Damián Escribano

Validation of an automated chemiluminescent immunoassay for salivary cortisol measurements in pigs

The aim of the current study was to validate an automated immunoassay for cortisol quantification in the saliva of pigs. The assay had intra- and interassay coefficients of variation lower than 16%, in all cases. The limit of detection was 0.016 µg/dl, and the lower quantification limit was 0.197 µg/dl. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution and recovery tests. In addition, this assay was used to quantify cortisol in 2 models of stress: 1 in which animals were immobilized with a nose-snare and 1 in which pigs were transported for a duration of 30 min. In both cases, a significant increase (P < 0.01) in salivary cortisol was detected after the stressful situation. Overall, the assay validated in the present study could be used for the evaluation of cortisol changes in stressful situations.

Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA359-379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra- and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 × 10-3 μg/ml and a limit of quantification of 24.5 × 10-3 μg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution (r = 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase (P < 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.

Response of salivary haptoglobin and serum amyloid A to social isolation and short road transport stress in pigs.

The possible use of serum amyloid A and haptoglobin (Hp) determination in saliva as stress markers in swine was investigated in this study. Firstly, a model of social isolation was followed. Significantly higher serum amyloid A concentrations were obtained in isolated animals (n=10) compared to grouped animals (n=10; P=0.036), in agreement with cortisol levels (P=0.015), while haptoglobin levels did not significantly change. Secondly, animals were subjected to short road transport. Cortisol and serum amyloid A levels significantly increased following road transport. Serum amyloid A levels were significantly high on arrival at the slaughterhouse and maximal at 30 and 60 min lairage (P<0.0001). Cortisol levels were only significantly elevated on arrival at the slaughterhouse (P<0.0001). These results indicate that salivary serum amyloid A (and not haptoglobin) determination is a potential biomarker for the assessment of complex stress in pigs, and that it has a more prolonged response than cortisol.

Different stressors elicit different responses in the salivary biomarkers cortisol, haptoglobin, and chromogranin A in pigs

Most commonly, salivary cortisol is used in pig stress assessment, alternative salivary biomarkers are scarcely studied. Here, salivary cortisol and two alternative salivary biomarkers, haptoglobin and chromogranin A were measured in a pig stress study. Treatment pigs (n = 24) were exposed to mixing and feed deprivation, in two trials, and compared to untreated controls (n = 24). Haptoglobin differed for feed deprivation vs control. Other differences were only found within treatment. Treatment pigs had higher salivary cortisol concentrations on the mixing day (P < 0.05). Chromogranin A concentrations were increased on the day of refeeding (P < 0.05). Haptoglobin showed a similar pattern to chromogranin A. Overall correlations between the salivary biomarkers were positive. Cortisol and chromogranin A were moderately correlated (r = 0.49, P < 0.0001), correlations between other markers were weaker. The present results indicate that different types of stressors elicited different physiological stress responses in the pigs, and therefore including various salivary biomarkers in stress evaluation seems useful.

Causes, consequences and biomarkers of stress in swine: an update

BackgroundIn recent decades there has been a growing concern about animal stress on intensive pig farms due to the undesirable consequences that stress produces in the normal physiology of pigs and its effects on their welfare and general productive performance. This review analyses the most important types of stress (social, environmental, metabolic, immunological and due to human handling), and their biological consequences for pigs. The physio-pathological changes associated with stress are described, as well as the negative effects of stress on pig production. In addition an update of the different biomarkers used for the evaluation of stress is provided. These biomarkers can be classified into four groups according to the physiological system or axis evaluated: sympathetic nervous system, hypothalamic-pituitary-adrenal axis, hypothalamic-pituitary-gonadal axis and immune system.ConclusionsStress it is a process with multifactorial causes and produces an organic response that generates negative effects on animal health and production. Ideally, a panel of various biomarkers should be used to assess and evaluate the stress resulting from diverse causes and the different physiological systems involved in the stress response. We hope that this review will increase the understanding of the stress process, contribute to a better control and reduction of potential stressful stimuli in pigs and, finally, encourage future studies and developments to better monitor, detect and manage stress on pig farms.

Changes in saliva biomarkers of stress and immunity in domestic pigs exposed to a psychosocial stressor

A combination of salivary biomarkers measured at the same time, reflecting the different systems that are involved in the stress mechanism, could be the best tool for its evaluation. In this study, changes in a panel of salivary biomarkers of stress and immunity including chromogranin A (CgA), IgA, cortisol, testosterone, haptoglobin (Hp) and C-reactive protein (CRP), were evaluated. A total of 14 (7 control and 7 test) crossbred Duroc × (Landrace × Large White) males of 190 days of age were used for this experiment. The stress mechanism was evaluated after applying a psychosocial stressor model in 7 pigs, based on isolation and regrouping. Our results show that after of isolation, there was a significant (P<0.05) increase in salivary CgA and IgA. However, after regrouping, there was a significant increase (P<0.05) in salivary cortisol, testosterone and CgA. Salivary Hp and CRP concentrations did not significantly change after applying this stress model. This panel of salivary biomarkers could be used as a practical and non-invasive tool for reflecting the activity of different physiology systems involved in the stress response.

Saliva chromogranin A in growing pigs: a study of circadian patterns during daytime and stability under different storage conditions.

Salivary chromogranin A (CgA) is considered to be a biomarker of activation of the sympatho-adrenomedullary system, and has recently been proposed as a useful indicator of the acute stress response in pigs. The aim of the present study was to determinate whether salivary CgA concentrations in healthy growing pigs exhibits any circadian pattern during the daytime, and to evaluate its stability under different storage conditions. A total of 80 pigs (40 in spring and another 40 in autumn) of two different ages and genders were used. To establish the circadian pattern, saliva samples were collected at 07.00, 11.00, 15.00 and 19.00 h on two consecutive days. Pooled samples were used for the stability study and were measured on the day of sampling and periodically for up to 360 days later. Samples were stored at 4 °C, -20 °C or -80 °C and the effect of repeated freezing and thawing was also evaluated. No circadian pattern was detected for salivary CgA in either season and there were no significant effects of gender or age. However, mean salivary CgA concentrations were significantly higher (P<0.0001) in the pigs sampled in autumn, compared to those sampled in the spring. Short term storage at 4 °C is recommended for up to 2 days, whereas frozen samples can be stored for 1 year at -20 °C or -80 °C, without substantial reduction in CgA values. In addition, samples can be frozen and thawed up to seven times without significant loss of the biomarker.

Growth promotion in pigs by oxytetracycline coincides with down regulation of serum inflammatory parameters and of hibernation-associated protein HP-27.

The growth promoting effect of supplementing animal feed with antibiotics like tetracycline has traditionally been attributed to their antibiotic character. However, more evidence has been accumulated on their direct anti‐inflammatory effect during the last two decades. Here we used a pig model to explore the systemic molecular effect of feed supplementation with sub therapeutic levels of oxytetracycline (OTC) by analysis of serum proteome changes. Results showed that OTC promoted growth, coinciding with a significant down regulation of different serum proteins related to inflammation, oxidation and lipid metabolism, confirming the anti‐inflammatory mechanism of OTC. Interestingly, apart from the classic acute phase reactants also down regulation was seen of a hibernation associated plasma protein (HP‐27), which is to our knowledge the first description in pigs. Although the exact function in non‐hibernators is unclear, down regulation of HP‐27 could be consistent with increased appetite, which is possibly linked to the anti‐inflammatory action of OTC. Given that pigs are good models for human medicine due to their genetic and physiologic resemblance, the present results might also be used for rational intervention in human diseases in which inflammation plays an important role such as obesity, type 2 diabetes and cardiovascular diseases.

Circadian pattern of acute phase proteins in the saliva of growing pigs.

The circadian rhythm of the acute phase proteins (APPs) haptoglobin (Hp) and C-reactive protein (CRP) was assessed in saliva samples from 18- and 21-week old pigs. Saliva was collected at 07.00, 11.00, 15.00 and 19.00 h on two consecutive days and the Hp and CRP concentrations were quantified using two species-specific, time-resolved immunofluorometric assays. Salivary Hp levels were significantly higher (P < 0.001) in the morning compared to late afternoon (0.68 and 0.37 μg/mL, respectively) although the magnitude of the difference was much lower than is produced by inflammatory conditions. No significant differences were observed in CRP concentrations. Although the concentration of both APPs was higher in the 21- compared to the 18-week old pigs (P < 0.0001), no differences were observed in the circadian rhythm of these APPs when the two age groups were compared. Animal gender did not influence the circadian pattern of either APP, although the mean salivary CRP levels were higher in females (P < 0.05).

Serum paraoxonase type-1 activity in pigs: assay validation and evolution after an induced experimental inflammation.

Paraoxonase 1 (PON1) is a serum enzyme synthesised and secreted primarily by the liver. It possesses anti-inflammatory properties limiting the production of pro-inflammatory mediators. The objectives of this study were to validate three spectrophotometric assays for the quantification of PON1 activity in pig serum, and to determine if PON1 activity in porcine behaves as a negative acute phase protein (APP), decreasing in inflammatory conditions. An analytical validation using three different substrates - 5-thiobutil butyrolactone (TBBL), phenylacetate (PA) and 4-(p)-nitrophenyl acetate (pNA) - was performed. In addition, inflammation was experimentally induced in five pigs by subcutaneous injection of turpentine oil, while five control pigs were left untreated. The treated pigs showed significant increases in CRP and decreases in albumin, indicating an inflammatory condition. The three substrates used would be suitable for PON1 activity measurements in serum samples, since they offer adequate precision (coefficients of variation<10%), sensitivity (0.01, 0.15, 0.02 U/mL for TBBL, pNA and PA respectively) and accuracy (r=0.99). In addition, PON1 behaves as a negative APP in pigs since a significant decrease (P<0.05) in its activity after 72 h of the induction of the inflammation was observed with all substrates.