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Conflicting Results in the Association Between Plasma
and Salivary Cortisol Levels in Foals Introduction Glucocorticoids are present in many biological fluids as a free fraction or bound to Corticoid Binding Globulins (CBG) (Matteri et al, 2000). There are conflicting claims regarding the validity of saliva as a biological fluid to measure cortisol in horses (Lebelt et al, 1996; McGreevy and Pell, 1998; van der Kolk et al, 2001). Measuring changes in salivary cortisol levels in normal horses and horses with Cushing’s disease van der Kolk and collaborators (2001) demonstrated the validity of saliva to assess adrenal function. Puzzling results were reported by McGreevy and Pell (1998) who suggested that plasma and salivary cortisol concentrations in horses showing oral stereotypies were correlated but this association was non-existent in control animals. Investigating the responses of foals to branding and foot-trimming Zanella et al (unpublished results) were unable to identify a relationship between plasma and salivary cortisol levels in foals. In several species, levels of cortisol in plasma and saliva are tightly correlated (Fenske, 1996). Cortisol found in blood consists of a fraction bound to corticoid binding globulin (CBG) and a free, unbound fraction. Free cortisol represents the biologically active fraction of this steroid hormone. Salivary cortisol reflects the unbound fraction found in plasma or serum and it passes readily through the parotid membrane (Riad-Fahmy, 1983; Horning Walker et al,1977). Unbound steroids transfer rapidly between plasma and saliva (Walker,1989; Scott et al 1990). Saliva flow-rate does not appear to influence saliva cortisol levels in different species (Hubert and de Jong-Meyer, 1989; Walker 1989, Scott et a, 1990). In horses, Lebelt et al (1996) reported that salivary and plasma total cortisol in stallions were correlated. We hypothesized that changes in salivary cortisol in foals would show a pattern that is correlated to that of plasma free and plasma total cortisol concentrations in foals. In addition, we anticipated that the lack of good sampling techniques provides an explanation for the failure in determining the association between salivary and plasma cortisol in foals. Methods The experiment was conducted on 10 Arabian foals, ranging from 2 to 3 months of age. The animals were randomly divided into two groups that were each sampled on three non-consecutive days. Each group was sampled once in the morning (between 7 and 9 AM) and once in the afternoon (between 3 and 5 PM). The animals were kept on pasture most of the time, but about an hour prior to sampling, the 5 subjects for the trial were brought into a barn and each mare and foal dyad was placed in individual box stalls. The stalls measured 3.048 m2 and the foals were familiar with these stalls since they were housed there until they were 2 to 6 weeks of age. Foals had been halter trained prior to this experiment. They were not wearing a halter when outside on pasture, but once inside the stall, a halter was put on to facilitate handling and sampling. Sampling methods For saliva sampling, an in-house constructed saliva collection device (SCD) was used. On average 17.5 of Tygon tubing was used and holes were cut in the tube. Gauze was cut into separate pieces, rolled and, using a metal wire, inserted in the tubes. At both ends of the tube, a suspender clip was attached which in turn was connected to a swivel-head clip using elastic ribbon that was adapted in length to comfortably fit the foal’s head. The swivel-head clips were attached to the halter and the tube was placed inside the foal’s mouth, and the animal immediately began to chew. After collection, the gauze was removed from the tube, folded in half and placed in 15ml conical tubes with the ends of the gauze pointing upward. The cap was placed on the tubes to keep the gauze in place and the samples were stored on ice. Blood sampling was performed using a 19 x 3/4” winged-infusion set that, after insertion into the jugular vein, was attached to a syringe and 5ml of blood was drawn. Approximately an hour prior to blood sampling, the site of venipuncture was clipped and covered with an anesthetizing cream to minimize the response to the initial puncturing of the skin. Restraint during blood sampling was kept to a minimum. The handler grasped the halter with one hand and with the other groomed the foal’s neck to direct attention away from the blood sampling procedure. After collection, tubes were kept on ice until they were transported to the lab to be processed. Experimental timeline A precise protocol was followed to be able to analyze all data in relation to all measurements taken. At time 0’, a first SCD was placed in the foal’s mouth and was removed at time 1’. This first collection device was intended to clear the mouth of food and saliva residues that were not associated to the experimental sampling. Right after the first SCD was removed, a second one was placed. At time 3’, a blood sample was taken and 4 ml were placed in a polypropylene tube with heparin (20 units per ml) while the remaining 1ml was placed in a test tube with no anti-coagulant. At time 6’, the second SCD was removed, thus the total duration to take the saliva sample was five minutes. Processing of the samples To process the saliva samples, we tightened the cap of the tube to keep the gauze in place as we centrifuged the samples at 1,000 x g for 5 minutes. The centrifuged saliva was collected from the tubes, aliquoted and stored in a freezer at –30 °C. Saliva samples were assayed using an Enzyme Immuno-Assay (DSL-10-67-1000 ActiveÔ Cortisol Enzyme Immunoassay kit, Diagnostic Systems Laboratory, Inc., Webster, TX 77598), originally developed to determine cortisol levels in human saliva samples. Blood samples were centrifuged for 2 minutes at 5,000 x g. The serum was stored at –30 °C. Blood samples collected with heparin were centrifuged at 2,000 X g for 15 min and the plasma aliquots were stored in the freezer at –30 °C. We used one aliquot to determine plasma and serum total cortisol using a radioimmunoassay, and the other aliquots were centrifuged in YM-100 Centricon filters (Millipore, Bedford, MA, Cat. No. 4211) with regenerated cellulose filter matrix and cut-off point for molecular weight of 100,000. This way, glucocorticoids bound to proteins remained on the filter membrane and the free fraction of cortisol passed through the filter. The filtrate was stored in the freezer until processing. The assay used to process the plasma free samples was the same EIA as was used for processing the saliva samples. Statistical analysis was performed using the repeated and random options of PROC MIXED of SAS. The residual correlation was examined, meaning the correlation adjusted for individual horse, group (whether they were sampled on Monday- Wednesday-Friday or on Tuesday-Thursday-Saturday), day, time and variables (the biological fluid source of cortisol used). Though both groups were sampled on different days there was no difference in treatment, and using a Chi Square test, we determined for each correlation whether the variance-covariance structures were deemed to be similar, and thus whether groups could to be analyzed together. Results and Discussion In total, each horse was sampled 6 times over 3 non-consecutive days. However, for some horses it was occasionally difficult to collect a blood sample due to extreme resistance. As a result, some samples were lacking or had insufficient volume to be included in the assay. In addition, though our saliva collection technique was considered to be overall quite efficient, we sometimes were unable to collect sufficient volume of saliva. One of the animals in the first group was dropped from the study because an insufficient number of samples was collected over the three sampling days. For the final assays, 44 samples were used to measure plasma free cortisol, 51 samples to measure plasma total cortisol, 37 samples to measure salivary cortisol, and 53 samples for serum total cortisol. Correlation salivary cortisol with filtered plasma cortisol (100,000 cut off) A total of 74 observations (37 salivary and 37 plasma filtered samples) were used in the correlation. The Chi-Square test showed that we could pool the data from group 1 and 2 because the correlations did not significantly differ (p>0.05). Due to the increased handling and the possibility that this may require a period of habituation, we expected the correlation on the first day of sampling for each group to differ from the other two days, but after performing a Chi Square test, this difference also proved to be non-significant (p>0.05). The effect of day and the day by time interaction affected both plasma and saliva measurements in a similar way, but were not found to be significant (p>0.1). Time of day (AM versus PM) had an influence on both plasma free cortisol (p=0.0168) and salivary cortisol (p=0.0509). Morning levels of cortisol for both measurements were higher than the afternoon levels. Morning levels of plasma free cortisol over all animals averaged 0.4345 ± 0.06007 mg/dl whereas saliva cortisol had a mean of 0.1004 mg/dl ± 0.01639 mg/dl. In the afternoon, average plasma free cortisol levels dropped to 0.2077 ± 0.0677 mg/dl and salivary cortisol levels to 0.05667 ± 0.01741 mg/dl. The overall correlation between salivary and plasma free cortisol levels was 0.701 (p<0.05). Correlation plasma total cortisol with filtered plasma cortisol A total of 86 observations (43 plasma total cortisol and 43 filtered plasma cortisol samples) were used in the correlation. Again, the Chi Square test showed that the variance-covariance structures of both variables were similar enough that we could pool the data for both groups (p>0.05). In addition, the effect we expected to see of the increased handling on the first day of sampling for both groups proved to be non-significant (p>0.05). Time had a significant effect on plasma free (p=0.0007) and plasma total cortisol (p=0.0061). The mean level of plasma free cortisol in the morning was 0.4410 ± 0.05171mg/dl versus 0.1919 ± 0.05270 mg/dl in the afternoon. Plasma total cortisol also decreased from morning to afternoon, 2.2549 ± 0.1678 mg/dl and 1.8529 ± 0.1706 mg/dl respectively. The correlation coefficient between plasma total and plasma free cortisol levels was 0.6242 (p<0.05). Correlation serum total cortisol with filtered plasma cortisol A total of 88 observations (44 for serum total and 44 for plasma free cortisol samples) were used in the correlation. In this correlation the Chi Square test rendered a significant result that indicated that group 1 and group 2 needed to be analyzed separately (p<0.05). For group 1 (40 observations: 20 for serum total and 20 for plasma free cortisol samples), the correlation coefficient (0.8323, p<0.05) was much higher than that of group 2 (0.6329, p<0.05). No time effect on the level of serum total cortisol was present in group 1 (p=0.1896), but morning plasma free cortisol levels were significantly higher (p=0.0466) in the morning (0.3421 ± 0.06971 mg/dl) than in the afternoon (0.1468 ± 0.06376 mg/dl). The correlation within group 2 used 48 observations (24 for serum total and 24 for plasma free cortisol samples). Both serum total and plasma free cortisol levels showed a significant effect of time (p<0.0001 and p=0.0176). The morning cortisol level for the serum total samples averaged 2.8571 ± 0.2619 mg/dl and decreased to an average of 1.8806 ± 0.2756 mg/dl in the afternoon. Plasma free cortisol levels decreased from 0.4975 ± 0.07086 mg/dl in the morning to 0.2199 ± 0.08415 mg/dl. Correlation salivary cortisol with plasma total cortisol A total of 72 observations were used in the correlation, 36 for salivary cortisol and 36 for plasma total cortisol samples. No difference was found between variance-covariance structures of the variables for group 1 or 2, and likewise there was no effect of increased handling on the first day of sampling for each group (p>0.05). As was found in the other correlations, there was a significant effect of time on salivary (p=0.0325) and plasma total cortisol levels (p=0.0301), with morning levels of cortisol being higher than in the afternoon. For plasma total cortisol the level averaged 2.4872 ± 0.1591 mg/dl and 1.9686 ± 0.1769 mg/dl respectively and 0.09870 ± 0.01499 mg/dl in the morning versus 0.04999 ± 0.01730 mg/dl during the afternoon for salivary cortisol. The correlation coefficient was 0.4056, but this was less significant compared to the other correlations discussed above (p<0.1). Correlation salivary cortisol with serum total cortisol A total of 74 observations were used in the correlation, 37 for salivary cortisol and 37 for serum total cortisol samples. No difference was found between variance-covariance structures of the variables for group 1 or 2, and likewise there was no effect of increased handling on the first day of sampling for each group (p>0.05). There was a significant effect of time on both salivary (p=0.0362) and serum total cortisol (p=0.0150) levels, with morning levels of cortisol being higher than in the afternoon. Morning serum total cortisol levels averaged 2.4170 ± 0.1911 mg/dl and 1.8731 ± 0.2069 mg/dl respectively and 0.09498 ± 0.01483 mg/dl versus 0.04815 ± 0.01738 mg/dl for salivary cortisol. The correlation coefficient was 0.5185 and, though this value is similarly low as in the previous correlation, the relationship is significant (p=0.0306). References Fenske M (1996) Measurement of salivary cortisol in guinea pigs. J Exp Anim Sci 38(1):13-19. Fuchs E, Kirschbaum C, Bensich D, Bieser A (1997) Salivary cortisol: a non-invasive measure of hypothalamo-pituitary-adrenocortical activity in the squirrel monkey, Saimiri sciureus. Lab Anim 31(4): 306-311. Horning MG, Brown L, Nowlin J, Lertratanangakoon K, Kellaway P, Zion TE (1977) Use of saliva in therapeutic drug monitoring. Clin Chem 22(2 PT. 1): 157-164. Hubert W, de Jong-Meyer R (1989) Emotional stress and saliva cortisol response. Report on the workshop conference “application of saliva in laboratory medicine.” J Clin Chem Clin Biochem 27(4): 235-237. Lebelt D, Schönreiter S, Zanella A J (1996) Salivary cortisol in stallions: the relationship with plasma levels, daytime profile and changes in response to semen collection. Pferdeheilkunde 12(4): 411-414. Matteri RL, Carroll JA, Dyer C J (2000) Neuroendocrine responses to stress. In Moberg JP, Mench J A (Eds) The Biology of Animal Stress. CABI Publishing, Oxford, UK, pp 43-76. McGreevy PD, Pell S (1998) The relationship between stereotypic behavior and plasma and salivary cortisol levels. In: Veisser I, Boissy A (Eds), Proceedings of the 32nd Congress of the International Society for Applied Ethology. Clermont-Ferrand, France. Riad-Fahmy D, Read G F, Walker R F (1983) Salivary steroid assays for assessing variation in endocrine activity. J Steroid Biochem 19(1A): 265-272. Scott EM, McGarrigle HHG, Lachelin GCL (1990) The increase in plasma and saliva cortisol levels in pregnancy is not due to the increase in corticosteroid-binding globulin levels. J Clin Endocrinol Metab 71(3): 639-644. Van der Kolk JH, Nachreiner RF, Schott HC, Refsal KR, Zanella AJ (2001) Salivary and plasma concentration of cortisol in normal horses and horses with Cushing’s disease. Eq Vet J 33(2): 211-213. Walker RF (1989) Salivary corticosteroids: clinical and research applications. Report on the workshop conference “application of saliva in laboratory medicine.” J Clin Chem Clin Biochem 27(4): 234-235. |
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