Can an Enrichment Programme with Novel Manipulative and Scent Stimuli Change the Behaviour of Zoo-Housed European Wildcats? A Case Study.

Objects and semiochemicals may be used as enrichment in zoos. Domestic cats release Fraction 3 of Facial Pheromone (F3) by rubbing the muzzle to convey relational and territorial information. We aimed to evaluate whether and how the introduction of novel objects and scent stimuli could change the behaviour of one group (N = 5 subjects) of adult European wildcats (Felis silvestris silvestris) hosted at Parco Natura Viva-Garda Zoological Park, Italy. We assessed the behavioural changes following the introduction of novel objects (blocks and rags) and scent (synthetic F3) via observations over four experimental conditions (baseline, rags, F3 rags, blocks) using continuous focal animal sampling. Our results showed that no behavioural differences were found between the different conditions and the baseline, except for the condition with blocks when significantly less exploration was observed. Between conditions, wildcats performed significantly less individual explorative, affiliative, and agonistic behaviours, but more individual inactivity, when exposed to rags after F3 administration. Our findings suggest that the enrichment programme did not substantively affect the behaviour of the zoo-housed wildcats. However, the behavioural differences recorded between conditions suggest that, while novel objects introduced as visual stimuli (blocks) do not affect the wildcat behaviour, novel manipulative objects (rags) might impact their behaviour. Moreover, the changes in affiliative and agonistic behaviours displayed during the condition with exposure to rags sprayed with F3 suggest that such semiochemical could play an appeasement role within this study group.

The Scent Enriched Primate.

Zoos worldwide play an important role in both in situ and ex situ conservation via efforts such as providing breeding programmes and reintroductions into the wild. Zoo populations are crucial as a buffer against extinction. However, a mismatch between the wild and zoo environments can lead to psychological as well as physiological health issues, such as stress, boredom, diabetes, and obesity. These problems, in turn, can impact the reproductive success of individuals. Consequently, some primate species have reduced breeding success when housed in zoos compared to their wild counterparts. To prevent the onset of behavioural, physiological, and cognitive negative effects and to continually improve the welfare of their animals, zoos widely implement different types of environmental enrichment. There are many forms enrichment can take, such as feeding, puzzles and training, but sensory enrichments, including implementing the use of scents, are currently understudied. Scent enrichments are less utilized despite multiple research studies showing that they may have positive effects on welfare for zoo-housed animal species, including non-human primates. Despite being traditionally considered to be microsmatic, various lines of evidence suggest that olfaction plays a larger role in primates than previously thought. This review therefore focuses on scent-based enrichment and the specifics of captive primates.

Primate Sex and Its Role in Pleasure, Dominance and Communication.

Sexual intercourse in the animal kingdom functions to enable reproduction. However, we now know that several species of non-human primates regularly engage in sex outside of the times when conception is possible. In addition, homosexual and immature sex are not as uncommon as were once believed. This suggests that sex also has important functions outside of reproduction, yet these are rarely discussed in sex-related teaching and research activities concerning primate behaviour. Is the human sexual experience, which includes pleasure, dominance, and communication (among others) unique, or do other primates also share these experiences to any extent? If so, is there any way to measure them, or are they beyond the rigour of scientific objectivity? What would be the evolutionary implications if human-like sexual experiences were found amongst other animals too? We comment on the evidence provided by our close relatives, non-human primates, discuss the affective and social functions of sex, and suggest potential methods for measuring some of these experiences empirically. We hope that this piece may foster the discussion among academics and change the way we think about, teach and research primate sex.

Zoo-Led Initiatives and Their Role in Lemur Conservation In Situ.

We examined wider society's ability to achieve biodiversity conservation and management targets using lemurs as a case study. We evaluated the impact on lemur conservation in situ by conservation initiatives led by European zoos in Madagascar exploring the European Association of Zoos and Aquaria's Conservation Database projects in terms of performed actions and achieved goals as well as communication to the public. We found that zoo-led conservation initiatives may have a positive impact on the ground and tend to achieve most conservation goals related to wild lemurs. However, we suggest that such conservation programmes should underline that they target further lemur species beyond the flagship charismatic species and that enhanced communication efforts would be beneficial to further raise public awareness.

Anogenital scent-marking signals fertility in a captive female Alaotran gentle lemur.

The Lake Alaotra gentle lemur (Hapalemur alaotrensis) is one of the 25 most endangered primates in the world and shows low success rate in captive breeding programmes. It is therefore vital to further understand its reproductive biology. We studied a captive troop consisting of five individuals hosted at Jersey Zoo during breeding and non-breeding periods over 1 year. We collected behavioural data (n = 318 h) using all occurrence of some behaviours and ad libitum sampling methods, as well as faecal (n = 54) and anogenital scent (n = 35) samples of the breeding female. We measured sex hormone levels using enzyme immunoassay technique and investigated the volatile component of odour signals using solid-phase microextraction and gas chromatography-mass spectrometry. We observed sexual and aggressive behaviours occasionally during the breeding period. Our regression analysis showed that only period significantly predicted rates of female anogenital scent-marking, whereby the female performed anogenital scent-marking more frequently during the breeding rather than the non-breeding period. In contrast, female hormone levels did not significantly explain variation in rates of neither male nor female olfactory, sexual and affiliative behaviours, suggesting that individuals' behaviour alone is not an effective indicator of the ovulation window. The volatile chemical profile of anogenital odour secretions changed over the study, with four compounds distinguishing the fertile window during the breeding period. In conclusion, our findings suggest that anogenital scent-marking may signal the reproductive status of captive female gentle lemurs.

The Impact of COVID-19 Zoo Closures on Behavioural and Physiological Parameters of Welfare in Primates.

Primates are some of the most cognitively advanced species held in zoos, and their interactions with visitors are complex. The COVID-19 pandemic provided a unique opportunity to understand the impact of zoo visitors on animals, in comparison to "empty zoos". This study sought to understand the impact of facility closures and subsequent reopenings on behavioural and physiological parameters of welfare in four primate species housed in the UK: bonobos (Pan paniscus) (n = 8), chimpanzees (Pan troglodytes) (n = 11), and western lowland gorillas (Gorilla gorilla gorilla) (n = 6) held at Twycross Zoo (TZ); and olive baboons (Papio anubis) (n = 192) held at Knowsley Safari (KS). Behavioural data were collected from April-September 2020 (KS) and November 2020-January 2021 (TZ). Faecal samples were collected during morning checks from October-November (TZ) and July-November 2020 (KS). Faecal glucocorticoid metabolites (FGMs) were measured using ELISA kits. Statistical analysis for behavioural observations was undertaken using general linear models. Enclosure usage was assessed using t-tests and Mann-Whitney U-tests as appropriate. Bonobos and gorillas spent less time alone when facilities were open to the public (p = 0.004, p = 0.02 respectively). Gorillas spent less time resting when the facility was open to the public (p = 0.04), and chimpanzees engaged in more feeding (p = 0.02) and engagement with enrichment (p = 0.03) when the zoo was open to the public than when it was closed. Olive baboons performed less sexual and dominance behaviour and approached visitor cars more frequently when the safari park was opened to the public than they did the ranger's vehicle during closure periods. There were no significant changes in physiological parameters for any of the study species. The results suggest variable impacts of the zoo closures on zoo-housed primates. We recommend future work that seeks to understand the impact of individual-level differences on "visitor effects" and that differences between animal experiences in zoos and safari parks are further explored in a range of species.

Chemical cues of identity and reproductive status in Japanese macaques.

Olfactory communication plays an important role in the regulation of socio-sexual interactions in mammals. There is growing evidence that both human and nonhuman primates rely on odors to inform their mating decisions. Nevertheless, studies of primate chemical ecology remain scarce due to the difficulty of obtaining and analyzing samples. We analyzed 67 urine samples from five captive female Japanese macaques (Macaca fuscata) and 30 vaginal swabs from three of these females using gas chromatography-mass spectrometry and examined the relationship between odor (compounds identified, richness, intensity, and diversity) and female identity as well as cycle phase. We found a total of 36 urine compounds of which we identified 31, and 68 vaginal compounds of which we identified 37. Our results suggest that urine and vaginal odor varied more between individuals than within cycle phases. However, we found that within a female cycle, urine samples from similar phases may cluster more than samples from different phases. Our results suggest that female odor may encode information about identity (vaginal and urine odor) and reproductive status (urine odor). The question of how conspecifics use female urine and vaginal odor remains open and could be tested using bioassays. Our results and their interpretation are constrained by our limited sample size and our study design. Nonetheless, our study provides insight into the potential signaling role of female odor in sexual communication in Japanese macaques and contributes to our understanding of how odors may influence mating strategies in primates.

Sexual Dimorphism in Crowned Lemur Scent-Marking.

Primates are traditionally considered to have a poor sense of smell. However, olfaction is important for non-human primates as demonstrated by conspicuous scent-marking behaviours in lemurs. We studied two pairs (n = 4) of crowned lemurs (Eulemur coronatus) housed at Colchester and Twycross zoos (UK) by combining behavioural observations and chemical analyses of scent-marks and glandular swabs. We recorded observations of olfactory behaviours for 201 h using instantaneous scan sampling. We investigated the volatile compounds of ano-genital odour secretions (n = 16) using solid-phase microextraction and gas chromatography-mass spectrometry. Males scent-marked most frequently, displaying ano-genital marking for allomarking, head marking for countermarking and wrist marking in specific areas of the enclosure. Females displayed ano-genital marking, predominantly on feeding devices. We detected a total of 38 volatile components in all male ano-genital scent-marks and 26 in all female samples of ano-genital odour secretions, including a series of esters, aldehydes, ketones, alcohols, terpenes, volatile fatty acids and hydrocarbons that have been identified in odour profiles of other primates. In conclusion, we found sexual dimorphism in crowned lemur scent-marking. Male head and wrist marking behaviours might play defensive territorial functions, while ano-genital marking would be related to socio-sexual communication as chemical mate-guarding. Female ano-genital marking might be involved in resource defense.

Olfactory signals and fertility in olive baboons.

Female primates signal impending ovulation with a suite of sexual signals. Studies of these signals have focussed on visual, and to a lesser extent, acoustic signals, neglecting olfactory signals. We aimed to investigate the information content of female olfactory signals in captive olive baboons (Papio anubis) and relate these to the female fertile period. We studied eight adult females living in four groups at the CNRS Station de Primatologie, Rousset-sur-Arc, France. We used vaginal cytology to detect ovulation. We investigated the volatile component of odour signals using solid-phase microextraction and gas chromatography-mass spectrometry. We found a total of 74 volatile compounds, of which we tentatively identified 25, including several ketones, alcohols, aldehydes, terpenes, volatile fatty acids and hydrocarbons that have been identified in odour profiles of other primates. Our results show that vaginal odour intensity differs with sexual cycle stage suggesting that odour might play a role in signalling female baboon fertility. We found differences in vaginal odour between females living in all-female and in mixed sex groups but we could not distinguish the effects of group composition, female age and identity. This study of olfactory signalling improves our understanding of how female primates advertise their sexual receptivity.

The effects of a zoo environment on free-living, native small mammal species.

One of the main threats to native species conservation is urbanisation. It is causing changes to natural habitats and species composition. Urban green spaces have shown to have conservation value for native species by providing safe spaces in urban areas. They typically contain a variety of habitats and plant species which is correlated with greater abundance and diversity of small mammal species. Zoos are a vital resource for animal conservation and, in some instances, could be considered as an urban green space for native species conservation. Their unique environment provides free-living, native species an abundance of resources including food and shelter. This project involved the live trapping of free-living small mammal species (<40 g) between enclosures in Dudley Zoological Gardens to study the effects of the zoo environment. There were no significant differences found between the total number of captures and trap proximity to enclosures. There was a significant difference in total captures found between different enclosure trapping areas. Generalized linear mixed models were fitted to the data and there were significant relationships between abundance and both habitat type and enclosure species. Habitats associated with semi-natural woodland had the greatest diversity and total captures of small mammals. Total captures were lower in trapping areas that were associated with predatory species. Similar to research on green spaces, habitat was an important factor determining abundance, but predator enclosures were a factor unique to zoos. This study illustrates the potential of zoos as an urban green space and for the study of small mammals.

Sampling and Analysis of Animal Scent Signals.

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

Effects of scent enrichment on behavioral and physiological indicators of stress in zoo primates.

Captive breeding is vital for primate conservation, with modern zoos serving a crucial role in breeding populations of threatened species and educating the general public. However, captive populations can experience welfare issues that may also undermine their reproductive success. To enhance the wellbeing of endangered zoo primates, we conducted a study to assess the effects of a new scent enrichment program on captive red-ruffed lemurs (Varecia rubra), black howler monkeys (Alouatta caraya), siamangs (Symphalangus syndactylus), lar gibbons (Hylobates lar) and orangutans (Pongo pygmaeus pygmaeus). We combined behavioral observations and fecal endocrinology analyses to evaluate the effects of a series of essential oils (benzoin, lavender, lemongrass) on five captive troops (N = 19) housed at Dudley Zoo & Castle and Twycross Zoo (UK). We recorded observations of natural species-specific and abnormal stress-related behaviors for 480 h using instantaneous scan sampling. We collected 189 fecal samples and measured the fecal cortisol concentrations using radioimmunoassay. We found a significant effect of the scent enrichment on behaviors, with red-ruffed lemurs and black howler monkeys reducing their social interactions, as well as red-ruffed lemurs and lar gibbons decreasing their stress-related behaviors after they were exposed to the series of essential oils. We also found that red-ruffed lemurs displayed a significant increase in fecal glucocorticoids following exposure to essential oils. Our contradictory findings suggest that the effects of this series of essential oils may change depending on the species-specific social lives and olfactory repertoires of primates. In conclusion, we cannot recommend using these essential oils widely with zoo primates without additional evaluation.

Effects of Rearing on the Behaviour of Zoo-Housed Chimpanzees (Pan troglodytes).

Early-life experiences may considerably affect the behavioural patterns of adult primates. Particularly, atypical rearing practices might lead to abnormal behaviours and social-sexual deficiencies in captive, adult non-human primates. We conducted behavioural observations of mother-reared (n = 5) and hand-reared (n = 6) adult chimpanzees in a social group at Parco Natura Viva, Italy. We used continuous focal animal sampling to collect behavioural data focusing on individual and social behaviours. We found that all study subjects performed individual and social species-specific behaviours. However, mother-reared chimpanzees performed locomotion and affiliative behaviours significantly more than hand-reared subjects. In addition to these species-typical behaviours, hand-reared chimpanzees showed significantly more abnormal behaviours than mother-reared subjects. Therefore, these findings suggest that hand-rearing could have wide-reaching effects on the behavioural repertoire in adult zoo-housed chimpanzees. Hence, even if sometimes human intervention in rearing may be necessary to ensure the survival of captive infant chimpanzees, our results suggest that zoo-housed chimpanzees might benefit from minimised human-animal interactions and exposure to conspecifics throughout their development. These suggestions should be implemented in regular husbandry practices.

Gum feeder as environmental enrichment for zoo marmosets and tamarins.

Tamarins and marmosets are small-bodied social callitrichines. Wild callitrichines feed on exudates, such as sap and gum; particularly, marmosets are mainly gummivores, while tamarins consume gums only occasionally and opportunistically. Zoo marmosets and tamarins are usually provided with gum arabic as an alternative to the exudates normally found in the wild. The aim of this study was to evaluate the effects of a gum feeder on the behavior and well-being of four zoo-managed callitrichines. We studied four cotton-top tamarins (Saguinus oedipus), four red-handed tamarins (S. midas), two pygmy marmosets (Cebuella pygmaea), and three Geoffroy's marmosets (Callithrix geoffroyi) housed at Parco Natura Viva (Italy). We conducted the study over two different periods, a baseline (control, without the gum feeder) and then a gum feeder (when the gum feeder was provided) period. We used continuous focal animal sampling to collect behavioral data, including durations of social and individual behaviors. We collected 240 min of observations per period per study subject, with a total of 3,120 min for all the subjects in the same period and of 6,240 min in both periods. We analyzed data by using nonparametric statistical tests. First, we found that the gum feeder promoted species-specific behaviors, such as exploration, and diminished self-directed behaviors, suggesting an enriching effect on tamarin and marmoset behavior. Moreover, in red-handed tamarins, the provision of the gum feeder reduced the performance of self-directed and abnormal behavior, specifically coprophagy. These results confirm that gum feeders are effective foraging enrichment tools for zoo marmosets and tamarins.

Sex differences in scent-marking in captive red-ruffed lemurs.

Primate chemical communication remains underappreciated, as primates are considered to rely on other sensory modalities. However, various lines of evidence suggest that olfaction plays an important role in primate societies, including the conspicuous scent-marking behavior of many strepsirrhines and callitrichines. Although lemurs typically show scent-marking, little is known about this behavior in red-ruffed lemurs (Varecia variegata rubra). We combined behavioral observations and semiochemistry analyses to improve our understanding of scent-marking in two captive troops housed at Dudley and Twycross zoos (UK). We collected olfactory behavioral observations by focusing on two family troops (N = 7) for 132 hr. We investigated the volatile compounds of ano-genital scent-marks using solid-phase microextraction and gas chromatography-mass spectrometry and compared volatile chemical profiles with features of the signaller. Males scent-marked most frequently and predominantly in specific meaningful areas of the enclosure, while within females the occurrence of scent-marking was related to their age. We found behavioral sexual dimorphism, with male predominantly depositing secretions via neck and mandible glands and females via ano-genital glands. We identified a total of 32 volatile components of ano-genital gland secretion, including compounds that have already been found in other mammals as sex pheromones and cues to fitness, in ano-genital scent-marks spontaneously left on filter paper by adult females. Our findings suggest that red-ruffed lemurs might use scent-marking to convey information about sex and female age, with male neck-marking behavior playing defensive territorial functions and ano-genital marking related to socio-sexual communication.

Sternal Gland Scent-Marking Signals Sex, Age, Rank, and Group Identity in Captive Mandrills.

Mandrills are one of the few Old World primates to show scent-marking. We combined ethological and chemical approaches to improve our understanding of this behavior in 3 zoo-managed groups. We observed the olfactory behavior performed by adults and adolescents (N = 39) for 775h. We investigated the volatile components of sternal scent-marks using gas chromatography-mass spectrometry and compared volatile profiles with traits of the signaler. Males marked more than females and within each sex the frequency of scent-marking was related to age and dominance status, but alpha males scent-marked most frequently and particularly in specific areas at the enclosure boundaries. We identified a total of 77 volatile components of sternal gland secretion, including compounds functioning as male sex pheromones in other mammals, in scent-marks spontaneously released on filter paper by 27 male and 18 female mandrills. We confirmed our previous findings that chemical profiles contain information including sex, male age and rank, and we also found that odor may encode information about group membership in mandrills. Our results support the hypotheses that scent-marking signals the status of the dominant male as well as playing territorial functions but also suggest that it is part of sociosexual communication.

Histology of the suprapubic and anogenital cutaneous glands in male cotton top tamarins (Saguinus oedipus).

In cotton top tamarins (Saguinus oedipus), scent glands have been mostly studied in females from museum collections. This work aims to extend the investigation to male specimens, introducing a novel source of skin samples. Two adult males from zoo populations, one intact and one castrated, were immediately frozen after natural death. Skin samples were later collected at the thawing onset, soaked with cold fixative and processed for light microscopy. Sebaceous units of scent glands showed phasic secretory activity in the intact male and marked fibrosis in the castrated male. It appears, therefore, that light microscopy samples from frozen tissues provide detailed features that can disclose distinctive traits in specimens characterized by different hormonal balances.

Fecal concentrations of cortisol, testosterone, and progesterone in cotton-top tamarins housed in different zoological parks: relationships among physiological data, environmental conditions, and behavioral patterns.

The aim of this investigation was to study the welfare of 3 captive groups of cotton-top tamarins housed in different zoological parks. Ethological observations were conducted during 1 year. In addition, fecal samples were collected and the concentrations of glucocorticoids, androgens, and progestogens were measured. Within each group, no significant differences in fecal cortisol concentrations were found between subjects. The fecal concentrations of testosterone and progesterone significantly differed depending on the sexes and ages of the tamarins. A significant association was found among hormone concentrations, exhibit dimensions, and group composition. A highly significant correlation was found between all hormones considered and the space available for each subject. Significant differences in behavioral patterns were observed among groups, including social-individual, affiliative-aggressive, and anogenital-suprapubic scent marking. Correlations between hormone measurements and behaviors were detected. In conclusion, this study confirmed the associations between some behaviors exhibited by these nonhuman primates and both cortisol and testosterone; these data also highlight the role played by progesterone in these behaviors.

Red-green color vision in three catarrhine primates.

The evolution of the red-green visual subsystem in trichromatic primates has been linked to foraging advantages, specifically the detection of either ripe fruits or young leaves amid mature foliage, and to the intraspecific socio-sexual communication, namely the signal of the male rank, the mate choice and the reproductive strategies in females. New data should be added to the debate regarding the evolution of trichromatic color vision. Three catarrhine primates were observed to achieve this goal. The research was performed on captive groups of vervet monkeys (Chlorocebus aethiops), pig-tailed macaques (Macaca nemestrina) and chimpanzees (Pan troglodytes) housed at Parco Natura Viva - Garda Zoological Park (Italy). Using pairs of red-green bags containing the same hidden reward in comparable outdoor enclosures, we recorded the choices by observed individuals (n = 25) to investigate the role of color cues in choosing an object. The results indicate that chimpanzees used red color as cue to choose an object that contains food by showing a preference toward red objects; in contrast, vervet monkeys and pig-tailed macaques do not demonstrate a clear choice based on the color of the object. Our findings highlight the importance of the foraging hypothesis but not rule out the potential role of the intraspecific socio-sexual communication and may serve to add useful information to the debate regarding the adaptive value of the evolution of color vision in order to fill a phylogenetic gap from Old World monkeys to humans. Future studies should address the role of socio-sexual communication, such as the selection of the reproductive partner of both high genetic quality and with compatible genes, to determine how this influenced the evolution of color vision in non-human primates.

Odour signals major histocompatibility complex genotype in an Old World monkey.

The major histocompatibility complex (MHC) is an extraordinarily diverse cluster of genes that play a key role in the immune system. MHC gene products are also found in various body secretions, leading to the suggestion that MHC genotypes are linked to unique individual odourtypes that animals use to assess the suitability of other individuals as potential mates or social partners. We investigated the relationship between chemical odour profiles and genotype in a large, naturally reproducing population of mandrills, using gas chromatography-mass spectrometry and MHC genotyping. Odour profiles were not linked to the possession of particular MHC supertypes. Sex influenced some measures of odour diversity and dominance rank influenced some measures of odour diversity in males, but not in females. Odour similarity was strongly related to similarity at the MHC, and, in some cases, to pedigree relatedness. Our results suggest that odour provides both a cue of individual genetic quality and information against which the receiver can compare its own genotype to assess genetic similarity. These findings provide a potential mechanism underlying mate choice for genetic diversity and MHC similarity as well as kin selection.