Journal of the American Association for Laboratory Animal Science Copyright 2017 by the American Association for Laboratory Animal Science
Vol 56, No 2 March 2017 Pages 181–189
Comparison of Saliva Collection Methods for the Determination of Salivary Cortisol Levels in Rhesus Macaques (Macaca mulatta), Cynomolgus Macaques (Macaca fascicularis), and African Green Monkeys (Chlorocebus aethiops) Kamala J Rapp-Santos,1,* Louis A Altamura,2 Sarah L Norris,3 Luis A Lugo-Roman,1 Pedro J Rico,1 and Christian C Hofer1 The ability to quickly and accurately determine cortisol as a biomarker for stress is a valuable tool in assessing the wellbeing of NHP. In this study, 2 methods of collecting saliva (a commercial collection device and passive drool) and the resulting free salivary cortisol levels were compared with total serum cortisol concentration in rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis) and African green monkeys (Chlorocebus aethiops) at 2 collection time points. Serum and salivary cortisol levels were determined using a competitive quantitative ELISA. In addition, both saliva collection methods were evaluated for volume collected and ease of use. Compared with passive drool, the experimental collection device was more reliable in collecting sufficient volumes of saliva, and the resulting salivary cortisol values demonstrated stronger correlation with serum cortisol concentration in all species and collection days except cynomolgus macaques on day 1. This saliva collection device allows quick and reliable sample collection for the determination of salivary cortisol levels. In addition, the results might provide a useful tool for evaluating hypothalamic-pituitary-adrenal axis activity or the physiologic stress reaction in NHP as well as a biomarker of psychologic stress states in a variety of situations. Abbreviations: SS, experimental collection device; PD, passive drool
Scientists have consistently recognized stress in laboratory animals in response to both invasive and routine procedures.3,11,30,31,35 Research-related manipulations, social housing arrangements, husbandry procedures, and even personnel entering an animal room can contribute to acute or chronic stress.3 The 2011 edition of the Guide for the Care and Use of Laboratory Animals as well as the 1985 amendments to the Animal Welfare Act place an emphasis on promoting the psychologic wellbeing of NHP in laboratory research.1,26 Specific recommendations such as social housing and the provision of environmental enrichment enhance NHP wellbeing by facilitating the expression of species-typical behaviors. In addition to the animal welfare considerations, stress may have an underestimated effect on research and can potentially confound study results. A quick, simple tool to assess stress in NHP would be valuable for evaluating potential influences and assessing the effects of enrichment and other interventions on animal wellbeing. Glucocorticoid measurement remains one of the most frequently evaluated biomarkers of stress in mammals, including NHP.2,3,28,34,36,48,51 In response to stress, the hypothalamic– pituitary–adrenal axis is activated to mobilize a variety of compounds for adaptive behavior and physiologic responses. These compounds include glucocorticoids and corticotropin-releasing
Received: 17 Aug 2016. Revision requested: 23 Sep 2016. Accepted: 24 Oct 2016. 1Veterinary Medicine Division, 2Diagnostic Systems Division,, and 3Office of Biostatistics, The United States Army Research Institute of Infectious Diseases, Fort Detrick, Maryland. *Corresponding author. Email: [email protected]
factor, the catecholamines epinephrine and norepinephrine, and immune factors such as cytokines and lymphocytes. These hormones and cytokines are often referred to as primary stress mediators.35 Cortisol is an important adrenal biomarker and analyte in stress research, exhibiting both normal variation in response to circadian rhythm as well as elevations in response to stress.15,21 Chronic activation of the hypothalamic–pituitary– adrenal axis has been associated with psychologic and physical pathology.42 For example, stress markedly affects metabolism and leads to suppression of the immune and reproductive systems.11,25 Repeated transient increases in cortisol and extremely high or low basal levels may further impair health.5 Measuring cortisol over time can reveal how animals respond to acute and chronic stressors, such as changes in their social or physical environments. Investigators have collected a variety of biosamples to measure cortisol, each with different advantages and disadvantages. Serum or plasma cortisol is one of the most widely used biomarkers for stress in mammals, including NHP.3,5,12,22,25,29,48 Unless a NHP is trained for voluntary blood sampling, serum cortisol assessment requires physical or chemical restraint to collect blood from a peripheral vein.42 A potential drawback to this method is that serum cortisol levels increase within minutes of a stressful event.3,23,42 Therefore, if samples are not collected immediately after restraint or sedation, the measurement likely reflects the animal’s reactivity to capture or restraint rather than baseline levels. Other noninvasive methods, such as urine and fecal cortisol measurement, reflect circulating steroid levels over several hours to days, whereas hair cortisol levels can reflect 181
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accumulation over months to years.8 In addition, fecal and urine cortisol are subject to potential degradation or contamination if samples are not collected immediately.33 Saliva collection represents an alternative method to measure cortisol and has several advantages over other biosamples. Cortisol is highly lipid-soluble and rapidly diffuses from the blood into the acinar cells of the salivary glands at a consistent 10% to 15% fraction of circulating levels.27,31 In contrast to serum or plasma, which allows only total cortisol measurement, salivary cortisol analysis represents the unbound, biologically active cortisol.31,50 In addition, free salivary cortisol levels appear to be independent of saliva flow rate.24,31 Another major advantage is that peak salivary cortisol levels lag behind serum levels after the onset of a stressor, reducing the potential confounders of researcher presence and physical restraint and allowing better assessment of baseline cortisol.31,33 Saliva collection represents a noninvasive, accurate, and simple method to assess acute stress in NHP in response to a variety of potential stimuli. Researchers have reported free salivary cortisol measurement in several species of NHP including squirrel monkeys, infant and adult rhesus macaques, Tibetan macaques, marmosets, gorillas, and orangutans.6,10,17,24,33,41,42,46,49 Salivary cortisol levels can vary considerably between species. For example, squirrel monkeys (Saimiri sciureus) have demonstrated salivary cortisol levels that are 100 times greater than those in humans,49 whereas other NHP species demonstrate levels similar to that of humans.4,6,10,24,42,46 Diurnal variability in cortisol has been demonstrated in male rhesus macaques, with values higher in the early morning and late afternoon than the middle of the day.24 Variation has also been observed in rhesus macaques according to male dominance rank, with lower-ranked animals having higher mean cortisol levels.24 Various studies report the use of several saliva collection substrates in NHP including cotton ropes, buds, and rolls.10,33,39,42 Steroid hormones show consistent and high-percentage recovery from cotton rope and rolls and do not appear to depend on salivary flow rate.24,31 However, one study suggested that low specimen volume may affect the percentage of cortisol recovered in humans,19 and others have demonstrated variable results in cortisol recovery depending on the collection technique.14,19,44 The first objective of the current study was to compare salivary cortisol levels, collection volume, and ease of use for 2 different methods of saliva collection—passive drool (PD) and a commercially available salivary collection device (SS)—in 3 NHP species commonly used in biomedical research. The second goal was to demonstrate the correlation between serum and salivary cortisol levels to determine whether salivary cortisol measurements accurately reflect serum cortisol. Finally, we sought to determine the range of cortisol levels in both the serum and saliva of anesthetized healthy adult cynomolgus macaques, rhesus macaques, and African green monkeys housed in a typical biomedical research setting. Although salivary cortisol ranges have been published for rhesus macaques,6,12,24,33 little information is available regarding cynomolgus macaques or African green monkeys. This information could be helpful in refining methods for voluntary saliva collection. Furthermore, these baseline values could be used to assess the effect of various husbandry and enrichment parameters on the overall wellbeing of NHP used in research.
Materials and Methods
Animals and husbandry. The study population consisted of 20 healthy, adult NHP from each of 3 species (Macaca mulatta, M. fascicularis, and Chlorocebus aethiops) with approximately equal
ratios of male and female animals. This study was conducted under an IACUC-approved protocol in an AAALAC-accredited facility and in compliance with the Guide,26 Animal Welfare Act and Regulations,1 Public Health Service Policy,40 and other federal statutes and regulations relating to animals and experiments involving animals. The housing environment was maintained at 69 to 75 °F (22.6 to 23.9 °C), with relative humidity of 30% to 70%, and on a 12:12-h light:dark cycle. A commercial diet (2050 Teklad Global 20% Protein Primate Diet, Harlan Laboratories, Frederick, MD) was provided to NHP twice daily, as well as fresh produce once a day. Chlorinated and filtered municipal water was provided without restriction through an automated watering system (Edstrom Industries, Waterford, WI). Manipulanda (Challenge Ball, Kong, football, and Dental Star [Bio-Serv, Frenchtown, NJ]) were available at all times in the cage as a form of physical enrichment, according to an established schedule as outlined by our institute’s husbandry and care program. Clinically healthy African green monkeys (n = 20; 10 female and 10 male; age, 15.3 ± 3.6 y [mean ± 1 SD]; weight, 5.2 ± 1.3 kg) were evaluated in this study. They originated from St Kitts and arrived at our facility at least 2 y prior to the start of this study. The study population was housed in 3 separate rooms within the same corridor within 4.3-ft2 cages (Allentown Caging Equipment, Allentown, NJ). At the start of the study, 8 of the 20 African green monkeys were housed in partial contact with a cagemate according to previous behavioral assessments. Animals in partial contact were housed next to a potential cagemate, with either a mesh or solid transparent divider to allow for protected contact. The remaining African greens were singly housed but had visual and auditory contact with conspecifics at all times. The clinically healthy cynomolgus macaques (n = 20; 10 female and 10 male; age, 9.0 ± 2.2 y; weight, 7.0 ± 2.6 kg) evaluated in this study arrived at our facility from either China or Cambodia at least 2 y prior to study, with the exception of one animal that arrived 2 mo prior to study. This population was housed in 3 different rooms within the same corridor. Most animals were housed in 4.3-ft2 cages (Allentown Caging Equipment), with visual and auditory contact with conspecifics at all times; 3 of the 20 animals were singly housed in 6.0-ft2 caging, given their size. Overall, 6 of the 20 animals were in full-contact housing with one cagemate, 5 were in partial contact, and 9 were singly housed in light of previous behavioral assessments. Animals in partial contact were housed next to a potential cagemate, with a mesh divider to allow for protected contact. Pairs in full contact were housed in double-wide caging, to allow appropriate floor space for each animal. Clinically healthy rhesus macaques (n = 20; 9 female and 11 male; age, 5.7 ± 1.3 y; weight, 6.5 ± 1.8 kg) were also evaluated in this study. They arrived to our facility from China at least 6 mo prior to the start of the study. This population was housed in 2 rooms within the same corridor. Most animals were housed in 4.3-ft2 cages (Allentown Caging Equipment), with visual and auditory contact with conspecifics at all times; however, 2 animals were singly housed in 6.0-ft2 caging because of their size. Overall, 14 macaques were in full-contact housing with one cagemate, 2 were in partial contact, and 4 were singly housed because of previous behavioral assessments. Animals in partial contact were housed next to a potential cagemate, with a mesh divider to allow for protected contact. Pairs in full contact were housed in double-wide caging, to allow appropriate floor space for each animal. All macaques were seronegative for Macacine herpesvirus 1, SIV, simian retrovirus type D, and simian T-lymphotropic
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leukemia virus. African green monkeys were seronegative for Cercopithecine herpesvirus 2, SA11 rotavirus, SIV, and simian T-lymphotropic leukemia virus. All animals were tested twice annually for Mycobacterium tuberculosis by tuberculin skin testing and remained negative throughout the study. Prior to study initiation, each NHP received baseline health assessment from a qualified veterinarian, including a physical exam, CBC, and blood chemistry. Physical exams revealed no significant health concerns for any of the NHP in this study. Study design. A 2×2 crossover study design was chosen to compare collection methods.7 During the first iteration of the experiment, animals were randomized into 1 of 2 collection sequence groups stratified by species and sex. One group was sampled by using the SS device first, followed by PD collection. In the other group, PD was collected first, followed by use of the SS. The experiment was repeated with the same animals 4 d later, with the sequences reversed from the original order to rule out potential effects of saliva collection order. In addition, at each sampling time point, one blood sample was collected to evaluate correlation between salivary and serum cortisol levels. All sample collection occurred in the morning between 0800 to 1200 to reduce variability associated with the natural diurnal cortisol variation.15,21 Although voluntary saliva collection techniques have been published for some NHP species, we sought to demonstrate sufficient correlation between salivary and serum cortisol levels from samples collected within a specified period, thus necessitating the sedation of NHP for sample collection. Sample collection and processing. Feed was withheld from animals for 12 h prior to all anesthetic procedures. To achieve adequate sedation, a mixture of ketamine (9.0 mg/kg) and acepromazine (0.1 mg/kg) was administered intramuscularly in the caudal thigh by using a 21- to 25-gauge, 3/8- to 1-in. needle. Approximately 5 to 10 min after sedation, blood samples were collected from the femoral vein, samples were centrifuged, and serum transferred to individually labeled cryovials for storage at –80 °C until needed for the assay. To account for the potential lag in salivary cortisol relative to serum cortisol, the first saliva sample was collected approximately 20 min after sedation and the second approximately 25 min after sedation. The SS device (SuperSAL Oral Fluid Collection Device, Oasis Diagnostics, Vancouver WA) uses a highly absorbent cylindrically shaped pad that can be compressed or centrifuged to collect the sample (Figure 1 A). The absorbent pad is approximately 3 inches long, has a sample volume indicator, and is attached to a 3-in. plastic handle. The pad was placed in the cheek pouch or along the side of the tongue for 5 min, and then was removed and placed in the supplied syringe for processing (Figure 1 B). The entire syringe was placed into a 15-mL polypropylene centrifuge tube. For comparison, the PD collection technique was also used to collect saliva. For this technique, the animal’s head was slightly draped over the edge of the exam table, and saliva was collected into a culture dish as it passively flowed out of the oral cavity for approximately 5 min. Saliva was removed from the culture dish by using a disposable plastic pipette and was placed directly into a 15-mL polypropylene centrifuge tube. All saliva samples were centrifuged (3000 × g for 5 min) to enhance extraction from the absorbent tube, transferred to individually labeled cryovials, and stored in a freezer (–80 °C) until needed for the assay. Measurement of serum and salivary cortisol levels. Serum and salivary cortisol values were obtained by using commercial competitive ELISA (Cortisol Parameter KGE008B, R and D Systems, Minneapolis, MN) with 50 µL saliva and 10 µL serum, to allow for duplicate analysis. Serum samples were pretreated (trichloroacetic acid and buffer) according to manufacturer’s
Figure 1. (A) The experimental saliva collection device and (B) placement of the device in the mouth of a sedated rhesus macaque. Note the blue line, indicating sufficient saliva volume.
recommendations, to remove potentially interfering proteins and protein-bound cortisol. To generate values that fell within the standard curve, serum samples were diluted 80-fold and saliva samples diluted 10- to 20-fold as needed. Absorbance was measured on a microplate reader (Infinite 200Pro, Tecan Systems, Mannedorf, Switzerland), and a 4-parameter logistic curve-fit was generated by using online software (MyAssays, www.myassays.org).37 Data analysis. Data were analyzed by using SAS Version 9.4 (SAS Institute, Cary, NC).45 Cortisol values and volume amounts were compared by using mixed-model ANOVA. If a saliva collection technique was unsuccessful, the volume was recorded as 0 and included in the analysis. Method, species, sequence, and period were treated as fixed effects. Subject was treated as a random effect. Posthoc Tukey tests were used for pairwise comparisons between species. Covariates such as sex, weight, pair status, age, cage position, and room order were also singly tested for influence on cortisol values. Correlation of serum and salivary cortisol values was assessed through Pearson productmoment correlation. Mean volume and cortisol concentrations are presented as mean ± 1 SD, and statistical significance was set at a P value of less than 0.05. 183
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Results
Assay performance. The mean intraassay coefficient of variation was 9.9% for serum and 11.2% for saliva. The mean interassay coefficient of variation was 11.7%. The manufacturer reported an average recovery from serum of 104% (range, 92% to 114%) and from saliva of 101% (range, 80% to 118%). The recovery of standard dilutions for this study ranged from 97% to 106%. The mean minimal detectable dose of cortisol was 0.071 ng/mL, according to manufacturer assessments. Saliva volume. In the African green monkey group, sufficient saliva samples (defined as greater than or equal to 0.05 mL) were collected by using SS from 36 of 40 attempts (90%) and by using the PD technique from 26 of 40 collection attempts (65%) over both days. The success rate was similar in cynomolgus macaques, with 36 successful attempts from a total of 40 (90%) by using SS and 27 of 40 (67.5%) by PD. In rhesus macaques, sufficient saliva samples were obtained from all attempts with SS, and from 28 of 40 (70%) by using the PD collection technique. For African green monkeys, the volume of saliva collected was greater from SS than PD at both day 1 (F1,18 = 8.66, P = 0.0087) and day 2 (F1,18 = 13.20, P = 0.0019). In the cynomolgus macaque group, the volume collected again was greater when using SS than PD, but only for day 1 (F1,18 = 8.74, P = 0.0085); no significant difference was observed on day 2 (F1,18 = 0.35, P = 0.5624). In rhesus macaques, the volume of saliva collected was greater from using SS than PD only on day 2 (F1,18 = 5.41, P = 0.0319). When comparing saliva volume between species, the only significant difference observed was when using SS during day 1 (F2,56 = 4.61, P = 0.0140). Tukey posthoc tests showed a significant difference between cynomolgus macaques and rhesus macaques, with greater volume collected from rhesus macaques (P = 0.0173). Saliva volume results are summarized in Table 1. In addition, period effects were nonsignificant for all species, methods, and iterations, indicating that the order of method collection did not have a significant effect on saliva volume. Free saliva cortisol. For African green monkeys, salivary cortisol values were greater in the samples collected by PD compared with SS, but significant differences occurred only on day 2 (F1,13.5 = 19.24, P = 0.0007). In the cynomolgus macaques, salivary cortisol values were greater in samples collected by PD compared with SS on day 1 (F1,11.5 = 15.60, P = 0.0021) and day 2 (F1,14.5 = 7.74, P = 0.0143). For rhesus macaques, no significant differences were observed for either iteration. Comparing salivary cortisol levels between species revealed no significant differences on either day when collected by PD. However, for saliva samples collected with SS, significant differences were found between species on day 2 (F2,53 = 6.95, P = 0.0021) but not day 1 (F2,52 = 2.02, P = 0.1430). Tukey posthoc tests showed a significant difference between cynomolgus and rhesus macaques, with greater salivary cortisol values in rhesus macaques (P = 0.002). Salivary cortisol results (ANOVA) are summarized in Table 2. Figure 2 summarizes the distribution of mean salivary cortisol values for each animal as obtained by using the SS and PD methods in each species. If only one saliva sample was collected by using the indicated method, that value was included rather than the mean. Period effects were nonsignificant for all species, methods, and iterations, indicating that order of method collection did not have a significant effect on cortisol values. Total serum cortisol. Serum was successfully collected from all 20 animals within each species. For African green monkeys, the mean serum cortisol was 384.8 ng/mL (1 SD, 123.3 ng/mL) on day 1 and 355.5 ng/mL (175.6 ng/mL) on day 2. For cynomolgus macaques, the mean serum was 386.0 ng/mL (108.9 ng/mL) on day 1 and 362.9 ng/mL (118.9 ng/mL) on day 2. Finally, in the
rhesus macaque group, the mean serum cortisol was 521.9 ng/ mL (162.3 ng/mL) on day 1 and 463.2 ng/mL (155.6 ng/mL) on day 2. On Day 1, rhesus macaques had significantly higher serum cortisol values than either African green monkeys (P = 0.0055) or cynomolgus macaques (P = 0.0059). No other significant differences were noted in serum cortisol levels between species. In addition, serum cortisol did not differ significantly between the 2 collection time points for any group. Serum and saliva cortisol correlation. Strong correlations were observed when comparing serum cortisol and salivary cortisol obtained by using SS in all species and collection days (r > 0.527), except for cynomolgus macaques on day 1 (r = 0.179; Figure 3). Less prominent correlations were obtained when comparing serum cortisol and salivary cortisol obtained from PD, with significant correlations only in the African green monkeys (r > 0.760; Figure 3). The correlation between salivary cortisol levels obtained by using the 2 saliva collection methods was very strong for all species and collection days (r > 0.747; Figure 3). Correlation results, including Pearson correlation coefficients and significance level, are summarized in Table 3. Covariate analysis. In the African green monkey group, the only significant covariate was cage position on day 1, when the salivary cortisol was greater in the NHP housed in the bottom cage compared with the top cage (F1,16.5 = 10.39, P = 0.0051). This effect was not observed on day 2 or for the serum cortisol values on either day. In the cynomolgus macaque group, sex and weight had significant effects on salivary cortisol levels measured on day 2. Female macaques had higher salivary cortisol levels than male (F1,27 = 33.64, P < 0.0001), and smaller cynomolgus macaques had higher salivary cortisol levels compared with larger animals (F1,17.1 = 8.24, P = 0.0105). Pair status had a significant effect on salivary cortisol on both days, with single-housed animals demonstrating lower salivary cortisol concentrations compared with partial or fully paired animals. None of the covariates examined was significant in the rhesus macaque group. Room entry order was specifically included as a covariate, because NHP further down on the random-order sample-collection list were exposed to more activity prior to sedation. For all 3 species, no significant effect of room entry order was noted on either serum or salivary cortisol (P > 0.25 for all groups).
Discussion
This study is the first to measure free salivary cortisol concentrations in samples from NHP species collected by using this commercially available human device and explores the relationship between salivary and serum cortisol concentrations with regard to the 2 collection techniques used. In addition, our study generated new data on salivary cortisol values in African green monkeys and cynomolgus macaques that—to our knowledge—have not yet been reported in the literature. One important conclusion from this study is that despite the measurement of a lower cortisol concentration from SS than PD, cortisol values obtained from SS samples were a better predictor of serum cortisol concentration than values obtained from PD samples. Previous studies conducted with human saliva samples similarly found that salivary cortisol concentrations were lower when obtained by using a cotton-based absorbent material and suggest that the absorbent material can reduce cortisol concentrations and create random errors.14,19,47 A potential reason for this effect may be the binding characteristics of the material used or of the plastic container or low sample volume.44 Regarding the collection device used in the current study, the manufacturer has claimed that the absorbent pad, which is a
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Table 1. Volume of saliva collected (mL; mean ± 1 SD; n = 20 samples per group) from 3 species of NHP by using either the experimental collection device (SS) or passive drool (PD) Day 1 SS
PD
Day 2 F1,18
Pa
SS
PD
F1,18
Pa
C. aethiops
0.36 ± 0.40
0.16 ± 0.24
8.66
0.0087
0.60 ± 0.62
0.27 ± 0.50
13.2
0.0019
M. fascicularis
0.88 ± 1.13b
0.55 ± 1.06
8.74
0.0085
0.79 ± 0.96
1.10 ± 2.96
0.35
0.5624
M. mulatta
0.25 ± 0.17b
0.17 ± 0.44
1.10
0.3088
0.54 ± 0.78
0.33 ± 0.47
5.41
0.0319
aANOVA group
bANOVA group
significance level significance level between species: P = 0.017
Table 2. Free saliva cortisol concentration (ng/mL; mean ± 1 SD; range) in samples from 3 species of NHP collected by using the experimental collection device (SS) or as passive drool (PD) Day 1 C. aethiops
M. fascicularis
M. mulatta
aANOVA group
bANOVA group
Day 2
SS
PD
F
Pa
SS
PD
F
n = 18
n = 11
1, 9.4 = 0.18
0.6799
n = 19
n = 12
1, 13.5 = 19.24
29.7 ± 24.9
34.0 ± 24.6
19.4 ± 13.0
42.4 ± 30.3
2.0–107.9
7.4–87.4
2.3–101.1
7.0–170.2
n = 18
n = 14
18.1 ± 34.6
31.6 ± 42.6
1.3–166.0
2.8–159.8
n = 20
n = 10
37.6 ± 28.9
35.0 ± 24.3
3.1–101.6
5.0–63.1
1, 11.5 = 15.6 0.0021
1.8, 41.0 = 0.01 0.9896
n = 18
n = 13
12.7 ± 10.7b
28.0 ± 28.3
1.0–64.8
2.1–97.2
n = 20
n = 18
30.8 ± 19.4b
30.7 ± 21.1
2.8–54.1
4.5–165.1
Pa 0.0007
1, 14.5 = 7.74 0.0143
1, 16.3 = 0.23 0.6371
significance level between methods significance level between species: P = 0.002
Figure 2. Free salivary cortisol values (ng/mL) obtained from samples collected by using the experimental collection device (SS) or passive drool (PD) for 3 species of NHP. Each value depicted represents the mean saliva cortisol concentration from days 1 and 2, obtained from samples collected by using the indicated method. For animals in which only a single cortisol value was obtained, only that value is depicted.
proprietary noncellulose material, does not significantly affect the recovery of salivary cortisol. However, the manufacturer mentions the potential effects of mucin aggregates on various proteins and states that the PD method is prone to increased deviation in analyte recovery because recovery is more highly dependent on the mucin concentration than on the collection device, which binds mucin aggregates.20 In addition, previous
studies did not investigate the effect of small saliva volumes obtained from the absorbent material of the collection device. The potential cause of the discrepancy between salivary cortisol levels in samples collected by using the 2 methods warrants further investigation. However, the data show that the salivary cortisol values obtained with SS demonstrate strong correlation with serum cortisol concentration, and the values obtained by SS and PD themselves are also strongly correlated. One consideration regarding the differences in correlation between the 2 collection methods is that obtaining adequate saliva volumes was more difficult when using the PD collection technique, resulting in more missing data points. The relatively limited data available to compare salivary cortisol levels obtained by PD with serum cortisol concentration may have resulted in weaker correlations. A single outlier heavily influenced the weak correlation observed between salivary cortisol levels obtained by using SS and serum cortisol concentration in the cynomolgus macaque group during day 1. The saliva from this animal contained a high salivary cortisol concentration when obtained by both SS and PD (153 ng/mL and 166 ng/mL, respectively), which were not reflected in the serum cortisol level. The volumes of saliva obtained from this animal were more than sufficient by both methods, and there was no evidence of gross contamination with blood or food material. Correlation of salivary and serum or plasma cortisol levels has been demonstrated in many species, including pigs, sheep, goats, horses, cattle, and humans.9,15,16,18,28,29,32,38,44 After stress onset, cortisol concentrations rise in parallel, with peak salivary cortisol levels lagging behind serum cortisol concentration.30 In dairy cattle, the salivary cortisol concentration peaked 10 min after the plasma cortisol concentration, whereas in dogs, the 185
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Figure 3. Relationship between total serum cortisol and free salivary cortisol collected with the experimental device (SS) in (A) C. aethiops, (B) M. fascicularis, and (C) M. mulatta. Correlation was significant in all cases except for M. fascicularis on day 1. Similarly, the relationship between total serum cortisol and salivary cortisol in passive drool (PD) is demonstrated for (D) C. aethiops, (E) M. fascicularis, and (F) M. mulatta. Correlation was significant only for C. aethiops on both days. Finally, the relationship between free salivary cortisol collected by using the experimental device and in passive drool is demonstrated for (G) C. aethiops, (H) M. fascicularis, and (I) M. mulatta. Correlation was very strong for in all cases; the Pearson product-moment correlation is reported for each comparison.
salivary cortisol concentration did not change within 4 min of restraint.23,32 In humans, the time lag has been reported to be several minutes to as long as 20 to 30 min.31,33,42 The results from this study demonstrated a greater mean volume of saliva by SS collection compared with PD in all cases except the cynomolgus macaque group on day 2. This aberrant result was due to a single outlier, which generated 13.5 mL saliva during PD collection. The maximal saliva volume collected by using SS was approximately 3.5 mL due to the size and absorbency limitations of the material. This feature
may be a potential limitation of SS that could be alleviated by use of multiple collection devices. The mean saliva volumes collected from the rhesus macaque group on day 1 were not significantly greater when collected by SS than PD. The lack of significance in this one group might reflect the relatively small saliva volumes collected by both methods. Regarding ease of use, SS was substantially less cumbersome, given that, once placed, the absorbent material required little attention until the end of the collection period. In contrast, PD required constant attention and effort, especially in animals that produced small
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Table 3. Correlation of serum and salivary cortisol levels in 3 NHP species from which saliva was collected by using the experimental collection device (SS) or as passive drool (PD) Day 1
Day 2
n
ra
Pb
n
ra
Pb
C. aethiops
18
0.685
0.002
19
0.708
Vol 56, No 2 March 2017 Pages 181–189
Comparison of Saliva Collection Methods for the Determination of Salivary Cortisol Levels in Rhesus Macaques (Macaca mulatta), Cynomolgus Macaques (Macaca fascicularis), and African Green Monkeys (Chlorocebus aethiops) Kamala J Rapp-Santos,1,* Louis A Altamura,2 Sarah L Norris,3 Luis A Lugo-Roman,1 Pedro J Rico,1 and Christian C Hofer1 The ability to quickly and accurately determine cortisol as a biomarker for stress is a valuable tool in assessing the wellbeing of NHP. In this study, 2 methods of collecting saliva (a commercial collection device and passive drool) and the resulting free salivary cortisol levels were compared with total serum cortisol concentration in rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis) and African green monkeys (Chlorocebus aethiops) at 2 collection time points. Serum and salivary cortisol levels were determined using a competitive quantitative ELISA. In addition, both saliva collection methods were evaluated for volume collected and ease of use. Compared with passive drool, the experimental collection device was more reliable in collecting sufficient volumes of saliva, and the resulting salivary cortisol values demonstrated stronger correlation with serum cortisol concentration in all species and collection days except cynomolgus macaques on day 1. This saliva collection device allows quick and reliable sample collection for the determination of salivary cortisol levels. In addition, the results might provide a useful tool for evaluating hypothalamic-pituitary-adrenal axis activity or the physiologic stress reaction in NHP as well as a biomarker of psychologic stress states in a variety of situations. Abbreviations: SS, experimental collection device; PD, passive drool
Scientists have consistently recognized stress in laboratory animals in response to both invasive and routine procedures.3,11,30,31,35 Research-related manipulations, social housing arrangements, husbandry procedures, and even personnel entering an animal room can contribute to acute or chronic stress.3 The 2011 edition of the Guide for the Care and Use of Laboratory Animals as well as the 1985 amendments to the Animal Welfare Act place an emphasis on promoting the psychologic wellbeing of NHP in laboratory research.1,26 Specific recommendations such as social housing and the provision of environmental enrichment enhance NHP wellbeing by facilitating the expression of species-typical behaviors. In addition to the animal welfare considerations, stress may have an underestimated effect on research and can potentially confound study results. A quick, simple tool to assess stress in NHP would be valuable for evaluating potential influences and assessing the effects of enrichment and other interventions on animal wellbeing. Glucocorticoid measurement remains one of the most frequently evaluated biomarkers of stress in mammals, including NHP.2,3,28,34,36,48,51 In response to stress, the hypothalamic– pituitary–adrenal axis is activated to mobilize a variety of compounds for adaptive behavior and physiologic responses. These compounds include glucocorticoids and corticotropin-releasing
Received: 17 Aug 2016. Revision requested: 23 Sep 2016. Accepted: 24 Oct 2016. 1Veterinary Medicine Division, 2Diagnostic Systems Division,, and 3Office of Biostatistics, The United States Army Research Institute of Infectious Diseases, Fort Detrick, Maryland. *Corresponding author. Email: [email protected]
factor, the catecholamines epinephrine and norepinephrine, and immune factors such as cytokines and lymphocytes. These hormones and cytokines are often referred to as primary stress mediators.35 Cortisol is an important adrenal biomarker and analyte in stress research, exhibiting both normal variation in response to circadian rhythm as well as elevations in response to stress.15,21 Chronic activation of the hypothalamic–pituitary– adrenal axis has been associated with psychologic and physical pathology.42 For example, stress markedly affects metabolism and leads to suppression of the immune and reproductive systems.11,25 Repeated transient increases in cortisol and extremely high or low basal levels may further impair health.5 Measuring cortisol over time can reveal how animals respond to acute and chronic stressors, such as changes in their social or physical environments. Investigators have collected a variety of biosamples to measure cortisol, each with different advantages and disadvantages. Serum or plasma cortisol is one of the most widely used biomarkers for stress in mammals, including NHP.3,5,12,22,25,29,48 Unless a NHP is trained for voluntary blood sampling, serum cortisol assessment requires physical or chemical restraint to collect blood from a peripheral vein.42 A potential drawback to this method is that serum cortisol levels increase within minutes of a stressful event.3,23,42 Therefore, if samples are not collected immediately after restraint or sedation, the measurement likely reflects the animal’s reactivity to capture or restraint rather than baseline levels. Other noninvasive methods, such as urine and fecal cortisol measurement, reflect circulating steroid levels over several hours to days, whereas hair cortisol levels can reflect 181
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accumulation over months to years.8 In addition, fecal and urine cortisol are subject to potential degradation or contamination if samples are not collected immediately.33 Saliva collection represents an alternative method to measure cortisol and has several advantages over other biosamples. Cortisol is highly lipid-soluble and rapidly diffuses from the blood into the acinar cells of the salivary glands at a consistent 10% to 15% fraction of circulating levels.27,31 In contrast to serum or plasma, which allows only total cortisol measurement, salivary cortisol analysis represents the unbound, biologically active cortisol.31,50 In addition, free salivary cortisol levels appear to be independent of saliva flow rate.24,31 Another major advantage is that peak salivary cortisol levels lag behind serum levels after the onset of a stressor, reducing the potential confounders of researcher presence and physical restraint and allowing better assessment of baseline cortisol.31,33 Saliva collection represents a noninvasive, accurate, and simple method to assess acute stress in NHP in response to a variety of potential stimuli. Researchers have reported free salivary cortisol measurement in several species of NHP including squirrel monkeys, infant and adult rhesus macaques, Tibetan macaques, marmosets, gorillas, and orangutans.6,10,17,24,33,41,42,46,49 Salivary cortisol levels can vary considerably between species. For example, squirrel monkeys (Saimiri sciureus) have demonstrated salivary cortisol levels that are 100 times greater than those in humans,49 whereas other NHP species demonstrate levels similar to that of humans.4,6,10,24,42,46 Diurnal variability in cortisol has been demonstrated in male rhesus macaques, with values higher in the early morning and late afternoon than the middle of the day.24 Variation has also been observed in rhesus macaques according to male dominance rank, with lower-ranked animals having higher mean cortisol levels.24 Various studies report the use of several saliva collection substrates in NHP including cotton ropes, buds, and rolls.10,33,39,42 Steroid hormones show consistent and high-percentage recovery from cotton rope and rolls and do not appear to depend on salivary flow rate.24,31 However, one study suggested that low specimen volume may affect the percentage of cortisol recovered in humans,19 and others have demonstrated variable results in cortisol recovery depending on the collection technique.14,19,44 The first objective of the current study was to compare salivary cortisol levels, collection volume, and ease of use for 2 different methods of saliva collection—passive drool (PD) and a commercially available salivary collection device (SS)—in 3 NHP species commonly used in biomedical research. The second goal was to demonstrate the correlation between serum and salivary cortisol levels to determine whether salivary cortisol measurements accurately reflect serum cortisol. Finally, we sought to determine the range of cortisol levels in both the serum and saliva of anesthetized healthy adult cynomolgus macaques, rhesus macaques, and African green monkeys housed in a typical biomedical research setting. Although salivary cortisol ranges have been published for rhesus macaques,6,12,24,33 little information is available regarding cynomolgus macaques or African green monkeys. This information could be helpful in refining methods for voluntary saliva collection. Furthermore, these baseline values could be used to assess the effect of various husbandry and enrichment parameters on the overall wellbeing of NHP used in research.
Materials and Methods
Animals and husbandry. The study population consisted of 20 healthy, adult NHP from each of 3 species (Macaca mulatta, M. fascicularis, and Chlorocebus aethiops) with approximately equal
ratios of male and female animals. This study was conducted under an IACUC-approved protocol in an AAALAC-accredited facility and in compliance with the Guide,26 Animal Welfare Act and Regulations,1 Public Health Service Policy,40 and other federal statutes and regulations relating to animals and experiments involving animals. The housing environment was maintained at 69 to 75 °F (22.6 to 23.9 °C), with relative humidity of 30% to 70%, and on a 12:12-h light:dark cycle. A commercial diet (2050 Teklad Global 20% Protein Primate Diet, Harlan Laboratories, Frederick, MD) was provided to NHP twice daily, as well as fresh produce once a day. Chlorinated and filtered municipal water was provided without restriction through an automated watering system (Edstrom Industries, Waterford, WI). Manipulanda (Challenge Ball, Kong, football, and Dental Star [Bio-Serv, Frenchtown, NJ]) were available at all times in the cage as a form of physical enrichment, according to an established schedule as outlined by our institute’s husbandry and care program. Clinically healthy African green monkeys (n = 20; 10 female and 10 male; age, 15.3 ± 3.6 y [mean ± 1 SD]; weight, 5.2 ± 1.3 kg) were evaluated in this study. They originated from St Kitts and arrived at our facility at least 2 y prior to the start of this study. The study population was housed in 3 separate rooms within the same corridor within 4.3-ft2 cages (Allentown Caging Equipment, Allentown, NJ). At the start of the study, 8 of the 20 African green monkeys were housed in partial contact with a cagemate according to previous behavioral assessments. Animals in partial contact were housed next to a potential cagemate, with either a mesh or solid transparent divider to allow for protected contact. The remaining African greens were singly housed but had visual and auditory contact with conspecifics at all times. The clinically healthy cynomolgus macaques (n = 20; 10 female and 10 male; age, 9.0 ± 2.2 y; weight, 7.0 ± 2.6 kg) evaluated in this study arrived at our facility from either China or Cambodia at least 2 y prior to study, with the exception of one animal that arrived 2 mo prior to study. This population was housed in 3 different rooms within the same corridor. Most animals were housed in 4.3-ft2 cages (Allentown Caging Equipment), with visual and auditory contact with conspecifics at all times; 3 of the 20 animals were singly housed in 6.0-ft2 caging, given their size. Overall, 6 of the 20 animals were in full-contact housing with one cagemate, 5 were in partial contact, and 9 were singly housed in light of previous behavioral assessments. Animals in partial contact were housed next to a potential cagemate, with a mesh divider to allow for protected contact. Pairs in full contact were housed in double-wide caging, to allow appropriate floor space for each animal. Clinically healthy rhesus macaques (n = 20; 9 female and 11 male; age, 5.7 ± 1.3 y; weight, 6.5 ± 1.8 kg) were also evaluated in this study. They arrived to our facility from China at least 6 mo prior to the start of the study. This population was housed in 2 rooms within the same corridor. Most animals were housed in 4.3-ft2 cages (Allentown Caging Equipment), with visual and auditory contact with conspecifics at all times; however, 2 animals were singly housed in 6.0-ft2 caging because of their size. Overall, 14 macaques were in full-contact housing with one cagemate, 2 were in partial contact, and 4 were singly housed because of previous behavioral assessments. Animals in partial contact were housed next to a potential cagemate, with a mesh divider to allow for protected contact. Pairs in full contact were housed in double-wide caging, to allow appropriate floor space for each animal. All macaques were seronegative for Macacine herpesvirus 1, SIV, simian retrovirus type D, and simian T-lymphotropic
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leukemia virus. African green monkeys were seronegative for Cercopithecine herpesvirus 2, SA11 rotavirus, SIV, and simian T-lymphotropic leukemia virus. All animals were tested twice annually for Mycobacterium tuberculosis by tuberculin skin testing and remained negative throughout the study. Prior to study initiation, each NHP received baseline health assessment from a qualified veterinarian, including a physical exam, CBC, and blood chemistry. Physical exams revealed no significant health concerns for any of the NHP in this study. Study design. A 2×2 crossover study design was chosen to compare collection methods.7 During the first iteration of the experiment, animals were randomized into 1 of 2 collection sequence groups stratified by species and sex. One group was sampled by using the SS device first, followed by PD collection. In the other group, PD was collected first, followed by use of the SS. The experiment was repeated with the same animals 4 d later, with the sequences reversed from the original order to rule out potential effects of saliva collection order. In addition, at each sampling time point, one blood sample was collected to evaluate correlation between salivary and serum cortisol levels. All sample collection occurred in the morning between 0800 to 1200 to reduce variability associated with the natural diurnal cortisol variation.15,21 Although voluntary saliva collection techniques have been published for some NHP species, we sought to demonstrate sufficient correlation between salivary and serum cortisol levels from samples collected within a specified period, thus necessitating the sedation of NHP for sample collection. Sample collection and processing. Feed was withheld from animals for 12 h prior to all anesthetic procedures. To achieve adequate sedation, a mixture of ketamine (9.0 mg/kg) and acepromazine (0.1 mg/kg) was administered intramuscularly in the caudal thigh by using a 21- to 25-gauge, 3/8- to 1-in. needle. Approximately 5 to 10 min after sedation, blood samples were collected from the femoral vein, samples were centrifuged, and serum transferred to individually labeled cryovials for storage at –80 °C until needed for the assay. To account for the potential lag in salivary cortisol relative to serum cortisol, the first saliva sample was collected approximately 20 min after sedation and the second approximately 25 min after sedation. The SS device (SuperSAL Oral Fluid Collection Device, Oasis Diagnostics, Vancouver WA) uses a highly absorbent cylindrically shaped pad that can be compressed or centrifuged to collect the sample (Figure 1 A). The absorbent pad is approximately 3 inches long, has a sample volume indicator, and is attached to a 3-in. plastic handle. The pad was placed in the cheek pouch or along the side of the tongue for 5 min, and then was removed and placed in the supplied syringe for processing (Figure 1 B). The entire syringe was placed into a 15-mL polypropylene centrifuge tube. For comparison, the PD collection technique was also used to collect saliva. For this technique, the animal’s head was slightly draped over the edge of the exam table, and saliva was collected into a culture dish as it passively flowed out of the oral cavity for approximately 5 min. Saliva was removed from the culture dish by using a disposable plastic pipette and was placed directly into a 15-mL polypropylene centrifuge tube. All saliva samples were centrifuged (3000 × g for 5 min) to enhance extraction from the absorbent tube, transferred to individually labeled cryovials, and stored in a freezer (–80 °C) until needed for the assay. Measurement of serum and salivary cortisol levels. Serum and salivary cortisol values were obtained by using commercial competitive ELISA (Cortisol Parameter KGE008B, R and D Systems, Minneapolis, MN) with 50 µL saliva and 10 µL serum, to allow for duplicate analysis. Serum samples were pretreated (trichloroacetic acid and buffer) according to manufacturer’s
Figure 1. (A) The experimental saliva collection device and (B) placement of the device in the mouth of a sedated rhesus macaque. Note the blue line, indicating sufficient saliva volume.
recommendations, to remove potentially interfering proteins and protein-bound cortisol. To generate values that fell within the standard curve, serum samples were diluted 80-fold and saliva samples diluted 10- to 20-fold as needed. Absorbance was measured on a microplate reader (Infinite 200Pro, Tecan Systems, Mannedorf, Switzerland), and a 4-parameter logistic curve-fit was generated by using online software (MyAssays, www.myassays.org).37 Data analysis. Data were analyzed by using SAS Version 9.4 (SAS Institute, Cary, NC).45 Cortisol values and volume amounts were compared by using mixed-model ANOVA. If a saliva collection technique was unsuccessful, the volume was recorded as 0 and included in the analysis. Method, species, sequence, and period were treated as fixed effects. Subject was treated as a random effect. Posthoc Tukey tests were used for pairwise comparisons between species. Covariates such as sex, weight, pair status, age, cage position, and room order were also singly tested for influence on cortisol values. Correlation of serum and salivary cortisol values was assessed through Pearson productmoment correlation. Mean volume and cortisol concentrations are presented as mean ± 1 SD, and statistical significance was set at a P value of less than 0.05. 183
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Results
Assay performance. The mean intraassay coefficient of variation was 9.9% for serum and 11.2% for saliva. The mean interassay coefficient of variation was 11.7%. The manufacturer reported an average recovery from serum of 104% (range, 92% to 114%) and from saliva of 101% (range, 80% to 118%). The recovery of standard dilutions for this study ranged from 97% to 106%. The mean minimal detectable dose of cortisol was 0.071 ng/mL, according to manufacturer assessments. Saliva volume. In the African green monkey group, sufficient saliva samples (defined as greater than or equal to 0.05 mL) were collected by using SS from 36 of 40 attempts (90%) and by using the PD technique from 26 of 40 collection attempts (65%) over both days. The success rate was similar in cynomolgus macaques, with 36 successful attempts from a total of 40 (90%) by using SS and 27 of 40 (67.5%) by PD. In rhesus macaques, sufficient saliva samples were obtained from all attempts with SS, and from 28 of 40 (70%) by using the PD collection technique. For African green monkeys, the volume of saliva collected was greater from SS than PD at both day 1 (F1,18 = 8.66, P = 0.0087) and day 2 (F1,18 = 13.20, P = 0.0019). In the cynomolgus macaque group, the volume collected again was greater when using SS than PD, but only for day 1 (F1,18 = 8.74, P = 0.0085); no significant difference was observed on day 2 (F1,18 = 0.35, P = 0.5624). In rhesus macaques, the volume of saliva collected was greater from using SS than PD only on day 2 (F1,18 = 5.41, P = 0.0319). When comparing saliva volume between species, the only significant difference observed was when using SS during day 1 (F2,56 = 4.61, P = 0.0140). Tukey posthoc tests showed a significant difference between cynomolgus macaques and rhesus macaques, with greater volume collected from rhesus macaques (P = 0.0173). Saliva volume results are summarized in Table 1. In addition, period effects were nonsignificant for all species, methods, and iterations, indicating that the order of method collection did not have a significant effect on saliva volume. Free saliva cortisol. For African green monkeys, salivary cortisol values were greater in the samples collected by PD compared with SS, but significant differences occurred only on day 2 (F1,13.5 = 19.24, P = 0.0007). In the cynomolgus macaques, salivary cortisol values were greater in samples collected by PD compared with SS on day 1 (F1,11.5 = 15.60, P = 0.0021) and day 2 (F1,14.5 = 7.74, P = 0.0143). For rhesus macaques, no significant differences were observed for either iteration. Comparing salivary cortisol levels between species revealed no significant differences on either day when collected by PD. However, for saliva samples collected with SS, significant differences were found between species on day 2 (F2,53 = 6.95, P = 0.0021) but not day 1 (F2,52 = 2.02, P = 0.1430). Tukey posthoc tests showed a significant difference between cynomolgus and rhesus macaques, with greater salivary cortisol values in rhesus macaques (P = 0.002). Salivary cortisol results (ANOVA) are summarized in Table 2. Figure 2 summarizes the distribution of mean salivary cortisol values for each animal as obtained by using the SS and PD methods in each species. If only one saliva sample was collected by using the indicated method, that value was included rather than the mean. Period effects were nonsignificant for all species, methods, and iterations, indicating that order of method collection did not have a significant effect on cortisol values. Total serum cortisol. Serum was successfully collected from all 20 animals within each species. For African green monkeys, the mean serum cortisol was 384.8 ng/mL (1 SD, 123.3 ng/mL) on day 1 and 355.5 ng/mL (175.6 ng/mL) on day 2. For cynomolgus macaques, the mean serum was 386.0 ng/mL (108.9 ng/mL) on day 1 and 362.9 ng/mL (118.9 ng/mL) on day 2. Finally, in the
rhesus macaque group, the mean serum cortisol was 521.9 ng/ mL (162.3 ng/mL) on day 1 and 463.2 ng/mL (155.6 ng/mL) on day 2. On Day 1, rhesus macaques had significantly higher serum cortisol values than either African green monkeys (P = 0.0055) or cynomolgus macaques (P = 0.0059). No other significant differences were noted in serum cortisol levels between species. In addition, serum cortisol did not differ significantly between the 2 collection time points for any group. Serum and saliva cortisol correlation. Strong correlations were observed when comparing serum cortisol and salivary cortisol obtained by using SS in all species and collection days (r > 0.527), except for cynomolgus macaques on day 1 (r = 0.179; Figure 3). Less prominent correlations were obtained when comparing serum cortisol and salivary cortisol obtained from PD, with significant correlations only in the African green monkeys (r > 0.760; Figure 3). The correlation between salivary cortisol levels obtained by using the 2 saliva collection methods was very strong for all species and collection days (r > 0.747; Figure 3). Correlation results, including Pearson correlation coefficients and significance level, are summarized in Table 3. Covariate analysis. In the African green monkey group, the only significant covariate was cage position on day 1, when the salivary cortisol was greater in the NHP housed in the bottom cage compared with the top cage (F1,16.5 = 10.39, P = 0.0051). This effect was not observed on day 2 or for the serum cortisol values on either day. In the cynomolgus macaque group, sex and weight had significant effects on salivary cortisol levels measured on day 2. Female macaques had higher salivary cortisol levels than male (F1,27 = 33.64, P < 0.0001), and smaller cynomolgus macaques had higher salivary cortisol levels compared with larger animals (F1,17.1 = 8.24, P = 0.0105). Pair status had a significant effect on salivary cortisol on both days, with single-housed animals demonstrating lower salivary cortisol concentrations compared with partial or fully paired animals. None of the covariates examined was significant in the rhesus macaque group. Room entry order was specifically included as a covariate, because NHP further down on the random-order sample-collection list were exposed to more activity prior to sedation. For all 3 species, no significant effect of room entry order was noted on either serum or salivary cortisol (P > 0.25 for all groups).
Discussion
This study is the first to measure free salivary cortisol concentrations in samples from NHP species collected by using this commercially available human device and explores the relationship between salivary and serum cortisol concentrations with regard to the 2 collection techniques used. In addition, our study generated new data on salivary cortisol values in African green monkeys and cynomolgus macaques that—to our knowledge—have not yet been reported in the literature. One important conclusion from this study is that despite the measurement of a lower cortisol concentration from SS than PD, cortisol values obtained from SS samples were a better predictor of serum cortisol concentration than values obtained from PD samples. Previous studies conducted with human saliva samples similarly found that salivary cortisol concentrations were lower when obtained by using a cotton-based absorbent material and suggest that the absorbent material can reduce cortisol concentrations and create random errors.14,19,47 A potential reason for this effect may be the binding characteristics of the material used or of the plastic container or low sample volume.44 Regarding the collection device used in the current study, the manufacturer has claimed that the absorbent pad, which is a
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Table 1. Volume of saliva collected (mL; mean ± 1 SD; n = 20 samples per group) from 3 species of NHP by using either the experimental collection device (SS) or passive drool (PD) Day 1 SS
PD
Day 2 F1,18
Pa
SS
PD
F1,18
Pa
C. aethiops
0.36 ± 0.40
0.16 ± 0.24
8.66
0.0087
0.60 ± 0.62
0.27 ± 0.50
13.2
0.0019
M. fascicularis
0.88 ± 1.13b
0.55 ± 1.06
8.74
0.0085
0.79 ± 0.96
1.10 ± 2.96
0.35
0.5624
M. mulatta
0.25 ± 0.17b
0.17 ± 0.44
1.10
0.3088
0.54 ± 0.78
0.33 ± 0.47
5.41
0.0319
aANOVA group
bANOVA group
significance level significance level between species: P = 0.017
Table 2. Free saliva cortisol concentration (ng/mL; mean ± 1 SD; range) in samples from 3 species of NHP collected by using the experimental collection device (SS) or as passive drool (PD) Day 1 C. aethiops
M. fascicularis
M. mulatta
aANOVA group
bANOVA group
Day 2
SS
PD
F
Pa
SS
PD
F
n = 18
n = 11
1, 9.4 = 0.18
0.6799
n = 19
n = 12
1, 13.5 = 19.24
29.7 ± 24.9
34.0 ± 24.6
19.4 ± 13.0
42.4 ± 30.3
2.0–107.9
7.4–87.4
2.3–101.1
7.0–170.2
n = 18
n = 14
18.1 ± 34.6
31.6 ± 42.6
1.3–166.0
2.8–159.8
n = 20
n = 10
37.6 ± 28.9
35.0 ± 24.3
3.1–101.6
5.0–63.1
1, 11.5 = 15.6 0.0021
1.8, 41.0 = 0.01 0.9896
n = 18
n = 13
12.7 ± 10.7b
28.0 ± 28.3
1.0–64.8
2.1–97.2
n = 20
n = 18
30.8 ± 19.4b
30.7 ± 21.1
2.8–54.1
4.5–165.1
Pa 0.0007
1, 14.5 = 7.74 0.0143
1, 16.3 = 0.23 0.6371
significance level between methods significance level between species: P = 0.002
Figure 2. Free salivary cortisol values (ng/mL) obtained from samples collected by using the experimental collection device (SS) or passive drool (PD) for 3 species of NHP. Each value depicted represents the mean saliva cortisol concentration from days 1 and 2, obtained from samples collected by using the indicated method. For animals in which only a single cortisol value was obtained, only that value is depicted.
proprietary noncellulose material, does not significantly affect the recovery of salivary cortisol. However, the manufacturer mentions the potential effects of mucin aggregates on various proteins and states that the PD method is prone to increased deviation in analyte recovery because recovery is more highly dependent on the mucin concentration than on the collection device, which binds mucin aggregates.20 In addition, previous
studies did not investigate the effect of small saliva volumes obtained from the absorbent material of the collection device. The potential cause of the discrepancy between salivary cortisol levels in samples collected by using the 2 methods warrants further investigation. However, the data show that the salivary cortisol values obtained with SS demonstrate strong correlation with serum cortisol concentration, and the values obtained by SS and PD themselves are also strongly correlated. One consideration regarding the differences in correlation between the 2 collection methods is that obtaining adequate saliva volumes was more difficult when using the PD collection technique, resulting in more missing data points. The relatively limited data available to compare salivary cortisol levels obtained by PD with serum cortisol concentration may have resulted in weaker correlations. A single outlier heavily influenced the weak correlation observed between salivary cortisol levels obtained by using SS and serum cortisol concentration in the cynomolgus macaque group during day 1. The saliva from this animal contained a high salivary cortisol concentration when obtained by both SS and PD (153 ng/mL and 166 ng/mL, respectively), which were not reflected in the serum cortisol level. The volumes of saliva obtained from this animal were more than sufficient by both methods, and there was no evidence of gross contamination with blood or food material. Correlation of salivary and serum or plasma cortisol levels has been demonstrated in many species, including pigs, sheep, goats, horses, cattle, and humans.9,15,16,18,28,29,32,38,44 After stress onset, cortisol concentrations rise in parallel, with peak salivary cortisol levels lagging behind serum cortisol concentration.30 In dairy cattle, the salivary cortisol concentration peaked 10 min after the plasma cortisol concentration, whereas in dogs, the 185
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Figure 3. Relationship between total serum cortisol and free salivary cortisol collected with the experimental device (SS) in (A) C. aethiops, (B) M. fascicularis, and (C) M. mulatta. Correlation was significant in all cases except for M. fascicularis on day 1. Similarly, the relationship between total serum cortisol and salivary cortisol in passive drool (PD) is demonstrated for (D) C. aethiops, (E) M. fascicularis, and (F) M. mulatta. Correlation was significant only for C. aethiops on both days. Finally, the relationship between free salivary cortisol collected by using the experimental device and in passive drool is demonstrated for (G) C. aethiops, (H) M. fascicularis, and (I) M. mulatta. Correlation was very strong for in all cases; the Pearson product-moment correlation is reported for each comparison.
salivary cortisol concentration did not change within 4 min of restraint.23,32 In humans, the time lag has been reported to be several minutes to as long as 20 to 30 min.31,33,42 The results from this study demonstrated a greater mean volume of saliva by SS collection compared with PD in all cases except the cynomolgus macaque group on day 2. This aberrant result was due to a single outlier, which generated 13.5 mL saliva during PD collection. The maximal saliva volume collected by using SS was approximately 3.5 mL due to the size and absorbency limitations of the material. This feature
may be a potential limitation of SS that could be alleviated by use of multiple collection devices. The mean saliva volumes collected from the rhesus macaque group on day 1 were not significantly greater when collected by SS than PD. The lack of significance in this one group might reflect the relatively small saliva volumes collected by both methods. Regarding ease of use, SS was substantially less cumbersome, given that, once placed, the absorbent material required little attention until the end of the collection period. In contrast, PD required constant attention and effort, especially in animals that produced small
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Table 3. Correlation of serum and salivary cortisol levels in 3 NHP species from which saliva was collected by using the experimental collection device (SS) or as passive drool (PD) Day 1
Day 2
n
ra
Pb
n
ra
Pb
C. aethiops
18
0.685
0.002
19
0.708
