Evaluation of the Effect of Sampling Time on Biomarkers of Stress, Immune System, Redox Status and Other Biochemistry Analytes in Saliva of Finishing Pigs.

This study aims to evaluate the possible variations due to the sampling time in the day in 26 analytes of pigs' saliva, related to stress, the immune system, redox status and other biomarkers related to metabolism and selected tissues and organs, in order to know the possible effects of the hour of the day in their interpretation. These analytes were measured in saliva obtained from a population of 40 clinically healthy pigs from 8 a.m. to 8 p.m., every 4 h in the same day. In our experimental conditions, daily variations were observed in cortisol, salivary α-amylase, total esterase activity, butyrylcholinesterase, lipase, adenosine deaminase isoenzyme 1, uric acid, superoxide dismutase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, lactate dehydrogenase, lactate and triglycerides. These changes appeared in both sexes, except for adenosine deaminase isoenzyme 1 and superoxide dismutase which only showed differences in females. In conclusion, this report indicates that, in the experimental conditions of this trial, the time of the day and sex can influence the values obtained in various salivary analytes in pigs. These variations should thus be taken into consideration for an adequate interpretation of these analytes when used for the evaluation of health and welfare in this species.

Low-cost do-it-yourself (DIY) mannequin for blood collection: A comprehensive evaluation about its use in teaching.

The use of mannequins to practice different clinical procedures in undergraduate students complies with the 3R principle (Replacement, Reduction, Refinement) without affecting professional competencies. However, commercial solutions are often expensive and therefore, not available for many schools. The main aim of this study was to describe the development and validation of an economical Do-It-Yourself mannequin for jugular blood collection from dogs. The use of the mannequin was evaluated by (1) assessment of the opinion of students and experts and (2) by conducting a pilot study where salivary biomarkers of stress were determined in students and dogs. The costs of the materials needed for the mannequin confection were less than 60 Euros and it was easily built in less than 1 h. The mannequin was very well accepted and scored by both, students and experts, being mostly liked a lot and considered it to be very useful for the practices (Scored 8, 9 or 10/10). Students that could first practice with the mannequin reported a self-perceived higher level of confidence and had lower levels of alpha-amylase in saliva after the procedure. Overall, the mannequin enables the initiation of blood sampling skills in agreement with 3R principles and is easy to perform, economically affordable and sustainable. This model could be adapted to other vein simulations and animal species, and has the potential to help students deal with stressful situations such as taking blood samples from a live animal.

Evaluation of the Effect of a Live Interview in Journalism Students on Salivary Stress Biomarkers and Conventional Stress Scales.

A career in journalism can be very stressful, as journalists frequently have to deal with uncontrolled situations such as conducting live interviews. Therefore, training is essential during their career, both for the development of communication skills and for the improvement of the real and effective capacity to perform the tasks of their professional activity. The aim of this study was to assess the levels of stress in students before and after a practical training in a professional television set using subjective (State-Trait Anxiety Inventory (STAI) and Likert scale) and objective (salivary cortisol and alpha-amylase) methods. The results indicate that a live interview produces stress in the students as revealed by increased concentrations of cortisol and alpha amylase in saliva. Furthermore, students with lower initial concentrations of these biomarkers obtained better grades in evaluation, suggesting that greater control of anticipatory stress could be associated with a better activity performance.

Changes in Saliva Analytes in Dairy Cows during Peripartum: A Pilot Study.

This pilot study aimed to study the possible changes in a profile of 26 salivary analytes in thirteen healthy dairy cows along their peripartum period. Analytes associated with the stress (salivary cortisol, salivary alpha-amylase, butyrylcholinesterase, and total esterase), inflammation (adenosine deaminase), oxidative status (total antioxidant capacity and the advanced oxidation protein products), and general metabolism (creatine kinase, γ-glutamyl transferase, urea, triglycerides, glucose, and lactate) varied along the sampling times. A positive correlation between the white blood cells counts, and the lipase, Trolox equivalent antioxidant capacity, advanced oxidation protein products, and lactate levels in saliva were observed at the delivery. A linear association between selected salivary analytes at different sampling times and the milk yield after calving was observed. In conclusion, in our experimental conditions, it was observed that the peripartum period in dairy cows can induce changes in salivary analytes. Some of them were associated with inflammatory status and the capacity of milk production after calving.

Changes Occurring on the Activity of Salivary Alpha-Amylase Proteoforms in Two Naturalistic Situations Using a Spectrophotometric Assay.

This study aimed to evaluate the changes in the activity of total salivary alpha-amylase (TsAA) and both the non-glycosylated and glycosylated salivary alpha-amylase proteoforms (NGsAA and GsAA, respectively) in physical and psychological stress models, estimated using a simple and easily set-up method. The method used was a spectrophotometric assay with 2-chloro-4-nitrophenyl-α-D-maltotriose (CNPG3) as a substrate, incubated with Concanavalin A (ConA) to remove most of the glycosylated protein from the sample. This method allowed the measurement of TsAA and estimation of NGsAA and GsAA activities with imprecision lower than 10%. When this method was applied to two different stress models, differences in the responses of the proteoforms were observed, with the NGsAA activity showing changes of higher magnitude after stress induction than the GsAA activity, and the highest correlation with the State-Trait Anxiety Inventory Scale in the Trier Social Stress Test (TSST). In conclusion, the activity of the two main sAA proteoforms can be easily estimated in saliva, and their measurement can provide additional information on TsAA activity in physical or psychological stress situations.

Changes in Saliva Analytes Associated with Lameness in Cows: A Pilot Study.

The possible changes in a panel of 21 salivary analytes on a population of cows with lameness before and after treating lameness by hoof trimming were analyzed. Then, the analytes that showed significant changes were studied in a larger population of cows with lameness and compared with healthy cows For this purpose, two groups of cows were made by a specialized veterinarian. One consisted of healthy cows with no external signs of diseases and no hematological or biochemical abnormalities, and showing no signs of lameness according to the numerical rating system of severity (NRS, 5-point scale); and the other composed of cows showing only lameness with a NRS of 3.1 ± 0.87 and a lesion scoring system (LSS, 4-point scale) of 3.3 ± 0.89. Both groups did not differ in parity (p = 0.140), days in milk (DIM) (p = 0.780), and body condition score (BCS) (p = 0.074). Initially, 21 biochemical analytes were determined in the saliva of six cows with lameness at the diagnosis time (T0) and twenty days after hoof trimming that successfully solved the lameness (TF). This exploratory study only showed significantly higher values in lipase (Lip) and total esterase (TEA) at T0 compared to TF (p < 0.001 and p = 0.034, respectively). When both analytes were measured in the additional five lame cows and the results of all the animals of the lame group (n = 11) were compared with the healthy group (n = 11), only TEA showed higher activities in the group of lame cows than healthy cows (p = 0.004). TEA was positively correlated with both NRS and LSS (r = 0.43, p = 0.004 and r = 0.35, p = 0.003). In conclusion, this study showed that cows with lameness in our experimental conditions had higher TEA values than healthy cows, and these values decreased after treatment. This is a pilot study, and further studies using a larger population of cows with lameness due to different causes and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows.

Changes in Salivary Analytes of Horses Due to Circadian Rhythm and Season: A Pilot Study.

This study aims to evaluate the circadian and circannual variations in a panel of analytes in horse saliva that have been previously described as biomarkers related to stress and disease, in order to interpret them correctly when they are measured in this species. This panel of analytes integrated cortisol, salivary alpha-amylase (sAA), lipase (Lip), total esterase (TEA), butyrylcholinesterase (BChE), adenosine deaminase (ADA), γ-glutamyl transferase (gGT), creatine kinase (CK), urea, total bilirubin, total protein (TP), and phosphorus. These analytes were measured in saliva obtained from a population of five clinically healthy mares from 06:30 to 20:30, every 2 h over two consecutive days in two different photoperiod seasons, winter and spring. The temperature and relative humidity did not change between the two consecutive days sampled in each sampled season, and no thermal discomfort was observed. Changes throughout the course of the day were observed for cortisol, sAA, TEA, BChE, ADA, and CK. However, a circadian pattern was only observed for cortisol, TEA, BChE, ADA, and CK. Moreover, the values obtained for sAA, Lip, and BChE were significantly different between seasons, with different daily rhythms for cortisol, TEA, BChE, and ADA depending on the season. In conclusion, this pilot study indicates that the time of the day and the season influence salivary analytes in horses, showing a rhythmic pattern for cortisol, TEA, BChE, ADA, and CK. These factors should thus be taken into consideration for the interpretation of analytes in horse saliva.

Variation of human salivary alpha-amylase proteoforms in three stimulation models.

OBJECTIVES: To evaluate the sAA proteoforms' expression during different stimulation situations. MATERIALS AND METHODS: This study evaluated the salivary alpha-amylase (sAA) proteoforms' behavior by western blot (WB) analysis and high-resolution mass spectrometry (LC-MS/MS) in different situations that produce increases in sAA activity. For this purpose, six healthy women with a similar body mass index, age, and fit, underwent different sAA stimulation tests, such as acetic acid stimulation, psychological stress using the standardized Trier social stress test, and physical effort using the Cooper treadmill test. RESULTS: The three models showed an increase in sAA activity. The WB demonstrated seven common bands observed in the six women (band one at 59 kDa, two at 56 kDa, three at 48 kDa, four at 45 kDa, five at 41 kDa, six at 36 kDa, and seven at 14 kDa), in which sAA protein was identified. The individual WB analysis showed that band two, which corresponded to the native non-glycosylated sAA proteoform, had a higher increase after the three sAA stimulation inducers, and this band was also the only proteoform correlated with sAA activity (r = 0.56, P = 0.001). In addition, when the label-free quantification analysis was performed, the different proteoforms showed different responses depending on the type of stimulation. CONCLUSIONS: This preliminary study showed that the diverse sAA proteoforms' expression depends on the different stimulation models. CLINICAL RELEVANCE: This study opens new perspectives and challenges for the use of the different alpha-amylase proteoforms as possible biomarkers in addition to the sAA activity.

Serum and salivary adiponectin dynamics in septic and non-septic systemic inflammation in a canine model.

Adiponectin is a 30 kDa protein hormone that has anti-atherogenic properties, being an insulin-sensitizing and anti-inflammatory molecule. Salivary adiponectin concentrations correlate positively with serum, thus, saliva was indicated as appropriate biofluid for its measurement in different clinical situations. However, inflammation was indicated as main confounding factor when evaluating the usefulness and the reliability of determination of salivary adiponectin. The aim of the present report was to evaluate the dynamics of salivary and serum adiponectin in systemic non-septic and septic inflammation using a dogs as a model. Forty bitches were enrolled. Seventeen dogs were healthy (group I, non-septic) and 23 bitches were diagnosed with pyometra (group II, septic). Ovariohysterectomy was performed for all animals. Saliva and blood samples were collected before (D0) and 3 (D3) and 10 (D10) days after ovariohysterectomy. At D0, Group I showed higher serum and salivary adiponectin than group II, although statistical significance was only detected in salivary adiponectin between the two groups at D0 (P = 0.001). In serum, adiponectin was higher on D0 than on D3 and tended to reach pre-surgery values on D10 in both groups. Salivary adiponectin showed similar behaviour to serum in Group I, while in group II salivary adiponectin concentrations were lowest on D0 and tended to increase on D3 and D10. The data obtained in present study describe for the first time the comparative behavior of salivary adiponectin in non-septic and septic inflammation. Salivary, and not serum, adiponectin seemed to mimic better the inflammatory and general health status.

Changes in Saliva Analytes Correlate with Horses' Behavioural Reactions to An Acute Stressor: A Pilot Study.

Acute stress induces an array of behavioural reactions in horses that vary between individuals. Attempts to relate behavioural patterns and physiological responses have not always given clear-cut results. Here, we measured the changes in a panel of salivary components: salivary alpha-amylase (sAA), lipase, total esterase (TEA), butyrylcholinesterase (BChE), adenosine deaminase (ADA), and cortisol, and their potential link with horses' behaviours after acute stress. Saliva samples were collected in nine riding horses subjected to a test consisting of opening an umbrella. Saliva sampling was obtained at a basal time point in the stall (T1), in the test indoor arena (T2), at a time of stress (T3), and 30 min (T4) and 60 min (T5) later. The horses' behaviour was recorded at T3 for 1 minute. sAA, lipase, TEA, and BChE showed significant changes along time, increasing at T3 for BChE, and decreasing at T4 for sAA and BChE. Butyrylcholinesterase appeared to be the most reliable predictor of behavioural responses, as it correlated with the index of emotionality, of laterality, and the occurrence of alarm signals, while sAA decreased when horses expressed quieter behaviours. These first results bring promising lines for novel, more precise physiological markers of acute stress in horses that can bridge the gap between behaviour and physiology.

Stability of selected enzymes in saliva of pigs under different storage conditions: a pilot study.

Stability of α-amylase (α-A), butyrylcholinesterase (BChE), lipase, adenosine deaminase (ADA) and total esterase activity (TEA) in two pools of porcine saliva was studied after 1 and 4 days at 4°C, and after 30, 90 and 360 days at -20° and -80°C. At 4°C, BChE, lipase and TEA were stable less than 1 day, α-A less than 4 days and ADA for up to 4 days. At -20°C, BChE and TEA were stable less than 30 days, α-A and lipase less than 90 days and ADA up to 360 days. At -80°C, TEA was stable less than 30 days, α-A and lipase less than 360 days, and BChE and ADA for up to 360 days.

Adenosine deaminase activity in pig saliva: analytical validation of two spectrophotometric assays.

We validated 2 assays for the measurement of adenosine deaminase (ADA) activity in the saliva of pigs: the Giusti-Galanti manual method (ADA-GG) and a commercial automated assay (Diazyme Laboratories; ADA-D). Intra-assay coefficients of variation (CVs) were <7 and 9%, and interassay CVs were <12 and 5%, for ADA-GG and ADA-D, respectively. Accuracy was measured by 2 methods: recovery and linearity-under-dilution. Recovery was 82.4-106.8% for ADA-GG, and 92.8-107.9% for ADA-D. Serial dilutions showed R2 > 0.95 and 0.99 for ADA-GG and ADA-D, respectively. Linear regression between the methods gave R2 = 0.997 ( p < 0.0001), and a Bland-Altman plot showed a proportional bias of 112 IU/L (95% confidence interval of -99 to 322 IU/L) for ADA-D. No significant differences were observed between the results obtained by either method in saliva or serum. ADA activity was much higher in porcine saliva than in serum. Salivary ADA activity was significantly higher in lame pigs compared to healthy animals. However, serum ADA activity was significantly lower in lame pigs.

Total esterase measurement in saliva of pigs: Validation of an automated assay, characterization and changes in stress and disease conditions.

An automated spectrophotometric method for total esterase activity (TEA) measurement in porcine saliva has been developed and validated, using 4-nitrophenyl acetate (4-NA) as substrate. The method was precise and accurate, with low limit of detection, and was able to measure samples with TEA activities up to 400IU/L without any dilution. In addition, the different enzymes contributing to TEA were characterized, being identified carbonic anhydrase VI (CA-VI), lipase, cholinesterase (ChE) and cholesterol esterase (CEL). TEA significantly increased (1.49-fold, P<0.01) in healthy pigs just after applying an acute stress stimulus consisting of nasal restraint, being lipase and cholinesterase the main responsible of this increase. TEA was significantly increased (1.83-fold, P<0.001) in a group of pigs with lameness; in this case, in addition to lipase and ChE, CA-VI also increased. The results found in this report indicate that TEA can be easily measured in porcine saliva with an accurate and highly reproducible automated method. Salivary TEA is mainly due to the activity of four enzymes: CA-VI, lipase, ChE and CEL, and these enzymes can change in a different way in situations of stress or disease.