Salivette kit

Fecal (n = 525) and salivary (n = 521) samples were collected monthly from January to December 2019 between 9:00 and 12:00 h, although some saliva data are missing due to sampling constraints. Fresh fecal samples were collected on the same day as saliva. The fecal ball was mixed by hand and two ~20 g subsamples were placed into two zip-lock plastic bags and frozen at −20 °C for separate GC and IgA extraction processes. Saliva was collected using a Salivette^® kit (Sarstedt Inc., Newton, NC, USA) [16 (link),24 (link)] by wiping a synthetic swab inside the buccal area for 30–60 s; collection was complete within 5 min or less. The mahout used food to get the elephant to open its mouth so the sample collector could swipe the mouth with a gloved hand, and rewarded it after collection was complete. Samples were kept in a 4 °C cool box for <8 h until centrifugation at 1500× g for 5 min at 15 °C. Two swabs were collected, and the saliva was pooled for an average volume of 500 µL (100–1500 µL) per sample. Saliva was stored frozen at −20 °C until analysis.

The collection of saliva samples for the analysis of the biomarkers was carried out between 10:00 a.m and 12:30 p.m. with the aim of obtaining stable samples that always responded to the same daily interval since many of the hormones found in saliva respond to a circadian pattern that can affect their presence [1 (link),46 (link),47 (link),48 (link),49 (link)]. Saliva was collected using a Salivette^® kit (SARSTEDT) with a synthetic fiber swab specially developed to detect C and T in saliva. To standardize the sample collection, all the participants were placed in the same time range without having eaten, drank (including caffeinated beverages), brushed their teeth, or practiced physical activity the hour prior to the collection of the salivary sample. Following the manufacturer’s recommendations, the subjects were instructed to place the swab in their mouth without touching it, to keep it in the mouth under the tongue without biting it for approximately 1 min, and return it to the container without handling it. Once collected, the samples were centrifuged for 5 min at 3000 rpm (FUGELAB-GB10 centrifuge) and stored at −20 °C. The centrifuged saliva was analyzed within a month after its collection. T and free C levels were measured using the luminescence immunoassay (IBL, Hamburg, Germany) [50 (link)].

Over the course of 2 days, participants provided saliva samples using Salivette kits (Sarstedt, Germany) upon waking, 30 min after waking, midafternoon (~1500 hours) and 30 min before bedtime. Samples were stored frozen at −20 °C until assayed by luminescence immunoassay reagents according to commercial instructions (Immuno-Biological Laboratories, Hamburg, Germany). Total daily cortisol production was calculated as the area under the curve with respect to ground (AUCg)^{20 (link)} using exact time of sample collection. Outlying values (+2 s.d.) were corrected using winsorizing in the larger sample from the parent study (N=135).^{19 (link)}