Porcine salivary analysis by 2-dimensional gel electrophoresis in 3 models of acute stress: A pilot study

Abstract

The purpose of this research was to study changes in the salivary proteome of healthy pigs in stressful situations to identify any potential new salivary biomarker of stress. Three groups of animals were subjected to 3 stress models: snaring restraint followed by simulated sampling of vena cava blood; brief transport by road; and restriction of movement in a digestibility cage. Saliva was obtained from each animal before and 15 and 30 min after the induction of stress. The samples from the animals that showed the greatest increase in salivary cortisol concentration were pooled and run on 2-dimensional gels. Coomassie Brilliant Blue R-250 was used for spot detection and mass spectrometry for spot identification. Statistical analyses showed that 2 proteins had significant differences in expression before and after the induction of stress. These proteins were identified as odorant-binding protein and fragments of albumin. Further studies will be necessary to confirm the value of using these proteins as salivary biomarkers of stress in pigs.

Introduction

Stress is one of the most important factors to consider in animal production for both animal-welfare and economic reasons. High levels of stress and poor welfare increase susceptibility to diseases, decrease life expectancy, impair growth and reproduction, damage the body, cause behavioral abnormalities (1), and decrease meat quality (2,3).

Biomarkers of stress are commonly sought in blood or serum. Proteomic studies of porcine serum have shown that some proteins, such as haptoglobin, annexin IV, apolipoprotein A-I, and fetuin, are biomarkers of welfare in these animals (4). In addition, other proteins, such as serum amyloid A, paraoxonase 1, and platelet-activating factor acetylhydrolase, may act as markers of an oxidative stress response in pigs, as has been postulated in other animal species (5). But blood sampling generates a substantial stress response by itself in pigs (6). For this reason, it is advisable to use samples whose collection is noninvasive and stress-free, such as saliva. Several studies have shown that saliva samples can be used for the measurement of various biologic analytes that are useful for stress monitoring, such as cortisol (7), chromogranin A (8), α-amylase (9), and immunoglobulin A (10). Currently, cortisol is the most common indicator of stress measured in farm animals, but its use as the only stress indicator can have some limitations (11,12). For example, the cortisol concentration depends not only on physical or psychological stress but also on environmental conditions and metabolic factors (12). Therefore, it would be of great value to find new and sensitive biomarkers in saliva that could be useful to evaluate stress and welfare in the production chain.

Proteomics has been used frequently in recent years to study biomarkers (13), and the results with saliva samples in pigs have been promising. A 2-dimensional gel electrophoresis (2-DE) map of porcine saliva has recently been published (14) and used to identify biomarkers of disease in growing pigs under field conditions (15).

The main objective of this research was to analyze possible changes in the salivary proteome of pigs suffering acute stress. The pigs were subjected to 3 experimental models of acute stress: snaring restraint followed by simulated sampling of vena cava blood; brief transport by road; and restriction of movement in a digestibility cage. The salivary proteome profiles before and after the experimental procedures were compared in a search for potential new biomarkers of stress. Data from this research could provide new tools to increase the knowledge about changes in the expression of salivary proteins in pigs under stress.

Materials and methods

Animals and housing

Twenty-eight crossbred Duroc × [Landrace × Large White] growing pigs (Sus scrofa domesticus) 3 to 4 mo of age from the experimental farm unit of the University of Murcia, Murcia, Spain, were used in the study. The pigs were given access ad libitum to a nutritionally balanced diet and water. They were housed in pens with a minimum space of 0.65 m² per animal (16) and an average temperature of 23°C. The 3 experimental groups were housed in different rooms of the farm and each animal was housed in a different pen to avoid the influence of applied psychological stress on the other animals.

Experimental procedures

The animals were divided into 3 groups. The 10 pigs in group I were subjected to snaring restraint followed by simulated sampling of vena cava blood; a nose sling was used for snaring and the lasso kept under tension manually for 5 min. The 10 pigs in group II were subjected to brief transport by road (40 min; 35 km) from the farm to the slaughterhouse. They were loaded into the trailer (which allowed 1.08 m² of space per pig) without food or drink and were mixed to break social hierarchy. No stops were made along the way. The 8 pigs in group III were placed in a digestibility cage 1 m above ground for 24 h; their space was sufficient only for turning in place, getting up, and lying down.

In all groups 3 saliva samples were taken from each animal; the 1st sample was taken before the induction of stress (at T0), and the remaining samples were taken 15 and 30 min later (at T15 and T30). The samples were obtained by introducing a small sponge into the pig’s mouth for at least 1 min with the help of a metal rod, as previously described (9). The sponges were placed in collection devices (Salivette; Sarstedt, Aktiengesellschaft and Company, Nümbrecht, Germany) and centrifuged at 4000 × g for 8 min to obtain the saliva. After centrifugation the saliva samples were stored at −80°C until analyzed.

All procedures involving the animals were approved by the University of Murcia Ethics Committee and followed the recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (16).

Salivary cortisol quantification

The salivary cortisol concentration was measured by means of the commercial kit Immulite 1000 Cortisol (Siemens Medical Solutions Diagnostics, Los Angeles, California, USA), validated for determinations in porcine saliva (7). An increase in concentration was interpreted as a positive stress response (17). The Friedman test was done to assess the time point at which an increase in concentration in the stressed animals was statistically significant. All statistical analyses were done with a statistical package GraphPad Prism, version 5.0 (GraphPad Software, La Jolla, California, USA). The significance level was set at P < 0.05.

Proteomic analysis

In each group the samples from the 4 pigs with the highest salivary cortisol values were selected for proteomic evaluation. The time points selected for evaluation were T0 and T15, the latter corresponding to the time at which the highest concentrations were obtained. To reduce the influence of biologic variation the saliva specimens at T0 and T15 from the selected pigs in each group were pooled, for 2 pools per group.

Before the pooling, the salivary protein concentration of each sample was determined by the RC DC (reducing-agent and detergent-compatible) Protein Assay (BioRad, Hercules, California, USA). Before electrophoresis, 150 μg of protein from the sample was treated with the 2-D Clean-Up Kit (GE Healthcare Europe, Freiburg, Germany) and resuspended in rehydration buffer (30 mmol/L Tris basic, 2 mol/L thiourea, 7 mol/L urea, and 4% wt/vol CHAPS). The salivary protein concentration was then determined again with the 2-D Quant Kit (GE Healthcare Europe). Each sample pool contained 50 μg of protein from each of the 4 pigs, for a total of 200 μg per pool.

Each 2-DE gel was run in duplicate. Each pool was diluted in rehydration solution (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [CHAPS], 60 mM dithiothreitol [DTT], 0.5% ampholytes [pH 3 to 10], and 0.002% bromophenol blue) and loaded onto an 18-cm-long immobilized pH gradient strip (IPG), pH 3 to 10, during the 20-h rehydration period, according to the instructions of the manufacturer (GE Healthcare Europe). Proteins were focused isoelectrically in the 1st dimension for a total of 60 000 Vh in a horizontal electrophoresis chamber (Ettan IPGphor 3 system; GE Healthcare Europe).

For the 2nd dimension the IPG strips were equilibrated for 15 min with 2% DTT equilibration solution [6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), and 0.05 M Tris-HCl (pH 6.8)] and then 15 min with 2.5% iodoacetamide (IAA) in equilibrate solution. Afterwards, SDS-polyacrylamide gel electrophoresis was done on homemade 12% polyacrylamide gels measuring 170 × 170 × 1 mm in a vertical electrophoresis chamber (Ettan DALTsix Electrophoresis System; GE Healthcare Europe) at 5 mA per gel for 2 h and 2 W per gel for 7 h at 23°C (18).

After electrophoresis the gels were fixed with a solution of 50% (v/v) ethanol and 10% (v/v) acetic acid and stained with Coomassie Brilliant Blue R-250 (19). Then 2-DE images were captured with the ImageScanner III System (GE Healthcare Europe) and analyzed by means of specific software (ImageMaster 2D Platinum 7.0; GE Healthcare Europe). Analysis included spot detection, landmarking, and spot matching of protein patterns for all gels in the set. Percentage spot volume was analyzed for a difference between the pigs in protein expression at T0 and T15 by a Wilcoxon signed-rank test and SPSS statistical software, version 10.0 (SPSS Inc., Chicago, Illinois, USA). Spots with a P-value < 0.05 were manually picked from the stained gels for identification by mass spectrometry (MS).

Analysis by mass spectronomy

Proteins within the chosen gel spots were first reduced and alkylated with DTT and IAA, respectively, and then digested to peptides with trypsin (Sigma-Aldrich, St. Louis, Missouri, USA) according to the protocol described by Shevchenko et al (20). The tryptic peptides were analyzed by capillary reversed-phase liquid chromatography coupled online with MS/MS. The column, BioBasic 18 (particle size 5 μm, pore size 300 Å, internal diameter 0.18 mm, and length 30 mm; Thermo Fisher Scientific, San Jose, California, USA), was connected to a Surveyor MS Pump Plus (Thermo Fisher Scientific) and then coupled with an LXQ Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific). The flow rate was set at 100 μL/min but split to a rate of 2 μL/min. Mobile phase A was 0.1% formic acid/2% methanol in water and B was 0.1% formic acid in methanol. The peptide samples were injected and gradient elution was done under the following conditions: 5% B in 2 min; a linear increase of 5% to 70% B in 18 min; 70% B for 10 min; and 5% B for 15 min. The mass spectrometer was operated in a data-dependent MS/MS mode in which the 15 most abundant peptide molecular ions in every scan were sequentially selected for collision-induced dissociation with a normalized collision energy of 34%. Dynamic exclusion was applied to minimize repeated selection of peptides previously selected. The capillary temperature and electrospray voltage were set to 200°C and 3.5 kV.

The resulting mass spectra were searched against the Swiss-Prot protein sequence database (453 320 sequences; released December 11, 2012) with Proteome Discoverer software, version 1.3 (Thermo Fisher Scientific), and the following search parameters: default charge states of 2+, 3+, and 4+; a maximum of 1 missed cleavage; and an average peptide mass tolerance of 1.5 Da. A fragmention search tolerance of 0.8 Da was permitted. A fixed modification was carbamidomethylation of cysteine and a variable modification oxidation of methionine. All data were searched against a decoy database. The peptide score cut-off for each run was automatically adjusted to ensure a 1% false-discovery rate throughout the experiments. A positive identification was assigned when 2 or more unique peptides were identified.

Results

The salivary cortisol values for the 4 animals in each group with the greatest response to the stressor stimulus are shown in Figure 1. The median values at T15 were significantly higher (P < 0.05) than those at T0 in all the experimental groups: in group I those values were 0.51 and 0.11 μg/dL, in group II 1.67 and 0.36 μg/dL, and in group III 0.92 and 0.30 μg/dL, respectively.

Figure 1.

Salivary cortisol concentrations in the 4 pigs in each group that showed the greatest response to 3 stressful situations: A — snaring restraint followed by simulated sampling of vena cava blood (group I); B — brief transport by road (group II); and C — restriction of movement in a digestibility cage (group III). Saliva samples were obtained before the induction of stress (at T0) and 15 and 30 min later (at T15 and T30). Each plot shows median (line within box), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers). Each asterisk indicates a statistically significant difference (*P < 0.05) from T0.

The 2-DE gels obtained (Figure 2) were quite homogeneous despite being created from pools of samples obtained after different types of stress, which could indicate that the pattern is quite stable despite the many factors that can affect the composition of saliva. The gels were manually aligned by defining 4 landmarks well distributed over each entire image that clearly represented the same protein form. After statistical analysis of the percentage spot volume of each of the 58 spots detected in all the gels, only 2 spots (nos. 7 and 22) appeared to be differentially regulated by the experimental stressor at T15 in comparison with T0; the same behavior of these 2 spots was observed with all 3 stress-induction methods (Table I). Spot 7 decreased more than 1.5-fold with stress, the greatest decrease being in group I. In contrast, spot 22 increased, the most pronounced change again being in group I; however, in general the observed changes in this spot were less than those observed in spot 7. With MS, spot 7 was identified as odorant-binding protein (OBP) and spot 22 as albumin.

Figure 2.

Representative protein patterns obtained by 2-dimensional electrophoresis of saliva samples from experimental groups I, II, and III at T0 (A) and T15 (B). Significant spots identified by mass spectrometry are circled; the numbering corresponds with that in Table I. MW — molecular weight; kDa — kilodaltons; pI — isoelectric point.

Table I.

Differential protein expression before and 15 min after (T0 and T15) the induction of 3 different types of stress in 3 groups of pigs as evaluated with 2-dimensional electrophoresis

Protein and pig group	Change in % spot volume, T0 → T15	Accession number in Swiss-Prot database	Theoretical MW (kDa)/pI	Sequence coverage (%)	Mascot score
7, odorant-binding protein		P81245	17.708/4.27	53.50	414.54
I	− 86%
II	− 42%
III	− 25%
22, albumin		P08835	69.692/6.08	45.47	1490.01
I	− 85%
II	− 16%
III	− 3%

MW — molecular weight; kDa — kilodaltons; pI — isoelectric point.

Discussion

Saliva is a fluid with many research and diagnostic possibilities, and its composition of thousands of proteins and peptides of glandular or blood origin is suggested to hold physiological and pathological information accessible by proteomic research (21). The state-of-the-art of salivary proteomics is in evolution, and a growing number of proteins will be investigated and discovered, with the goal of maintaining and improving livestock productivity and welfare (22). A recent publication from proteomics research suggested new porcine salivary biomarkers of disease conditions (15). However, to our knowledge no proteomic studies of the saliva of pigs under stress had been reported before our study.

The choice of the stressful stimuli for this study was based on existing literature. Restraint with a nose sling, a technique for temporarily restraining pigs that is commonly used in veterinary practice, represents an acute stressor (23,24). Similarly, poor environmental conditions during road transport are noted in the literature to be very stressful to animals (25,26). Digestibility cages are normally used in studies of metabolism and nutritional evaluation (27,28); the restriction of movement causes stress to the animal.

For proteomics purposes, the saliva samples from each group and time point were pooled. It has been postulated that the use of pools in proteomics studies could result in some loss of information in comparison with the use of single samples (29). However, pooling is a valid and potentially valuable method to detect a common pattern of protein expression in the analysis of limited amounts of sample (30–32) and to eliminate the biologic variation between individuals (33). In addition, the total protein concentration of porcine saliva is very low, 70 times less than the concentration in other biologic samples, such as serum. Thus, in our experience, large-volume samples are required to reach appropriate concentrations for proteomic analysis. Therefore, the use of individual samples of low amounts of saliva can be a limitation.

Differential expression of proteins in saliva samples from pigs subjected to stress was observed in 2 gel spots. One spot was identified as OBP; this protein appeared to be down-regulated in all 3 groups after stress was applied. Other researchers have found a decreased in OBP after the application of a stressor in bovine bronchoalveolar lavage fluid (34) and in porcine saliva from diseased animals (15).

The OBPs, low-molecular-weight soluble proteins, are a subclass of the lipocalin family (35,36). They are highly concentrated in the nasal mucus of vertebrates (37), are excreted in biologic fluids, such as saliva (38), and are thought to be involved in the release and modulation of chemical messages with pheromonal activity through a still-undefined mechanism (37,39).

Several hypotheses about why the OBP concentration decreases in stressful conditions could be made. Stress is known to be a factor in decreased reproductive activity and therefore could also lower the values of related proteins such as OBP. However, the mechanisms by which stress impacts reproduction and OBP concentration are not clearly understood (40). Also, the increase in oxidative products associated with stress could contribute to the decrease in OBP concentration (33).

Currently there are several commercial enzyme-linked immunosorbent assay kits for the detection of OBP in serum, plasma, and biologic fluids from humans. However, whether there is cross-reactivity with porcine OBP and whether these methods are sensitive enough to detect the concentrations of OBP in saliva have not been confirmed. For this reason, it would be necessary to perform analytical studies and clinical validation of these kits before they could be used for porcine OBP quantification in saliva. If any of these kits were analytically validated, they could be used for studies of stress involving large number of animals.

It is common that some proteins appear as multiple-spot chains or fractions in different parts of the gel in salivary 2-DE gel images (15). In the present study, statistical analysis revealed changes in spot 22, an albumin fragment. Other studies had similarly observed differential expression of albumin fragments in situations of altered homeostasis, such as disease (15) or oxidative stress (41). Thus, some authors claim that albumin is a biomarker of the welfare of pigs (4). It could be that high concentrations of cortisol can lead to an increase in albumin production, as has been reported for dogs (42). Therefore, it might be thought that in this study the high levels of cortisol caused by induced stress increased the albumin levels.

This salivary proteomic study had some limitations, such as the use of pooled samples and the low number of animals; thus, it should be considered preliminary. However, one can conclude from the results that acute stress involves changes in saliva proteins such as OBP and fractions of albumin. Further studies involving more animals and other stress conditions should be done to assess the value of these proteins as biomarkers for evaluating and monitoring stress in pigs under field conditions.