Measurement of haptoglobin in saliva of cows: Validation of an assay and a pilot study of its potential application.

In recent years, the use of saliva as a matrix for the measurement of biomarkers of health and welfare is gaining importance due to its non-invasive collection. Haptoglobin (Hp) is an acute-phase protein involved in the inflammatory response and changes in its concentration can provide information about the health status of the animals. This study aimed to develop and validate an assay based on luminescent amplification (AlphaLISA technology) for the measurement of Hp in bovine saliva and to study the possible changes in different inflammatory situations such as peripartum period and lameness. The assay proved to be accurate, reliable, and sensitive for the measurement of Hp in cow saliva (coefficient of variation (CV) 7.57%; coefficient of determination (R2) 0.992; recovery test 105.15%; lower limit of quantification (LLQ) 7.9 ng/ml). Significant differences were observed between Hp levels in saliva of cows before (13 days before) and after (7 and 20 days after) calving and at the moment of calving (p < 0.0001), and between lame and healthy cows (p < 0.008). In conclusion, this assay can detect Hp in a precise, sensitive, and accurate way in saliva of cows. Future studies with a larger population and different disease conditions should be conducted to determine the potential of Hp as an inflammatory biomarker in cow saliva.

Automated assays for trace elements and ferritin measurement in saliva of pigs: Analytical validation and a pilot application to evaluate different iron status.

The aim of this study was to validate automated methods to measure iron (Fe), zinc (Zn), copper (Cu) and ferritin in pig saliva samples. A complete analytical validation was performed of all assays. In addition, these methods were applied to saliva of Fe supplemented (n = 22) and non-supplemented (n = 20) piglets. All assays were able to measure these biomarkers in pig saliva with adequate precision, accuracy and high sensitivity and, in case of trace elements without needing a deproteinization pre-process. The group of piglets supplemented with Fe presented significantly higher levels of ferritin and Zn in saliva. In conclusion, the automated assays evaluated were able to measure Fe, Zn, Cu and ferritin in saliva of pigs, and in case of trace elements, they have the advantage of not needing a deproteinization pre-treatment and thus these analytes can be measured in a simple and fast manner.

Basics for the potential use of saliva to evaluate stress, inflammation, immune system, and redox homeostasis in pigs.

The use of saliva as a biological sample has many advantages, being especially relevant in pigs where the blood collection is highly stressful and painful, both for the animal and the staff in charge of the sampling. Currently one of the main uses of saliva is for diagnosis and detection of infectious diseases, but the saliva can also be used to measure biomarkers that can provide information of stress, inflammation, immune response and redox homeostasis. This review will be focused on the analytes that can be used for such evaluations. Emphasis will be given in providing data of practical use about their physiological basis, how they can be measured, and their interpretation. In addition, some general rules regarding sampling and saliva storage are provided and the concept of sialochemistry will be addressed. There is still a need for more data and knowledge for most of these biomarkers to optimize their use, application, and interpretation. However, this review provides updated data to illustrate that besides the detection of pathogens in saliva, additional interesting applicative information regarding pigs´ welfare and health can be obtained from this fluid. Information that can potentially be applied to other animal species as well as to humans.

Changes in saliva analytes during pregnancy, farrowing and lactation in sows: A sialochemistry approach.

Salivary biomarkers were studied in 17 healthy Large White sows from early gestation to the end of lactation. Saliva samples were obtained at 34 ± 3 days from insemination (G30), 24 ± 4 days before farrowing (G90), within the first 24 h after farrowing (L1) and at the end of a lactation period of 21 days (L21). The measurements in saliva included stress-related biomarkers (cortisol, chromogranin A, α-amylase, butyrylcholinesterase [BChE] and lipase [Lip]), inflammatory biomarkers (adenosine deaminase isoenzymes 1 [ADA1] and 2 [ADA2], and haptoglobin [Hp]) and oxidative stress biomarkers (cupric reducing antioxidant capacity, trolox equivalent antioxidant capacity, ferric reducing ability, uric acid, advanced oxidation protein products [AOPP] and hydrogen peroxide [H2O2]), as well as routine biochemistry analytes (aspartate aminotransferase [AST], alkaline phosphatase [ALP], γ-glutamine transferase [GGT], lactate dehydrogenase [LDH], creatine kinase [CK], urea, creatinine, triglycerides, lactate, calcium and phosphorus). The main changes were observed at farrowing, with increases in biomarkers of stress (cortisol and BChE), inflammation (ADA isoenzymes and Hp) and oxidative stress (AOPP and H2O2), as well as muscle and hepatic enzymes (CK, AST, ALP, GGT and LDH). Lactate and triglycerides increased at the end of gestation and remained at high concentrations until the end of lactation. Lip was higher in gestation than at lactation. Thus, changes in biomarkers of stress, immune function, oxidative stress, hepatic and muscle integrity, and energy mobilization occur in sow saliva during pregnancy, farrowing and lactation. These changes, caused by physiological conditions, should be taken into consideration when these biomarkers are used for the evaluation of sow health and welfare.

Effect of reduction and alkylation treatment in three different assays used for the measurement of oxytocin in saliva of pigs.

Oxytocin is a hormone that is increasingly being used for welfare evaluation in animals. Although several types of samples have been used for oxytocin measurement, saliva can be a suitable option for pigs producing less stress than blood sampling. In this study, 3 different methods for oxytocin measurements, 2 based on alphaLISA technology (one with a monoclonal and other with a polyclonal antibody) and one commercially available kit, were compared in saliva of pigs. These methods were used in saliva samples obtained from female pigs at 3 different days during gestation and lactation, with and without a reduction/alkylation (R/A), which is a procedure for breaking the links between oxytocin and proteins of the sample. The assays showed a different behavior after the R/A procedure, with no significant changes in the oxytocin results in case of the alphaLISA monoclonal method, a significant decrease with the alphaLISA polyclonal method, and a significant increase with the commercial kit. Although all assays showed a similar tendency in detecting the changes in oxytocin during gestation and lactation, they showed changes of different magnitude and statistical signification. This report indicates that different assays can measure different forms of oxytocin present in saliva and can have a different behavior after R/A of the sample and when are used to measure oxytocin in gestation and lactation.

Measurement of cortisol, cortisone and 11ß-hydroxysteroid dehydrogenase type 2 activity in hair of sows during different phases of the reproductive cycle.

Two sensitive assays based on AlphaLISA technology were developed and validated for the measurement of cortisol and cortisone in hair of pigs, that also enabled estimation of 11ß-hydroxysteroid dehydrogenase type 2 activity. These assays were applied to hair samples from sows (n = 32) collected at 5 days before, and at 23 and 59 after farrowing, in reproductive cycles in two different periods: spring-summer (n = 16) and winter-spring (n = 16). The assays were precise (imprecision <12%) and accurate (recovery range, 80-115%) for cortisol and cortisone determination. Hair cortisone concentrations and the cortisone/cortisol ratio (an estimate of 11ß-hydroxysteroid dehydrogenase isoenzyme type 2 activity) increased after farrowing more than cortisol, being these changes of higher magnitude during periods of higher atmospheric temperature. The measurement of hair cortisone concentrations and estimations of the activity of the 11ß-hydroxysteroid dehydrogenase isoenzyme type 2, measured by the assays developed in this study, are complementary biomarkers to hair cortisol, and can increase at periods associated with stress, such as farrowing and lactation, especially at high atmospheric temperatures. .

Oxytocin in saliva of pigs: an assay for its measurement and changes after farrowing.

Oxytocin is a hormone of interest in reproduction, but also in the field of psychology and behavior, being considered as a biomarker of positive emotions. Saliva can be a noninvasive way to measure oxytocin, which is very useful in species such as the pig where blood collection can produce a high degree of stress. In this study, a new assay for oxytocin measurement was developed, analytically validated, and used to measure possible changes in oxytocin in saliva of female pigs at different days after farrowing. The assay showed an adequate accuracy and precision and does not need a previous extraction step. In addition, oxytocin concentrations were significantly higher at day 1 of lactation than at day 9 after farrowing, but levels increased at day 20 again. This assay can contribute to a wider use of oxytocin measurements in pigs as it is a noninvasive sampling procedure that minimizes stress.

Evaluation of new biomarkers of stress in saliva of sheep.

Some routine handling procedures can produce stress in farm animals, and an adequate control of these stressors is important to avoid the negative effects on animal health and production. The measurement of biomarkers in saliva can be a suitable tool for the evaluation and control of stress. In this report, lipase, butyrylcholinesterase (BChE), total esterase (TEA) and adenosine deaminase (ADA) activities in the saliva of sheep were evaluated as biomarkers of stress. For this purpose, they were measured after inducing stress by facing a dog (experiment 1) and shearing (experiment 2), and comparing them to other stress salivary biomarkers such as α-amylase (sAA) and cortisol, as well as heart rate (HR). Each analyte was measured at the basal time, and during and just after the end of the stressful stimulus, and at various times for the first hour after the period of stress induction. Values were compared with those obtained from a control group. Lipase was the only analyte that showed significant changes between the stress and the control group in both experiments. Although TEA and ADA increased after stress, no significant differences were seen compared with the control group. Lipase was correlated highly with sAA and HR, in experiment 1; and correlated moderately with cortisol and HR in experiment 2. Lipase showed the greatest percentage increase after the stressful stimuli and less overlap with the control group in the two experiments. From the results of this study it can be concluded that lipase, TEA, BChE and ADA are enzymes present in the saliva of sheep and that they can be measured by using simple and fast colorimetric methods. Further studies should be undertaken with regard to the possible application of lipase as a biomarker of stress in sheep.