Influence of vitamin E and carcass feeding supplementation on fecal glucocorticoid and androgen metabolites in male black-footed ferrets (Mustela nigripes).

In recent years, the ex situ population of the endangered black-footed ferret (Mustela nigripes; ferret) has experienced a decline in normal sperm morphology (from 50% to 20%), which may be linked to inbreeding depression and/or a dietary change. We examined the effects of adding carcass and vitamin E to the diet on stress and reproductive biomarkers in male ferrets (n = 42 males including 16 juveniles and 26 adults) housed at the U.S. Fish and Wildlife National Black-footed Ferret Conservation Center (Carr, CO, USA). Fecal samples (3x/week) were collected from November and December (pre-breeding season, no diet change), February through May (breeding season, diet change) and June (post-breeding season, diet change) and analyzed for fecal glucocorticoid metabolites (FGM) via a corticosterone enzyme immunoassay (EIA). A subset of samples from adult males (n = 15) were analyzed for fecal androgen metabolites (FAM) via a testosterone EIA. We first used a linear mixed effects model to identify the important fixed effects among meat treatment, vitamin E treatment, age class (juvenile or adult), and all possible interactions on each hormone. We then examined the important factor's effects across seasons using the non-parametric Friedman test. We found that age did not influence (p = 0.33) FGMs; however there was a significant effect of meat treatment on FGM (p = 0.04) and an effect of vitamin E on FAMs (p<0.10). When fed carcass, FGMs declined (p<0.001) from pre- to the during the breeding season time period, but was similar (p>0.05) between during and post-breeding season periods. Males that were not fed carcass had higher (p<0.05) FGMs during the breeding season compared to pre- and post-breeding season and FGMs were lower (p<0.05) in the post-breeding season compared to pre-breeding season. Males fed with carcass had lower (p<0.001) FGM than males that were not fed carcass during both the pre-breeding and the breeding season but not during the post-breeding season (p>0.05). Males supplemented with vitamin E had higher (p<0.001) FAM than non-supplemented males during the breeding season only. For both groups, FAM was highest (p<0.05) during the breeding season. In conclusion, adding carcass to the diet can reduce glucocorticoid production and adding vitamin E can increase testosterone during the breeding season, which may influence reproductive success.

Introduction

Nutrition plays an important role in animal welfare as it can affect both physical and mental health. Proper nutrition for some species, like grazers, may be easier to replicate ex situ, but for other species, such as specialist carnivores, it may be more difficult to meet specific dietary needs [1,2]. Although it can be challenging to maintain healthy populations of specialized carnivores outside of their natural environment, for the black-footed ferret (ferret; Mustela nigripes) the ex situ population is the only reason why the species did not go extinct. Sylvatic plague (plague, Yersinia pestis), which is an introduced bacterial disease spread by fleas, along with habitat loss and the reduction of its natural prey, the prairie dog (Cynomys spp.), has resulted in the ferret’s demise [3,4]. In the mid-1980s, the remaining ferret population was removed from the wild to initiate an ex situ breeding program in an effort to prevent extinction. Thirty-four years of successful management has led to the production of more than 9,600 black-footed ferrets at six facilities led by the United States Fish and Wildlife Service (USFWS) through a Species Survival Plan® (SSP; managed through AZA) and includes USFWS’s National Black-footed Ferret Conservation Center (Colorado), Smithsonian Conservation Biology Institute (Virginia), Louisville Zoological Garden (Kentucky), Cheyenne Mountain Zoo (Colorado), Phoenix Zoo (Arizona), and Toronto Zoo (Ontario, Canada). Of those ferrets born, more than 4,400 have been released into 30 reintroduction sites across North America’s Great Plains in the past 29 years [5]. However, the population is now facing reproductive challenges. Since 2000, the percentage of normal spermatozoa has declined from ~50% to 25%, and pregnancy success has decreased from 60% to 36% [6,7]. It is presumed that because there are no novel genes to bring into the population through natural means that this decline is related to inbreeding depression. Additionally, higher inbreeding coefficient (F) in ferrets correlated with lower ejaculate volume, sperm forward progression, and normal sperm [7]. Santymire and coauthors [7] also found a positive relationship between litter size and both normal sperm acrosome and sperm motility, demonstrating that having normal sperm is important for producing larger litters. There is evidence, however, that wild-born ferrets have improved semen quality and larger testes [8,9]. The latter results suggest that environmental conditions, rather than genetics, may be limiting ferret reproductive success.

Another factor that may affect ferret reproductive success is diet. In 2001, the Black-footed Ferret SSP switched from a diet consisting of 60% mink pellets and 40% rabbit meat (60/40 diet), which was based on domestic ferret (Mustela putorius furo) production diet, to a commercially available diet, Toronto Small Carnivore diet (TOR), which was produced by Milliken Meat Products Ltd. (Markham, Ontario, Canada). TOR is a soft diet formulated for carnivores that is composed of horsemeat supplemented with vitamins and minerals. It contains no bones, cartilage, organs, skin or connective tissue (millikenmeat.com/products) [10,11]. A previous study evaluated the impact of various diets including supplementing the TOR diet with whole carcass (hamster or prairie dog) and/or vitamin E on seminal traits in 55 male ferrets [12]. Results indicated that TOR had excessively high concentrations of vitamin A (~43,000 IU/kg), which can compete with the absorption of vitamin E [13-16]. Specifically, vitamin E may protect testosterone production in the Leydig cells by reducing the incidence of lipid peroxidation and/or increasing the anterior pituitary hormones, luteinizing hormone, and follicle stimulating hormone as observed in rats supplemented with the antioxidant [17]. Testosterone is important for the production of spermatozoa, thus, reproductive success. Without supplementation of vitamin E and/or carcass, ferrets on the TOR diet had lower sperm concentrations, testes volume, and sperm motility indices [12].

Supplementing carnivores’ diet with whole prey or carcasses can also stimulate natural behaviors [18-20], and may reduce stereotypies [21,22] and be more enriching [20,23]. Incorporating dietary enrichment items like live prey, carcass, bones, frozen fish or spices [24] can reduce the effect of stressors by lowering aggression, encouraging positive social interactions, and promoting natural food acquisition [20]. The reduction of stressors in the ex situ environment is important for maintaining animal health. In response to long-term or repeated stressors, the hypothalamus-pituitary-adrenal (HPA) axis will produce glucocorticoids (GCs), which are steroidal hormones such as cortisol and corticosterone, to help the body return to homeostasis through energy mobilization [25]. However, chronic production of GCs can lead to suppression of normal behaviors, the immune system, and reproduction [26]. Therefore, monitoring GC production can provide some insight on how the environment is impacting animals. Because analyzing GCs via blood sampling can be stressful, non-invasive methods, such fecal glucocorticoid metabolite (FGM) analysis, are often used to monitor changes in stress physiology in wildlife [27]. FGM analysis is an important tool for monitoring the stressors of the ex situ environment especially for species that may be particularly sensitive [28].

In this study, we examined stress and reproductive biomarkers to determine the effects of vitamin E and whole carcass supplementation on fecal glucocorticoid (FGM) and androgen metabolite (FAM) production in ferrets. Our hypotheses were: 1) whole prey items added to the diet would lower FGM, and 2) because of the anti-oxidative properties of vitamin E, FGM would be lower and FAM would increase in ferrets fed a diet supplemented with vitamin E.

Methods

This research was reviewed and approved by Lincoln Park Zoo’s Research Committee (proposal #2007–005) and United States Fish and Wildlife Service (Carr, CO). No animals were anesthetized nor euthanized during this study. All animal studies conformed to the Guide for Care and Use of Laboratory Animals.

Animals and dietary treatments

Ferrets (n = 42 males; ~1 kg; 1 to 3 years old) were housed individually at the National Black-footed Ferret Conservation Center (FCC; Carr, CO) in (1.0 m x 1.3 m x1.0 m) indoor cages. Lighting was both natural (provided by skylights) and artificial (via fluorescent illumination; set to natural photoperiod). Ferrets were fed 75–100 g of TOR, which was handled according to manufacturer’s recommendations, daily and were provided water ad libitum. Males were assigned randomly to one of the following four diet treatments, including 16 juveniles (one year olds) and 26 adults (two and three year olds, which is the prime breeding age [29]),: 1) horsemeat diet with no supplementation (control); 2) horsemeat diet + vitamin E (D-α-tocopherol; 400 IU/kg diet dry matter basis (DMB); Stuart Products, Inc., Bedford, TX); 3) horsemeat diet + vitamin E (400 IU/kg diet DMB) + carcass item (two hamsters or prairie dogs pieces weighing approximately 75 to 100 g per week); 4) horsemeat diet + carcass item (two hamsters or prairie dogs pieces weighing approximately 75 to 100 g per week). The hamsters were from a colony that was raised at FCC. The prairie dogs came from a partnership between the U.S. Fish and Wildlife Service and local county government agencies in Colorado who removed prairie dogs in accordance with their land management plans. Complete dietary analyses of TOR, hamster and prairie dog carcasses are described in Santymire et al. [12]. A subset of samples from adult males (n = 15) was used to evaluate the effects of supplementation of vitamin E and/or carcass on FAM, even though previous research demonstrated that age (1 to 5 years of age) did not affect semen quality and testosterone concentrations [30].

Sample collection and processing

Fecal samples were collected in the morning three times a week from November and December (pre-breeding season), February through May (breeding season) and June (post-breeding season). The diet change occurred in January and continued through June for all treatment groups. All samples were stored at -20°C until processing. Fecal samples were shipped to and processed at Lincoln Park Zoo’s endocrinology laboratory (Chicago, IL, USA). Feces were processed using previously described methods [31]. Briefly, samples were dried on a lyophilizer (Thermo Fisher Scientific, Inc., Waltham, MA) and were then crushed using a rubber mallet. The powdered samples were weighed (0.02 ± 0.002g) and 0.5 ml of 90% ethanol: distilled water was added to each aliquot. Samples were mixed (Glas-Col, Terre Haute, IN) with the ethanol for 30 mins and centrifuged (1,500 rpm; 20 minutes). The supernatant was then poured into clean test tubes. Residual feces were resuspended in another 0.5 ml of 90% ethanol, vortexed for 30 seconds, and centrifuged for 15 minutes. The supernatant was added to the first extract and was evaporated with air and heat (60°C). The extracts were reconstituted in 0.5 ml of phosphate-buffered saline (0.2M NaH2PO4, 0.2Ms Na2HPO4, NaCl) before analysis.

Hormone metabolite analysis

We analyzed the samples for FGM via a corticosterone enzyme immunoassay (EIA) that was previously validated for ferrets [31]. Briefly, the corticosterone antiserum (CJM006; provided by C. Munro, University of California, Davis, CA) and horseradish peroxidase (HRP; provided by C. Munro) were used at dilutions of 1:6,000 and 1:20,000, respectively. Antiserum cross-reactivities for corticosterone were previously described [32]. Biochemical validations of parallelism between binding inhibition curves of fecal extract dilutions and the corticosterone standard and significant recovery (>90%) of exogenous corticosterone (1.95–1,000 pg/50 μl) has been previously published [31]. Assay sensitivity was 1.95 pg/50 μl and intra- and inter-assay coefficients of variation were <10%.

FAMs were analyzed using a testosterone EIA. The polyclonal antiserum (R156/7) and HRP were provided by C. Munro and used at dilutions of 1:10,000 and 1:30,000, respectively. Antiserum cross-reactivities for testosterone were previously described [32]. The testosterone EIA was validated for ferrets by demonstrating: 1) parallelism between binding inhibition curves of fecal extract dilutions (1:160–1:10,240) and the testosterone standard (R2 = 0.994); and 2) significant recovery (> 90%) of exogenous testosterone (1.17–300 pg/50 μl) added to fecal extracts (1:10,000; y = 1.48x − 2.00, R² = 0.997). Assay sensitivity was 2.3 pg/50 μl and intra- and inter-assay coefficients of variation were <10%.

Data analysis

We first used a linear mixed effects model to identify the important fixed effect(s) among carcass treatment, vitamin E treatment, age class (juvenile or adult; FGM only), and all possible interactions between these fixed effects on each hormone. We included individual males nested within months nested within seasons (pre-, during, and post-breeding season) as random effects. We log-transformed the response variables (FAM and FGM) for analysis. We fit these models in R version 3.5.2 [33] using the package nlme [34]. Models were fit using the REML method and assuming no within-group correlations. We estimated effects of the random factors using variance components analysis and tested significance using likelihood ratio tests. We measured the goodness of fit of the models by examining the correlation between model predictions and the observed data. We then examined the effects over time (across seasons) for the treatment identified as important from these models using a Friedman test (a non-parametric repeated measures ANOVA) and the Student-Newman-Keuls method for multiple comparisons using Sigma Stat (version 11.0; Systat Software, Inc.; Chicago, IL). Finally, we examined the effects of the important treatments within each season, using a t-test if data were normal or Mann-Whitney U Rank Sum test otherwise. For all statistical analyses, we used the significance threshold of p<0.05.

Results

The mixed effects models showed no significant interactions among the three fixed effects for either FGM (Table 1) or FAM (Table 2). All three random effects were included in the models as the likelihood ratio tests indicated removing any of them would significantly reduce model fit (p<0.05 in all cases). The models showed good fit to the dataset, with R2 values for the correlation between observed and predicted values being 0.68 and 0.77 for FGM and FAM, respectively (Table 3).

Table 1

Model parameters from the fitted linear mixed effects model for FGM.

Fixed effects	Value	Standard Error	DF	t-value	p-value
(Intercept)	8.06	0.10	3499	77.73	0.00
Carcass treatment	0.15	0.07	280	2.04	0.04
Vitamin E treatment	0.03	0.07	280	0.45	0.65
Age class	0.08	0.08	280	0.97	0.33
Carcass * Vitamin E	0.08	0.10	280	0.81	0.42
Carcass * Age class	0.03	0.12	280	0.26	0.80
Vitamin E * Age class	-0.17	0.11	280	-1.50	0.13
Carcass * Vitamin E * Age class	-0.12	0.16	280	-0.78	0.44
Random effects	Standard Deviation	% Variance explained
Season	0.15	17.9
Month within season	< 0.001	0.73
Male within month within season	0.31	36.4
Residual	0.39	45.7

Table 2

Model parameters from the fitted linear mixed effects model for FAM.

Fixed effects	Value	Standard Error	DF	t-value	p-value
(Intercept)	8.94	0.37	643	24.01	0.00
Carcass treatment	-0.02	0.14	95	-0.12	0.90
Vitamin E treatment	0.25	0.14	95	1.85	0.07
Carcass * Vitamin E	-0.22	0.20	95	-1.08	0.28
Random effects	Standard Deviation	% Variance explained
Season	0.60	32.3
Month within season	0.22	12.0
Male within month within season	0.46	24.9
Residual	0.57	30.9

Table 3

Goodness of fit statistics for the two models.

	AIC	BIC	logLikelihood	R^2	t-value	DF	p-value
FGM	4406.71	4481.58	-2191.36	0.68	57.48	3791	< 2.2e-16
FAM	1503.53	1540.43	-743.77	0.77	33.47	746	< 2.2e-16

For FGM, there was a significant effect of carcass treatment (p<0.05) but no effect of vitamin E or age class (Table 1). Variation between different measurements for each male within a month explained 36.4% of the variation not explained by the fixed effects, and variation between seasons explained 17.9% of the variation (Table 1). Because there were no interactions (p = 0.42) between the carcass and vitamin E treatments, the four treatment groups were pooled into two groups of carcass and no carcass. In the pre-breeding season before supplementation began, males in the carcass treatment had lower (U = 129,424.0, p = 0.004) FGM than non-supplemented males even though individuals were randomly selected for each treatment. Once the carcass was fed during the breeding season, FGMs declined (p<0.001) in males who received carcass; however, males that did not receive carcass had higher (p<0.05) FGMs (Fig 1). Comparing the treatments, FGM was lower (U = 517,553.5, p<0.001) in the carcass fed males in than males who were not fed carcass during the breeding season. In the post-breeding season, FGM were comparable (U = 15,245.0, p = 0.67) between treatments; however, while FGM for carcass fed males was similar (q = 0.951, p>0.05) between during and post-breeding season periods, non-supplemented males had lower FGMs in the post-breeding season compared to pre- (q = 11.118, p<0.05) and during (q = 10.734, p<0.05) the breeding season (Fig 1).

Fig 1

Mean (± SEM) fecal glucocorticoid metabolites (FGM) across season (pre-, during and post-breeding season) and treatment (carcass vs. no carcass).

Different superscripts (a, b, c) depict differences (P < 0.05) across time in ferrets fed carcass. Different symbols (square, triangle, circle) depict differences (P < 0.05) across time in non-supplemented ferrets. Finally asterisks indicate differences (P < 0.05) between treatments within each time period.

For FAM, there was an effect of vitamin E at the level of p = 0.07, and no effect of carcass treatment (p>0.05; Table 2). Variation between measurements within a month and between different seasons accounted for most of the unexplained variation (24.9% and 32.3%, respectively; Table 2). Because there were no interactions (p = 0.28) between the carcass and vitamin E treatments, the four treatment groups were pooled into two groups of vitamin E and no vitamin E supplementation. In the pre-breeding season before supplementation began, males in the vitamin E treatment had lower (U = 6594.0, p = 0.026) FAM than non-supplemented males. When supplementation began in the breeding season, males receiving vitamin E had higher (t557 = -3.91, p<0.001) FAMs than males that were not supplemented, while both treatment groups had increases in FAM (vitamin E, q = 2.907, p<0.001; no vitamin E, q = 4.289, p<0.05) compared to the pre-breeding season (Fig 2). In the post-breeding season, supplementation of vitamin E had no effect (U = 641.0, p = 0.127) on FAM, but FAM was the lowest (compared to pre-breeding:q = 6.261, p<0.05; during: q = 6.483, p<0.05) for the vitamin E group during this time, while males that did not receive vitamin E had similar FAM as pre-breeding (q = 2.236, p>0.05), but lower than the breeding season (q = 4.585, p<0.05; Fig 2).

Fig 2

Mean (± SEM) fecal androgen metabolites (FAM) across season (pre-, during and post-breeding season) and treatment (vitamin E vs. no vitamin E).

Different superscripts (a, b, c) depict differences (P < 0.05) across time in ferrets supplemented with vitamin E. Different symbols (square, triangle) depict differences (P < 0.01) across time in non-supplemented ferrets. Finally, asterisks indicate differences (P < 0.05) between treatments within each time period.

Discussion

Our goal was to determine the effects of supplementing whole carcass and vitamin E on ferret stress and reproductive physiology. Our previous research examined the relationship between vitamin E and carcass supplementation on ferret seminal characteristics, and determined that the addition of the carcass twice a week improved semen traits and testes volume [12]. Here we specifically wanted to examine the effects on hormonal production. In general, lower psychological stress could reduce HPA axis activity, promote health, and increase reproduction [35,36]. Overall, we found an effect of carcass feeding but not vitamin E on FGMs. For FAMs, there was an effect of vitamin E supplementation but no effect of feeding carcass.

For FGMs, we found ferrets that were fed carcass twice weekly had lower FGM during the breeding season compared to the pre-breeding season. Interestingly, males that were not fed carcass had increased FGM during the breeding season. Initially before the supplementation began, the non-carcass treatment group had significantly higher FGMs (~8% higher). This is was an artifact of random selection of individuals for each treatment. However, upon carcass supplementation, the difference between the treatment groups increased (~18% higher) during the breeding season. This increase in difference could be a result of increased time spent foraging and a reduction of abnormal behaviors; however, we were unable to measure behavior to determine exactly how the carcass directly affected time spent foraging. Previously, environmental enrichment reduced FGM in ferrets, but this finding was sex and age dependent with juvenile males demonstrating greater decline in FGM compared to adult males and females [31]. Here, we only used males in the study and did not observe a difference in response between juveniles and adults. As all ferrets had lower FGM during the post-breeding season, FGM may be driven by the breeding season. However, the carcass supplementation did lower FGM enough during breeding season that was not significantly different than post-breeding season.

Naturally, the breeding season may be more stressful for the ferret as this is the time that wild males would set up territories and compete with conspecific males to gain access to females coming into estrus [37,38]. Although physical competition does not occur ex situ, males are housed in the same room as other males and females; therefore, there may be perceived conflict with other males especially when females are coming into estrus. Poessel and colleagues [31] also observed an increase in ferret FGM as animals entered the breeding season. Romero [39] reviewed the literature and found that most species, especially amphibians, reptiles and birds, have higher baseline glucocorticoids during the breeding season; in general, mammals did not strictly follow this pattern.

Whole carcass-supplementation stimulated natural foraging behavior and maintained dental health in other carnivores, such as African lions (Panthera leo) [35], fishing cats (Felis viverrina) [40] and Sumatran tigers (Panthera tigris sumatrae) [35]. Furthermore, based on ferret museum specimens, individuals fed TOR had a higher prevalence of calculus and periodontal disease than counterparts fed the 60/40 diet; both had poorer dental health than wild ferrets [41]. Anecdotally, by adding carcass twice a week to the diet, the incidence of tartar and gingivitis in ferrets was reduced [42]. Improving the ferret’s oral health may explain why FGMs were lower for carcass-fed males during the breeding season compared to the non-supplemented ferrets. Previous studies have indicated a correlation between HPA activation and periodontal disease (reviewed by [43]). Glucocorticoid-mediated suppression of the immune system, specifically T-cell inhibition, increases susceptibility to periodontal disease [44]. In the rat, inflammatory responses to gingivitis resulted in increased HPA activation and higher circulating corticosterone concentrations [45]. Future studies should incorporate other biomarkers of stress, such as measuring oxidative stress, which is a result of an imbalance of antioxidants and pro-oxidants like reactive oxygen species (ROS) [46]. ROSs damage tissues and cells and can lead to infertility by affecting spermatozoa structure and function [47-50]. However, we did not observe a reduction in FGMs with the addition of vitamin E, which is an antioxidant that helps to minimize the impact of ROS [47]; therefore, the change in FGMs may be related to something other than inflammation.

In a previous study, male ferrets fed carcass were significantly heavier than males that were not supplemented [12]. This increase in weight did not affect overall body condition or reproductive success [12]; therefore, it is difficult to state whether it was a positive or negative change. However, the change in FGMs, observed here, could be a reflection of shifts in the ferrets’ metabolic rate. Glucocorticoids, as the name suggests, are hormones that stimulate the production of readily available glucose to support the body in overcoming challenges whether they are psychological or physiological [26]. The increased FGMs for males that did not receive carcass supplementation may signal a shift in metabolic rate to maintain energy balance during the breeding season. Future research should include measuring thyroid hormones (TH), including thyroxine (T4) and triiodothyronine (T3), which are important biomarkers of stress and energy balance. THs regulate many metabolic and ontogenetic pathways and there is communication between HPA and the H-P-Thyroid (HPT) axes [reviewed by 51]. Specifically for mammalian species, an acute HPA stimulate may result in lower TH production, which can affect metabolic rates [51]. Therefore, by analyzing both THs and FGMs we can better understand ferret homeostasis [52].

As observed previously in the ferret and in closely related species including the domestic ferrets (Mustela putorius furo) and Siberian polecats (M. eversmanni) [53], FAM values peaked during the breeding season for all treatments. However, we observed higher FAMs in males supplemented with vitamin E compared to the non-supplemented males during the breeding season. Vitamin E is known to play a role in steroidal hormone production and even in activating spermatogenesis [17,54]. Specifically, vitamin E stimulates the production of pituitary gonadotropins increasing plasma testosterone [17]. Vitamin E supplementation on rat Leydig cells resulted in increased testosterone production [55]. And vitamin E also reduces lipid peroxidation in the semen and improves sperm motility in men [56], stallions [57], sheep [58] and boars [59]. Our previous research examined the relationship between vitamin E and carcass supplementation on ferret seminal characteristics, showing that the addition of the carcass twice a week improved semen traits and testes volume [12]. Interestingly, in the current study we found no effect of carcass feeding and no interaction between vitamin E and carcass feeding on FAM production.

While we observed a decrease in FGMs during the breeding season when the ferret was supplemented with carcass and an increase in FAMs with supplemental vitamin E, a previous report found no effect on siring abilities [12]. Boosting reproductive success in the ferret recovery program is our primary conservation goal [60] as pregnancy success declined from 80% in 1990s to 36% across the SSP [61]. Normal spermatozoa in male ejaculate has declined from 50% to 25% [6]. Since demonstrating the importance of normal spermatozoa and sperm motility for pregnancy in the ferret [7], we have been investigating factors that could be attributing to ferret infertility. Declining reproductive rates may be related to inbreeding depression. The ferret population is a closed population with no opportunity to introduce novel genes to the species, at least not naturally. However, we have observed an improvement in semen quality in wild-born males [8,9]. Specifically, wild-born ferrets have nearly double the percent of normal spermatozoa in the ejaculate, while ex situ-born males living in the wild for at least one year still have a comparable quality to the ex situ population [62]. Therefore, we surmise that an environmental condition, such as diet, housing, lighting and/or other husbandry factors, may be affecting ferret physiology and reducing reproductive success. These effects may be altered by being born into wild living conditions, but they cannot be reversed in ex situ-born individuals. Because diet may be the variable that is leading to infertility in the ferret population, our next step is to conduct a multi-year study across several generations of the ex situ population to test whether we observe similar trends noted in wild populations.

Another important finding is that variation in FGMs between multiple measurements within a month for each male explained 36.4% of the variation not explained by the fixed effects. For FAM analysis, variation among measurements within a month and among different seasons accounted for relatively high levels of the unexplained variation (32.3% and 24.9%, respectively). This variability over time demonstrates the importance of collecting multiple, frequent samples from individuals when using fecal hormone metabolite analysis particularly for understanding stress physiology.

Conclusions

In conclusion, this study is the first to examine the effects of vitamin E and carcass supplementation on hormone concentrations in the ferret. While FAMs increased in all males, ferrets supplemented with vitamin E had significantly higher FAMs than the non-supplemented males during the breeding season. Furthermore, the addition of carcass to the ferret diet resulted in lower FGMs during the breeding season compared to males not fed carcass, which had increased FGMs compared to pre-breeding season values. The reduction in FGMs for the carcass-fed males may be attributed to improved dental health and/or the enriching nature of feeding on the carcass; however, this effect did not continue in the post-breeding season. This finding suggests that FGMs are affected by breeding season but we cannot distinguish between changes in FGM due to metabolic changes and/or psychological stress. Further research is needed to determine the effects of carcass feeding on ferret metabolism and stress physiology by examining other biomarkers, such as THs. Neither an increase in FAMs nor a decrease in FGMs during the breeding season influenced reproductive success [12]. Our results justify the need to continue studying the effects of diet on reproductive and stress physiology in the ferret. We suggest conducting a long-term study that extends over multiple years to determine the effects of hormonal changes on ferret reproductive biology.

Fecal hormone metabolite data including age class, diet treatment and season.

(XLSX)

Click here for additional data file.

28 May 2020

PONE-D-20-09363

Diet supplementation reduced fecal glucocorticoid metabolites and increased androgen metabolites in male black-footed ferrets (Mustela nigripes)

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: No

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

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: Abstract

Line 32: enzyme immunoassay (no “s”)

Line 39: Remove the extra “the”; explicitly state that age was not significant so the reader is not looking for those data in the lines that follow

Line 41: Does “supplemented” refer to vitamin E or carcass? The abstract needs to be more consistent throughout so that it is clear what “supplemented” is referring to in each sentence.

Introduction

Line 63: No need to repeat “muscle”

Line 63-64: The authors have already stated it is difficult to replicate.

Line 75: Suggest “improving physical health” (vs. dental alone)

Line 78: Do the authors mean “psychological” here (since physiological was discussed in the previous paragraph and the current paragraph seems to focus on behavior)?

Line 102: normal sperm acrosome

Line 106: Please quantify “nearly coincides”

Line 119-122: The authors did not really examine stress or psychological enrichment. They examined adrenal and reproductive biomarkers, and the first hypothesis should be revised to state simply that FGM would be lower in ferrets fed whole prey items.

Overall, this section is well-written, interesting, and presents good reason for conducting the study.

Methods

Lines 135-137: Perhaps this can be combined with the sentences at the start of this section to consolidate?

Line 138: Sample size was stated in the previous paragraph but with less detail regarding sample sizes of the age groups. Consolidate all this information in the same sentence.

Line 143: Presumably, “adult” is the 2 and 3 year-olds? The authors have not yet talked about the samples, so it might be clearer to state simply “a subset of adult males…was used to evaluate…” Lastly, there are 4 treatment groups, but this subset in which FAM will be measured appears to be divided into only 2, yet is to be used for evaluating effects of vit. E and/or carcass. If it is for evaluating vit. E plus either carcass or no carcass, perhaps in that case the authors can state, “a subset of adult males in groups 2 and 3 was used to evaluate…”. Otherwise, there would seem to be two missing groups from this subset (no vit E + carcass; no vit E no carcass)? Indeed, the results suggest the comparison of importance is between vitamin E versus no vitamin E, so is that really group 3 vs. group 4 were vitamin E supplementation differs but both receive carcass?

Line 149: Were all these samples collected at the same time of day or, if not, have previous publications already demonstrated that the circadian rhythm in glucocorticoid concentrations does not appear in fecal samples collected at different times of day (i.e., the excretory lag time is sufficiently long to “average out” the daily variation in FGM concentrations)? If not collected at the same time of day and if not previously demonstrated that FGM do not vary across the day, perhaps this accounts for some of the important but unexplained variation in the statistical models mentioned in the discussion?

Line 150: It is unfortunate there could not be a breeding season, no diet change combination.

Line 151: Samples were stored at minus 20C

Line 154: were dried

Line 158: It would be clearer to state the residual fecal material was resuspended rather than the “samples” to avoid confusion with the first supernatant that was decanted.

Line 161: 0.5 ml

Line 165: immunoassay

Line 167: Remove extra “(“

Line 171, 172; Line 180, 181: Please provide standard values in pg/ml (or similar units) or provide assay details of how many µl are added per well so the reader is able to do the necessary calculations if they wish to repeat the methods. Also provide assay sensitivity in pg/ml or similar.

Line 174: Sample size already described earlier in this section

Line 174-175: analyzed by or analyzed using

Line 187-188: It was described earlier that juveniles were not sampled for the FAM analysis, only adults, so presumably this statement is irrelevant?

Clarification is needed in this section with respect to the experimental design for the FAM analysis. Otherwise, only minor edits to clarify details are needed.

Results

Line 222: Suggest “from pre-breeding season to breeding season time periods”

Line 220-231: I think these data will be more easily understood by the audience if the authors take us through the “story” told by these data chronologically. Currently, it describes what happens to each group of males (carcass-supplemented or not) separately, then compares them within the seasons, and we have to go back and remember what the differential responses were across those seasons. It could be better to say, “in pre-breeding season, males that would be fed carcass in the coming months had X FGM concentrations compared to those that would not be supplemented. Once carcass was fed, the males receiving it experienced X whereas those not receiving it experienced Y, and these concentrations differed from each other. By post-breeding season…” I think this will better communicate the message about FGM differences to the audience than the way it is currently presented.

Line 232: Suggest stating p = 0.07 rather than p < 0.10

Line 232-243: Again, I suggest laying these data out chronologically to better communicate the differences. Also, from Table 2, it looks like carcass had no effect on FAM and did not interact with vitamin E, but Figure 2 suggests there was no carcass supplementation going on at all. I suspect Figure 2 should state that treatment was “(carcass + vitamin E vs. carcass with no vitamin E)”. I was unclear about the break-down of the FAM groups in the methods as well. In addition to clarifying this in the methods, it would be helpful to state explicitly in the results that the described results for vit. E on FAM occurred for those on carcass in the same manner as they occurred for those not on carcass (or were only assessed for those on carcass?).

Overall, the data are all here, but the way they are organized leaves the reader to try and piece together a complete picture from the components, which I fear could result in losing the point the authors will want to make. If these paragraphs could be re-written to better focus the reader on the critical differences at the critical points in time, the authors will have the readers “on board” by the time we get to the discussion. The figures are quite nice and allow the audience to interpret the data (except it is still unclear if the vitamin E data are for animals that were on carcass, not on carcass, or both).

Discussion

Line 264-282: These previous findings provide the initial rational for testing supplemental carcass feeding, but perhaps this could be more briefly summarized since it was already mentioned in the introduction as well? The study itself does not generate any dental data, yet the topic kicks-off and dominates the first 20 lines of the discussion.

Line 282-288: While interesting, this deals with skeletal changes as a result of space/activity, which has nothing to do with the present study.

Line 288-293: Again, while that might be interesting to consider, none of the data presented in this study speaks to morphological changes, so this musing seems unnecessary.

Line 294-297: It could be reasonable that whole carcass is more psychologically enriching, but no behavioral data were presented in this study. There is no pre-carcass behavioral data (or non-FGM physiological data that could be used as an indicator of stress) to support the notion that the ferrets were stressed to begin with, nor were any behavioral (or other) data recorded to support the notion that stress was reduced by carcass feeding. The authors acknowledge the absence of this data in subsequent lines and suggest instances in other studies where behavior was improved by carcass feeding, but the suggested conclusion in ferrets is entirely based on supposition.

Glucocorticoids are metabolic hormones, and while the psychological stress response can require a change in metabolism, metabolic change associated with breeding condition and associated with a change in diet/digestion (both of which are featured in this study) can occur in the absence of a change in psychological state. The authors go too far in concluding that their data suggest a reduction in stress when the only parameter measured is glucocorticoid metabolites. The authors have demonstrated a change in metabolism, and as it was in the direction opposite of those ferrets that did not receive carcass during the breeding season, the change does indeed appear to be a result of the carcass supplementation, but the effects of that supplementation are unknown at this point in terms of why it caused a change in metabolism and what it means for the ferrets. Instead of a change in psychological stress, couldn’t FGM have been reduced because there was more bone and fibrous tissue to attempt to digest and less protein and simple carbohydrate? It would be very helpful to have weight data on these different groups to see what effect the diet change had on their weight gain/loss. Perhaps this information is available in the ZIMS records and can be analyzed to provide some insight here? Another option to consider if the authors feel strongly that the change in FGM is indeed related to stress is to test another biomarker for stress. Markers of oxidative stress have been measured using fecal samples in other species—possibly try Arbor Assays K059?

Line 311-313: If improved oral health is tied to carcass supplementation, and carcass supplementation continued in the post-breeding season, then shouldn’t FGM continue to be lower in the carcass group than the non-carcass group in the post-breeding season? Instead, they are the same. This suggests that the effects of carcass supplementation on FGM concentrations were strongest or most relevant to the biology of the ferret during breeding season in particular. It would seem that a relationship to oral health should be fairly consistent across the year, no? The authors do bring up the interesting idea of glucocorticoid-mediated suppression of the immune system in subsequent lines. I wonder if perhaps there is literature on how the immune response of ferrets during the breeding season is up- or down-regulated normally and if that could then have any relationship to the differential FGM response of the ferrets in this study?

Line 323: What is the situation in the managed population? Are males and females paired individually according to genetics or is there some opportunity for competition/choice?

Line 336: Is it possible to add the no-effect-of-carcass data to the FAM graph? It is interesting that although FGM increased in the non-carcass group whereas FGM decreased in the carcass group, the carcass group did not manage to have higher FAM (since FGM might suppress FAM). Perhaps FAM were higher in the carcass group, it just was not statistically significant, especially since the authors noted an improvement in semen characteristics in a previous study of carcass supplementation?

Line 328-347: Within this paragraph, the connection between the fact that FAM increased more during the breeding season in the ferrets that received vitamin E supplementation than those that did not (line 335-336) and the mechanism for that relationship and beneficial effects related to semen (lines 343-347) gets lost by the intervening information about the lack of relationships in this study between carcass and FAM and between vitamin E and FGM. The important vitamin E-FAM relationship probably needs to be in its own paragraph, while the absent carcass-FAM and vitamin E-FGM relationships can be in a separate paragraph. Your topic sentences for the paragraph beginning on line 367 could be topic sentences for these paragraphs, especially since they are not related to the subsequent lines of the paragraph in which they currently appear (370-375).

Line 360-364: It sounds like the issue might be an organizational development issue. Have you considered estrogens in the TOR diet or in the water source?

Line 369-373: Perhaps all this variation that is important but unexplained by the fixed effects also suggests, along with your fecundity data, that some other fixed effect must be tested in order to solve this ferret fertility issue.

Line 379-380: Again, I really do not think the authors can point to what about carcass feeding contributed to the lower FGM in carcass-fed ferrets with this data set and with the discussion points that are presented. If attributed to improved dental health or enriching qualities, I would think those effects should have continued into the post-breeding season. The authors should consider what could be special about the physiology and metabolism of the ferrets during the breeding season in particular that could better explain this interesting and possibly important finding.

The discussion brings up a lot of interesting points, but the authors need to discuss the data in this study a bit more thoroughly. There seems to be something missing from how the authors are thinking about the change in FGM that could be better explored. Possibly more data needs to be gathered (weight, other biomarkers of stress, even behavioral notes from keepers), and at this time the most that can be said is that a change occurred but additional study is necessary to understand why and to understand its potential effects. The authors should be very careful about concluding that there was any change in psychological stress—it is possible but remains unsupported at this time. The part of the discussion related to FAM could be better organized to draw attention to the essential finding(s) and how that relates to previous studies but does not fully solve the ferret reproduction issue.

The study on the whole is interesting and important, but clarification is necessary in the experimental design, and a more focused interpretation of the data presented by this study so that it neither over-states nor under-states possible conclusions is needed. Alternative interpretations should be sought for the FGM data as those provided in the discussion do not seem to adequately explain the findings.

Reviewer #2: Introduction -

1. The intro is far too long with unnecessary information. The authors don't include any measures about dentition or dental health so it is not needed in the intro. There were also no measures of behavior; therefore, much of Lines 61 - 81 are simply extra words and could be shortened substantially without losing any integrity of the paper's intent.

2. There should be some information included regarding the use of FGM and the link with behavior and stress. There are other more common measures of stress so some lit review provided regarding this link would be useful to the reader. The words "psychologically enriching" is rather vague and would be very difficult to prove.

Materials and Methods

1. Line 133. What is meant by "commercially available"? How is the diet formulated? Formulated to what recommendations, I would assume feline?

2. If the paper is titled "Diet supplementation", additional information should be provided about the diet inclusive of actual dietary analyses, particularly for vitamin E. Why were there no analyses of diets provided or any information about the diet itself? Were all the lots of meat diet the same? There is substantial variation in the concentrations of vitamin E in diets and certainly in whole prey depending on the diet of the prey item. It is unclear how this was or was not controlled for in this study and is a major fault considering the title. There also was no measurement of vitamin E from the ferrets so without either, it is not possible to draw a substantial conclusion about vitamin E as you have no idea if the requirement was met in first place.

2. Are there recommendations for ferrets or are all diets formulated for cats?

3. Clarify how long the seasons lasted in days? The post breeding season was shorter than the other 2 by a month. Why were 2 months not included for that season? Is that an appropriate amount of time for supplementation?

3. Since you had an age class, why not block the study by age?

Discussion

1. What is the relevance of lines 272 - 320 to the paper? It's a lot of extra words that are not important to this manuscript as none of that was measured here. There is indication that dental health was improved but it wasn't measured. It should not be included unless data are included. This is an entire section of the paper that is not substantiated by any data included in the study presented and should be removed. It is a nice lit review for the value of whole prey but is not relevant.

2. Starting with line 321: How are FGM's related to "stress"? Rather than spend so much time on 272-320 that means nothing to the measures of the study, it seems that there could be value in this particular measure but it is hardly discussed. If FGM's are a reliable measure of stress then this is very important for animal managers and could definitely be used to enhance the diets of managed carnivores. It's not clear in current form how FGM's are related to stress and this could really enhance this paper. and is only one measure of Why were other measures of "stress" not considered such as inflammatory markers, cortisol, etc...?

3. It would be beneficial for the authors to discuss the 2 main measures (FGM and FAM) as indicators of reproductive success? There is some indication that one (FGM) is related to stress while FAM is related to testosterone? With this being a generally read publication, it would be valuable to provide some rationale as to these 2 measures. Why they were selected and how they are more directly related to reproductive success.

4. The "no carcass" pre and breeding season values look like the error bars overlap? Therefore, how are those significantly different?

Conclusions

1. Your conclusions are not substantiated with the data you collected. You only measured FGM and FAM and both were only weakly linked to stress and reproductive success. The authors simply restated the results that FGM reduced with whole prey and FAM increased with E then there were subjective assumptions made about enrichment, dental health, feeding ecology and visitor perceptions...none of which were measures and conclusions cannot be drawn from the 2 measures obtained.

General

1. Is there any information on these values (FAM/FGM) during these time frames (pre, breeding and post breeding) while not on any supplementation (what are typical)? That would really strengthen the data if one could look at it comparatively. I assume that's why there was no diet change in the post breeding but what is it normally and how do those values change seasonally as a whole?

2. The title is misleading as "diet supplementation". It might be better as "Influence of supplemental vitamin E and whole prey on..."

**********

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Reviewer #2: No

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Submitted filename: review for PONE-D-20-09363.docx

Click here for additional data file.

4 Aug 2020

The authors want to thank the Reviewers and the Editor. We appreciate the thoroughness of the review. All of these comments that have made this manuscript stronger. Our responses are in bold below each comment.

Abstract

Line 32: enzyme immunoassay (no “s”)

Corrected

Line 39: Remove the extra “the”; explicitly state that age was not significant so the reader is not looking for those data in the lines that follow

Corrected and added a statement about age (Lines 44-45)

Line 41: Does “supplemented” refer to vitamin E or carcass? The abstract needs to be more consistent throughout so that it is clear what “supplemented” is referring to in each sentence.

We changed “supplemented” to “fed carcass” throughout abstract and left “supplemented” to refer to vitamin E supplementation only.

Introduction

Line 63: No need to repeat “muscle”

Corrected

Line 63-64: The authors have already stated it is difficult to replicate.

We removed the phrase in the sentence.

Line 75: Suggest “improving physical health” (vs. dental alone)

Agreed and corrected

Line 78: Do the authors mean “psychological” here (since physiological was discussed in the previous paragraph and the current paragraph seems to focus on behavior)?

Agreed and corrected

Line 102: normal sperm acrosome

Corrected

Line 106: Please quantify “nearly coincides”

Corrected

Line 119-122: The authors did not really examine stress or psychological enrichment. They examined adrenal and reproductive biomarkers, and the first hypothesis should be revised to state simply that FGM would be lower in ferrets fed whole prey items.

Agreed and corrected

Overall, this section is well-written, interesting, and presents good reason for conducting the study.

Thank you

Methods

Lines 135-137: Perhaps this can be combined with the sentences at the start of this section to consolidate?

Corrected

Line 138: Sample size was stated in the previous paragraph but with less detail regarding sample sizes of the age groups. Consolidate all this information in the same sentence.

Corrected

Line 143: Presumably, “adult” is the 2 and 3 year-olds? The authors have not yet talked about the samples, so it might be clearer to state simply “a subset of adult males…was used to evaluate…” Lastly, there are 4 treatment groups, but this subset in which FAM will be measured appears to be divided into only 2, yet is to be used for evaluating effects of vit. E and/or carcass. If it is for evaluating vit. E plus either carcass or no carcass, perhaps in that case the authors can state, “a subset of adult males in groups 2 and 3 was used to evaluate…”. Otherwise, there would seem to be two missing groups from this subset (no vit E + carcass; no vit E no carcass)? Indeed, the results suggest the comparison of importance is between vitamin E versus no vitamin E, so is that really group 3 vs. group 4 were vitamin E supplementation differs but both receive carcass?

The reviewer makes an excellent point. To clarify the difference between FGM and FAM groups, we removed the “A subset (n=15)” sentence because it is stated below in the Methods describing FAM analysis. We also removed the carcass vs no carcass because they are also part of the 4 diet treatments as we investigated the interactions of vitamin E and carcass supplementation. These changes will make it clearer to the reader.

Line 149: Were all these samples collected at the same time of day or, if not, have previous publications already demonstrated that the circadian rhythm in glucocorticoid concentrations does not appear in fecal samples collected at different times of day (i.e., the excretory lag time is sufficiently long to “average out” the daily variation in FGM concentrations)? If not collected at the same time of day and if not previously demonstrated that FGM do not vary across the day, perhaps this accounts for some of the important but unexplained variation in the statistical models mentioned in the discussion?

Yes, all of the samples were collected the same time of day. We have added a sentence to the methods to clarify the time of day (Line 166).

Line 150: It is unfortunate there could not be a breeding season, no diet change combination.

Control males were included on this analysis (Group 1). Line 150 is describing the timing (breeding season/pre- or post-breeding season) of the diet change.

Line 151: Samples were stored at minus 20C

Corrected

Line 154: were dried

Corrected

Line 158: It would be clearer to state the residual fecal material was resuspended rather than the “samples” to avoid confusion with the first supernatant that was decanted.

Corrected

Line 161: 0.5 ml

Corrected

Line 165: immunoassay

Corrected

Line 167: Remove extra “(“

Corrected

Line 171, 172; Line 180, 181: Please provide standard values in pg/ml (or similar units) or provide assay details of how many µl are added per well so the reader is able to do the necessary calculations if they wish to repeat the methods. Also provide assay sensitivity in pg/ml or similar.

Corrected

Line 174: Sample size already described earlier in this section

Corrected

Line 174-175: analyzed by or analyzed using

Corrected

Line 187-188: It was described earlier that juveniles were not sampled for the FAM analysis, only adults, so presumably this statement is irrelevant?

We deleted that sentence.

Clarification is needed in this section with respect to the experimental design for the FAM analysis. Otherwise, only minor edits to clarify details are needed.

To clarify, the only difference between the FGM and FAM analyses was age. No juveniles were included on the FAM because of the confounding effect of it being their first breeding season.

Results

Line 222: Suggest “from pre-breeding season to breeding season time periods”

Corrected

Line 220-231: I think these data will be more easily understood by the audience if the authors take us through the “story” told by these data chronologically. Currently, it describes what happens to each group of males (carcass-supplemented or not) separately, then compares them within the seasons, and we have to go back and remember what the differential responses were across those seasons. It could be better to say, “in pre-breeding season, males that would be fed carcass in the coming months had X FGM concentrations compared to those that would not be supplemented. Once carcass was fed, the males receiving it experienced X whereas those not receiving it experienced Y, and these concentrations differed from each other. By post-breeding season…” I think this will better communicate the message about FGM differences to the audience than the way it is currently presented.

Thank you for the suggestion, we have reorganized the FGM paragraph using your suggestions.

Line 232: Suggest stating p = 0.07 rather than p < 0.10

Corrected

Line 232-243: Again, I suggest laying these data out chronologically to better communicate the differences. Also, from Table 2, it looks like carcass had no effect on FAM and did not interact with vitamin E, but Figure 2 suggests there was no carcass supplementation going on at all. I suspect Figure 2 should state that treatment was “(carcass + vitamin E vs. carcass with no vitamin E)”. I was unclear about the break-down of the FAM groups in the methods as well. In addition to clarifying this in the methods, it would be helpful to state explicitly in the results that the described results for vit. E on FAM occurred for those on carcass in the same manner as they occurred for those not on carcass (or were only assessed for those on carcass?).

For both FGM and FAM we have added a sentence that states that because there were no interactions between vit E and carcass supplementation, for each hormone we condensed the 4 treatment groups into 2: Carcass vs. no carcass for FGM and Vit E and no vit E for FAM. We also did reorganize the FAM results as suggested.

Overall, the data are all here, but the way they are organized leaves the reader to try and piece together a complete picture from the components, which I fear could result in losing the point the authors will want to make. If these paragraphs could be re-written to better focus the reader on the critical differences at the critical points in time, the authors will have the readers “on board” by the time we get to the discussion. The figures are quite nice and allow the audience to interpret the data (except it is still unclear if the vitamin E data are for animals that were on carcass, not on carcass, or both).

Thank you, hopefully now with your helpful suggestions we have clarified the analysis and results.

Discussion

Line 264-282: These previous findings provide the initial rational for testing supplemental carcass feeding, but perhaps this could be more briefly summarized since it was already mentioned in the introduction as well? The study itself does not generate any dental data, yet the topic kicks-off and dominates the first 20 lines of the discussion.

We have greatly reduced this text. Some of the diet information was put into the introduction. The rest was either deleted or used to support our findings.

Line 282-288: While interesting, this deals with skeletal changes as a result of space/activity, which has nothing to do with the present study.

This text has been deleted.

Line 288-293: Again, while that might be interesting to consider, none of the data presented in this study speaks to morphological changes, so this musing seems unnecessary.

This text has been deleted.

Line 294-297: It could be reasonable that whole carcass is more psychologically enriching, but no behavioral data were presented in this study. There is no pre-carcass behavioral data (or non-FGM physiological data that could be used as an indicator of stress) to support the notion that the ferrets were stressed to begin with, nor were any behavioral (or other) data recorded to support the notion that stress was reduced by carcass feeding. The authors acknowledge the absence of this data in subsequent lines and suggest instances in other studies where behavior was improved by carcass feeding, but the suggested conclusion in ferrets is entirely based on supposition.

Glucocorticoids are metabolic hormones, and while the psychological stress response can require a change in metabolism, metabolic change associated with breeding condition and associated with a change in diet/digestion (both of which are featured in this study) can occur in the absence of a change in psychological state. The authors go too far in concluding that their data suggest a reduction in stress when the only parameter measured is glucocorticoid metabolites. The authors have demonstrated a change in metabolism, and as it was in the direction opposite of those ferrets that did not receive carcass during the breeding season, the change does indeed appear to be a result of the carcass supplementation, but the effects of that supplementation are unknown at this point in terms of why it caused a change in metabolism and what it means for the ferrets. Instead of a change in psychological stress, couldn’t FGM have been reduced because there was more bone and fibrous tissue to attempt to digest and less protein and simple carbohydrate? It would be very helpful to have weight data on these different groups to see what effect the diet change had on their weight gain/loss. Perhaps this information is available in the ZIMS records and can be analyzed to provide some insight here? Another option to consider if the authors feel strongly that the change in FGM is indeed related to stress is to test another biomarker for stress. Markers of oxidative stress have been measured using fecal samples in other species—possibly try Arbor Assays K059?

Since we can’t state the exact effects of the whole carcass we have modified our suggestion of the effect on stress and added information about weight changes due to the dietary treatments, which was published in Santymire et al., 2015. We also added the suggestions of adding another biomarker of stress, like thyroid hormonal and oxidative stress analysis, and that glucocorticoid changes may be an indicator of metabolic changes and not psychology stress.

Line 311-313: If improved oral health is tied to carcass supplementation, and carcass supplementation continued in the post-breeding season, then shouldn’t FGM continue to be lower in the carcass group than the non-carcass group in the post-breeding season? Instead, they are the same. This suggests that the effects of carcass supplementation on FGM concentrations were strongest or most relevant to the biology of the ferret during breeding season in particular. It would seem that a relationship to oral health should be fairly consistent across the year, no? The authors do bring up the interesting idea of glucocorticoid-mediated suppression of the immune system in subsequent lines. I wonder if perhaps there is literature on how the immune response of ferrets during the breeding season is up- or down-regulated normally and if that could then have any relationship to the differential FGM response of the ferrets in this study?

There is no literature on seasonal immune response changes in ferrets; however, we do state that both glucocorticoid production may more closely related to breeding season. We also added information on how FAMs increase too and because testosterone is known to suppress the immune system, perhaps the carcass provides a “distraction” by increasing foraging time and oral health (Lines 403-407).

Line 323: What is the situation in the managed population? Are males and females paired individually according to genetics or is there some opportunity for competition/choice?

Males and females are kept in the same room. Individuals are paired based on genetics (using a pedigree analysis). There are no opportunities for physical competition or mate choice. To clarify the statement, we added “wild” before “males”. In captivity, males still may have this competitive nature since they are sharing the same room as females. We have also added a sentence with this information.

Line 336: Is it possible to add the no-effect-of-carcass data to the FAM graph? It is interesting that although FGM increased in the non-carcass group whereas FGM decreased in the carcass group, the carcass group did not manage to have higher FAM (since FGM might suppress FAM). Perhaps FAM were higher in the carcass group, it just was not statistically significant, especially since the authors noted an improvement in semen characteristics in a previous study of carcass supplementation?

Because our model found no interaction effects of vitamin E and carcass feeding and no effect of carcass, we grouped these data by vitamin E supplementation for analyses and for the figure. We believe that this is the most appropriate way of grouping these results.

Line 328-347: Within this paragraph, the connection between the fact that FAM increased more during the breeding season in the ferrets that received vitamin E supplementation than those that did not (line 335-336) and the mechanism for that relationship and beneficial effects related to semen (lines 343-347) gets lost by the intervening information about the lack of relationships in this study between carcass and FAM and between vitamin E and FGM. The important vitamin E-FAM relationship probably needs to be in its own paragraph, while the absent carcass-FAM and vitamin E-FGM relationships can be in a separate paragraph. Your topic sentences for the paragraph beginning on line 367 could be topic sentences for these paragraphs, especially since they are not related to the subsequent lines of the paragraph in which they currently appear (370-375).

We have reorganized these paragraphs to make this connection.

Line 360-364: It sounds like the issue might be an organizational development issue. Have you considered estrogens in the TOR diet or in the water source?

Yes, we are investigating phytoestrogens in the TOR because it is composed of horse meat and the horses may be fed legumes. Preliminary findings are showing increased phyto E2s in captive males compare to wild males and females and captive females. We aren’t ready to discuss this in this paper, though.

Line 369-373: Perhaps all this variation that is important but unexplained by the fixed effects also suggests, along with your fecundity data, that some other fixed effect must be tested in order to solve this ferret fertility issue.

Agreed. We are currently making these dietary changes over generations and are investigating gene expression and have some interesting findings that we will be publishing after another year of data collection.

Line 379-380: Again, I really do not think the authors can point to what about carcass feeding contributed to the lower FGM in carcass-fed ferrets with this data set and with the discussion points that are presented. If attributed to improved dental health or enriching qualities, I would think those effects should have continued into the post-breeding season. The authors should consider what could be special about the physiology and metabolism of the ferrets during the breeding season in particular that could better explain this interesting and possibly important finding.

We have pointed out that the lower FGM did not remain significant in the post-breeding season and suggest that FGM changes may be more related to metabolism. We added a sentence suggesting adding the analysis of other biomarkers of stress like oxidative stress thyroid hormones.

The discussion brings up a lot of interesting points, but the authors need to discuss the data in this study a bit more thoroughly. There seems to be something missing from how the authors are thinking about the change in FGM that could be better explored. Possibly more data needs to be gathered (weight, other biomarkers of stress, even behavioral notes from keepers), and at this time the most that can be said is that a change occurred but additional study is necessary to understand why and to understand its potential effects. The authors should be very careful about concluding that there was any change in psychological stress—it is possible but remains unsupported at this time. The part of the discussion related to FAM could be better organized to draw attention to the essential finding(s) and how that relates to previous studies but does not fully solve the ferret reproduction issue.

We have made these suggested changes and believe that the discussion focuses more on this study’s findings and that FGM changes may be a reflection of other factors such as metabolic rate vs. psychology stress. We also reorganized the FAM discussion to focus on this study’s findings.

The study on the whole is interesting and important, but clarification is necessary in the experimental design, and a more focused interpretation of the data presented by this study so that it neither over-states nor under-states possible conclusions is needed. Alternative interpretations should be sought for the FGM data as those provided in the discussion do not seem to adequately explain the findings.

Thank you and your suggestions have helped us improve the discussion.

Reviewer #2: Introduction -

1. The intro is far too long with unnecessary information. The authors don't include any measures about dentition or dental health so it is not needed in the intro. There were also no measures of behavior; therefore, much of Lines 61 - 81 are simply extra words and could be shortened substantially without losing any integrity of the paper's intent.

We have reduced these two paragraphs down to one.

2. There should be some information included regarding the use of FGM and the link with behavior and stress. There are other more common measures of stress so some lit review provided regarding this link would be useful to the reader. The words "psychologically enriching" is rather vague and would be very difficult to prove.

We have removed “psychologically” enriching. And we added a paragraph about monitoring stress physiology of ex situ wildlife.

Materials and Methods

1. Line 133. What is meant by "commercially available"? How is the diet formulated? Formulated to what recommendations, I would assume feline?

TOR was defined in the introduction. We deleted “commercially available” since we state the company that makes it. We also added to the introduction that TOR was specifically formulated for the ferret.

2. If the paper is titled "Diet supplementation", additional information should be provided about the diet inclusive of actual dietary analyses, particularly for vitamin E. Why were there no analyses of diets provided or any information about the diet itself? Were all the lots of meat diet the same? There is substantial variation in the concentrations of vitamin E in diets and certainly in whole prey depending on the diet of the prey item. It is unclear how this was or was not controlled for in this study and is a major fault considering the title. There also was no measurement of vitamin E from the ferrets so without either, it is not possible to draw a substantial conclusion about vitamin E as you have no idea if the requirement was met in first place.

In the Santymire et al. 2015 where the effect of this diet study on seminal characteristics is described, the diet analysis is included. We added a statement referring to that paper for dietary analysis in the methods (Lines 159-160).

2. Are there recommendations for ferrets or are all diets formulated for cats?

For the ferret, we follow the dietary recommendations for the mink, which is also used in the Santymire et al. 2015 paper as a guideline for the dietary recommendations.

3. Clarify how long the seasons lasted in days? The post breeding season was shorter than the other 2 by a month. Why were 2 months not included for that season? Is that an appropriate amount of time for supplementation?

We believe that by listing the months, the readers will understand how long each “season” lasted. It would be ideal to collect samples for longer in the post-breeding season, but we were unable to collect samples past June because of kit production which requires a lot more staff time. Additionally, because the ferret is experience reproductive issues and we were wondering if GC production was suppressing reproduction, investigating stress physiology in the post-breeding season would not be helping to answer this question. Therefore, we did not prioritize staff time for fecal samples collection for this study.

3. Since you had an age class, why not block the study by age?

We included age class in the model for FGM but did not find it to be a significant predictor of FGM levels. We only measured FAM in adults and therefore did not include age class in that analysis.

Discussion

1. What is the relevance of lines 272 - 320 to the paper? It's a lot of extra words that are not important to this manuscript as none of that was measured here. There is indication that dental health was improved but it wasn't measured. It should not be included unless data are included. This is an entire section of the paper that is not substantiated by any data included in the study presented and should be removed. It is a nice lit review for the value of whole prey but is not relevant.

We have reduced this section and included our findings supporting them with the literature.

2. Starting with line 321: How are FGM's related to "stress"? Rather than spend so much time on 272-320 that means nothing to the measures of the study, it seems that there could be value in this particular measure but it is hardly discussed. If FGM's are a reliable measure of stress then this is very important for animal managers and could definitely be used to enhance the diets of managed carnivores. It's not clear in current form how FGM's are related to stress and this could really enhance this paper. and is only one measure of Why were other measures of "stress" not considered such as inflammatory markers, cortisol, etc...?

Based on Reviewer 1’s comments, we have added how changes in FGMs might be a reflection of metabolism and added information about body weight changes. We also suggested that changes in FGM may be more related to the season than just the carcass feeding alone. We also suggesting investigating other biomarkers of stress like oxidative stress.

3. It would be beneficial for the authors to discuss the 2 main measures (FGM and FAM) as indicators of reproductive success? There is some indication that one (FGM) is related to stress while FAM is related to testosterone? With this being a generally read publication, it would be valuable to provide some rationale as to these 2 measures. Why they were selected and how they are more directly related to reproductive success.

We have added statements about the effect of GCs and testosterone on reproduction in the introduction.

4. The "no carcass" pre and breeding season values look like the error bars overlap? Therefore, how are those significantly different?

The SEM bars do not overlap in the pre- and breeding seasons between the carcass and no carcass treatments. The asterisks do correctly indicate that there is a significant difference.

Conclusions

1. Your conclusions are not substantiated with the data you collected. You only measured FGM and FAM and both were only weakly linked to stress and reproductive success. The authors simply restated the results that FGM reduced with whole prey and FAM increased with E then there were subjective assumptions made about enrichment, dental health, feeding ecology and visitor perceptions...none of which were measures and conclusions cannot be drawn from the 2 measures obtained.

In the conclusions, we have made suggestions for other studies to help determine why FGMs were reduced using other biomarkers. We have emphasized that these hormonal changes did not affect reproduction in the ferret and therefore need additional research to determine the etiology of infertility in the ferret.

General

1. Is there any information on these values (FAM/FGM) during these time frames (pre, breeding and post breeding) while not on any supplementation (what are typical)? That would really strengthen the data if one could look at it comparatively. I assume that's why there was no diet change in the post breeding but what is it normally and how do those values change seasonally as a whole?

We did have a control group (no vitamin E and no carcass feeding) to compare to these treatments and found that there were no effects or interactions. This treatment group would have defined what is “normal”.

2. The title is misleading as "diet supplementation". It might be better as "Influence of supplemental vitamin E and whole prey on..."

We have changed the title to: Influence of supplementation of vitamin E and carcass feeding on fecal glucocorticoid and androgen metabolites in male black-footed ferrets (Mustela nigripes)

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Submitted filename: Response to reviewers.docx

Click here for additional data file.

25 Aug 2020

PONE-D-20-09363R1

Influence of vitamin E and carcass feeding supplementation on fecal glucocorticoid and androgen metabolites in male black-footed ferrets (Mustela nigripes)

PLOS ONE

Dear Dr. Santymire,

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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Reviewer #1: Introduction

Line 112: So, the problems with reproduction, at least from the perspective of semen quality, pre-dated the diet change, i.e., were unrelated to the diet change? If so, while it is still very important to get the diet as good as it can be for numerous reasons, this timing suggests the specific change made in 2001 is not the culprit for reproductive decline (though perhaps it is not helping the situation either).

Line 115: Missing the “)” after “Canada”

Methods

Lines 137: Seems to be something missing from the second half of this sentence

Lines 148-152: Groups are much clearer now—thank you

Line 163-164: The way it is currently presented with the parenthetical notes, it still seems like the control group was not sampled February-June, but I see your reply in the reviewer notes. I suggest stating that samples were collected from all 4 groups at each time point but no diet changes were initiated in treatment groups until February.

Results

Much clearer—thank you.

Discussion

Line 301: Remove “is largely”

Line 301-302: Suggested revision for clarity: “However, the carcass supplementation did lower FGM enough during breeding season that is was not significantly different than post-breeding season.”

Line 363-364: Intro states 60% to 35%

Line 376: Again, because the decline in reproduction was noted a year before the diet change, stating that “diet may be the variable that is leading to infertility” could be a bit strong. It might not help, to be sure, but I agree a more detailed study is required.

Line 401: I believe the authors mean to say “because glucocorticoids can suppress the immune response” based on the rest of the sentence discussing carcass addition and its potential benefits.

This section is better organized, the take-home-messages more clearly stated, and the statements better supported than in my first review. Alternative explanations are appropriately noted. Just a thought: If vit E supplementation begins in pre-breeding season as males are preparing for increased spermatogenesis (v. starting after the fact during breeding season), perhaps there might be a better outcome for reproduction…

Reviewer #2: Thank you for the changes that were made. Overall, it reads much better. However, I am still struggling with the Introduction. There are few reasons a paper should have a 3 1/2 page introduction. The first 3 paragraphs do not outline the problem at all but instead discuss nutrition, diet formulation and dental health. The gist of the paper's background very nicely starts on Line 88 with a very nice presentation of the history and issues. That then follows very nicely with the need to evaluate vitamin E starting on line 122. It seems that right after Line 128 you could add a bit about the behavior impact/stress on reproduction (starting from Line 71) then introduce the concept of whole prey. That would allow your ending objective statement to flow much better. Regardless the first 2 paragraphs do not benefit your paper in any way and take away from the actual problem that you don't start talking about until Line Line 88. This is about trying to enhance the diets for the BFF program not formulate diets and discuss nutrition for all zoo managed carnivores.

Line 67: What is "processed"? Do you mean basically a TOR that you define later (line 115)? A definition of processed should be included...is that simply ground meat that is complete? I think you mean TOR but you should define it sooner if you are referring to it here.

Line 141 should read Animals and Diet since that section is not just about the animals or it could be Animals and Treatments

Line 150: Add "diet" between Kg and Dry Matter Basis

Line 150 - 152: It is unclear if treatments 3 and 4 have the same whole/prey composition. Is "two hamsters or prairie dogs

pieces/week (3) the same thing as carcass item twice weekly (4)? Was there any attempt made to control/standardize the weight of carcass items for these 2 treatments. As it's currently written it doesn't appear to be the same.

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Submitted filename: review for PONE-D-20-09363_R1.docx

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5 Oct 2020

in bold are our responses.

Reviewer #1: Introduction

Line 112: So, the problems with reproduction, at least from the perspective of semen quality, pre-dated the diet change, i.e., were unrelated to the diet change? If so, while it is still very important to get the diet as good as it can be for numerous reasons, this timing suggests the specific change made in 2001 is not the culprit for reproductive decline (though perhaps it is not helping the situation either).

Yes, the males collected from in 2001 were for artificial insemination program because they weren’t siring on their own and were genetically valuable. This might have been due to poor semen quality. We have removed this sentence in the introduction.

Line 115: Missing the “)” after “Canada”

Corrected

Methods

Lines 137: Seems to be something missing from the second half of this sentence

Corrected

Lines 148-152: Groups are much clearer now—thank you

You are welcome. Thank you for the helpful comments.

Line 163-164: The way it is currently presented with the parenthetical notes, it still seems like the control group was not sampled February-June, but I see your reply in the reviewer notes. I suggest stating that samples were collected from all 4 groups at each time point but no diet changes were initiated in treatment groups until February.

We have clarified sample collection and when diet change occurred.

Results

Much clearer—thank you.

You are welcome. Thank you for the helpful comments.

Discussion

Line 301: Remove “is largely”

Corrected

Line 301-302: Suggested revision for clarity: “However, the carcass supplementation did lower FGM enough during breeding season that is was not significantly different than post-breeding season.”

Corrected

Line 363-364: Intro states 60% to 35%

That was since 2000. Originally, whelping rates were around 80%. We clarified the sentence to make that point clear.

Line 376: Again, because the decline in reproduction was noted a year before the diet change, stating that “diet may be the variable that is leading to infertility” could be a bit strong. It might not help, to be sure, but I agree a more detailed study is required.

Hopefully, with the clarification about the males 2001 being AI males because they were not siring on their own, demonstrates why we are investigating diet further. With this several generations diet study, we hope to show results that suggest more than correlation, but causation (if possible without laboratory experimentation).

Line 401: I believe the authors mean to say “because glucocorticoids can suppress the immune response” based on the rest of the sentence discussing carcass addition and its potential benefits.

We did mean “testosterone”. We stated this because GCs went down during the breeding season but testosterone increased with the addition of carcass. The increased testosterone did not seem to improve reproductive success. So our point was that high testosterone has its costs, but the carcass might help to mitigate them along with providing enrichment/a distraction. But, we removed it and kept the conclusions more general.

This section is better organized, the take-home-messages more clearly stated, and the statements better supported than in my first review. Alternative explanations are appropriately noted. Just a thought: If vit E supplementation begins in pre-breeding season as males are preparing for increased spermatogenesis (v. starting after the fact during breeding season), perhaps there might be a better outcome for reproduction…

Thanks! We hope our 3 year diet change study will address this thought/idea.

Reviewer #2: Thank you for the changes that were made. Overall, it reads much better. However, I am still struggling with the Introduction. There are few reasons a paper should have a 3 1/2 page introduction. The first 3 paragraphs do not outline the problem at all but instead discuss nutrition, diet formulation and dental health. The gist of the paper's background very nicely starts on Line 88 with a very nice presentation of the history and issues. That then follows very nicely with the need to evaluate vitamin E starting on line 122. It seems that right after Line 128 you could add a bit about the behavior impact/stress on reproduction (starting from Line 71) then introduce the concept of whole prey. That would allow your ending objective statement to flow much better. Regardless the first 2 paragraphs do not benefit your paper in any way and take away from the actual problem that you don't start talking about until Line Line 88. This is about trying to enhance the diets for the BFF program not formulate diets and discuss nutrition for all zoo managed carnivores.

We have greatly reduced the first three paragraphs in the introduction. We made the suggested organizational changes, but kept a couple of sentences, such as the lead into nutrition’s importance to welfare, difficulty of specialized carnivore diets and the how the addition of prey items may reduce stress. We wanted some of the literature to set up the difficulties of feeding BFF, which is a specialized carnivore, and why adding carcass could affect GCs (through behavioral modifications).

Line 67: What is "processed"? Do you mean basically a TOR that you define later (line 115)? A definition of processed should be included...is that simply ground meat that is complete? I think you mean TOR but you should define it sooner if you are referring to it here.

Based on your suggested intro re-organization, this sentence has been deleted.

Line 141 should read Animals and Diet since that section is not just about the animals or it could be Animals and Treatments

Corrected

Line 150: Add "diet" between Kg and Dry Matter Basis

Corrected

Line 150 - 152: It is unclear if treatments 3 and 4 have the same whole/prey composition. Is "two hamsters or prairie dogs

pieces/week (3) the same thing as carcass item twice weekly (4)? Was there any attempt made to control/standardize the weight of carcass items for these 2 treatments. As it's currently written it doesn't appear to be the same.

Yes, it was the same. And, yes, weight of the carcass item was standardized. We clarified this in the text.

________________________________________

Submitted filename: Response to reviewers revision I 100520.docx

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8 Oct 2020

Influence of vitamin E and carcass feeding supplementation on fecal glucocorticoid and androgen metabolites in male black-footed ferrets (Mustela nigripes)

PONE-D-20-09363R2

Dear Dr. Santymire,

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Govindhaswamy Umapathy, PhD

Academic Editor

PLOS ONE

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13 Oct 2020

PONE-D-20-09363R2

Influence of vitamin E and carcass feeding supplementation on fecal glucocorticoid and androgen metabolites in male black-footed ferrets (Mustela nigripes)

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