Using scent to enhance captive welfare and breeding in a zoo-housed endangered lemur species. Sara Fontani University of Wolverhampton Gale Glendewar Durrell Wildlife Conservation Trust – Jersey Zoo Georgia Callagan University of Wolverhampton Anna Beatrice Costantini University of Wolverhampton Giovanna Marliani University of Wolverhampton Matthew Palframan University of Wolverhampton Stefano Vaglio (  S.Vaglio@wlv.ac.uk ) University of Wolverhampton Article Keywords: Posted Date: September 5th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3304474/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/27 Abstract The Alaotran gentle lemur is one of the most endangered primates in the world and shows a low success rate in captive breeding programmes. We tested a novel scent enrichment, made up of a synthesized mixture potentially conveying information about female fertility, on four unsuccessful breeding pairs (n = 8) hosted at Jersey, Birmingham, London (UK) and Mulhouse (France) zoos. We evaluated the effects of the scent enrichment combining behavioural observations (n = 515 hours) with faecal endocrinology (cortisol and testosterone measurements) (n = 180 samples). We did not nd any signi cant change in a liative behaviours, while aggressive and abnormal behaviours were rarely observed. However, we found a signi cant increase of both male and female olfactory behaviours during the enrichment condition, with females performing these behaviours signi cantly less than males. We also found that sexual behaviours signi cantly increased over the enrichment period. Conversely, we did not nd any signi cant change related to enrichment in cortisol and testosterone levels. Our ndings show little effectiveness by our scent enrichment, which is likely due to shortfalls with regards to fertile odour sampling. However, our results also highlight that biologically relevant odour signals may trigger natural species-speci c behaviours, with potential implications for management and conservation breeding of zoo-based endangered species. Introduction With almost 60% of primate species currently classi ed as endangered or critically endangered by the International Union for Conservation of Nature (IUCN)1, primate conservation is now of vital importance2, with zoos playing a major role in conservation, research, education, and outreach3. The maintenance of the genetic variation of zoo populations, especially those involved in captive breeding (e.g., European Association of Zoos and Aquaria – EAZA Ex situ programmes – EEP) and reintroduction programmes, is imperative to halt biodiversity loss4,5. However, several endangered primate species are currently showing a low success rate in captive breeding, which impair them from serving as a buffer against extinction (reviewed in Elwell and Vaglio6). Particularly, numerous captive lemur populations are struggling, in terms of abundance and demographic trend, almost as much as their wild counterparts and currently would not support reintroduction actions into the wild7,8. The need of captive animals to express natural behaviours has been acknowledged by many studies9,10. In the zoo environment, the lack of stimuli and the repetitive routine can lead to boredom11 and to the display of stereotypic behaviours12, as well as endocrinological disfunction13, which may be linked with decreased reproductive tness of captive populations14. Nevertheless, captivity is a human-controlled environment, and it is therefore possible to enhance captive breeding via evidence-based facilitation of reproductive behaviours and environmental enrichments7. Environmental enrichment can be described as a motor, cognitive, sensory, and social stimulation that boosts animals’ psychological and physiological welfare status promoting a wide range of natural Page 2/27 species-speci c behaviours through a more naturalistic environment15,16. Enrichment may also enhance resiliency to stress, helping captive animals to cope with adverse stimuli17, and ultimately improve their breeding success14. Nowadays several types of enrichment (e.g., feeding, sensory and cognitive) are implemented to improve animal welfare18 in modern zoos, but various lines of evidence suggest that they can also affect the reproductive success in different species, from shes to mammals (reviewed in Elwell and Vaglio6). Primates are generally considered microsmatic mammals, relying more on visual and vocal rather than olfactory cues19. However, olfactory signalling plays a crucial role in socio-sexual communication in strepsirrhine primates20,21. For instance, scent-marking behaviour (i.e, the releasing of semiochemical signals on a substrate22) may have numerous functions23, conveying information about age, rank, reproductive status, site, individual and group identity24,25. Moreover, in many mammal species, including primates, sexual pheromones may advertise female fertility and elicit male behavioural and physiological responses26, comprising species showing courtship displays for pre-mating isolation, such as rhesus monkeys (Macaca mulatta) and even humans27. Odour may also provide females with information about male quality as potential mates (e.g., strepsirrhine primates28 and cheetahs29). Chemicals have therefore great potential as tools to trigger olfactory and sexual behaviours in lemur species28. Although evidence shows that scents can facilitate mate choice and improve the chances for reproductive success in mammal species, such as striped dunnarts (Sminthopsis macroura)30 and harvest mice (Micromys minutes)31, studies on the effects of olfactory enrichments in primates are still scarce6. Furthermore, despite scent enrichment can be used to manipulate the environment in different ways32, promoting active species-speci c behaviours17,33, most studies have focused on anthropogenic scents, such as spices or essential oils, rather than testing biologically relevant scents34, which in turn may also impact positively on reproductive success35. We focused on the gentle lemur (Hapalemur alaotrensis), one of the world’s 25 most endangered primates, with an estimated population of around 2,500 individuals in the wild36, which is currently listed on the Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). To guarantee the survival of this critically endangered species, since 1990 their captive population is managed by an EEP37. However, EAZA gentle lemur population consists of only around 70 adult individuals and breeding has recently declined in many European zoos, with only few active breeding pairs38. In this context, it has become crucial to improve our understanding of their reproductive biology and enhance their well-being and breeding success39. The overarching aim of this study was to design and test a new scent enrichment to enhance the wellbeing and breeding success of captive gentle lemurs. To achieve this, we investigated the chemical pro le of the anogenital odour secretions of a successfully breeding female39, then reproduced the chemical mixture in our semiochemistry laboratory and tested it with four unsuccessful breeding pairs. Speci cally, Page 3/27 to assess the effects of the scent enrichment we combined behavioural observations (focusing on a liative, aggressive, abnormal, olfactory, and sexual behaviours) with faecal endocrinology (focusing on stress and sex hormones). In this study, we tested the following hypotheses and predictions: 1. Our newly designed scent enrichment reduces stress levels. We predicted that stress-related behaviours (i.e., abnormal and aggressive) would decrease, species-speci c behaviours (i.e., a liative and olfactory) would increase, and cortisol levels would decrease after the enrichment condition. 2. Our newly designed scent enrichment triggers sexual behaviors. We predicted that male and female sexual behaviours, including male mating behaviours, and male testosterone levels would increase during and after the enrichment condition. Materials and Methods Subjects, housing, and study protocol We studied four captive unsuccessful breeding pairs (i.e., biologically able to reproduce but never successfully breeding as a pair) of gentle lemurs (N = 8), hosted at Birmingham Wildlife Conservation Park (UK), Parc Zoologique & Botanique de Mulhouse (France), Jersey Zoo – formerly Durrell Wildlife Park (Channel Islands) and ZSL London Zoo (UK). We collected behavioural and physiological (e.g., hormone levels) data from June 2022 to February 2023 (Table 1). All troops were housed in indoor enclosures (heated to 25–28°C) and had access to outdoor enclosures. Table 1 Study subjects and sampling periods. Site Name Sex Age at study start Period of sampling Birmingham Zoo Zoma Male 14 years, 11 months, 1 week 27/06/22–10/08/22 Bozy Female 12 years, 3 months, 2 weeks Kwic Male 8 years, 1 month, 3 weeks Manon Female 12 years, 1 month, 3 weeks Brian Male 13 years, 7 months, 3 weeks Bala Female 17 years, 5 months, 2 weeks Rocky Male 15 years, 6 months, 3 weeks Hazo Female 3 years, 7 months, 3 weeks Mulhouse Zoo Jersey Zoo London Zoo Study protocol Page 4/27 25/07/22–26/08/22 19/09/22–21/10/22 09/01/23–10/02/23 We divided the study period into three phases: pre- enrichment (i.e., before enrichment condition) (10 days), enrichment (i.e., during enrichment condition) (6 days), post- enrichment (after enrichment condition) (10 days). We carried out behavioural observations and faecal sampling every study day from early morning to early afternoon (~8AM-1PM, 5 hours per day) over 6 days per week. We assessed the effects of the enrichment combining the observation of sexual behaviours and behavioural indicators of welfare (e.g., explorative olfactory behaviours, a liative behaviours, aggressive, and abnormal behaviours)40–42 and faecal endocrinology (e.g., faecal cortisol levels in both males and females and testosterone levels in males). Odour sampling We investigated the volatile component of odour signals using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) techniques, as previously described by Fontani et al.39. Brie y, we introduced a 65 µm polydimethylsiloxane/divinylbenzene SPME syringe needle through the vial septum and exposed the bre to the headspace above the sample in the vial for 15 min at 40°C. We analysed the adsorbed volatile analytes of all samples using a 5975C mass spectrometer (Agilent Technologies) EI, 70 eV, coupled directly to a 7890B gas chromatograph (Agilent Technologies) equipped with a fused silica HP5-MS UI capillary column (Agilent Technologies) 30 m × 0.25 mm crossbonded 5%phenyl-95% dimethylpolysiloxane, lm thickness 0.25 µm. We maintained the injector and transfer line temperatures at 270°C and 280°C, respectively. We made injections in splitless mode (purge valve opened after 1 min) with a constant ow of helium carrier gas of 1 ml min − 1. We started the oven temperature program at 45°C for 2 min, then raised it by 4°C min − 1 to 170°C, and nally by 20°C min − 1 to 300°C 40. We assessed possible environmental contamination via blank analyses using an empty 10 ml vial (Supelco) and control swabs following the same procedure as for the samples and conditioned the bre at 260°C pre-injection for 5 min and 260°C post-injection for 20 min to avoid any possible carry-over effects. We analysed all samples in a short period of time to minimize inter-assay variability. We overlaid chemical pro les from control swabs on animal chemical pro les to identify compounds that did not derive from the animals and removed these from the swab results. We rst tentatively identi ed eluted compounds by comparing the experimental spectra with those of the mass-spectral library in ChemStation (Agilent Technologies) and NIST Database (National Institute of Standards and Technology), version MSD F.01.01.2317 (Agilent Technologies). We accepted a putative identi cation if the minimum matching factor was higher than 90%. After that, we carried out the unequivocal identi cation of the key compounds distinguishing the fertile window of the breeding female using the same swabs and vials as for lemur sample collection and then comparing these compounds with standard compounds injected and analysed by applying the same SPME and GC-MS protocol43. Scent enrichment We prepared standard dilutions using similar methods to other authors who identi ed volatile organic compounds in owl monkeys (Aotus spp.)44. Brie y, we diluted each chemical compound (2-heptanone; 3Page 5/27 heptanone; 3-octanone; 4-methyl, 3-hexanone) separately, placing 1.5mL of HPLC grade methanol in 15 mL test tube, adding 5µL of compound and 3.5mL of de-ionised water, and then we vortexed for 15 seconds to dissolve the compound in the mixture. We compared both the retention times of key compounds and standards and the overall patterns of the mass spectra. We accepted the identi cation only if both the parameters were satis ed. 2-heptanone, 3-heptanone and 3-octanone, as well as benzaldehyde and decanal, were commercially available (Agilent) while 4-methyl, 3-hexanone was synthesized in our chemistry laboratory. Once the identi cation was certain, we added 1 mL of each diluted compound into a new test tube and vortexed for 30 second to produce the scent mixtures to test as olfactory enrichment. Finally, we presented the enrichment to the study subjects applying the same protocol as in our prior study45. Brie y, we used white cotton sheets cut into 75 cm long and 5 cm wide strips, which were soaked with 20 drops of scent mixture diluted with 12 ml of cold boiled water. Newly soaked cotton strips were prepared each enrichment day. We placed 2 unscented (controls) and 6 scented strips on the climbing frames both indoor and outdoor (Figs. 1, 2), and removed them at the end of the period of observations every day. We randomized the locations of both scented and unscented cotton strips to avoid habituation on a daily basis. Behavioural data collection We collected behavioural data using all occurrences of some behaviours and ad libitum sampling methods46 and focused on a liative, aggressive, abnormal, olfactory, and sexual behaviours (Table 2). We recorded a total of 515 hours of observations over the study period. For each study subject, we determined the relative frequency of each behavioural category, calculating the number of behaviours performed out of the total hours of observation. Page 6/27 Table 2 Ethogram of selected behaviours for the study subjects modi ed by Fontani 39. Behaviours Description Olfactory behaviours Brachial scent marking Scratching object with lower dentition, then rubbing spot on brachial glands (on arms). Only males. Ano-genital scent marking Rubbing object with genitalia, then sit-rubbing repeatedly whilst depositing urine Territorial marking Grabbing, biting, chasing, lunging, confrontation display to conspeci cs Tail-scent marking Standing on hind legs with tail bent towards them, rubbing object on insides of wrists and tail simultaneously. Sniff genitals Place the nose less than 3 cm from the anogenital area of a conspeci c and lick it Sniff substrate Inspecting, sni ng, touching, biting, licking a substrate for at least 2 seconds Sexual behaviours Ano-genital selfgrooming Grooming of genital area, using ngers or mouth Follow Male approaches female from behind and follows closely (less than 1 m) Penil erection The subject shows a conspicuously erect red penis. Mating calls Female produces distinct singly or in series call, while soliciting copulation and during mating Attempt mounting Male approaches female, clasps, orients body for copulation; female chatters at and/or cuffs the male, male releases female Copulation Male approaches, female crouch, male introduces sperm into the female's reproductive tract A liative behaviours Grooming Using ngers or mouth to pick through the coat, removing any foreign bodies from a conspeci c Proximity Two or more individuals are in physical contact or stay no more than 10 cm away from each other, resting or sleeping Aggressive behaviours Intimidation The subject emits a short vocalization toward a conspeci c to warn it not to come closer Page 7/27 Behaviours Description Chase The subject chases a conspeci c; chasing him on the ground or scrambling to reach him Bite The subject bites a conspeci c Abnormal behaviours Self-scratching The subject rubs its own bot at a fast pace Pacing The subject walks back and forth in a distinct, unchanged pattern though the enclosure We performed the inter-observer reliability test to measure the degree of agreement in the behaviour’s identi cation between the two observers (A.B.C. & G.C.) at the zoological facilities47. Speci cally, we used Cohen’s Kappa coe cient to measure the agreement between the observers and obtained 83%, which is considered “almost perfect”48. Faecal hormone sampling and measurements We collected a total of 180 faecal samples over the study period (Table 3). We collected the samples every morning during the behavioural observations, right after defecation was observed, when the identity of the study subject was certain. As diurnal secretion patters of hormones, such as cortisol and testosterone, may be detected in faecal samples (especially for small-bodied species), we restricted the sampling period to approximately the same time of the day49. We stored the samples in a − 20◦C freezer on site immediately after sampling. At the end of the study period, we transferred the sample to the Rosalind Franklin Science Centre – University of Wolverhampton using a cold bag with ice packs to avoid any risk of defrosting. Page 8/27 Table 3 Number of faecal samples collected per study subject. Site Name Sex Fecal samples Birmingham Zoo Zoma Male 23 Bozy Female 21 Kwic Male 28 Manon Female 27 Brian Male 26 Bala Female 21 Rocky Male 18 Hazo Female 16 Mulhouse Zoo Jersey Zoo London Zoo Total 180 Hormone analyses We used a freeze-drying machine (Christ R, Beta 1–8 LSC plus, Osterode am Harz, Germany) to lyophilize the faecal samples for 72 h, and then we pulverized them using a pestle and mortar. We sieved the faecal powder through a stainless-steel strainer, aperture 250 mic, to separate the faecal residue from any brous material. With regards to extraction, we followed the methods of our prior study39. Brie y, we extracted 0.05–0.1 g of faecal powder in 3 ml of 80% methanol using a 15 ml plastic tube and vortexing it for 15 min with a multi-tube vortexer (Grant Instruments R, Multi-Vortexer V-32, Cambridge, UK). Right after centrifugation for 20 min at 3,300 xg, we stored the supernatant at − 20◦C. When analysing faecal hormones, we considered the time course of hormones metabolite excretion relative to the production and circulation of the native hormones49,50. We measured faecal cortisol and testosterone levels using commercially available enzyme linked immunosorbent assay (ELISA) kits (Enzo Life Sciences® Cortisol, ADI-900-071, New York, USA and DetectX® Testosterone K032-H5W, Arbor Assays R, USA) following kits instructions. Before analysis, we diluted all the samples 1:1 with the assay buffer provided by the kits. We assayed all standards and faecal samples in duplicates, with samples showing a coe cient of variation (CV) exceeding 15% being re-analysed51. We analysed assay data applying a 4-parameter logistic tting programme (MyAssays R, Brighton, UK). Concentrations were expressed as pg/mg. Mean intra-assay coe cient of variation for cortisol, tested on four quality control samples (2 males and 2 females) with eight replicates within a single assay plate, was 7.77% ± 1.27, while for testosterone, tested on three control samples (all males), was 9.35% ± 2.57. Mean inter-assay coe cient of variation, tested on the same samples measured with four replicates across three assay plates, was 15.04% ± 5.21 for cortisol and 5.96% ± 1.42 for testosterone. Statistical analyses Page 9/27 We performed statistical analyses using R studio software (version 4.2.2)52 to evaluate the in uence of enrichment on behaviour and hormonal levels. To investigate the relationship between faecal hormonal levels and enrichment, we used the R package “lme4” to run a Linear Mixed Model (LMM)53. For this LMM analysis, the faecal hormonal concentration (cortisol or testosterone) was set as the outcome variable, and study phase (before, during and after the enrichment) and sex (only for cortisol), and their interaction between variables as xed variables. Before running the statistical analyses, we applied a logarithmic transformation to the variable of cortisol, and we excluded two outliers from testosterone analysis. Similarly, to test the effect of the scent enrichment on the behaviour of study subjects, we performed different LMM where the frequency of sexual and olfactory behaviours were the outcome variables, and study phase, sex, and their interactions were the xed variables. The same model was used for a liative behaviours; however, since these behaviours were reciprocal, only the study phase was set as a xed variable. We applied a natural logarithmic + 1 transformation to olfactory behaviours, and we used Yeo-Jhonson transformation to normalize the sexual behaviours, while a liative behaviours were not transformed. In all models, we considered the zoo as a nested factor and, with exception for a liative behaviours’ model, we set subject ID as a random effect to control for multiple sampling. We checked the normality of residuals for each model through the Shapiro-Wilk test and visually through the histogram of residuals. Statistical signi cance was set at p-value < 0.05. Ethics statement The study followed the institutional and international guidelines for the care and use of captive animals, involving non-invasive methods for obtaining behavioural data and faecal samples from the gentle lemurs. Moreover, the study was conducted in compliance with the CITES and approved by the Life Sciences Ethics committee at the University of Wolverhampton (UK) and the Ethics committees at Jersey Zoo (Channel Islands), Parc Zoologique & Botanique de Mulhouse (France), Birmingham Wildlife Conservation Park and ZSL London Zoo (UK). We also con rm that our research work was consistent with the ARRIVE guidelines for ethical treatment of non-human primates. Results Odour results We identi ed a total of 78 distinct peaks in 35 swab samples of gentle lemur anogenital secretions that were not present in the control swabs. These compounds included a series of hydrocarbons and fatty alcohols, organic aliphatic acid esters and carboxylic acid, aldehydes, and aliphatic ketones. Speci cally, a small pool of compounds was only present in the chemical pro les of anogenital odour samples collected during the fertile window. Four compounds were unequivocally identi ed as 2-heptanone, 3heptanone, 3-octanone, and 4-methyl 3-hexanone. A typical chromatogram from the ovulation window is shown in Fig. 3. Behavioural results Page 10/27 For behavioural analysis, we considered a liative, sexual, and olfactory behaviours of both male and female lemurs, as abnormal and aggressive behaviours were rarely observed (abnormal behaviours: mean frequency rate 0.02 N/hr, aggressive behaviours: mean frequency rate 0.05 N/hr). The LMM analysis of a liative behaviours did not show a signi cant relationship (p-value > 0.05) between the frequency of a liative behaviours and the study phase (Table 4). Table 4 Results of the LMM examining whether the frequency of a liative behaviours was signi cantly predicted by the study phase. SE = standard error; df = degree of freedom. Outcome Predictor Estimate SE df t-value Pr(>|t|) A liative behaviours (Intercept) 16.934 5.782 3.310 2.929 0.054 Post- enrichment 0.463 1.841 97.001 0.252 0.802 Pre- enrichment 2.796 1.832 97.000 1.527 0.130 However, we found that the frequency of olfactory behaviour was signi cantly higher during the enrichment condition compared to the pre (β ± SE = -0.490 ± 0.124, t-value = -3.959, p-value < 0.001) and post-enrichment conditions (β ± SE = -0.353 ± 0.124, t-value = -2.842, p-value = 0.005) (Table 5, Fig. 4). Additionally, the frequency of olfactory behaviours was signi cantly lower in females than males (β ± SE = 0.500 ± 0.206, t-value = 2.428, p-value = 0.029) (Table 5, Fig. 5). Table 5 Results of the LMM examining whether the frequency of olfactory behaviours was signi cantly predicted by research phase, sex, and their interaction. SE = standard error; df = degree of freedom; * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Outcome Predictor Estimate SE df tvalue Pr(>|t|) Olfactory behaviours (Intercept) 1.217 0.146 13.971 8.362 < 0.001 *** Post- enrichment -0.353 0.124 194.034 -2.842 0.005 ** Pre-enrichment -0.490 0.124 194.016 -3.959 < 0.001 *** Sex male 0.500 0.206 13.971 2.428 0.029* Post- enrichment : Sex male 0.235 0.176 194.034 1.334 0.184 Pre- enrichment : Sex male 0.281 0.175 194.016 1.606 0.110 Similarly, the frequency of sexual behaviours signi cantly increased (β ± SE = 0.605 ± 0.211, t-value = 2.865, p-value = 0.0046) during the enrichment condition compared to the pre-enrichment condition. The post-enrichment condition also showed a higher frequency of sexual behaviours than the pre-enrichment condition with a signi cant tendency (β ± SE = 0.323 ± 0.184, t-value = 1.754, p-value = 0.081) (Table 6, Page 11/27 Fig. 6). Interestingly, in this case, the display of sexual behaviours was not signi cantly in uenced by the sex of the study subjects (Table 6). Table 6 Results of the LMM examining whether the frequency of sexual behaviours was signi cantly predicted by research phase, sex, and their interaction. SE = standard error; df = degree of freedom; ** = p < 0.01. Outcome Predictor Estimate SE df tvalue Pr(>|t|) Sexual behaviours (Intercept) -0.154 0.316 7.4581 -0.487 0.640 Phase enrichment 0.605 0.211 194.006 2.865 0.0046 ** Pre-enrichment 0.323 0.184 194.016 1.754 0.081 Sex male -0.367 0.446 7.458 -0.822 0.436 Post- enrichment : Sex male 0.152 0.299 194.006 0.51 0.611 Pre- enrichment : Sex male 0.310 0.260 194.017 1.191 0.235 Endocrinological results The LMM analysis indicated no signi cant relationship between faecal cortisol and testosterone levels and the study phase (p-value > 0.05). Moreover, there was no signi cant in uence observed by sex or the interaction between sex and the research phase on faecal cortisol levels (p-value > 0.05) (Tables 7 and 8). Table 7 Results of the LMM examining whether faecal cortisol level was signi cantly predicted by study phase, sex, and their interaction. SE = standard error; df = degree of freedom. Outcome Predictor Estimate SE df t-value Pr(>|t|) Faecal Cortisol (Intercept) 4.068 0.201 3.972 20.229 < 0.001 Post- enrichment -0.049 0.096 168.464 -0.517 0.606 Pre- enrichment 0.081 0.092 168.642 0.874 0.384 Sex male -0.036 0.118 12.788 -0.308 0.763 Post- enrichment : Sex male -0.006 0.132 168.495 -0.043 0.966 Pre- enrichment : Sex male -0.139 0.128 168.693 -1.083 0.280 Page 12/27 Table 8 Results of the LMM examining whether faecal testosterone level was signi cantly predicted by the research phase. SE = standard error; df = degree of freedom. Outcome Predictor Estimate SE df t-value Pr(>|t|) Faecal Testosterone (Intercept) 24.24 3.673 3.466 6.6 0.0045 Post- enrichment -0.183 1.474 87.004 -0.124 0.9015 Pre- enrichment 0.997 1.451 87.028 0.687 0.4938 Discussion Studies of olfactory enrichment are less frequent than those of other types of enrichment, particularly on primate species, and their results are contradictory. Nevertheless, recent evidence suggest that more attention should be paid on species-speci c biologically relevant scents, which may in uence both welfare status and mating behaviours in captive populations (reviewed in Elwell and Vaglio6). The effectiveness of scent enrichment programmes depends on the target species54, with differences also been found between sexes55, as male and female individuals may respond differently towards conspeci c scents, while using them as reproductive and mate quality cues56. In this study, we predicted that our newly designed scent enrichment, based on the fertile odour of a breeding female gentle lemur, would reduce stress levels, and trigger sexual behaviours in both male and female lemur counterparts. We observed aggressive behaviours (e.g., intimidation and bite) rarely and mostly during feeding. These behaviours were performed mainly by females toward males; this is not surprising as the gentle lemur is a female dominant species in which aggressive behaviours may occur in inter-sexual interactions, especially in the context of feeding57. With regards to abnormal behaviours, we recorded very little pacing behaviour and only performed by the female hosted at Mulhouse Zoo. Pacing was exclusively observed before feeding time and, accordingly to other authors (e.g.,58) may be interpreted as an anticipatory behavioural pattern. Particularly, anticipatory behaviours are usually exhibited by animals during the appetitive phase before acquiring a food resource59 and has been observed in a wide range of species when animals are cared for by humans60–63. The lack of aggressive and abnormal behaviours during the entire study period can be interpreted as an indicator of good welfare status in the study subjects, already before the scent enrichment program. As predicted, males performed olfactory behaviours more frequently than females. Particulalry, we found a signi cant increase in rates of olfactory behaviours during the enrichment condition. This is consistent with the ndings of other authors64 in captive Javan gibbons (Hylobates moloch), even if they recorded a Page 13/27 rapid decrease of the interest for the new olfactory stimulus already after the rst day of treatment. On the contrary, studies on other primate species (e.g., gorillas (Gorilla gorilla gorilla)34, ring-tailed lemurs (Lemur catta)65) showed no signi cant effects of scent enrichment programmes on the rates of olfactory behaviours. We also found that males performed these behaviours more frequently than females. This is not surprising as male gentle lemurs’ scent-mark more often than females66, and it could be explained by the fact that the olfactory enrichment was based on the female fertile odour55. By contrasts, we did not nd any signi cant increase in a liative behaviours (e.g., grooming and proximity). Grooming has been studied extensively throughout the animal kingdom and plays a key role in social bonding for many primate species (reviewed in Dunbar67). Moreover, a study carried out on 44 species of wild primates68 demonstrated that social grooming is related with group size. Our results may, therefore, depend on the group composition as we observed troops composed by only two individuals, where the need to strengthen the ties within group members is less important. Additionally, we can speculate that a lack of increase in proximity could be due to an increase in other behaviours, such as olfactory behaviours. We did not nd any signi cant decrease in cortisol levels after the enrichment condition. This may be due to several factors. First, cortisol is commonly used to measure both acute69 and chronic stress70 levels, and it is often related to group instability71,72, aggression patterns73,74 and displacement activities75,76 in primate species. As we observed stress-related behaviours very rarely during the study period, we may assume that the baseline was made up of study subjects with already good well-being and, in turn, the potential for impact by the scent enrichment was limited. Then, a previous study on rhesus monkeys77 showed that, although environmental enrichment may lead to behavioural improvements, it does not always affect adrenal function. Additionally, other authors78 found that in a single-housed orangutan (Pongo pygmaeus) cortisol metabolites signi cantly decreased over the long period to lower levels than those found during the pre-enrichment condition; this could indicate that a longer data collection after the enrichment condition could have led to different results in our study. We found a signi cant increase in sexual behaviours, performed by both sexes, during the enrichment condition, with effects lasting afterwards. Speci cally, the increased sexual behaviours were anogenital self-grooming and erection, which have been identi ed as sexual behaviours in other mammal species (e.g., male and female rats (Rattus norvegicus)79, male rodents80). This is consistent with ndings on rhesus monkeys81 when introducing oestrogen-treated females in a previously established group. This study found an increase in both male and female sexual behaviours, which remained elevated after the removal of the treated females, also suggesting that the presence of these females may induce sexual behaviours in the other females of the group. On the other hand, contrary to what we predicted, we did not observe any occurrence of mating behaviour. This suggests that our scent enrichment was not fully effective, even though the fact that our study was carried out during their non-breeding period might have played a role. Particularly, the chosen volatile compounds and their proportions in the mixture would not have mimicked the actual female fertile odour due to the study limitations (see below). Page 14/27 We did not nd any signi cant change in male testosterone levels, contrary to the expected signi cant increase in male testosterone levels as endocrinological response to the exposition to female fertile odour. Prior studies showed that in other primate species (e.g., stump-tailed macaques (Macaca arctoides)82, common marmosets (Callithrix jacchus)83, including humans84), males exposed to the odour of an ovulating female display higher testosterone levels in comparison with males exposed to the scent of a non-ovulating female or a control scent. Moreover, the interaction with oestrogen-treated females increased testosterone levels in male rhesus monkeys even outside the breeding season81. Our results may be due to several factors. According to the “challenge hypothesis”85, in multi-male multifemale groups access to breeding females is often regulated by a dominance hierarchy, with males competing to attain and maintain a high dominant rank, while male testosterone levels and aggression rates are higher during periods of social instability, rather than during the mating season86,87. Also, other authors found that in species with low aggression levels and little competition associated with access to mates (e.g., muriquis (Brachyteles arachnoides)88 and moustached tamarins (Saguinus mystax)89), testosterone uctuations are not associated with the breeding season, supporting the hypothesis that the absence of male competition over sexual access to females may explain the absence of testosterone variations. Although aggressive interactions have occasionally been observed in male gentle lemurs competing for a fertile female90, the social stability and the lack of male competitors in our study groups (i.e., pairs of individuals) may explain the absence of substantial uctuations in testosterone levels. Additionally, captive data suggest that relatively low concentrations of circulating testosterone are generally adequate to maintain male reproductive function91, while elevated testosterone levels imply costs92, including increased metabolic rates and immunosuppression93, which would prove unnecessary in captive settings. Research limitations We certainly have to acknowledge some major limitations that could have impacted on the e cacy of the proposed scent enrichment programme. First, we focused on a small sample size (i.e., one successful breeding female, four unsuccessful breeding pairs). Then, due to di culties with odour sampling (i.e., lack of odour secretions on the anogenital area of the breeding female and swab contaminants likely covering key compounds) we might have missed a few key compounds. Finally, due to the small pool of samples, we have had to mix the compounds in 1:1 proportion, which is not re ecting the real ratio of the anogenital odour of the fertile female gentle lemur. Conclusions and perspectives This was the rst study which aimed to improve both well-being and reproduction in captive gentle lemurs using a resynthesized biological scent as olfactory enrichment. Despite the major limitations highlighted above, and the lack of mating behaviour triggered in male gentle lemurs outside of the breeding period, our ndings show that natural biologically relevant odour have potential to induce species-speci c behaviours as well as affect sexual behavioural patterns. Page 15/27 Further investigations on the volatile chemical pro le of the gentle lemur female fertile odour, as well as an enlarged samples size when testing the re ned mixture of compounds conveying information about female fertility, are needed to improve the e cacy of this scent enrichment and hopefully enhance reproductive success in zoo-housed gentle lemurs, ultimately with impact on best practices adopted by host zoos and extended to other institutions through changes in management policies released by European Association of Zoos & Aquaria’s Studbook Keeper and Taxon Advisory Group’s Coordinator for the target species. Declarations Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgments We are grateful to Durrell Wildlife Conservation – Jersey Zoo (especially Eluned Price, Rachel Cowen and primate keepers), Birmingham Wildlife Conservation Park (especially Les Basford, Gareth Davies, and primate keepers), Parc Zoologique & Botanique de Mulhouse (especially Brice Lefaux and primate keepers), ZSL London Zoo (especially Lewis Rowden and primate keepers) for their support to the project and assistance with sample collection. We thank Wild Place Project – Bristol (especially Will Walker, Daniella Pierce-Butler, and primate keepers) for their help with the pilot study. We also thank Keith Holding for assisting with chemical analyses and David Luckhurst for helping with the endocrinology laboratory setting up at the Rosalind Franklin Science Centre—University of Wolverhampton, Stefano Kaburu for assisting with statistical analyses, and Pier Attilio Accorsi for advising with the interpretation of the physiological results. Furthermore, we thank Wolverhampton University’s Animal Behaviour and Wildlife Conservation Group members for their constructive comments and suggestions. Author contributions SF: study conception and design, training of research assistants in data collection and endocrinological laboratory work, writing original draft, project administration. GG: study conception and design. GC: data collection and analysis. ABC: data collection and analysis. GM: statistical analysis, writing original draft. MP: chemical laboratory work. SV: study conception and design, chemical laboratory work, writing and reviewing original draft, funding acquisition, project administration, supervision. All authors read and approved the nal manuscript. Funding This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement [no. 890341 to SF and SV] and the Primate Society of Page 16/27 Great Britain Captive Care Grant [round 2021 to SF]. Lab work and publication fees were funded by the University of Wolverhampton’s Research Investment Fund scheme – Phase 4 [to SV]. Additional information Competing interests. The authors declare no competing interests. References 1. IUCN. The IUCN Red List of Threatened Species. Version 2022-2. https://www.iucnredlist.org/ (2022). 2. Estrada, A. et al. Impending extinction crisis of the world’s primates: Why primates matter. Sci Adv 3, e1600946 (2017). 3. Mellor, D. J., Hunt, S. & Gusset, M. Caring for Wildlife: The World Zoo and Aquarium Animal Welfare Strategy. (Gland: WAZA Executive O ce, 2015). 4. Lacy, R. C. 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Figure 4 Page 25/27 Frequency of olfactory behaviours (N/hr) at pre- enrichment (“pre”), during the enrichment (“enr”) and post- enrichment (“post”). The bar within the box represents the median, the borders of the box are upper quartiles, the bottom and top whiskers signify the lowest and highest cases within 1.5 times interquartile range (IQR), and outliers are shown through black full circles. * signi cance at p < 0.01, ** signi cance at p < 0.001. Figure 5 Frequency of olfactory behaviours (N/hr) considering gender of individuals (female and male). The bar within the box represents the median, the borders of the box are upper quartiles, the bottom and top whiskers signify the lowest and highest cases within 1.5 times interquartile range (IQR), and outliers are shown through black full circles. * signi cance at p < 0.05. Page 26/27 Figure 6 Frequency of sexual behaviour (N/hr) at pre- enrichment (“pre”), during the enrichment (“enr”) and postenrichment (“post”). The bar within the box represents the median, the borders of the box are upper quartiles, the bottom and top whiskers signify the lowest and highest cases within 1.5 times interquartile range (IQR), and outliers are shown through black full circles. ** signi cance at p < 0.01. Page 27/27