30ff)

Lines 33-38: That could be described shorter (especially as collection and transport from distant locations might be a problem in wildlife, but unlikely in domestic pigs, or?)

As suggested, the sentence has been shortened to assist the reader (although not common, longer transportation of domestic livestock occurs in SA) (line 35ff)

Line 52: stereotypic

The sentence has been corrected (line 52)

Line 67: Your focus is domestic livestock – so would not be another review better suited here?

Somewhere in the introduction, it might be worth mentioning that pigs are an especially stress susceptible species.

Alternative references have been provided. We also added a statement about the comparatively high stress susceptibility of pigs (line 47f)

Line 85: ..on the stability of….

The sentence has been corrected (line 84f)

Line 90: I wonder when females of this breed get sexually mature, but 14 weeks old animals are too young.

In this breed puberty is reached between 115 and 160 days and they usually deliver their first set of piglets at about one year of age.

Line 94: normally written in italics and with 2 words: ad libitum

The sentence has been corrected (line 94)

Line 103: 50 IU (10 µg) there is a space between a number and its dimension (many other cases too – e.g. line 134; 138, etc..).

We apologize for this oversight and added respective spaces between given numbers and their dimensions throughout the manuscript.

Line 104: Add country! Switzerland?

We added the country of origin (Switzerland)( line 105)

Line 106: Were samples from all defecations collected?

Samples were taken as per scheduled timelines throughout the experiment as described in line 118ff. During the indicated period, samples from all voided faeces were collected (line 119).

Here and elsewhere: I suggest to replace the hyphen with a word space.

Corrected

Line 114: Salivette, or?

Yes, corrected (line 116)

Line 117-118: I guess the portion was taken from the core of each samples, and then mixed? Or why is it important to take it from the core of a well-mixed sample?

We apologize for the confusion. Yes, the portion was taken from the core of a sample, and then mixed. We adjusted the statement accordingly (line 120f)

Line 125: .. between defecation and freezing, or? For stability it is less important when the sample was collected!

Between defecation and freezing, we adjusted the text accordingly (line 127)

Line 128-129: I can imagine what was performed, but wording is unclear.

The sentence has been rephrased (line 130ff)

Line 142: via a cortisol enzyme immunoassay…. against cortisol-3-CMO:BSA.

The sentence has been corrected (line 144f)

Line 149: “Suited” is better than “reliable”. How would you define the latter? Most important is an EIA, which can monitor expected increases/decreases in adrenocortical activity well.

Corrected (line 151).

Line 151 onwards: Four different EIAs were tested – please add EIA to each of the descriptions (e.g. 11-oxoaetiocholanolone I EIA) - I guess GB-spelling is chosen, so aetiocholanolone is correct.

The sentence has been corrected (line 154ff)

Lines 156-157: not possible to write by [number] – need to write the authors and then [].

The sentence has been corrected (line 158ff)

Line 159: delete “fGCM”

The sentence has been corrected (line 161)

Line 175-176: Peak fGCM concentrations following ACTH administration were expressed as percentage increase above baseline concentrations.

The sentence has been corrected (line 177f)

Line 183-186: How many samples per animal were present (I guess only baseline samples were used)?

The missing information was added (line 186ff). Only baseline samples were used for this part of the study.

Line 187: post defecation.

The sentence has been corrected (line 190)

Line 188: mean value … (no hormones in the faeces).

The sentence has been corrected (line 191)

Line 188/189: Unclear what the authors mean here? Why distribution?

The sentence has been rephrased (line 191ff)

Line 195: ..occuring

The wording has been corrected (line 198)

Line 209: enzyme immunoassays (EIAs)

The sentence has been corrected (line 212)

As stated in the manuscript, all four assays tested revealed an overall increase in fGCM concentrations above 100% post ACTH injection and thus appear suited for monitoring alterations in fGCM concentrations in sows. Although the two 11-oxoaetiocholanolone EIAs tested show a higher overall increase after ACTH administration compared to the 5α-pregnane-3β,11β,21-triol-20-one EIA, the pre-injection baseline as well as the post-injection peak values for the three animals tested were most comparable when using the 5α-pregnane-3β,11β,21-triol-20-one EIA. We further knew that with at least the 11-oxoaetiocholaonolone I EIA, fGCM concentrations were not stable after defecation (a 175% increase after 4 h, 375% after 24 h) (Möstl et al. 1999). That’s why we finally decided to continue with the 5α-pregnane-3β,11β,21-triol-20-one EIA.

We adjusted the related statement which now reads ‘However, the 5α-pregnane-3β, 11β,21-triol-20-one EIA showed the most comparable baseline and peak values for all three animals and was chosen for the analysis of the remaining faecal samples.’ (line 207ff)

Line 213: (range: 215.7…) – but the 205.7 given in Table 1 is lower, why that?

Once the decision was made to continue with the 5α-pregnane-3β, 11β,21-triol-20-one EIA, the entire ACTH sample set (including the samples from the three animals used for EIA selection) were (re)analysed, which resulted in slight alterations in pre-injection baseline and post-injection peak values for the three animals already tested.

Only the individuals injected with Synacthen are shown in this figure, not the control individuals injected with saline. We added another figure depicting the two profiles of the control animals.

Line 232 (235) – what does “V” mean?

The V value is the sum of signed ranks (typically referred to as W). It has now been replaced by the corresponding Z value (lines 235, 238).

Line 248: first time of “GIT” – spell it out!

Thanks for pointing that mistake out, we now refer to ‘gastro intestine transit (GIT) systems’ (line 251)

Line 249: add [8] to [14]

The reference has been added (line 252)

Line 250: “suited” is better than “sufficient”

The sentence has been corrected (line 253)

Line 251: [20] they also used a dexamethasone suppression test, and the assay was found unsuited, based (at least partly) on the fact that the suppression was not well reflected in fGCM levels.

The information has been added (line 254ff)

Line 250/251: Those studies did not perform ACTH test or biological validations “to measure fGCM concentrations” – please reword.

The sentence has been rephrased (line 254ff)

Line 256: .. a cortisol assay (using an antibody against cortisol-3-CMO:BSA) …

The sentences has been corrected (line 260f)

Line 259: barrows? Did you mean “farrows”?

Barrow can be used to describe castrated male pigs

Line 263: no hyphen in case of enzyme immunoassay. “glucocorticoid metabolites” – and also reword the rest (see comments above).

The sentence has been rephrased (line 269)

Line 265-267: Please reword. I wonder, but cannot find a sustained (up to 48 h) peak in Fig 2?

The sentence has been rephrased (line 270ff)

Line 268: baseline levels between 221.5 and 246.3 ng/g: I’m confused, as this is the third interval given for the baseline levels (Tab 1; line 213), and all are different?

We sincerely apologize for the confusion. As explained above, the different values stated in Tab1 compared to line 213 result from a re-measurement of the sub-sample set originally used for the assay screening. The values mentioned in line 268 are only referring to the animals treated with ACTH and not all six study animals (as referred to in line 213). To assist the reader we are now referring to all six animals here as well (line 272ff).

Line 269 – repetition of line 266 (180% increase)?

The sentence has been corrected (line 272ff).

Line 270: delete one full stop.

The mistake has been corrected (line 275).

Line 272: .. and the levels did not reach baseline after 24 hours [32].

The sentence has been corrected (line 277f).

Line 275: “fGCM recovery time”?

The sentence has been rephrased (line 279ff).

Line 276: .. and the appearance of their metabolites in the faeces [33].

The sentence has been corrected (line 281f).

Lines 283-286: I suggest adding “other species” and “different measurement method” as the most likely reasons for the observed differences.

The information has been added (line 289f).

LINE 288: “were” instead of “was”

The sentence has been corrected (line 294).

Line 294: .. when evaluating stressors in pigs using saliva as sample matrix.

The sentence has been corrected (line 299ff).

Line 296: ..might be a more suited..

The sentence has been corrected (line 302)

Line 299 “steroid concentrations”

The sentence has been corrected (line 305)

The paragraph has been shortened (lines 304ff)

Line 322 onwards: I would prefer more straight forward applications in immature pigs (too many potential things/possibilities) – but see also further comments below!

In light of the aligned comments below we rephrased the conclusion section to address the various points raised

Lines 326-327: Don’t think that this statement is true. Your aim was validating such a non-invasive tool, but the importance must be beyond the tool.

The sentence has been removed

We agree with the reviewer and added a statement concerning the limitations (line 318ff).

References: Not all words should be capitalized in the title [correct for 6, 9, 12,15, 17, 22, 28, 33, 36, 46].

The references have been corrected

Line 337: last accessed?

The reference has been corrected (line 341).

Line 346: delete the 1 after cattle.

The reference has been corrected (line 351).

Line 379: Domestic Animal Endocrinology

The reference has been corrected (line 390).

Line 388: the journal is missing.

The reference has been corrected (line 399).

Line 393: “Suppl 2” is missing.

The reference has been corrected (line 405).

Line 395: Veterinary Research Communications

The reference has been corrected (line 408).

Line 452: “ethological”

The reference has been corrected (line 457).

Line 453: better: “last accessed” than “updated”.

The reference has been corrected (line 459)

Fig 1 and 2: Smaller symbols for the 4 pigs would make the picture clearer, also allowing adding the missing two.

We have changed both figures accordingly and added additional figures with the control animals.

Unfortunately we don’t have any data on this point and can only speculate about an additional stressor perceived on that day.

We have changed figure 3 accordingly

We thank the reviewer for the encouraging words.

Attachment

Submitted filename: Response to reviewer.docx

Click here for additional data file.^{(25.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0234971.r003

Decision Letter 1

Henrik Oster

20 Apr 2020

PONE-D-19-24903R1

Non-invasive monitoring of adrenocortical function in female domestic pigs using saliva and faeces as sample matrices

PLOS ONE

Dear Dr Wolf,

==============================

- fix the methods descriptions

- compare saliva and faeces data

- fix minor issues with legends etc.

==============================

We would appreciate receiving your revised manuscript by Jun 04 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Henrik Oster, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: Thank you – the revised paper is significantly improved now. The authors have sufficiently addressed my points; thus, I think the paper is suitable for publication now. Only a few minor errors are left for correction (typos; etc… - see details below), before it should go to the production department.

The hyphen of “enzyme-immunoassay” was only partly deleted – so there is now an inconsistent situation. Please also delete it in the remaining cases (lines 144 and 153)

The 11-oxoaetiocholanolone is sometimes still present in its US-spelling (please change “e” to “ae”; lines 154 and 155)

Thanks for adding data of the control animals (Fig. 2b). I’ve only one question left now here: The mentioned increase after saline injection (60% above baseline; lines 219/220) is present on Day 0 – when were the samples collected in relation to the injection time – I wonder if that really reflects the saline injection? If too close, I suggest deleting that sentence.

Line 260: please delete the first “”an assay using”

The problem with the capitalized title words is not fully resolved – please change all occurrences in refs 6.; 8; 10; 18 and 35 (correct only with the first word after a “:”).

Line 401: A typing error was introduced: “acpacity”.

Ref 33: “American”

Ref 34: The end was lost during revision; please add: Wiener Tierärztliche Monatsschrift. 2010;97: 259–262.

Reviewer #3: The revised paper is well-written and the goals of the study are clear and well-justified. The results of the studies described will be of use to others that would like to implement non-invasive fecal glucocorticoid metabolite measures with domestic pigs. The methods appear sound, although there are some details that are missing or presented in a confusing manner that makes it difficult to interpret the significance of the results. There are important changes to address to help readers interpret the study design and results, however these should be relatively easy to address.

Introduction:

The background information is clearly presented and the motivation for the current study is clear. The study objectives are sound.

Methods:

Overall, the methods are well-explained and easy to follow. The different sub-studies are well-designed, and the sampling regime was good.

There are three sub-studies in the current work: (1) document post-ACTH and post transport salivary/fecal corticoid peak concentrations and timing, (2) compare morning vs. evening salivary/fecal corticoid concentrations, and (3) determine relative degradation of glucocorticoid metabolites in feces kept in ambient conditions. These three goals/sub-studies are not presented as distinct, separate studies in the methods and results, and this makes it difficult at times to elucidate the sample size and sampling/analytical methods associated with each result. One way to clarify the methods would be to provide salivary and fecal sampling times relative to each manipulation (e.g. ACTH, transport, time of day). This is well done for the degradation study, but difficult to understand for the ACTH/transport studies. Please also see comments about Results below.

One example of this confusion: there is disagreement in the description of when fecal samples were collected in the methods vs. how data are presented in Figure 2. In the Methods, the authors indicated that fecal samples were collected during the same 7.5-hour continuous window on a daily basis (8:00-15:30 h). However, in Figure 2, there are data points that indicate that samples were collected every 12 hours rather than every 24 hours (i.e. the data points after ACTH injection). This should be clarified in both the Methods and the Results. For example, the authors should indicate when ACTH injections were administered relative to fecal sample collections.

In the above example, there is another concern because a peak in fGCM is more readily documented if samples are collected more frequently (as they appear to have been done post- vs. pre-ACTH injection). Specifically, when samples are collected more frequently, there is more chance to catch a true peak without averaging a peak sample with other lower-concentration samples collected during a wider sampling window. Thus, if the sampling regime became more frequent after the ACTH injection compared to before the injection, then there is some concern about methodological bias leading to a greater chance of documenting peak fGCM after vs. before ACTH injection. The authors should clarify this.

It is good to see that the authors compared multiple EIAs.

To compare morning vs. afternoon salivary cortisol and fGCM it would be more appropriate to compare corticoid concentrations from samples that are collected at more distinct time points. In the present design, the authors compare ‘morning’ to ‘afternoon’ corticoid concentrations by comparing corticoid concentrations from samples collected between 8:00-12:00 h (morning) vs. 12:01-18:00 h (afternoon). These are not particularly distinct time frames – the morning samples may consist of a majority of samples collected near 11:00 h while afternoon samples could be primarily from 12:30 h. To truly determine if there is a circadian rhythm, it would be best to compare samples that are collected at the beginning vs. the end of the active period. To truly analyze if there is a circadian rhythm in fGCM, the authors should compare mean fGCM from 8:00-12:00 h vs. 14:00-18:00 h, or better yet, compare fGCM from samples collected 8:00-10:00 vs. 16:00-18:00 h. As currently conducted, this analysis favors the authors hypothesis that fGCM measures will fluctuate less across the day compared to salivary cortisol measures.

Were all animals equally represented in the morning vs. afternoon samples? This should be clarified.

The authors should indicate the sample size involved in each statistical analysis. And to indicate how often each animal was represented in each data set (e.g. was animal ID controlled for in analyses that included multiple samples from the same individual?).

In the current study, blood samples were not collected to allow for a comparison of fGCM concentrations to blood cortisol concentrations as a validation step. However, both saliva and feces were collected from the same six pigs. Can the authors include an analysis to compare individual salivary and fecal corticoid measures? In other words, were pigs that had high baseline or post-ACTH salivary cortisol levels the same pigs that had high fGCM concentrations at these times? This is not necessary, but it would provide additional validation of the fGCM method used in the current study.

Results:

Please include sample size in all reported result that included an average or median value (in particular for ‘Effect of Collection Time’ and ‘Stability test for fGCM concentrations’ results).

Line 216: Clarify that the results in this section are from the 5α-pregnane-3β,11β,21-triol-20-one EIA. In addition, explain how the average baseline fGCM level was calculated. How many samples were included from each female? Were individual female means calculated on each day or across multiple days, and then an average taken from all individual female averages? Please indicate how differential sampling from individual females was accounted for in the calculations, or at least provide information on how much each female contributed to the sample size.

Figure 2 legend: Indicate that values in figure are from the 5α-pregnane-3β,11β,21-triol-20-one EIA.

Line 241: “Within the first hour,” should read “Within the first hour at ambient temperature,”

Discussion:

The authors reference two papers that previously used fecal corticoid measures in domestic pigs with ‘mixed results’. Given the relative importance of these prior papers relative to the current manuscript, the authors should tell us more about why the methods used in the current study led to clear results, while the prior work led to mixed results. What were the mixed results in prior studies? What methods did they use? And why were those prior studied unsuccessful while the current study was successful? These are important aspects that should be clarified.

Otherwise, the Discussion appropriate compares results of the current study to results from other relevant studies.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Rupert Palme

Reviewer #3: No

PLoS One. 2020 Jun 25;15(6):e0234971. doi: 10.1371/journal.pone.0234971.r004

Author response to Decision Letter 1

12 May 2020

Response to editor and reviewers

PONE-D-19-24903R1

Non-invasive monitoring of adrenocortical function in female domestic pigs using saliva and faeces as sample matrices

Dear Dr Wolf,

- fix the methods descriptions

- compare saliva and faeces data

- fix minor issues with legends etc.

We look forward to receiving your revised manuscript.

Kind regards,

Henrik Oster, Ph.D.

Academic Editor

PLOS ONE

We thank the editor for the opportunity to resubmit our revised manuscript. As suggested, we have addressed the comments from the reviewers and edited the manuscript accordingly if required.

Review Comments to the Author

We thank the reviewer and hope we have addressed the remaining points raised.

The hyphen of “enzyme-immunoassay” was only partly deleted – so there is now an inconsistent situation. Please also delete it in the remaining cases (lines 144 and 153)

We have now corrected the spelling (lines 153 & 162)

The 11-oxoaetiocholanolone is sometimes still present in its US-spelling (please change “e” to “ae”; lines 154 and 155)

We have now corrected the spelling (lines 163 & 164)

We agree with the reviewer and omitted the statement

Line 260: please delete the first “”an assay using”

We have corrected the sentence (line 277)

The problem with the capitalized title words is not fully resolved – please change all occurrences in refs 6.; 8; 10; 18 and 35 (correct only with the first word after a “:”).

Line 401: A typing error was introduced: “acpacity”.

Ref 33: “American”

Ref 34: The end was lost during revision; please add: Wiener Tierärztliche Monatsschrift. 2010;97: 259–262.

We have now corrected the references

We thank the reviewer for the valuable input and hope that our revised version sufficiently addresses the respective points raised.

Introduction:

The background information is clearly presented and the motivation for the current study is clear. The study objectives are sound.

Methods:

Overall, the methods are well-explained and easy to follow. The different sub-studies are well-designed, and the sampling regime was good.

To assist the reader, we have now added more information concerning the timeline of events and sampling regime, and subdivided the methods section further into the specific parts of the study, namely the monitoring of transport and the effect of sampling time.

We apologise for the confusion and have now added a more detailed description of the sampling in the methods section. We now also indicate that ACTH administration took place in the early morning of day 0 (line 103). We also want to point out that Figure 2 shows the median fGCM levels per day for each individual, and only on the 2 days after injection we show fGCM values of all individual samples to better visualize the time when the peak concentrations were found.

We apologise for the misunderstanding, the sampling regime over the course of the ACTH challenge did not change, as samples were taken from all faeces voided between 8:00 and 18:00h. We now indicate that in the revised version of the manuscript (line 123). This should indicate, that there is no bias between the samples collected before and after ACTH injection.

It is good to see that the authors compared multiple EIAs.

We agree with the reviewer that this is a valuable point, originally, we used fGCM values from samples for the ‘afternoon’ sample set collected between 12:45 and 18:00 for this analysis. However, when excluding samples collected before 16:00, the mean of the afternoon samples appears comparable to the mean including the early afternoon samples (235.6 vs 240.8 ng/g DW; W=5134, p=0.738). All morning samples were collected between 8:00 and 10:00am, and fGCM concentrations did not differ significantly regardless of comparing steroid values from the morning samples with those from the more broader (12:45 – 18:00) or stricter (16:00 – 18:00) afternoon sample set. We therefore decided to keep the currently presented approach. However, we are willing to exchange it with the one representing the more rigorous time bouts if requested.

Were all animals equally represented in the morning vs. afternoon samples? This should be clarified.

All individuals are represented equally in morning and afternoon samples as indicated in the methods section (line 132ff & 194ff). 6 morning samples were compared to 6 afternoon samples from the respective days. It is therefore not necessary to control for individual bias within the analyses.

Salivary GC concentrations, similar to GC concentrations in blood, provide a valuable insight into GC fluctuations over a small timeframe by indicating what is going on in the organism seconds to minutes before the sample was collected (Anestis 2010). They are therefore also prone to additional factors like diurnal rhythm of GC production. Faecal samples on the other hand provide a more cumulative picture of GCM concentrations, providing an insight into a time depth of up to 40 hours, depending on the size of the organism (Touma et al. 2004; Anestis 2010; Behringer & Deschner 2017). As animals, even if kept under standardised conditions, might perceive various occurring stressors differently, the cumulative signal depicted in fGCM concentrations will most likely differ from that point-in-time GC value revealed in a saliva sample. We therefore don’t think that a comparison of salivary GC and fGCM concentrations would be reasonable and thus haven’t included any additional information in the revised version of the manuscript. However, we did a respective comparison of individual baseline sGC/fGCM levels, and as expected the animals with the highest or lowest sGC concentrations do not have the highest or lowest fGCM levels. Interestingly, and another possible reason for the mismatch might be that especially the fGCM baseline values only vary minimally between the individuals (216 – 246 ng/g DW) and could thus be considered equal.

Results:

The sample size per individual for the analyses of the collection time is indicated in the methods, we now added the total numbers (line 198). The sample size for the stability test is 24 as already indicated in the methods (line 138).

The EIA name has been added to the sentence. ‘To establish a baseline individual median fGCM concentrations were calculated using pre- ACTH administration samples’ (line 185). 9 samples per individual (collected 5 days prior to ACTH injection) were used to establish individual baseline levels. We have added more information (line 99ff).

Figure 2 legend: Indicate that values in figure are from the 5α-pregnane-3β,11β,21-triol-20-one EIA.

The information has been added to the figure legend (line 234)

Line 241: “Within the first hour,” should read “Within the first hour at ambient temperature,”

The sentence has been corrected (line 255)

Discussion:

Additional information concerning the used methods and outcome in these studies has been added to the discussion (line 270ff)

Otherwise, the Discussion appropriate compares results of the current study to results from other relevant studies.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(23.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0234971.r005

Decision Letter 2

Henrik Oster

8 Jun 2020

Non-invasive monitoring of adrenocortical function in female domestic pigs using saliva and faeces as sample matrices

PONE-D-19-24903R2

Dear Dr. Wolf,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Henrik Oster, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: The authors have addressed the majority of my comments and concerns on the original revised manuscript.

The description of the methods and the number of samples involved in each analysis has improved.

I appreciate the authors’ legitimate concerns with comparing salivary and fecal corticoid levels. Unfortunately, they do not take full advantage of the data that they have; there are ways to control for salivary corticoid moment-to-moment variation to legitimize comparison with more integrated fecal measures. For example, salivary levels could be adjusted for time of day, or an integrated salivary corticoid metric (e.g. AUC) post-ACTH injection could be compared to post-ACTH fecal measures, etc. I understand that the data set is small, and individual variability in corticoid production may be relatively limited given the housing conditions, thus limiting the possibility of finding statistically significant results.

For the time of day analysis of fecal corticoids, I would include the more stringent time of day analysis.

Minor:

- There are 5 (not 6) days of fecal sample collections prior to the ACTH injection day.

- Page 6, line 131: It would help to indicate how many saliva samples were collected pre- vs. post-ACTH injection – as was done for fecal samples.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: Yes: Sonia A. Cavigelli

PLoS One. doi: 10.1371/journal.pone.0234971.r006

Acceptance letter

Henrik Oster

12 Jun 2020

PONE-D-19-24903R2

Non-invasive monitoring of adrenocortical function in female domestic pigs using saliva and faeces as sample matrices

Dear Dr. Wolf:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Henrik Oster

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

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PERMALINK

Non-invasive monitoring of adrenocortical function in female domestic pigs using saliva and faeces as sample matrices

Tanja E Wolf

Norbert Mangwiro

Folorunso O Fasina

Andre Ganswindt

Roles

Abstract

Introduction

Material and methods

Study animals and housing

Monitoring of transport and ACTH challenge test

Sample collection

Effect of collection time and degradation study

Steroid extraction

Steroid analysis

Data analysis

Results

ACTH challenge test

Fig 1.

Table 1. Baseline and peak fGCM concentrations, and overall median increase (%) for samples from the three animals tested after ACTH injection with four enzyme immunoassays (EIAs).

Fig 2.

Effect of transport on fGCM

Effect of collection time

Stability test for fGCM concentrations

Fig 3. Relative change (%) of faecal glucocorticoid metabolite concentrations (mean +/- SE) in pigs over time since defecation (0, 1, 2, 4, 8, 16, 24, and 48h).

Discussion

Conclusion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Govindhaswamy Umapathy

Roles

Author response to Decision Letter 0

Decision Letter 1

Henrik Oster

Roles

Author response to Decision Letter 1

Decision Letter 2

Henrik Oster

Roles

Acceptance letter

Henrik Oster

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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