Methods of collection for salivary cortisol ...

Hormones and Behavior 55 (2009) 163–168

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Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y h b e h

Methods of collection for salivary cortisol measurement in dogs☆ Nancy A. Dreschel a,⁎, Douglas A. Granger b a b

Department of Dairy and Animal Science, Pennsylvania State University, University Park, PA 16802, USA Behavioral Endocrinology Laboratory, Department of Biobehavioral Health, Pennsylvania State University, University Park, PA 16802, USA

a r t i c l e

i n f o

Article history: Received 19 August 2008 Revised 22 September 2008 Accepted 22 September 2008 Available online 7 October 2008 Keywords: Dog Canine Salivary cortisol Methods Measurement Stress

a b s t r a c t Salivary cortisol has been increasingly used as a measure of stress response in studies of welfare, reaction to stress and human–animal interactions in dogs and other species. While it can be a very useful measure, there are a number of saliva collection issues made evident through studies in the human and animal fields which have not been investigated in the canine species. Collection materials and the volume of saliva that is collected; the use of salivary stimulants; and the effect of food contamination can all dramatically impact cortisol measurement, leading to spurious results. In order to further examine the limitations of the collection method and the effects of collection material and salivary stimulant on salivary cortisol levels, a series of clinical, in vitro and in vivo studies were performed. It was found that there is a large amount of inter- and intra-individual variation in salivary cortisol measurement. Beef flavoring of collection materials leads to unpredictable variability in salivary cortisol concentration. Using salivary stimulants such as citric acid also has the potential to affect cortisol concentration measurement in saliva. Hydrocellulose appears to be a useful collection material for salivary cortisol determination. Recommendations for collection materials and use of salivary stimulants are presented. © 2008 Elsevier Inc. All rights reserved.

Introduction Salivary cortisol has been increasingly used as a measure of stress response in dogs (Beerda et al., 1996, 1998, 1999; Vincent and Michell, 1992) and has found particular favor in canine studies of welfare (Coppola et al., 2005), reaction to stress challenges (Bergeron et al., 2002; Dreschel and Granger, 2005; Horváth et al., 2007) and human– animal interactions (Jones and Josephs, 2006). While plasma cortisol levels are the most indicative of the activity of the hypothalamicpituitary-adrenal (HPA) system, blood collection is an invasive procedure, requiring skilled technical capabilities; a compliant subject; and sample collection, processing and storage capabilities. Handling and venipuncture have been shown to increase canine blood cortisol levels 20 min later (Hennessy et al., 1998). Because saliva sampling is non-invasive and salivary cortisol is highly correlated with plasma cortisol (Beerda et al., 1996), salivary cortisol can be used as a measure of HPA activity at convenient and meaningful times of the day in naturalistic settings (Kirschbaum and Hellhammer, 1994). Although handling of the animal is required, saliva seems to be tolerated well by many domestic animals. Kobelt et al. (2003) showed that up to 4 min could be taken to collect a saliva sample from a dog without the effect of handling being reflected in cortisol concentrations.

☆ In the interest of full disclosure, Dr. Granger is the founder and president of Salimetrics LLC (State College, PA). ⁎ Corresponding author. E-mail address: [email protected] (N.A. Dreschel). 0018-506X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2008.09.010

While salivary cortisol is a useful measure, a number of constraints associated with its collection and measurement have been made evident through studies in the human and animal fields. In particular, collection materials, the volume of saliva collected, the use of salivary stimulants, and the effect of food contamination have all been shown to dramatically impact cortisol measurement in humans, leading to spurious results (Granger et al., 2007). Although many of the new assays require a small volume of saliva sample (as little as 25 μl) for cortisol measurement, there may be difficulty in obtaining even this amount from small dogs or less compliant subjects. This limits the number of tests that can be run and the ability to run tests in duplicate for validity. Recent research also indicates that low volume samples result in a disproportionately lower percentage of cortisol retrieved from absorbent materials, resulting in the potential for considerable error variance in measurement (Harmon et al., 2007). This effect was most pronounced in cotton braided rope. In order to ensure adequate sample volume, previous studies in dogs have used salivary stimulants, such as citric acid, to induce salivation. Techniques described include sprinkling a “few pellets” (Beerda et al., 1998) or a “number of crystals” (Bergeron et al., 2002) of citric acid on a dog's tongue, or wiping the dog's tongue and palette with a citric acid soaked cotton ball (Kobelt et al., 2003). However, the use of citric acid has been shown to artificially increase the levels of both cortisol and testosterone measured in human saliva, likely related to an increase in sample acidity (Schwartz et al., 1998; Granger et al., 2004). Because it is known that canine saliva has buffering capabilities to protect the individual against ingestion of more acidic

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substances (Reece, 1994), the effect of citric acid stimulation on measurement of canine salivary cortisol levels is unknown. The materials used to collect saliva also have the potential to interfere with hormone testing. Typical collection of saliva in dogs involves the collector holding a cotton dental rope in the dog's mouth for 1–2 min. The saliva is then extracted by compressing the saturated rope in a 5 ml syringe (can be performed on-site) or centrifuging in a Salivette device (Sarstedt, NC). Although cotton-based sample collection methods have been shown to interfere with a variety of salivary biomarkers in humans, cortisol levels seem unaffected (Shirtcliff et al., 2001). As stated before, however, it can be difficult to obtain a good percentage yield of saliva from a cotton rope, introducing small sample issues. Surgical ophthalmic sponges made from hydrocellulose are effective at absorbing saliva and give a good percentage yield when centrifuged (Harmon et al., 2007). In humans, a high correlation between cortisol measurement using hydrocellulose and passive drool has been noted (Granger et al., 2007). Hydrocellulose has not been previously tested as a collection material for canine saliva for cortisol determination. It has been suggested by participants in previous studies (Dreschel and Granger, 2005) that flavoring the cotton rope used would aid in saliva collection from dogs. However, animal or plant origin food particles in the mouth may contain products that cross-react with the antibodies used in salivary immunoassays and lead to spurious results in the measurement of many hormones, including cortisol in human samples (Magnano et al., 1989; Shirtcliff et al., 2001). Whether the small amount of product used in flavoring a cotton rope for saliva collection would affect cortisol measurement is unknown. While the effects of salivary stimulants, food contamination and different collection media have been investigated in human saliva, the effects of these factors on saliva collection for cortisol measurement in dogs have not been investigated. This paper seeks to close these gaps in the literature by describing a series of clinical, in vitro and in vivo studies of collection methods and materials for salivary cortisol measurement in dogs. In all the studies, saliva was collected from adult (over the age of 1 year), healthy dogs weighing at least 9 kg. Female dogs were excluded if they were in estrus or pregnant. All of the dogs used were privately owned animals with often unknown birthdates and breed backgrounds. In each study, owners were asked not to have fed their dogs in the 20 min prior to sample collection. All studies were performed with the approval of the Pennsylvania State University Institutional Animal Care and Use Committee. Study 1: Collection of saliva in a clinical setting The first study reported here had a threefold purpose. First, a quantity of canine saliva was desired for further in vitro testing of collection methods. Second, the ease with which skilled professionals were able to obtain saliva samples in a clinical setting was examined. Third, the effect of perceived subject “stress” or “excitement” on the ease of collection and volume of sample collection was studied. The clinical veterinary setting was chosen because of the large number of patients presented, the already established handling and technical skills of the veterinarians and technicians, and the range of expected temperaments of animals presenting to the hospital. Methods Three veterinarians and three veterinary technician/assistants working at an active small animal hospital collected saliva samples from healthy adult dogs presenting for wellness and vaccination appointments. The primary investigator (ND), a veterinarian working at the clinic, instructed one other veterinarian, a technician and a veterinary assistant on collecting samples, and provided written instructions and explanations that were posted near the sample collection materials which were distributed throughout the hospital.

The trained individuals then trained the other veterinarian and assistant who also participated. Saliva samples were obtained from 23 adult dogs of pure and mixed-breed backgrounds ranging in age from 1.5 to 12 years. After gaining owner permission, samples were obtained by holding a 7.5 cm cotton dental rope in the subject's mouth for 1–2 min (with the mouth held closed if necessary) and the rope was placed in a salivette. The sample collector recorded the time of collection, and rated each dog subjectively on a scale of 1–5 on the following perceived characteristics: ease of handling (1 — “very easy” to 5 — “very hard”), excitement of dog (1 — “very mellow”, 5 — “very excited”), and “stress” of dog (1 — “not stressed”, 5 — “majorly stressed”). The samples were immediately placed in the clinic freezer at −18 °C and frozen until transport on ice to the Behavioral Endocrinology Laboratory at Penn State University where they were stored at −40 °C until testing. At the time of testing, the samples were thawed and spun in a centrifuge at 3000 rpm for 15 min. An approximate volume was estimated for each sample based on comparison to a graduated tube of the same size. Samples with large volumes were selected for use in a subsequent study. Descriptive statistics were performed on the quantity of saliva obtained, ease of collection and characteristics of the subjects. Correlational analyses were performed to determine if there were relationships between the volume of saliva obtained, the ease of collection and the relative excitement or stress of the dog. A 2 × 2 (collector × characteristic) ANOVA was performed to determine if there were differences in amount collected or ease of collection if the sampler were a veterinarian or a veterinary assistant/technician. Results of Study 1 The volume of saliva collected ranged from 0 to 1500 μl, (M = 351.1 μl, SE = .48). The mean “ease” of collection recorded was 1.6 (SE = .52), the mean “stress” of the dogs was 2.2 (SE = .52), and the mean “excitement” of the subjects was 2.9 (SE = .54). There were no correlations between perceived ease of sampling, “stress” of dog, “excitement” of dog and the amount of sample collected. There were also no significant differences in amounts collected or ease of collection if the collector were a veterinarian or veterinary technician/assistant. Conclusions The mean volume of saliva collected was fairly high. All but one sample yielded enough to perform a salivary cortisol assay. There was an inherent selection bias for good-natured, easily-handled dogs from which to obtain samples, as the veterinary staff selected which dogs to sample. It is no surprise, then, that the mean “ease” of sample collection was relatively low (“easy”). It is interesting to note that there was no correlation between perceived “stress” and perceived “excitement” in dogs, and that both of these characteristics fell in the middle range. Many dogs presenting to veterinary clinics are perceived to be either “stressed” or “excited” and are often both. None of these factors influenced the volume of saliva collected. Veterinarians and veterinary assistants/technicians were equally adept at collecting adequate saliva samples from this population. This study indicates that by the method described here, with relatively little training, veterinarians, veterinary technicians and veterinary assistants are able to collect adequate quantities of saliva to measure salivary biomarkers in a clinical setting. Study 2: In vitro determination of the effect of method collection on salivary cortisol measurement The purpose of the second study was to determine, in vitro, if citric acid stimulation interferes with cortisol measurement in canine saliva, and, if so, to determine at what concentration of citric acid this effect is


seen. Hydrocellulose and beef-flavored cotton ropes as collection materials were also tested for interference with cortisol measurement. Methods Each sample from six dogs (1 neutered male and 5 spayed females including 2 Bassett hounds and 4 dogs of unknown mixed breeding) collected as part of the previously described study was aliquoted into six microcentrifuge tubes and refrozen at −40 °C before cortisol testing. The samples were thawed and assayed for salivary cortisol using a highly-sensitive enzyme immunoassay kit (Salimetrics, State College, PA). The test uses 25 μl of saliva (for singlet determinations), has a range of sensitivity from .007 to 1.8 μg/dl, and average intra- and inter-assay coefficients of variation of less than 10% and 15% respectively. Method accuracy, determined by spike recovery, and linearity, determined by serial dilution are 105% and 95%. All samples were performed in duplicate and those with coefficients of variation greater than 15% were repeated. Control samples containing 80 μl of saliva from each subject were tested for cortisol and for pH using visual pH strips (colorpHast Indicator strips pH 0–14, EM Science, Gibbstown, NJ). To determine the effect of citric acid on cortisol measurement, .01 g of food grade citric acid crystals was added to 100 μl of saliva from each subject to create a .1 g/ml solution. Serial dilution then yielded two additional concentrations of .01 g/ml and .001 g/ml citric acid/saliva. The pH of each concentration was measured using visual pH strips. Cortisol levels were measured at each concentration of citric acid. To examine the effect of collection material on cortisol concentration, 120 μl of saliva from each subject was absorbed with a BD hydrocellulose eye sponge. The handle of the sponge was cut short and the sponge was placed handle down in a microcentrifuge tube and re-spun. Saliva was harvested and tested for cortisol concentration. Beef-flavored dental rope was prepared in advance by soaking a cotton dental rope in a solution prepared by mixing one food grade beef-bouillon cube with 1 cup of boiling water as per instructions on the bouillon label. The cotton rope was then dried by baking in an oven at 60 °C for 2 h. The flavored dental rope was cut into 7.5 cm lengths and the ends were discarded. Two hundred μl of saliva from each subject was absorbed into the dental rope and re-extracted by pressing out of a 5 ml BD syringe. Cortisol was measured in each sample. Examination of the cortisol data revealed positively skewed distributions. Natural log transformation was performed to establish normal distributions prior to analysis. As appropriate, analyses were conducted using the transformed values, but for ease of interpretation, the values in the text and figures are raw scores. Paired sample t-tests were performed to compare the control procedure with the experimental procedure. For ease of comparison with the follow-up studies, non-parametric Wilcoxon signed-rank tests were performed on the skewed data. The SPSS statistical software package (SPSS, Inc., Chicago, IL) was used for all analyses. A concordance correlation coefficient to determine the reproducibility of scores using each method was calculated for all pairs (Lin, 1989). The concordance correlation coefficient evaluates agreement for two responses on a continuous scale and will have a value of 0 to 1.0 with 1.0 indicating perfect concordance between the two variables.

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coefficient for the beef-flavored rope collection method was .92 and the concordance coefficient for the hydrocellulose method was 1.0. The addition of citric acid decreased salivary pH from a mean of 8.4 at control to 1.8 at .1 g/ml, 3.2 at .01 g/ml and 5.5 at .001 g/ml (see Fig.1). Cortisol at the .1 g/ml level could not be measured, as it was off the scale (N1.8 μg/dl). The mean cortisol at the .01 g/ml dilution was .85 μg/dl (SE = .37) and at the .001 g/ml citric acid dilution the mean was .18 μg/dl (SE = .05). The difference between cortisol measurement at .01 g/ml and at control was significant (p b .01), but there was not a significant difference between the .001 g/ml level and the control (Fig. 1). The concordance coefficient for the .01 g/ml citric acid concentration was .16 and the concordance coefficient for the .001 g/ml citric acid concentration was 1.0. Conclusions This study clearly shows the potential limitations of using citric acid as a stimulant for saliva collection for cortisol measurement. As little as .01 g/ml of citric acid caused a significant drop in pH and a corresponding significant increase in measured salivary cortisol concentration. Thirty individual crystals of citric acid weigh approximately .01 g. Because the quantity of saliva typically collected from a dog's mouth is in the .1–.5 ml range, there is easily the possibility of achieving a .01 g/ml concentration with a “pinch” of citric acid placed in a dog's mouth. Lower concentrations (.001 g/ml, approximately 1–4 crystals of citric acid per ml) did not significantly lower the pH concentration and did not have an effect on salivary cortisol measurement in vitro. This study did show a high concordance between measurements taken before and after passing the saliva through a beef-flavored cotton rope. However, there was a significant difference between the measurements with a higher cortisol concentration measured in the beef-flavoring condition than in the control condition. Hydrocellulose appeared in vitro to be a useful collection medium. There was not a significant difference between the salivary cortisol concentration measured before and after the saliva was passed through a hydrocellulose sponge. The concordance coefficient was also very high for this measure. Study 3: In vivo determination of the effect of collection methods on salivary cortisol measurement Based on the findings of the previous study, an in vivo challenge was designed to determine if salivary stimulation with citric acid or

Results of Study 2 The mean salivary cortisol for the control samples was .17 μg/dl (SE = .06). The mean cortisol with the beef-flavored rope was .20 μg/dl (SE = .06) and the mean cortisol from the samples passed through the hydrocellulose was .15 μg/dl (SE = .05). There was a significant difference between the control (cotton) and the beef-flavored rope values (p b .01). There was no significant difference between the mean values of the control and the hydrocellulose. The concordance

Fig. 1. The addition of citric acid to canine saliva causes a decrease in salivary pH and an increased cortisol measurement in vitro (error bars represent SEM).

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sampling with beef-flavored cotton ropes or hydrocellulose swabs would have an effect on cortisol measurement in the live animal setting. In particular, this study sought to determine if there are mechanisms within the live animal system to counteract the negative consequences of using citric acid stimulation. In addition, empiric descriptions of the dogs' acceptance of different methods of saliva collection were obtained. Methods Saliva samples were collected from 24 purebred dogs (18 females and 6 males representing six sporting-dog breeds — 6 German shorthaired pointers, 5 English pointers, 3 English setters, 2 Springer spaniels, 2 Brittany spaniels, and 6 Labrador retrievers) by the researcher at a breeding and training kennel. Eight subjects (2 males and 6 females per group, with the breeds distributed as evenly as possible over the groups) were enrolled in each experimental condition (citric acid, hydrocellulose, beef flavoring). Control saliva samples were collected from all dogs using a cotton dental rope as previously described. For each dog the control sample was collected first, followed by the experimental sample, to avoid contamination between the experimental procedures and the control. All samples were immediately put on ice and frozen. One subject in the beefflavoring condition did not have an adequate volume of sample to measure control cortisol. To determine the effect of citric acid on cortisol measurement in the field, the inside of the dog's mouth and gums was swabbed using a citric acid coated cotton ball as described by Kobelt et al. (2003). The cotton balls were prepared by adding 1.25 ml of a 5% citric acid solution to one half of a cotton wool ball and drying in an oven at 60 °C for 3 h. Immediately following the swabbing of the dog's mouth, saliva was collected using a plain cotton rope, placed in a salivette, and frozen. To examine the effect of collection material on cortisol measurement, saliva was collected using a hydrocellulose eye sponge (BD) (DeWeerth et al., 2007). The sponge was held by the handle and held in the dog's mouth for approximately 30–60 s which was adequate for full saturation of the sponge. The handle of the sponge was then cut so that it could be placed handle side down in a microcentrifuge tube and frozen. Beef-flavored dental ropes prepared as described above were held in the subject's mouth for 1–2 min to allow for saturation of the collection material. They were then placed in salivettes and frozen. Salivary cortisol was measured in all samples and the pH was measured in the control and citric acid samples as described previously in “in vitro determination of the effect of collection methods on salivary cortisol measurement”. Concordance correlation coefficients were calculated for each pair of measurements. Because the data was skewed and could not be normalized through transformation, differences between the control and experimental samples were examined with the non-parametric Wilcoxon signed rank test. Results of Study 3 The mean salivary cortisol values in the control and experimental samples are shown in Fig. 2. There were no significant differences between the controls and any of the experimental conditions in the Wilcoxon signed rank test. The concordance coefficient for hydrocellulose and control in the kennel dogs was .53. The concordance coefficient for the measurements following swabbing with citric acid and the control was .63. The concordance coefficient for the measurement from the beef-flavored rope and the control was .3. Graphs of the mean salivary cortisol in the experimental and control samples are seen in Fig. 2. There was not a significant difference between the control pH (M = 8.5, SD = .53) and the experimental pH (M = 8.7, SD = .92) for those dogs whose mouths were swabbed with the citric acid cotton ball.

Fig. 2. There is no statistically significant difference in cortisol measurement in canine saliva collected with beef-flavored ropes or hydrocellulose swabs or when salivation is stimulated with citric acid in vivo.

Empirically, the dogs resisted having their mouths swabbed with the citric acid-flavored cotton ball before saliva collection, but this did not interfere with the researcher's ability to collect saliva. The dogs readily chewed on the beef-flavored cotton ropes and also chewed on the unflavored cotton ropes, although without as much enthusiasm. The hydrocellulose swabs were particularly easy to use, as the handles fit within the interdental space in the dogs' mouths and they were able to close their mouths without chewing on the swab or stick. Conclusions This experiment sheds light on the use of citric acid for saliva stimulation in dogs. Using the technique of wiping the inside of the dog's mouth with a citric acid soaked cotton ball did not cause a decrease in pH of the dog's saliva. In fact, in several cases, the pH increased following the procedure, indicating a buffering response of the dogs' saliva. However, despite the absence of a significant pH change, there was a very low concordance in measurement between the control condition and the experimental condition. Dogs resisted having their mouths wiped with the cotton swab but it did increase saliva flow and allow for a second sample to be collected. There was no difference in the volumes collected before and after swabbing the dogs' mouths. Non-parametric testing did not show significant differences in cortisol concentration measured in unflavored cotton rope vs. beefflavored rope or hydrocellulose, but there was little correlation between the measurements taken with the various methods. There was also a very low concordance coefficient and a large standard error in the measurements between the control and the beef-flavored rope conditions as well as between the hydrocellulose and cotton rope conditions. Qualitatively, some of the dogs did seem to enjoy chewing on the beef-flavored rope more than the plain cotton rope and the hydrocellulose sorbettes were quite easy to manipulate in the dogs' mouths. Study 4: Consistency in cortisol measurement across time using various methods of saliva collection Because of the lack of concordance between different measures which was not reflected by significant differences between the measures in the previous study, a fourth study was performed. It was postulated that because the samples were taken within minutes of each other in the in vivo study, the collection of the first control


sample may have interfered with the collection of the subsequent samples. The final in vivo study looked specifically at the reliability of test measures at two collection times spaced at a 20 minute interval when no “stressor” was taking place. Methods Saliva was collected from six privately owned dogs (5 neutered males and one spayed female, age range approximately 1.8–9 years, M = 4.4 years) by their owners in their own homes. Samples were collected using three different methods at six distinct time points. Each method was repeated two times, with a period of 20 min occurring between collection times. The hydrocellulose sorbette, beefflavored rope and citric acid stimulation methods were used as described in the previous study. Owners were instructed to choose a time of day when their animal was at rest and not stimulated by other activity. At least 1 h elapsed between collections using different methods. Samples were frozen and stored at −40 °C until testing. Samples were assayed for salivary cortisol using an ELISA test (Salimetrics, State college, PA) as previously described. Data from samples previously collected using cotton rope at 20 minute intervals on a control day for a previous study were also used (Dreschel and Granger, 2005). This data was analyzed and compared to the other collection techniques. Concordance coefficients were calculated for each pair of measurements. The Wilcoxon signed rank non-parametric test was used to detect differences in cortisol concentration between the time points within each experimental condition. Results of Study 4 There were no significant differences using the Wilcoxon signed rank test between cortisol concentrations at the 20 minute time points for any of the samples. The concordance coefficient over time for hydrocellulose was .88, for citric acid it was .49 and for the cotton rope alone, it was .36. The concordance coefficient for the beef-flavored rope was 0.

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served primarily as their own controls and there was not a comparison of cortisol levels between subjects, it is hoped that inter-individual variation between breeds and environments would not affect the method-focused results. Beef-flavored ropes, though well accepted by canine subjects, introduce unpredictable variability to cortisol concentration measurement. This is consistent with previous research findings that food particles can contain products which interfere with cortisol measurement. Ropes flavored by this method are not reliable for saliva collection for cortisol determination. Whether different concentrations of beef flavoring or different types of flavoring would fail to interfere with the immunoassay is not known. Citric acid in small amounts does not appear to affect salivary cortisol measurement to a significant degree in the dog. Although the in vitro study illustrates the potential effects of high levels of citric acid, the dog's mouth in vivo appears to have a buffering capability that counteracts the acidity of this salivary stimulant. Anecdotally, dogs resisted having their mouths swabbed with the citric acid cotton ball and significantly more saliva was not obtained with the citric acid than with cotton rope alone. However, in cases where larger volumes would be useful, citric acid stimulation is unlikely to affect cortisol concentration variability. Hydrocellulose as a collection material appears to be very effective for canine saliva collection. The concordance with cotton rope was high, particularly in the controlled in vitro study. One concern with this technique is obtaining adequate sample volume with which to run the assays, as the swabs themselves are small. It is recommended that researchers use two or more swabs at the same time and reinforce the importance to those collecting the sample to leave the swabs in the dog's mouth for an adequate length of time to allow for full saturation. While there are limitations to the use of salivary cortisol, many of these limitations may be overcome by planning, careful instruction to those collecting samples and research design. Because saliva samples may be collected in the absence of the researcher, adherence to collection methods and times takes on a new importance. The researcher is dependent on those collecting samples to adhere to procedures that have been specified. It is important for the researchers to be aware of these limitations so that their results can be properly interpreted.

Conclusion Acknowledgments From previous research (Dreschel and Granger, 2005), it was known that there was no significant difference in samples taken with cotton rope at 20 minute intervals on a control day in a pet's own home. In this study, we also found no significant differences in measurements taken at 20 minute intervals using any of the collection techniques. We did, however, find a very low concordance for beefflavored rope using this technique. The concordance for hydrocellulose was quite high. General conclusions and recommendations for salivary collection materials and techniques for cortisol determination Previous research has shown that there are potential limitations in the use of various collection methods and media for salivary cortisol determination. The evidence from the studies described in this article support specific recommendations for the collection of canine saliva for cortisol measurement. It is also clear that there is a great deal of inter- and intra-individual variation in salivary cortisol levels. Some of this variation is likely inherent in individual animals. In addition, variation exists in these studies because different individuals collected the samples, different breeds were involved and animals of different (and often unknown) ages were included. With the exception of the first study, in which the ability to collect saliva in a non-home environment was part of the study, saliva collections took place at the dogs' own places of residence, often by their owners. As the dogs

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