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«Item type Thesis or dissertation Authors Davis, Nicolas Citation Davis, N., Schaffner, C. M., & Smith, T. E. (2005). Evidence that zoo visitors ...»

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Nieves et al., (2005); Cuarón et al.,(2008) 3 Collins and Dubach (2000a, 2000b, 2001); Nieves, et al., (2005); Rylands et al. (2000; 2008); Urbani, et al., (2008) 4 Groves (1989); Froehlich, et al., (1991) ; Sampaio, et al., (1993); Medeiros et al., (1997); Collins and Dubach (2000a, 2000b, 2001); Nieves, et al., (2008; 2005); Mittermeier, et al., (2008) immediate replacement with a new breeding adult male. In summary, the group was comprised of one adult male, who was changed in March 2008, four or five adult females, zero to two sub adult males, zero to two sub adult females and one to three infants (see Table 2.3).

Figure 2.4 Photo of Ateles geoffroyi rufiventris at Chester Zoo (Mar and All).

There were 16 births over the eight years, although half of these were either stillborn or did not survive the first 10 days. Of the remaining eight births, one individual was killed during a fight at six months in 2004 and one was euthanized due to poor health at 15 months, leaving six surviving offspring. Other changes in the group, which impact on the data presented in the forthcoming chapters, included a two year old that was born prior to the study period that had to be euthanized in 2000 after being lethally attacked by another spider monkey and in 2002 an adult female was euthanized following prolonged serious health problems.

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2.1.5 Enclosure details The animals were housed in an indoor enclosure measuring 138 m² (11.5m x 12m x to a height of 5m), which was furnished with many ropes, logs, branches, hammocks and various enrichment devices to encourage arboreal behaviour (Figures

2.5 and 2.6). The animals also had access to an outside enclosure measuring 950 m² (60m x 55m x 30m), which contained several trees, large shrubs and 8m pine poles interconnected with ropes or webbing (Figures 2.7 and 2.8). Plants growing in the enclosure included alder (Alnus glutinosa), laurel (Prunus laurocerasus), bamboo (Sasa), poplar (P. canescens), apple (Malus domestica), leylandii (Cupressocyparis leylandii) and buddleia (Buddleja albiflora). During the study period there were some changes to the outdoor enclosure with new poles and ropes installed.

2.16 Husbandry The subjects had free access to both indoor and outdoor enclosures throughout the year, except during sustained cold periods of below 0°C when they did not have night-time access to the outside enclosure. The indoor area was connected to the outdoor area by two tunnels (3m x 0.8m x 0.8m), which passed over

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Figure 2.6 Plan of the general arrangement of the indoor enclosure at Chester Zoo showing the position of the tunnels used during urine collection and separation area.

Figure 2.7 Photograph of the spider monkey outdoor enclosure area at Chester Zoo.

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Figure 2.8 Plan of the general arrangement of the outdoor enclosure showing various vegetation, poles and pool and its relationship with the indoor enclosure.

a keeper area. The animals were normally maintained as one group; however there was the potential to separate animals at the back of the indoor enclosure when required (12m x 2.5m x 4m). This area was used in particular for separations or introductions of individuals and for particular husbandry reasons. Public viewing was achieved inside through four large viewing windows (2.4m x 2.2m each) and from along two sides of the outside enclosure. The tunnels and vegetation did provide a number of areas of privacy where the monkeys could escape from public view if they chose to do so.

The bedding consisted of a deep litter floor covering of wood bark and was cleaned daily, and entirely replaced every 12 – 18 months. Typically the monkeys received three feeds each day. The morning feed consisted of 600g of Primate pellet (Wildlife Feeds, England) and a supplemented protein such as eggs or mealworms.

They were also fed approximately 2 kg of a variety of fruit and vegetables twice a day at varying times, initially supplemented with bread and Vionate vitamin and mineral powder (Sherleys) although this was later removed from their diet in February 2004. Food was presented in a variety of ways throughout the indoor and outdoor enclosure. For example, food could be placed in various hanging log feeders, baskets, sacks and on top of the roof to encourage natural arboreal feeding behaviour.

Water was available ad libitum in the indoor and outdoor enclosures from pools, with the indoor pool changed daily.

2.2 General methods for urine collection

I collected urine three to four times a week between 0700 and 0800 hrs for all subjects for a period of seven years from February 2000 to September 2006 and then again from November 2007 to April 2008. A small proportion of samples were collected by other researchers over this time. Collection coincided with the daily husbandry regime when the monkeys were vacated from their inside enclosure for cleaning. This was advantageous in that no additional potentially stressful routine was required to collect the samples that could have affected the results. During this time the animals typically rested and waited in the tunnels over the keeper area (see Figure 2.6).

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I was able to stand under the tunnels and opportunistically collect urine in aluminium trays without requiring any formal training of monkeys, although successful voids were met with a vocal reward. This approach however did mean that urine samples were not collected from all individuals for each session. The samples were then transferred into labelled plastic vials, logged and immediately stored at – 20˚C until assayed. The animals typically woke at around 06:45 when the lights came on; therefore the samples were nearly always the first void of the day. Samples were collected from all individuals with a total of 4889 samples collected over the whole study period for the various studies (see Table 2.4).

2.3 Assay validation

Previous studies have demonstrated that urine samples can be an effective non-invasive medium by which to measure hormones in Neotropical primates (French, et al., 1996; Schaffner & French, 2004; T. E. Smith & French, 1997a) and specifically in spider monkeys (Campbell, et al., 2001) [typically using an enzymeimmunoassay (EIA)]. There is no previous literature on using EIA for quantifying cortisol in spider monkeys, therefore it was necessary to immunologically and biologically validate the assay (Buchanan & Goldsmith, 2004). The immunological validation is assessed through the demonstration of specificity, accuracy, precision and sensitivity (Diamandus & Christopoulos, 1996; Reimers & Lamb, 1991).

Specificity is the assay’s freedom from interference from other substances other than the one being studied. It can be determined by examining the parallelism of serial dilutions of the study samples with the standard solutions to establish whether the substance in the samples is immunologically identical to the substance in the standard solution (Reimers & Lamb, 1991).

Accuracy of an assay can be determined by adding known amounts of the target hormone to several samples. If the assay is quantitatively accurate the quantity of the hormone that the assay recovers (i.e. measures) should equal the amount added. The percentage of hormone that is recovered can then be calculated. This can be demonstrated by plotting the quantity added against quantity recovered, and the slope of the line should approximate 1 (Reimers & Lamb, 1991). Precision is a measure of the assay’s variability and is expressed as the coefficient of variation (CV) based on replicate measurements of a known sample (quality control) (Reimers & Lamb, 1991). Both the within assay variation (intra-assay) and between assay variation (inter-assay) should be reported. Sensitivity is the smallest amount of unlabelled hormone that can be distinguishable from the absence of hormone (Reimers & Lamb, 1991). To improve sensitivity it may be necessary to increase the volume of the sample, incubation time or temperature.

The biological validation of an assay can be demonstrated by determining whether the assay detects biologically meaningful changes in hormones. For example, it can involve verifying the presence of diurnal variation in the excretion of cortisol metabolites. Plasma cortisol is known to follow a diurnal pattern of excretion in diurnal mammals whereby cortisol levels peak in the early morning when the animals awaken and then gradually decrease throughout the day to reach the lowest point when the animals retire (Coe & Levine, 1995; Crockett, et al., 2000) and has been demonstrated in the plasma of many primate species (Abbott, et al., 2003; M. R.

Clarke, Harrison, & Didier, 1996; Gust, et al., 2000; Saltzman, Schultz-Darken, & Abbott, 1996). A similar diurnal pattern of urinary cortisol excretion has also been reported in several species (Anestis & Bribiescas, 2004; Coe & Levine, 1995;

McCallister, Smith, & Elwood, 2004; Muller & Lipson, 2003; T. E. Smith & French, 1997a).

2.3.1 Aims The first aim of this study was to validate an EIA for the measurement of cortisol in the urine of Colombian black-faced spider monkeys (Ateles geoffroyi rufiventris) in order that the activity of their HPA axis could be measured. This is the first time an enzyme-immunoassay has been carried out for the genus of Ateles.

Immunological validation is assessed through the demonstration of accuracy, specificity, precision and sensitivity (Diamandus & Christopoulos, 1996). To ensure the assay detects biologically meaningful changes in cortisol the validation also evaluated whether levels of cortisol excreted in the urine followed the typical circadian pattern of diurnal animals evident in the plasma and urine of many other primate species (Abbott, et al., 2003; M. R. Clarke, et al., 1996; Gust, et al., 2000;

Saltzman, Schultz-Darken, Wegner, Wittwer, & Abbott, 1998).

2.3.2 Methods Urine samples were collected from all members of the spider monkey group throughout the study period. Change in group composition are summarised in Table

2.2 with samples assayed from a total of eight adults. The demographics of the group are also summarised in Table 2.3.

Levels of excreted cortisol were measured in the selected urine samples using a modified EIA applied previously by Smith and French (1997a) in Wied’s marmosets (Callithrix kuhlii) to quantify excreted urinary cortisol. The cortisol antibodies and horseradish peroxide conjugated cortisol were supplied by the University of California (Davis, USA) while all other chemicals were supplied by Sigma-Aldrich (Poole, Dorset, UK). The assays were carried out on 96 well Microtiter plates (Maxisorp, NUNC™) and samples were run in duplicate (See Appendix A for template). The stock solutions for the cortisol antibody [R4866, raised against a steroid bovine albumin (BSA) in rabbit] and the cortisol horseradish peroxide (Hrp) were stored in dilutions of 1:50 in deionised water and 1:100 in EIA phosphate buffer solution (PBS) respectively, and stored in the freezer at -20°C in small aliquots. The antibody was diluted to 1:12,000 in 0.05M coating buffer for the purposes of the assay (1.59 g Na2CO3, 2.93g NaHCO3, 1L dH20, pH 9.6). This antibody has reported to have cross reactivities of 96% with prenisolone, 66% with prednisone, 60% with cortisone, 2.5% with corticosterone and 1% with various other steroids (Ziegler, et al., 1995).

The antibody (Ab) was made up to the working dilution of 1:12,000 and 50 µl was coated to each of 94 wells. The two remaining wells were used to determine non-specific binding (NSB) and were coated with 50 µl of carbonate buffer to act as a control for non-antibody binding. The plates were then tapped eight times on each side to allow an even coating of the antibody. They were then covered with an adhesive plate sealer and incubated for 12-18 hours in the refrigerator at around 4°C.

The following day all plates, samples and buffers were allowed to reach room temperature. Eppendorf tubes (1.8 ml volume) were then labelled and arranged in the tube racks corresponding to the template (see Appendix A) of the samples to be run.

The samples were run at a working dilution of 1:512. This dilution was made by diluting samples to 1:64 by mixing 10 μl of the urine sample newly defrosted at room temperature in 630 μl of distilled water, followed by making a 1:8 dilution by adding 100 μl of the 1:64 dilutions to 700 μl of distilled water. Distilled water was maintained at room temperature. The working dilution had been previously calculated as that dilution which showed approximately 50% binding against the standard curve, following previous protocols (McCallister, et al., 2004; T. E. Smith & French, 1997a).

The excess antibody was then emptied before each plate was washed six times using the plate washer, three times in each direction to minimise potential drift across the plate (10:1 EIA wash solution: 87.7 g NaCl [1.5M], 5 ml Tween 20 [0.5%], 1L dH20, 350 µl/well). Immediately after the plates were washed, 50 µl of EIA PBS were added to all wells, followed by the 50 µl of the standards diluted in dH20 (n = 10; 1.95-1,000 pg; Sigma, St. Louis, MO) and the samples (50 µl) in duplicate in the appropriate wells. Finally, 50 µl of cortisol Hrp (Batch#12/18/03) diluted to the working solution of 1:22,000 were added to all wells. The plates were then sealed again and left to incubate for 2.5 to 3 hours at room temperature in the dark. The assays were carried out in a laboratory and the temperatures were maintained at a steady 21°C.

Following incubation the plates were washed six times as before, and then 100 µl of the EIA substrate solution were added to all wells (12.5 ml EIA citrate buffer [0.05 m, pH 4.0:9.61 g citric acid (anhydrous), 1 l dH20], 125 µl EIA ABTS [40 Mm, 2,2’AZINO-bis (3 ethylbenzthmoline-6-sulfonic acid) Diammonium salt],

0.329 g ABTS, 15 ml dH20, pH to 6.0; 40 µl EIA H202 [2.0%, 0.5 M, 500 µl H202 (30%) 8 M, 7.5 ml dH20]). The plates were then left on a plate shaker to develop before being read using a microplate reader (Dynatech MR700) and the software Revelation version 4.22. The plates were left until the optimal density at 405 nm for the control wells with no hormone (Bo) measured 1.0. Any sample with a coefficient of variation (CV) greater than 10% was repeated.

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