Zoo Biology 33: 144–149 (2014)
BRIEF REPORT
Using Odor Cues to Elicit a Behavioral and Hormonal Response in Zoo‐Housed African Wild Dogs Michelle L. Rafacz,1,2* and Rachel M. Santymire2 1 2
Department of Science and Mathematics, Columbia College, Chicago, Illinois Davee Center for Epidemiology and Endocrinology, Lincoln Park Zoo, Chicago, Illinois Olfactory enrichment, like odor cues, can positively affect behavior, reproductive success, and stress physiology in zoo‐housed species. Our goal was to determine if odor cues were enriching to the African wild dog (AWD; Lycaon pictus), a species with a complex social structure and a highly developed sense of smell. Our objectives were to: (1) examine changes in activity levels and stress hormone physiology in response to fecal odor cues from natural competitor and natural/unnatural prey species; and (2) determine whether these odor cues could function as effective enrichment for zoo‐housed AWDs. Over a 6‐month period, fecal samples were collected from two males (AWD 1: dominant, AWD 2: subordinate), fecal glucocorticoid metabolites (FGMs) were validated using an ACTH‐challenge, and hormones were analyzed for FGMs by enzyme immunoassay. Behavioral observations were conducted using scan‐sampling, and contact and proximity were recorded. AWDs were presented with three fecal odor cues: LION (competitor), CATTLE (unnatural prey), and GAZELLE (natural prey). Only the GAZELLE cue elicited an increase in activity (10.6%) in both individuals and increased positive social behaviors with higher frequencies of affiliative, submissive, and dominant behavior. AWD 1 demonstrated lower (P < 0.05) FGMs than AWD 2 both before and after all odor cues, and FGMs decreased (P ¼ 0.08) in AWD 2 after all cues. We conclude that exposure to natural prey odor cues may be used as effective enrichment for AWDs, and that changes in stress hormone physiology in response to © 2013 Wiley Periodicals, Inc. odor cues may be dependent on social rank in this species. Zoo Biol. 33:144–149, 2014.
Keywords: Lycaon pictus; environmental enrichment; fecal glucocorticoids
INTRODUCTION Environmental enrichment can enhance welfare of captive animals through provisioning of stimuli needed for optimal physiological and psychological well‐being [Shepherdson, 1998] and has gained popularity in the last few decades [Mellen and MacPhee, 2001; Young, 2003; Swaisgood and Shepherdson, 2005]. Although it cannot be proven that certain behaviors result from stress [Carlstead, 1996], abnormal behavior has been associated with elevated cortisol levels [Mason, 1991; Carlstead, 1998]. Multiple studies, primarily with carnivores, have supported the role of enrichment in increasing both activity and species‐ appropriate behaviors in zoo‐housed species [Forthman et al., 1992; Carlstead et al., 1993a,b; Shepherdson et al., 1993; Shepherdson et al., 1998; Wielebnowski et al., 2002a,b; Bashaw et al., 2003]. The African wild dog (AWD; Lycaon pictus) is the most endangered carnivore in Africa [Woodroffe et al., 2007; IUCN, 2013] due to human persecution for livestock predation, habitat fragmentation, and competition with other
© 2013 Wiley Periodicals, Inc.
large carnivores like lions [Panthera leo; Creel and Creel, 1998; Buettner et al., 2007]. The AWD has complex social dominance hierarchies for each sex within the pack [Creel and Creel, 2002]. Although the sense of smell is highly developed and critical for hunting in this species [Estes and Wilson, 1991], only one study [Price, 2010] has examined the effect of olfactory enrichment on activity levels in AWDs. Other studies have shown that novel and biologically meaningless scents like essential oils are beneficial to the welfare of certain species [Wells, 2009]. However, no studies Correspondence to: Michelle L. Rafacz, Columbia College Chicago, 623 S. Wabash Ave., 500‐E, Chicago, IL 60605. E‐mail: [email protected]
Received 24 July 2012; Revised 29 August 2013; Accepted 26 November 2013
DOI: 10.1002/zoo.21107 Published online 23 December 2013 in Wiley Online Library (wileyonlinelibrary.com).
Odor Cues as Enrichment in African Wild Dogs 145
have specifically investigated the effect of biologically relevant fecal odor cues as enrichment on both behavior and stress physiology in this species. In this study, the fecal odor cues used came from animals living in the same area of the zoo as the AWDs, and AWDs were provided with a weekly enrichment schedule that included novel olfactory stimulation. In this study, our objectives were to: (1) examine changes in activity levels and stress hormone physiology in response to fecal odor cues from natural competitor and natural/unnatural prey species; and (2) determine whether these odor cues could function as effective enrichment for zoo‐housed AWDs.
Tampa, FL) was used to identify individual samples. Fecal glucocorticoid metabolite (FGM) concentrations were extracted and assayed using a corticosterone enzyme immunoassay (EIA; polyclonal antiserum CJM006) as previously reported [Santymire and Armstrong, 2009]. To validate the stress analysis, an adrenocorticotrophic (ACTH)‐challenge was conducted on AWD 1 [Monfort et al., 1998; Santymire et al., 2012]. Briefly, fecal samples were collected in the morning for 7 days prior to injection, followed by an intramuscular injection of ACTH (10 IU/kg; Monument Pharmaceuticals, Monument, CO, USA). Then, every subsequent fecal sample was collected for 7 days post‐injection. Data Analysis
METHODS Subjects Two zoo‐born male AWDs (12 year old brothers; AWD 1: dominant male, AWD 2: subordinate male), distinguished by distinctive markings, were housed together at Lincoln Park Zoo (LPZ; Chicago, IL) in an exhibit that consisted of an irregularly shaped outdoor space (approximately 225 m2) with several naturalistic structures. The AWDs were on display from approximately 10:00 until 17:00 hr and had access to an off‐exhibit indoor holding area. Males were fed a meat‐based diet with water provided ad libitum. Research was approved through the LPZ Research Committee and adhered to all legal requirements for research use in the United States. Odor Cues and Behavioral Measurements Behavioral data were collected 1.5 months prior to the experimental portion of the study using instantaneous scan‐ sampling methods with a 1‐min fixed interval [Altmann, 1974] conducted by co‐author (MR) three times per week. Observation sessions consisted of one continuous 60‐min session (11:00–12:00 hr) during cue presentation, followed by a 30‐min follow‐up session (14:00–14:30 hr), once the odor cue was removed [Freeman, 2005]. An ethogram modified from Price [2010] for African wild dogs and Maisch [2005] for Dholes was used (Table 1). Fecal samples were collected from the LPZ’s lions, cattle (Bos taurus), and Grant’s gazelle (Gazella granti) to serve as odor cues for the AWDs. The LION cue served as the “competitor” cue, whereas the CATTLE and GAZELLE served as “unnatural” and “natural” prey cues, respectively. Each odor cue was presented to the AWDs over three consecutive days behind a chain‐linked fence where they could smell but not access the cue. The order of cue presentation was randomized, resulting in the LION cue being presented first, followed by CATTLE and then GAZELLE. Behavioral and hormonal data were also collected for 1.5 months following presentation of the last odor cue. Fecal Sample Collection and Analysis Fecal samples were collected daily and stored at 20°C until processing. A fecal marker (Gordon Food Service,
Behavior categories were collapsed into “active,” “inactive,” or “social,” and the mean percentage of time spent in each category was calculated for the AWDs [Price, 2010] (Table 1). Paired t‐tests were used to compare overall mean (SEM) FGMs for each AWD before and after all odor cues were presented. For all odor cues, mean percentage of time spent “active” and FGMs were compared among “Pre‐Cue” (1 week prior to cue), “During‐Cue,” and “Post‐Cue” (1 week after cue) using Repeated Measures ANOVA and Holm–Sidak post‐hoc multiple comparisons test. One way ANOVA was used to compare overall mean activity and FGMs for both AWDs before versus after all odor cues were presented. The total number of occurrences of affiliative and submissive behaviors for both AWDs was also calculated and compared for each cue using RM ANOVA and Holm–Sidak post‐hoc tests. Sigma Stat (Systat Software Inc., Point Richmond, CA) was used for all statistical tests, and P < 0.05 was considered significant. For the ACTH‐ challenge, all pre‐ACTH FGMs were averaged and compared to values post‐ACTH injection to determine its effects on adrenocortical activity [Santymire et al., 2012].
RESULTS The ACTH‐challenge, which was used for biological validation of the stress response, resulted in an increase of FGMs around 48 hr post‐ACTH injection, which resulted in a159% increase (90.1 3.1 ng/g) over the pre‐ACTH injection mean (56.7 1.7 ng/g). However, after falling back to 79.2 1.9 ng/g, FGM (174.3 4.6 ng/g) peaked once again 5 days post‐injection with a 307% increase. All hormonal comparisons were adjusted to reflect the lag time of approximately 48 hr. Mean activity level did not vary (P > 0.05) between prior to and after all cues for AWDs. Both AWDs were more (P < 0.05) active during GAZELLE cue than before the cue (Table 2). Activity levels were similar (P > 0.05) for LION or CATTLE cues (Table 2). Both AWDs displayed more (P < 0.05) affiliative behavior to each other during GAZELLE cue compared to other cues (Fig. 1). Additionally, AWD 2 displayed submissive behavior towards AWD 1 two Zoo Biology
146 Rafacz and Santymire TABLE 1. Types of behaviors recorded in the study and their descriptions Active versus in‐active category State behaviors Rest‐lying down Rest‐standing Rest‐sitting
Inactive Inactive Active
Locomote Pace Forage Orient‐public Out of view Other Event behaviors Self‐groom Stalk Dig Elimination Scent‐mark Inspect‐feces Affiliation‐sniff/greet Affiliation‐play‐invitation Affiliation‐play‐chase Affiliation‐wrestle Affiliation‐pulse‐whine Submission Submission‐whine Submission‐beg Regurgitate/share food Aggression‐lunge Aggression‐chase Aggression‐pace Aggression‐vocalize Aggression‐bite
Active Active Active Inactive or active Inactive Inactive or active Active Active Active Active Active Active Social Social Social Social Social Social Social Social Social Social Social Social Social Social
Description Body in contact with the ground, no movement (lying down or sitting) Quadrepedal upright stance Back part of body in contact with the ground, usually occurs when animal is scratching itself Forward locomotion (walk, trot, or run) Walking back and forth over the same, small area Ingesting solid food or water Engaging, reacting to public (aggressive and non‐aggressive actions) Animal not visible Any behavior not otherwise categorized Licking, scratching own body, rolling and squirming on back Ears erect, forward, body tense, and moving slowly with attention focused forward Scratching ground with one or both front paws to make a depression Urinating or defecating Rubbing genitals or neck on object or urinating in same spot as another dog Sniffing or tasting feces as another dog defecates, usually accompanied by flehmen Sniffing or licking another dog, including anogenital region to greet Stamping or bowing on forelegs with ears up, facing other dog Chasing another dog, usually with ears forward and not piloerect Standing on hind legs, front legs on other dog, usually silent and with open mouth Whining softly, rapidly in presence of other dogs Lowered head, nose to ground, ears back, tail wagging, rolling on to side, back Long, high‐pitched whining, dog crouching sideways to other dog Whining, cringing, tail wagging, falling over to expose underside, for food Regurgitating food to share with other dogs Advancing towards another dog, piloerect, ears back Chasing another dog, usually with ears back and piloerect Walking parallel to other dogs with stiff foreleg pars, head down, piloerect Growling or barking with short, loud, hoarse vocalization Snapping jaws shut on legs, belly, or anus to halt other dog
Asterisks indicate behaviors that were recorded as either “active” or “inactive” (depending on context) or neither category.
and five times during LION and GAZELLE cues, respectively, with no submissive behavior occurrences during CATTLE cue. AWD 1 did not show any submissive behaviors toward AWD 2; however, AWD 1 displayed dominant behavior towards AWD 2 once, twice, and five times during LION, CATTLE, and GAZELLE cues, respectively. AWD 1 had lower (P < 0.05) overall FGMs (67.0 2.6 ng/g) than AWD 2 (94.4 2.8 ng/g), and AWD 1 demonstrated lower (P < 0.05) FGMs than AWD 2 both before and after all odor cues. Although not significant, there was a tendency (P ¼ 0.08) for the FGMs to decrease in AWD
2 after all cues (Fig. 2). AWD 1 demonstrated lower (P < 0.05) FGMs after versus before the LION odor cue, whereas AWD 2 demonstrated higher (P < 0.05) FGMs after GAZELLE cue than before or during the cue (Table 3). DISCUSSION This study is the first to use feces from competitor and prey species as odor cues to elicit both behavioral and hormonal responses in the AWD. Only one other study [Price, 2010] used the effect of scent (animal blood trail) to
TABLE 2. Mean (SEM) percentage of time spent “active” for AWD 1 (dominant) and 2 (subordinate) before (Pre‐Cue), during (During‐Cue), and after (Post‐Cue) presentation of each odor cue (LION, CATTLE, GAZELLE) AWD 1 Odor cue Lion Cattle Gazelle
AWD 2
Pre‐Cue
During‐Cue
Post‐Cue
Pre‐Cue
During‐Cue
Post‐Cue
4.4 0.0 1.1 0.0 1.5 0.4a
2.2 0.6 5.3 2.0 13.4 3.6b
3.3 1.7 6.1 3.9 1.3 0.5a
5.6 1.1 2.2 0.1 0.8 0.5a
4.4 1.3 3.0 1.3 10.1 2.7b
4.8 3.2 3.9 2.8 0.9 0.4a
Different superscripted letters represent differences (P < 0.05) in mean activity levels within each individual. Zoo Biology
Odor Cues as Enrichment in African Wild Dogs 147
status as natural prey for the AWDs. If the effect was simply due to novelty of scents, one might expect all fecal odor cues to elicit the same increase in overall activity. Additionally, the GAZELLE cue increased occurrences of affiliative, submissive, and dominant behavior consistent with species‐specific natural behaviors [Creel and Creel, 2002]. Previous studies have demonstrated a similar effect of feeding enrichment on activity in other canids (maned wolves, Chrsocyon brachyurus [Cummings et al., 2007], bush dogs, Speothos venaticus [Ings et al., 1997], and felids [Skibiel et al., 2007]). An ACTH‐challenge was used to validate the use of FGM analysis in AWDs. We observed a 1.6‐ to 3‐fold increase in FGMs after 48 hr post‐ACTH. A previous study reported a 10‐ to 30‐fold‐increase 30 hr post‐ACTH [Monfort et al., 1998]. Differences may be a result of the age of our AWD (12 years old), since age can reduce adrenal function [Touma and Palme, 2005], differences in diet (i.e., fiber content) [Wasser et al., 1993], differences in extraction method and analysis, or amount of ACTH administered (i.e., our 350 IU vs. 400 IU) [Monfort et al., 1998]. Although the 307% increase in FGMs 5 days after the ACTH‐challenged also may have resulted from age or differences in digestion [Millspaugh and Washburn, 2004], the procedure would need to be repeated for a more precise explanation. Although activity was not affected during the LION cue, a decrease in FGMs for AWD 1 was observed, suggesting that this cue reduced the hormonal stress response for the dominant individual. Along with increases in activity for both AWDs during the GAZELLE cue, AWD 2 demonstrated higher FGMs, followed by a return to Pre‐ Cue concentrations. AWD 1, on the other hand, demonstrated a decrease in FGMs (though not significant). Even though the natural prey cue may have increased activity in both AWDs, these findings suggest that behavior does not necessarily cause a hormonal response. The relationship between hormones and behavior, however, may become more influential once social rank is considered. For AWD 1, perhaps a decrease in FGMs is associated with knowledge that prey is close by and food will not be difficult to obtain. In contrast, for AWD 2 an increase in FGMs may be related to subordinate status. Lower social rank could mean having to work harder for a smaller share of prey or dealing with aggression from the more dominant individuals, which could contribute to a greater hormonal stress response [Creel, 2001].
Fig. 1. Mean (SEM) number of occurrences of affiliative behavior observed for AWD 1 and 2 during presentation of each odor cue. Asterisks represent differences (P < 0.05) in affiliative behavior between the three odor cues for both African wild dogs (AWD).
Fig. 2. Overall mean (SEM) fecal glucocorticoid metabolite (FGM) concentrations (ng/g wet feces) for AWD 1 (dominant) and 2 (subordinate) before versus after all odor cues were presented. Asterisks represent differences (P < 0.05) between dogs with timeframe.
increase activity levels in AWD. While other studies have demonstrated the effectiveness of biologically irrelevant scents as enrichment, no studies have specifically investigated biologically meaningful scents like fecal odor cues as enrichment in AWDs. While the GAZELLE cue elicited a 10.6% increase in activity in both individuals, neither the LION nor the CATTLE cue elicited increased activity, suggesting that such a finding may be related to the gazelle’s
TABLE 3. Mean (SEM) fecal glucocorticoid metabolite (FGM) concentrations (ng/g wet feces) for AWD 1 (dominant) and 2 (subordinate) before (Pre‐Cue), during, and after (Post‐Cue) presentation of each odor cue (Lion, Cattle, Gazelle) AWD 1 Odor cue Lion Cattle Gazelle
Pre‐Cue
AWD 2
During‐Cue
84.1 8.2 57.0 24.5 94.8 46.7 a
45.4 10.8 69.0 9.3 44.7 13.3
ab
Post‐Cue 39.3 5.5 67.7 8.7 52.0 17.3 b
Pre‐Cue 109.5 11.9 101.6 10.0 68.5 11.4
a
During‐Cue
Post‐Cue
111.7 14.9 125.8 15.3 108.2 13.7b
129.1 14.8 98.6 15.6 44.3 10.3a
Different superscripted letters represent differences (P < 0.05) in mean FGM concentrations within each individual. Zoo Biology
148 Rafacz and Santymire Following presentation of all odor cues at the conclusion of the study, there was a trend for declining FGMs in AWD 2 but not in AWD 1. This suggests that the use of fecal odor cues as enrichment may be most beneficial for lower ranking individuals of a pack species like the AWD. The stress hormone response may be dependent on social rank, which was also evidenced by the competitor and prey odor cues that evoked contrasting hormonal stress responses between the two AWDs. It is also important to note that social stress present in species with social dominance hierarchies may have had an effect on the hormonal stress response. Therefore, further investigation is warranted. Overall, AWD 1 had lower FGMs than AWD 2. This finding contrasts with evidence from multiple taxa suggesting that dominant individuals tend to have higher glucocorticoid concentrations than subordinate individuals [Creel, 2001]; however most of these are studies with wild populations. One must also take into account that the presence of receptive females or otherwise unstable situations may also affect FGM concentrations, as has been shown in primates [Setchell et al., 2010]. In this study, however, the AWDs were in a stable social situation for at least a decade, and no females were present whatsoever. Although results of the present study are intriguing and may indeed present an effective use of fecal odor cues as environmental enrichment for AWDs, they are also preliminary and warrant further investigation. CONCLUSIONS 1. Overall, this study demonstrated that competitor and prey
fecal odor cues may be employed as effective environmental enrichment tools that can increase activity levels and potentially reduce stress hormone concentrations in AWDs and possibly other canids and carnivores, more generally. 2. Natural prey odor cues increased species‐appropriate social behavior in the AWDs, including affiliatve and submissive behaviors. 3. Dominance rank and social stress may have an effect of hormonal stress response to odor cues in this species. 4. Future studies should focus on larger groups of AWDs, other canids, or other carnivores more generally, to further investigate the effect of such odor cues on behavioral and hormonal responses.
ACKNOWLEDGEMENTS The authors thank the LPZ, especially Jill Gossett and the Regenstein African Journey Animal Care staff. We also thank Diana Armstrong, Steve Thompson, and the LPZ veterinary staff. REFERENCES Altmann J. 1974. Observational study of behavior: sampling methods. Behaviour 49:227–266.
Zoo Biology
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Zoo Biology
BRIEF REPORT
Using Odor Cues to Elicit a Behavioral and Hormonal Response in Zoo‐Housed African Wild Dogs Michelle L. Rafacz,1,2* and Rachel M. Santymire2 1 2
Department of Science and Mathematics, Columbia College, Chicago, Illinois Davee Center for Epidemiology and Endocrinology, Lincoln Park Zoo, Chicago, Illinois Olfactory enrichment, like odor cues, can positively affect behavior, reproductive success, and stress physiology in zoo‐housed species. Our goal was to determine if odor cues were enriching to the African wild dog (AWD; Lycaon pictus), a species with a complex social structure and a highly developed sense of smell. Our objectives were to: (1) examine changes in activity levels and stress hormone physiology in response to fecal odor cues from natural competitor and natural/unnatural prey species; and (2) determine whether these odor cues could function as effective enrichment for zoo‐housed AWDs. Over a 6‐month period, fecal samples were collected from two males (AWD 1: dominant, AWD 2: subordinate), fecal glucocorticoid metabolites (FGMs) were validated using an ACTH‐challenge, and hormones were analyzed for FGMs by enzyme immunoassay. Behavioral observations were conducted using scan‐sampling, and contact and proximity were recorded. AWDs were presented with three fecal odor cues: LION (competitor), CATTLE (unnatural prey), and GAZELLE (natural prey). Only the GAZELLE cue elicited an increase in activity (10.6%) in both individuals and increased positive social behaviors with higher frequencies of affiliative, submissive, and dominant behavior. AWD 1 demonstrated lower (P < 0.05) FGMs than AWD 2 both before and after all odor cues, and FGMs decreased (P ¼ 0.08) in AWD 2 after all cues. We conclude that exposure to natural prey odor cues may be used as effective enrichment for AWDs, and that changes in stress hormone physiology in response to © 2013 Wiley Periodicals, Inc. odor cues may be dependent on social rank in this species. Zoo Biol. 33:144–149, 2014.
Keywords: Lycaon pictus; environmental enrichment; fecal glucocorticoids
INTRODUCTION Environmental enrichment can enhance welfare of captive animals through provisioning of stimuli needed for optimal physiological and psychological well‐being [Shepherdson, 1998] and has gained popularity in the last few decades [Mellen and MacPhee, 2001; Young, 2003; Swaisgood and Shepherdson, 2005]. Although it cannot be proven that certain behaviors result from stress [Carlstead, 1996], abnormal behavior has been associated with elevated cortisol levels [Mason, 1991; Carlstead, 1998]. Multiple studies, primarily with carnivores, have supported the role of enrichment in increasing both activity and species‐ appropriate behaviors in zoo‐housed species [Forthman et al., 1992; Carlstead et al., 1993a,b; Shepherdson et al., 1993; Shepherdson et al., 1998; Wielebnowski et al., 2002a,b; Bashaw et al., 2003]. The African wild dog (AWD; Lycaon pictus) is the most endangered carnivore in Africa [Woodroffe et al., 2007; IUCN, 2013] due to human persecution for livestock predation, habitat fragmentation, and competition with other
© 2013 Wiley Periodicals, Inc.
large carnivores like lions [Panthera leo; Creel and Creel, 1998; Buettner et al., 2007]. The AWD has complex social dominance hierarchies for each sex within the pack [Creel and Creel, 2002]. Although the sense of smell is highly developed and critical for hunting in this species [Estes and Wilson, 1991], only one study [Price, 2010] has examined the effect of olfactory enrichment on activity levels in AWDs. Other studies have shown that novel and biologically meaningless scents like essential oils are beneficial to the welfare of certain species [Wells, 2009]. However, no studies Correspondence to: Michelle L. Rafacz, Columbia College Chicago, 623 S. Wabash Ave., 500‐E, Chicago, IL 60605. E‐mail: [email protected]
Received 24 July 2012; Revised 29 August 2013; Accepted 26 November 2013
DOI: 10.1002/zoo.21107 Published online 23 December 2013 in Wiley Online Library (wileyonlinelibrary.com).
Odor Cues as Enrichment in African Wild Dogs 145
have specifically investigated the effect of biologically relevant fecal odor cues as enrichment on both behavior and stress physiology in this species. In this study, the fecal odor cues used came from animals living in the same area of the zoo as the AWDs, and AWDs were provided with a weekly enrichment schedule that included novel olfactory stimulation. In this study, our objectives were to: (1) examine changes in activity levels and stress hormone physiology in response to fecal odor cues from natural competitor and natural/unnatural prey species; and (2) determine whether these odor cues could function as effective enrichment for zoo‐housed AWDs.
Tampa, FL) was used to identify individual samples. Fecal glucocorticoid metabolite (FGM) concentrations were extracted and assayed using a corticosterone enzyme immunoassay (EIA; polyclonal antiserum CJM006) as previously reported [Santymire and Armstrong, 2009]. To validate the stress analysis, an adrenocorticotrophic (ACTH)‐challenge was conducted on AWD 1 [Monfort et al., 1998; Santymire et al., 2012]. Briefly, fecal samples were collected in the morning for 7 days prior to injection, followed by an intramuscular injection of ACTH (10 IU/kg; Monument Pharmaceuticals, Monument, CO, USA). Then, every subsequent fecal sample was collected for 7 days post‐injection. Data Analysis
METHODS Subjects Two zoo‐born male AWDs (12 year old brothers; AWD 1: dominant male, AWD 2: subordinate male), distinguished by distinctive markings, were housed together at Lincoln Park Zoo (LPZ; Chicago, IL) in an exhibit that consisted of an irregularly shaped outdoor space (approximately 225 m2) with several naturalistic structures. The AWDs were on display from approximately 10:00 until 17:00 hr and had access to an off‐exhibit indoor holding area. Males were fed a meat‐based diet with water provided ad libitum. Research was approved through the LPZ Research Committee and adhered to all legal requirements for research use in the United States. Odor Cues and Behavioral Measurements Behavioral data were collected 1.5 months prior to the experimental portion of the study using instantaneous scan‐ sampling methods with a 1‐min fixed interval [Altmann, 1974] conducted by co‐author (MR) three times per week. Observation sessions consisted of one continuous 60‐min session (11:00–12:00 hr) during cue presentation, followed by a 30‐min follow‐up session (14:00–14:30 hr), once the odor cue was removed [Freeman, 2005]. An ethogram modified from Price [2010] for African wild dogs and Maisch [2005] for Dholes was used (Table 1). Fecal samples were collected from the LPZ’s lions, cattle (Bos taurus), and Grant’s gazelle (Gazella granti) to serve as odor cues for the AWDs. The LION cue served as the “competitor” cue, whereas the CATTLE and GAZELLE served as “unnatural” and “natural” prey cues, respectively. Each odor cue was presented to the AWDs over three consecutive days behind a chain‐linked fence where they could smell but not access the cue. The order of cue presentation was randomized, resulting in the LION cue being presented first, followed by CATTLE and then GAZELLE. Behavioral and hormonal data were also collected for 1.5 months following presentation of the last odor cue. Fecal Sample Collection and Analysis Fecal samples were collected daily and stored at 20°C until processing. A fecal marker (Gordon Food Service,
Behavior categories were collapsed into “active,” “inactive,” or “social,” and the mean percentage of time spent in each category was calculated for the AWDs [Price, 2010] (Table 1). Paired t‐tests were used to compare overall mean (SEM) FGMs for each AWD before and after all odor cues were presented. For all odor cues, mean percentage of time spent “active” and FGMs were compared among “Pre‐Cue” (1 week prior to cue), “During‐Cue,” and “Post‐Cue” (1 week after cue) using Repeated Measures ANOVA and Holm–Sidak post‐hoc multiple comparisons test. One way ANOVA was used to compare overall mean activity and FGMs for both AWDs before versus after all odor cues were presented. The total number of occurrences of affiliative and submissive behaviors for both AWDs was also calculated and compared for each cue using RM ANOVA and Holm–Sidak post‐hoc tests. Sigma Stat (Systat Software Inc., Point Richmond, CA) was used for all statistical tests, and P < 0.05 was considered significant. For the ACTH‐ challenge, all pre‐ACTH FGMs were averaged and compared to values post‐ACTH injection to determine its effects on adrenocortical activity [Santymire et al., 2012].
RESULTS The ACTH‐challenge, which was used for biological validation of the stress response, resulted in an increase of FGMs around 48 hr post‐ACTH injection, which resulted in a159% increase (90.1 3.1 ng/g) over the pre‐ACTH injection mean (56.7 1.7 ng/g). However, after falling back to 79.2 1.9 ng/g, FGM (174.3 4.6 ng/g) peaked once again 5 days post‐injection with a 307% increase. All hormonal comparisons were adjusted to reflect the lag time of approximately 48 hr. Mean activity level did not vary (P > 0.05) between prior to and after all cues for AWDs. Both AWDs were more (P < 0.05) active during GAZELLE cue than before the cue (Table 2). Activity levels were similar (P > 0.05) for LION or CATTLE cues (Table 2). Both AWDs displayed more (P < 0.05) affiliative behavior to each other during GAZELLE cue compared to other cues (Fig. 1). Additionally, AWD 2 displayed submissive behavior towards AWD 1 two Zoo Biology
146 Rafacz and Santymire TABLE 1. Types of behaviors recorded in the study and their descriptions Active versus in‐active category State behaviors Rest‐lying down Rest‐standing Rest‐sitting
Inactive Inactive Active
Locomote Pace Forage Orient‐public Out of view Other Event behaviors Self‐groom Stalk Dig Elimination Scent‐mark Inspect‐feces Affiliation‐sniff/greet Affiliation‐play‐invitation Affiliation‐play‐chase Affiliation‐wrestle Affiliation‐pulse‐whine Submission Submission‐whine Submission‐beg Regurgitate/share food Aggression‐lunge Aggression‐chase Aggression‐pace Aggression‐vocalize Aggression‐bite
Active Active Active Inactive or active Inactive Inactive or active Active Active Active Active Active Active Social Social Social Social Social Social Social Social Social Social Social Social Social Social
Description Body in contact with the ground, no movement (lying down or sitting) Quadrepedal upright stance Back part of body in contact with the ground, usually occurs when animal is scratching itself Forward locomotion (walk, trot, or run) Walking back and forth over the same, small area Ingesting solid food or water Engaging, reacting to public (aggressive and non‐aggressive actions) Animal not visible Any behavior not otherwise categorized Licking, scratching own body, rolling and squirming on back Ears erect, forward, body tense, and moving slowly with attention focused forward Scratching ground with one or both front paws to make a depression Urinating or defecating Rubbing genitals or neck on object or urinating in same spot as another dog Sniffing or tasting feces as another dog defecates, usually accompanied by flehmen Sniffing or licking another dog, including anogenital region to greet Stamping or bowing on forelegs with ears up, facing other dog Chasing another dog, usually with ears forward and not piloerect Standing on hind legs, front legs on other dog, usually silent and with open mouth Whining softly, rapidly in presence of other dogs Lowered head, nose to ground, ears back, tail wagging, rolling on to side, back Long, high‐pitched whining, dog crouching sideways to other dog Whining, cringing, tail wagging, falling over to expose underside, for food Regurgitating food to share with other dogs Advancing towards another dog, piloerect, ears back Chasing another dog, usually with ears back and piloerect Walking parallel to other dogs with stiff foreleg pars, head down, piloerect Growling or barking with short, loud, hoarse vocalization Snapping jaws shut on legs, belly, or anus to halt other dog
Asterisks indicate behaviors that were recorded as either “active” or “inactive” (depending on context) or neither category.
and five times during LION and GAZELLE cues, respectively, with no submissive behavior occurrences during CATTLE cue. AWD 1 did not show any submissive behaviors toward AWD 2; however, AWD 1 displayed dominant behavior towards AWD 2 once, twice, and five times during LION, CATTLE, and GAZELLE cues, respectively. AWD 1 had lower (P < 0.05) overall FGMs (67.0 2.6 ng/g) than AWD 2 (94.4 2.8 ng/g), and AWD 1 demonstrated lower (P < 0.05) FGMs than AWD 2 both before and after all odor cues. Although not significant, there was a tendency (P ¼ 0.08) for the FGMs to decrease in AWD
2 after all cues (Fig. 2). AWD 1 demonstrated lower (P < 0.05) FGMs after versus before the LION odor cue, whereas AWD 2 demonstrated higher (P < 0.05) FGMs after GAZELLE cue than before or during the cue (Table 3). DISCUSSION This study is the first to use feces from competitor and prey species as odor cues to elicit both behavioral and hormonal responses in the AWD. Only one other study [Price, 2010] used the effect of scent (animal blood trail) to
TABLE 2. Mean (SEM) percentage of time spent “active” for AWD 1 (dominant) and 2 (subordinate) before (Pre‐Cue), during (During‐Cue), and after (Post‐Cue) presentation of each odor cue (LION, CATTLE, GAZELLE) AWD 1 Odor cue Lion Cattle Gazelle
AWD 2
Pre‐Cue
During‐Cue
Post‐Cue
Pre‐Cue
During‐Cue
Post‐Cue
4.4 0.0 1.1 0.0 1.5 0.4a
2.2 0.6 5.3 2.0 13.4 3.6b
3.3 1.7 6.1 3.9 1.3 0.5a
5.6 1.1 2.2 0.1 0.8 0.5a
4.4 1.3 3.0 1.3 10.1 2.7b
4.8 3.2 3.9 2.8 0.9 0.4a
Different superscripted letters represent differences (P < 0.05) in mean activity levels within each individual. Zoo Biology
Odor Cues as Enrichment in African Wild Dogs 147
status as natural prey for the AWDs. If the effect was simply due to novelty of scents, one might expect all fecal odor cues to elicit the same increase in overall activity. Additionally, the GAZELLE cue increased occurrences of affiliative, submissive, and dominant behavior consistent with species‐specific natural behaviors [Creel and Creel, 2002]. Previous studies have demonstrated a similar effect of feeding enrichment on activity in other canids (maned wolves, Chrsocyon brachyurus [Cummings et al., 2007], bush dogs, Speothos venaticus [Ings et al., 1997], and felids [Skibiel et al., 2007]). An ACTH‐challenge was used to validate the use of FGM analysis in AWDs. We observed a 1.6‐ to 3‐fold increase in FGMs after 48 hr post‐ACTH. A previous study reported a 10‐ to 30‐fold‐increase 30 hr post‐ACTH [Monfort et al., 1998]. Differences may be a result of the age of our AWD (12 years old), since age can reduce adrenal function [Touma and Palme, 2005], differences in diet (i.e., fiber content) [Wasser et al., 1993], differences in extraction method and analysis, or amount of ACTH administered (i.e., our 350 IU vs. 400 IU) [Monfort et al., 1998]. Although the 307% increase in FGMs 5 days after the ACTH‐challenged also may have resulted from age or differences in digestion [Millspaugh and Washburn, 2004], the procedure would need to be repeated for a more precise explanation. Although activity was not affected during the LION cue, a decrease in FGMs for AWD 1 was observed, suggesting that this cue reduced the hormonal stress response for the dominant individual. Along with increases in activity for both AWDs during the GAZELLE cue, AWD 2 demonstrated higher FGMs, followed by a return to Pre‐ Cue concentrations. AWD 1, on the other hand, demonstrated a decrease in FGMs (though not significant). Even though the natural prey cue may have increased activity in both AWDs, these findings suggest that behavior does not necessarily cause a hormonal response. The relationship between hormones and behavior, however, may become more influential once social rank is considered. For AWD 1, perhaps a decrease in FGMs is associated with knowledge that prey is close by and food will not be difficult to obtain. In contrast, for AWD 2 an increase in FGMs may be related to subordinate status. Lower social rank could mean having to work harder for a smaller share of prey or dealing with aggression from the more dominant individuals, which could contribute to a greater hormonal stress response [Creel, 2001].
Fig. 1. Mean (SEM) number of occurrences of affiliative behavior observed for AWD 1 and 2 during presentation of each odor cue. Asterisks represent differences (P < 0.05) in affiliative behavior between the three odor cues for both African wild dogs (AWD).
Fig. 2. Overall mean (SEM) fecal glucocorticoid metabolite (FGM) concentrations (ng/g wet feces) for AWD 1 (dominant) and 2 (subordinate) before versus after all odor cues were presented. Asterisks represent differences (P < 0.05) between dogs with timeframe.
increase activity levels in AWD. While other studies have demonstrated the effectiveness of biologically irrelevant scents as enrichment, no studies have specifically investigated biologically meaningful scents like fecal odor cues as enrichment in AWDs. While the GAZELLE cue elicited a 10.6% increase in activity in both individuals, neither the LION nor the CATTLE cue elicited increased activity, suggesting that such a finding may be related to the gazelle’s
TABLE 3. Mean (SEM) fecal glucocorticoid metabolite (FGM) concentrations (ng/g wet feces) for AWD 1 (dominant) and 2 (subordinate) before (Pre‐Cue), during, and after (Post‐Cue) presentation of each odor cue (Lion, Cattle, Gazelle) AWD 1 Odor cue Lion Cattle Gazelle
Pre‐Cue
AWD 2
During‐Cue
84.1 8.2 57.0 24.5 94.8 46.7 a
45.4 10.8 69.0 9.3 44.7 13.3
ab
Post‐Cue 39.3 5.5 67.7 8.7 52.0 17.3 b
Pre‐Cue 109.5 11.9 101.6 10.0 68.5 11.4
a
During‐Cue
Post‐Cue
111.7 14.9 125.8 15.3 108.2 13.7b
129.1 14.8 98.6 15.6 44.3 10.3a
Different superscripted letters represent differences (P < 0.05) in mean FGM concentrations within each individual. Zoo Biology
148 Rafacz and Santymire Following presentation of all odor cues at the conclusion of the study, there was a trend for declining FGMs in AWD 2 but not in AWD 1. This suggests that the use of fecal odor cues as enrichment may be most beneficial for lower ranking individuals of a pack species like the AWD. The stress hormone response may be dependent on social rank, which was also evidenced by the competitor and prey odor cues that evoked contrasting hormonal stress responses between the two AWDs. It is also important to note that social stress present in species with social dominance hierarchies may have had an effect on the hormonal stress response. Therefore, further investigation is warranted. Overall, AWD 1 had lower FGMs than AWD 2. This finding contrasts with evidence from multiple taxa suggesting that dominant individuals tend to have higher glucocorticoid concentrations than subordinate individuals [Creel, 2001]; however most of these are studies with wild populations. One must also take into account that the presence of receptive females or otherwise unstable situations may also affect FGM concentrations, as has been shown in primates [Setchell et al., 2010]. In this study, however, the AWDs were in a stable social situation for at least a decade, and no females were present whatsoever. Although results of the present study are intriguing and may indeed present an effective use of fecal odor cues as environmental enrichment for AWDs, they are also preliminary and warrant further investigation. CONCLUSIONS 1. Overall, this study demonstrated that competitor and prey
fecal odor cues may be employed as effective environmental enrichment tools that can increase activity levels and potentially reduce stress hormone concentrations in AWDs and possibly other canids and carnivores, more generally. 2. Natural prey odor cues increased species‐appropriate social behavior in the AWDs, including affiliatve and submissive behaviors. 3. Dominance rank and social stress may have an effect of hormonal stress response to odor cues in this species. 4. Future studies should focus on larger groups of AWDs, other canids, or other carnivores more generally, to further investigate the effect of such odor cues on behavioral and hormonal responses.
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