Design and test of novel scent enrichments to enhance breeding of zoo-housed lemurs

Ethical considerations

The use cases/studies followed the institutional and international guidelines for the care and use of captive animals, involving non-invasive methods for obtaining behavioural data, faecal and odour samples from the lemurs. Moreover, the studies were conducted in compliance with the Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora and approved by the Life Sciences Ethics Committee (LSEC) at the University of Wolverhampton (UK) (REC numbers LSEC/201819/CY/166 and LSEC/202021/SV/52) and the Ethics Committees at Jersey Zoo (Channel Islands), Parc Zoologique & Botanique de Mulhouse (France), Birmingham Wildlife Conservation Park, Dudley Zoo & Castle, Shaldon Zoo – Wildlife Trust, Twycross Zoo, and ZSL London Zoo (UK). The authors made all possible efforts to ameliorate harm to animals, which was achieved by using non-invasive methods to collect samples (including behavioural data and biological samples such as anogenital odour secretions and faeces) from the study subjects.

Study subjects and housing

We studied four pairs of gentle lemurs (Hapalemur alaotrensis) (N = 8), hosted at Birmingham Wildlife Conservation Park (UK), Parc Zoologique & Botanique de Mulhouse (France), Jersey Zoo (Channel Islands) and ZSL London Zoo (UK) (Fontani et al., 2022), and four small groups of red ruffed and black-and-white ruffed lemurs (Varecia spp.) (N=15) at Dudley Zoo & Castle, Shaldon Zoo – Wildlife Trust, and Twycross Zoo (UK) (Elwell et al., unpublished data). All study troops were housed in indoor enclosures (heated to 25-28°C) and had access to outdoor enclosures.

Study protocol

We divided the study period into three phases: pre- enrichment (i.e., before enrichment condition – two weeks), enrichment (i.e., during enrichment condition – one week), post- enrichment (i.e., after enrichment condition – two weeks). We carried out behavioural observations and faecal sampling every study day from early morning to early afternoon, as they are more active in the morning (~8AM-1PM, 5 hours per day), over five days per week. We assessed the effects of the enrichment combining the observation of sexual behaviours (including mating) and faecal endocrinology (e.g., faecal testosterone levels in males).

Odour sampling and investigation

We collected anogenital odour samples by rubbing 10 times a sterile cotton swab around the wall of the vulva, using steady pressure, as described by Vaglio et al. (2021a). Moreover, we exposed control swabs to the air to identify any compounds that did not derive from the lemurs. We placed all samples and controls into sterile vials and immediately stored them in a −20°C freezer at the zoo. We then transferred the vials to the Rosalind Franklin Science Centre, University of Wolverhampton, using a freezer box with ice packs to avoid any risk of defrosting, for laboratory analyses.

We investigated the volatile component of odour signals using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) techniques, as described by Walker & Vaglio (2021). Briefly, we introduced a 65 μm polydimethylsiloxane/divinylbenzene SPME syringe needle through the vial septum and exposed the fibre 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, film 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 flow of helium carrier gas of 1 ml/min. We started the oven temperature programme at 45 °C for 2 min, then raised it by 4 °C /min to 170 °C, and finally by 20 °C/min 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 fibre 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 profiles from control swabs on lemur chemical profiles to identify compounds that did not derive from the lemurs and removed these from the swab results.

We tentatively identified 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 identification if the minimum matching factor was higher than 90%. After that, we carried out the unequivocal identification of the key compounds distinguishing the fertile window of the breeding female comparing these compounds with standard compounds injected and analysed by applying the same SPME and GC-MS protocol (Elwell et al., unpublished data).

Scent enrichment

Briefly, we diluted each chemical compound separately, placing 1.5 mL of HPLC grade methanol (Fisher Chemical, Cat. number 10499560) in 15 mL test tube, adding 5 μL of compound and 3.5 mL 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 identification only if both the parameters were satisfied. Once the identification 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.

We then presented the enrichment to the study subjects applying the protocol described in Vaglio et al. (2021b). Briefly, 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 (Figure 1) and removed them at the end of observations every study day. To avoid habituation, we randomized the locations of both scented and unscented cotton strips daily.

Figure 1. Male gentle lemur interacting with scent enrichment in the outdoor enclosure at Birmingham Wildlife Conservation Park.

Photo by Georgia Callagan with permission.

Behavioural data collection

We collected behavioural data using all occurrences of some behaviours, instantaneous scan, and ad libitum sampling methods (Altmann, 1974) and focused on sexual behaviours (Table 1). 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.

We performed the inter-observer reliability test to measure the degree of agreement in the behaviour identification by the different observers at the zoo facilities (Wark et al., 2021). Specifically, we used Cohen’s Kappa coefficient to measure the agreement between the observers.

Faecal hormone sampling and measurements

We collected faecal samples every morning during study days (i.e., when behavioural observations were conducted), right after defecation was observed, when the identity of the study subject was certain. As diurnal secretion patters of hormones, such as testosterone, may be detected in faecal samples (especially for small-bodied species), we restricted the sampling period to approximately the same time of the day (Hodges & Heistermann, 2011). We stored the samples in a −20 °C freezer on site immediately after sampling. At the end of the study period, we transferred the samples to the Rosalind Franklin Science Centre – University of Wolverhampton using a cold bag with ice packs to avoid any risk of defrosting.

Hormone analyses

We used a freeze-drying machine (Beta 1–8 LSC plus, Christ R) 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 fibrous material. With regards to extraction, we followed the methods described in Fontani et al. (2022). Briefly, we extracted 0.05–0.1 g of faecal powder in 3 ml of 80% methanol (Scientific Laboratory Supplies, Cat. number CHE2536) using a 15 ml plastic tube and vortexing it for 15 min with a multi-tube vortexer (Multi-Vortexer V-32, Grant Instruments R). Right after centrifugation for 20 min at 3,300 ×g, 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 hormones (Hodges & Heistermann, 2011; Wheeler et al., 2013). We measured faecal testosterone levels using commercially available enzyme-linked immunosorbent assay (ELISA) kits (DetectX^® Testosterone K032-H5W, Arbor Assays R, USA, Cat. number K032-H5) 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 coefficient of variation (CV) exceeding 15% being re-analysed (Macagno et al., 2020). We analysed assay data applying a 4-parameter logistic fitting programme (MyAssays R, – open access, available online at https://www.myassays.com/index.html). Concentrations were expressed as pg/mg. Mean intra-assay coefficient of variation for testosterone, tested on three control samples (all males), was 9.35% ± 2.57. Mean inter-assay coefficient of variation, tested on the same samples measured with four replicates across three assay plates, was 5.96% ± 1.42 for testosterone.

Gentle lemur case study – Odour results

We unequivocally identified four compounds (2-heptanone; 3-heptanone; 3-octanone; 4-methyl 3-hexanone) that were only present in the chemical profiles of anogenital odour samples collected during the fertile window of the breeding period. A representative chromatogram from the fertile window is shown in Figure 2.

Figure 2. Example chromatogram from female gentle lemur (Hapalemur alaotrensis), anogenital odour sample from fertile period.

The peaks of the key compounds are pointed out with a red arrow.

Gentle lemur case study – Behavioural results

The frequency of male sexual behaviours significantly 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 significant tendency (β ± SE = 0.323 ± 0.184, t-value = 1.754, p-value = 0.081).

Gentle lemur case study – Endocrinological results

We did not find any significant relationship between faecal testosterone levels and the study period (p-value > 0.05).

Ruffed lemur case study – Odour results

We unequivocally identified 12 compounds (benzaldehyde; 1-hexanol, 2-ethyl; benzyl alcohol; dihydromyrcenol; 1-octanol; 2-phenyl-2-propanol; tetrahydrolinalool; linalool; nonanal; menthol; decanal; 2-phenoxyethanol) as being key to the breeding period (i.e., including compounds present only in the breeding period and compounds with much higher relative abundance over the breeding period) in the chemical profiles of anogenital odour samples collected during the breeding period. A typical chromatogram from the breeding period is shown in Figure 3.

Figure 3. Example chromatogram from female red ruffed lemur (Varecia variegata rubra), anogenital odour sample from breeding period.

The peaks of the key compounds are pointed out with a red arrow.

Ruffed lemur case study – Behavioural results

There was a significant effect of period on male sexual behaviours. Ruffed lemurs increased their sexual behaviours while the scent enrichment was present (Est = 2.069; SE = 1.008; z = 2.052; P = 0.040). In comparison, sexual behaviours continued throughout the study after scent exposure and were highest during the post-enrichment period (Est = 1.931; SE = 0.486; 3.978; P < 0.001) (Figure 4).

Figure 4. The mean count per hour of sexual behaviours by male ruffed lemurs (Varecia spp.) during each study period.

BW indicates black-and-white ruffed lemurs (Varecia variegata variegata); RR indicates red ruffed lemurs (Varecia variegata rubra). A * indicates a significant difference (*: P = 0.05-0.01; **: 0.01 > P = 0.001; ***: P < 0.001).

We found that mating behaviours specific to the breeding season were triggered in males once exposed to the scent enrichment. Overall, there was a significant effect of period on mounting in males and this was highest while the enrichment was present (Est = 2.998; SE = 0.817; z = 3.671; P < 0.001; R2 = 0.228).

Ruffed lemur case study – Endocrinological results

We found a large, but not significant (p-value > 0.05), increase of mean faecal testosterone concentration in the post-enrichment condition following scent exposure (Figure 5).

Figure 5. The mean faecal testosterone concentration (pg/mg) of ruffed lemurs (Varecia spp.) during each study period.

BW indicates black-and-white ruffed lemurs (Varecia variegata variegata); RR indicates red ruffed lemurs (Varecia variegata rubra). A * indicates a significant difference (*: P = 0.05-0.01; **: 0.01 > P = 0.001; ***: P < 0.001).