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Gender influence on the salivary protein profile of finishing pigs
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Gender influence on the salivary protein profile of finishing pigs ⁎
Ana M. Gutiérreza, , Ana Montesa, Cándido Gutiérrez-Panizoa, Pablo Fuentesc, Ernesto De La Cruz-Sánchezb a b c
Department of Animal Medicine and Surgery, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, Espinardo, Murcia, Spain Department of Physical Activity and Sport, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, San Javier, Murcia, Spain Cefu S.A., Alhama de Murcia, Murcia, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Gender Saliva Lipocalin Pig Proteome
A study on gender differences in the normal range of biomarkers in porcine saliva samples has the scope for further attention. In the present study, the salivary protein profiles of age-matched healthy male and female finishing pigs were compared. The levels of salivary adenosine deaminase (ADA) activity, haptoglobin (Hp) and C-reactive protein (CRP) have been quantified in 32 male and 32 female pigs to ensure the presence of gender effect on the median levels of salivary biomarkers. Moreover, the total salivary protein content was quantified and compared. The overall salivary protein distribution was compared with SDS-PAGE in 14 male and 14 female pigs and the possible gender influence in the salivary protein profile was analysed by 2DE in 6 male and 6 female pigs. Statistically significant differences were observed in the median values of Hp, CRP, and ADA between male and female pigs (p < 0.005). Although the total salivary protein content was not different between the sexes, the salivary protein distribution and profile showed specific gender differences in three proteins of the lipocalin family: the odorant-binding protein, salivary lipocalin and lipocalin 1. These proteins have been related to animal immune status and should be further explored as possible porcine salivary biomarkers. Significance: The biological relevance of the reported research is based on the possible gender influence on the discovery of salivary biomarkers in porcine production. As differences have been reported in the salivary protein distribution in male pigs in comparison to that of female pigs, the normal-range values, according to gender, of the newly discovered biomarkers should be explored and defined prior to its application in the porcine production system. A hormonal sexual influence is highly hypothesized.
1. Introduction Gender influence, which has been widely reported in porcine production, is varied from its effects on the performance and dietary feed intake values, with better parameters in sows than barrows [1], to differences in the levels of several biomarkers of health status. As an example, the normal-range levels of acute phase proteins (APP) have been reported to be higher in females than males in the serum [2,3] and saliva samples [4]. Moreover, the effects of gender have been postulated for a long time on the stress markers such as salivary cortisol [5], with higher average basal concentrations in barrows in comparison to gilts. However, no gender difference has been reported in other stress biomarkers, such as Chromogranin A [6]. The importance of gender annotations in research studies is widely discussed in order to provide homogeneous data or to reduce any possible gender effect. Although, the explanations about gender influence on the levels of salivary biomarkers or on the protein distribution
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in porcine saliva have been studied less. Unlike the relative stability of genome, the proteome is a dynamic entity that is affected by environmental, genetic, and epigenetic factors. Therefore, in order to catalogue the range of proteins in a specific tissue or biological fluid, it is desirable to analyse the variations caused by sexually dimorphic gene expression (male/female), as it has been reported in porcine muscle proteins [7]. Moreover, it has been published that 5.6% of liver proteins are differentially expressed between the males and females when the liver proteome was analysed by iTRAQ [8]. Taking into account that saliva is composed of a mixture of fluids from different origins - oral fluids, expectorated secretions, serum and blood derivates, bacteria, fungi and viruses and food debris [9] - differences in the salivary protein profile should be also expected. The saliva is a body fluid that the field of swine herd health management is increasingly interested in. The oral fluid testing was postulated as the optimal sample collection procedure to overcome the shortage of timely information on the circulation of pathogens, since it
Corresponding author. E-mail address: [email protected] (A.M. Gutiérrez).
https://doi.org/10.1016/j.jprot.2017.11.023 Received 31 July 2017; Received in revised form 20 November 2017; Accepted 25 November 2017 1874-3919/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Gutierrez, A., Journal of Proteomics (2017), https://doi.org/10.1016/j.jprot.2017.11.023
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2.3. Sampling procedure
offers an opportunity to easily collect group-level disease data [10]. Moreover, it has been postulated that the measurement of several salivary biomarkers in individual saliva samples has high diagnostic value for disease detection [11]. Thus, detailed information of porcine saliva at the molecular level, including gene and proteome composition, seems to be of great value. The salivary proteomic analyses reported until now in pigs have been performed exclusively in males, avoiding the possible gender influence on the analysis. However, no evidence about the possible variation of salivary protein profiles in female pigs in comparison to their male counterparts has been published. The present study is focused on the analysis of possible gender differences in the salivary protein profiles of pigs at the last stage of the production system. The overall salivary protein distribution and profile have been compared in agematched male and female pigs using one- and two- dimensional gel electrophoresis. Moreover, the proteins that appeared differentially regulated in one gender have been identified. The study could serve as a basic data for future studies on salivary proteomics.
The saliva samples were obtained prior to the veterinary clinical examination by introducing a sponge, of approximately 1 cm × 1 cm in size, clipped to a thin metal rod, in the mouth of the pigs individually for 1–2 min. The sponges were placed in specific tubes (Salivette tubes, Sarstedt, Nümbrecht, Germany) and stored on ice until processing at the laboratory, for not > 2 h. The saliva was processed by centrifugation of the tubes at 3000 × g for 10 min at the laboratory, and was stored in 2 identical aliquots, for biomarker quantification and proteomics analysis respectively, at − 80 °C until analysis. Two experienced veterinarians collected the saliva samples from all the animals at 2 consecutive days, at approximately 10:00 h a.m., in the beginning of February. The average ambient temperature in the finishing porcine unit at the time of sampling was 19.08 °C. 2.4. Salivary protein measurements The salivary levels of two acute phase proteins, Hp and CRP, were quantified in all the saliva samples for two purposes. First, to obtain an overall objective inflammatory-infection condition assessment and to identify any possible subclinical disorder, and second, to ensure the gender effect observed in previous studies [11]. For Hp and CRP quantification, previously validated homemade time-resolved immunofluorometric assays were used [4]. In addition, the activity of ADA in the saliva samples was quantified as a novel biomarker of immune status, by using an adaptation of a commercially available enzymatic assay, as recently published [12]. Moreover, the total salivary protein content of all the samples was determined according to Bradford [13]. To test if the values came from a Gaussian distribution, the Kolmogorov-Smirnov normality test was performed for each parameter quantified. As the results did not meet the normal distribution criteria, the median values obtained in males and females were compared with a non-parametric test, the Mann-Whitney test, with a specific statistical software (Graph Pad Prism 5, Graph Pad Software Inc. La Jolla, United States). The spearmen correlation coefficients were calculated among Hp, CRP, ADA and the total protein measurements in the saliva samples.
2. Material and methods 2.1. Sample size estimation and power analysis The sample size of the study has been estimated, according to the guidelines of the Ethical Committee for Animal Research in the University of Murcia, using specific statistical tools (http://bit.ly/ 2eco822). For an expected mean statistical power of 80% and an alpha level of 0.05, the analysis suggested a sample size of 29 animals/ group. Taking into account any possible experimental contingencies, an extra 10% has been added, making the total sample size of 64 animals (32 animals/group). 2.2. Animal characterization A total of 64 Duroc × Large White commercial pigs, from the same farm located in the southeast of Spain, were included in the study. The inclusion criteria were as follows: - Pigs should be at the last stage of the finishing process. - Animals should belong to animal-units with similar ages. - Randomly selection of animals until 32 male and 32 female animals were sampled. - All animals with any abnormal behaviour or any clinical sign of disease after veterinary inspection were excluded.
2.5. Salivary protein distribution analysis Afterwards, the saliva samples from 14 male and 14 female pigs were randomly selected and subjected to the SDS-PAGE analysis according to the method of Laemmli [14]. Briefly, 5 μg of the total saliva samples was reduced with dithiothreitol (DTT) at 95 °C and separated with 140 × 140 × 1.5 mm gradient gels (10–15% T, 2.7% C) in a vertical electrophoresis chamber (GE Healthcare, Life Sciences, Munich, Germany). The gels were silver-stained, to visualise the overall protein distribution [15], digitalized with an ImageScanner II (GE Healthcare Life Sciences, Uppsala, Sweden) and analysed by using a specific software (Image Quant TL, GE Healthcare Life Sciences, Uppsala, Sweden). The relative % volume of the different bands in the gel was used for protein distribution comparison as the normalized value. To evaluate the band % volume differences between the sexes, a non-parametric Mann-Whitney test was used with specific statistical software (Graph Pad Prism 5, Graph Pad Software Inc. La Jolla, United States) since the results did not meet the normal distribution criteria after the Kolmogorov-Smirnov normality test.
The animals were given ad libitum access to a nutritionally balanced commercial diet (3300 kcal ED/kg, 3.1% fibre, 16% protein, and 5% fat). Water was constantly available. The pigs were housed in unit-pens in groups of 10 with a minimum of 0.65 square meters per animal (Directive 2001/88/CE). Each unit was composed of 10 pens. All the procedures involving animals were approved by the Murcia University Ethics Committee and followed the recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Council of Europe, ETS no. 123). All the methods were performed in accordance with the relevant ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and regulations. All animals were subjected to a general clinical veterinary examination at the farm. The parameters taken into account during the examination includes the detailed observation of the patient pig and the other pigs in the group and their environment, the general aspect of the individual animals and the annotation of any clinical sign of disease or abnormal behaviour. If any alteration was observed in a pig, it was directly excluded from the analysis. Only one male pig was excluded in the experimental phase of the study, since a mild rectal prolapse was observed during its physical examination.
2.6. 2-DE analysis The saliva samples from 6 male and 6 female pigs were randomly selected from those subjected to the SDS-PAGE to perform a 2DE analysis. From the selected samples, 30 μg of the total freeze-dried saliva proteins were dissolved in rehydration buffer (8M Urea, 2% CHAPS, 2
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and 362.6 U/L (with 233.8 and 455.1 as the 25th and 95th percentiles respectively) respectively in males, and 24.67 ng/mL (with 9.17 and 39.80 as the 25th and 95th percentiles respectively) and 614.9 U/L (with 428.3 and 739.6 as the 25th and 95th percentiles respectively) respectively in females. No differences were found in the level of total salivary protein between the male and female pigs. The median values oscillated between 1.23 mg/mL (with 0.93 and 1.70 as the 25th and 95th percentiles respectively) in males and 1.35 mg/mL (with 0.94 and 1.75 as the 25th and 95th percentiles respectively) in females. A positive correlation was reported between the different markers analysed (Table 1). The highest coefficients observed among the ADA, Hp and CRP measurements were 0.596 and 0.462 respectively.
20 mM DTT and 0.5% IPG buffer pH 3–11 NL (GE Healthcare Life Sciences, Munich, Germany)) and swollen into immobilized pH gradient strips with 11 cm long nonlinear gradients pH 3–11 (GE Healthcare Life Sciences, Munich, Germany) as reported before [16]. In brief, the first-dimensional separation was carried out under reducing and denaturing conditions in a Multiphor II electrophoresis chamber (GE Healthcare, Life Sciences, Munich, Germany) using a multistep gradient protocol with a maximum of 3500 V until 12 kVh at 20 °C was reached. For the second dimension, the strips were equilibrated with 2% DTT solution, followed by 2.5% of iodoacetamide solution, and the SDS-PAGE was carried out at 15 °C and 25 mA/gel for 4–5 h in homemade gradient gels. After silverstaining, the 2DE gel images were digitalized and evaluated by using specific software (Image Master 2D Platinum 7.0, GE Healthcare Life Sciences, Uppsala, Sweden). The analysis included spot detection, landmarking, and spot matching of protein patterns of all the gels in the set. Two sets were performed, male and female. The relative % of spot volumes from each set was statistically compared by using a ttest with the software mentioned above. To explore the similarity of the salivary 2D protein profile in males and females, a scatter plots analysis for matched spots was performed, including linear regression and correlation coefficient calculations, by using the 2D software detailed above. Moreover, a multiple discriminant analysis with a stepwise method was employed in order to obtain the discriminant coefficients between the genders of each differentially expressed spot, deriving an equation for the linear combination of the modelled variables that have enabled the discrimination. A stepwise method (Wilk's lambda) was used for the analysis. At each step, the variable that minimized the overall Wilk's lambda was entered. The SPSS statistical package was employed to perform the analysis (SPSS 19.0). The differentially abundant spots that discriminate between genders with a high efficacy have already been identified in 2D pig saliva gels in a previous study [17]. In brief, the protein identification was performed by searching the peptide mass fingerprints (PMF) and MS/MS spectra against the Swiss Prot database with “Sus scrofa” as the taxonomy. A mass spectrometer (Bruker Ultraflex II mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany)), based on matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF-TOF) mass spectrometry (MS), was used for spectra acquisition in the MS and MS/MS modes. For database searches, the processed MS/MS spectra was searched in the Swiss-Prot database or in the NCBInr using the following search parameters - no limitation in taxonomy, global modifications: carbamido-methylation on cysteine, variable modifications: oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid, MS tolerance 100 ppm, MS/MS tolerance 1 Da, one missed cleavage allowed. The identifications were considered statistically significant when p < 0.05 and scores ≥ 60. To retrieve additional function information on proteins, a publicly available website was used - the UniProtKB database (http://www. uniprot.org/) for “Sus scrofa” proteins.
3.2. Salivary protein distribution and 2D pattern analysis
3. Results
Slight differences in the overall protein distribution of porcine saliva were observed between the male and female finishing pigs by SDSPAGE (Fig. 2). Only a band, at approximately 14.4 kDa, was differentially regulated according to gender, with higher values in males than in females. A further 2D analysis was performed in order to search for any possible difference that could be masked in the SDS-PAGE. First, spots were detected and analysed in the 2D gels (6 from saliva of females and 6 from saliva of male pigs) and a total of 77 matched spots were recorded. To the analysed gel similarities, the scatter plots for matched spots were performed. A high level of similarity in the salivary 2D protein pattern was obtained within groups in both female and male pigs, with coefficients of correlation of 0.90 and 0.95 respectively. The overall salivary matched spots data of the 12 animals (6 female and 6 male pigs) included in the 2D analysis were statistically filtered and resulted in 15 regulated protein spots (Fig. 3) out of the 77 matched spots analysed. The regulated protein spots were defined as those spots which appeared statistically differentially expressed in males in comparison to females (Table 2). A discriminant analysis of the differentially regulated spots showed 4 spots that differentiate the porcine gender with a confidence of 100% when analysed together. The results of the stepwise method are summarized in Table 3. The eigenvalue was 948.826 (> 1) and the canonical correlation was rc = 0.999 (> 0.35). The Wilk's lambda is 0.001, p < 0.000. Thus, the following function explains correctly the variation: F = 2.832 (spot 21) + 11.693 (spot 27) + 16.279 (spot 81) − 14.759 (spot 82). The cut-off for discriminant classification was 27.551 (Fig. 4). 100% of the original grouped cases have been correctly classified, and 100% of the cross-validated grouped cases have also been correctly classified, with 100% sensitivity and specificity. The predictive accuracy of positive and negative values is 100%. The affected spots of the discriminant analysis were identified by MS as the odorant-binding protein, salivary lipocalin and lipocalin 1 [17]. According to the database, these three unique identified proteins, that appeared differentially regulated in the saliva of male and female pigs, belong to the lipocalin family.
3.1. Salivary protein measurements
4. Discussion
The overall results of all the salivary measurements performed can be seen in Fig. 1. Statistically significant differences were observed for Hp, CRP and ADA measurements between males and females, with higher values in females. The biggest differences were observed in the CRP and ADA values. The median values of salivary Hp were 0.53 μg/mL (with 0.32 and 1.06 as the 25th and 95th percentiles respectively) in males and 0.93 μg/mL (with 0.56 and 1.96 as the 25th and 95th percentiles respectively) in females. The salivary CRP and ADA values were 3.63 ng/ mL (with 1.76 and 11.85 as the 25th and 95th percentiles respectively)
The differences in physiology between males and females extend far beyond the differences in reproductive functions [18]. Elevated immune responses and the higher incidence of autoimmune diseases in female humans and animals have been known for a long time [19]. In the last decade, the use of salivary markers of immune response is of great importance in porcine production. Moreover, saliva has been considered as a powerful tool for porcine surveillance programs [20]. However, the possible gender influence on porcine saliva as a biological fluid is minimal. We have studied the salivary proteomic profile in male and female 3
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Fig. 1. Concentrations of CRP (a), Hp (b), ADA (c) and total protein content (d) in the saliva samples of clinically healthy male and female pigs. The plot shows the median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) and outliers (°). The asterisk represents the statistically significant differences from each group. *level of significance p < 0.05. **level of significance p < 0.01.
in comparison to the female pigs by 2DE. The resulting data were treated by a stepwise discriminant analysis in order to ascertain the efficacy of these spots in classifying the animals according to their gender. Four of the differentiated expressed spots, 21, 27, 81 and 82, can discriminate between groups; therefore, the proposed model correctly describes group differences and it can be used for determining the animal gender. The MS analysis in a previous study identified those four spots as three unique proteins: the odorant-binding protein, lipocalin-1, and salivary lipocalin, which belong to the lipocalin family. Since no additional protein validation technique has been performed, it should be desirable to validate the protein identifications with appropriate methods such as western blot or ELISA in further studies, and could be considered as one of the main limitations of our study.
Table 1 Spearman correlation coefficients between the different salivary measurements performed: Hp, CRP, ADA and total protein content*: statistically significant (p < 0.01).
Total protein content Hp ADA
Hp
ADA
CRP
0.458*
0.327* 0.596*
0.141 0.354* 0.462*
pigs for the first time in a group of healthy finishing pigs with differences in the concentration of salivary immune markers, specifically Hp, CRP and ADA. After the 2DE-MS approach, a total of 9 spots were successfully identified as differentially regulated in the saliva of males
Fig. 2. Detailed view of the salivary protein distribution in male and female pigs after SDS-PAGE.
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Fig. 3. Detailed views of the spots differentially regulated from the 2DE patterns obtained in a healthy female (a1) and male (a2) pig. Protein spots successfully identified by MALDI-TOF/ TOF are marked with red circles (numbering corresponds with the data in Table 2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Detailed list of the spots differentially expressed in male or female pigs after 2DE. The statistical data obtained using a 2DE software (ImageMaster™ 2D Platinum 7.0, GE Healthcare Life Sciences, Munich, Germany) and a statistical software (SPSS 19.0). Mean: mean value (% spot volume). The list was sorted according to the p values. Acc. no. = accession number. PMF = identified as peptide mass fingerprint. MSMS = identified as tandem mass spectrometry. *Identification according to previously reported [17]. Match spot no
p value
*Protein Identification
Mean ♀
Mean ♂
Difference (SE)
t ratio
68 27 17 18 87 92 21 81 62 123 80 7 82 56 12
0.0002 0.0006 0.0045 0.0060 0.0100 0.0114 0.0117 0.0157 0.020 0.029 0.0304 0.032 0.0424 0.043 0.046
No identification Odorant-binding protein Lipocalin 1 Lipocalin 1 Salivary lipocalin Salivary lipocalin Lipocalin 1 Salivary lipocalin No identification No identification Salivary lipocalin No identification Salivary lipocalin No identification No identification
0.48 1.48 1.66 2.59 1.56 1.25 1.28 0.60 0.49 1.46 0.75 0.73 1.69 0.65 0.82
0.18 0.55 0.80 1.00 0.52 0.32 0.49 0.20 0.16 0.50 0.39 0.32 0.80 0.21 0.37
0.30 0.93 0.86 1.59 1.04 0.92 0.79 0.39 0.32 0.95 0.37 0.40 0.89 0.44 0.44
6.11 5.46 3.91 3.70 3.35 3.26 3.25 3.05 2.88 2.65 2.63 2.58 2.41 2.40 2.35
(0.04) (0.17) (0.22) (0.43) (0.31) (0.28) (0.24) (1.3) (0.11) (0.36) (0.14) (0.15) (0.37) (0.18) (0.19)
Table 3 The spots selected by stepwise discriminant analysis. There is only one discriminant function to determine the groups. λ Wilks = Wilks's lambda; df = degree freedom; p = level of significance. Step
Entered variable
Removed variable
λ Wilks Statistic
1 2 3 4 5 6
Spot Spot Spot Spot
68 27 82 81 Spot 68
Spot 21
0.176 0.101 0.059 0.005 0.005 0.001
df1
1 2 3 4 3 4
df2
1 1 1 1 1 1
df3
8 8 8 8 8 8
Exact F Statistic
df1
df2
p
37.450 31.132 31.629 243.614 382.097 1186.033
1 2 3 4 3 4
8 7 6 5 6 5
0.000 0.000 0.000 0.000 0.000 0.000
Odorant-binding protein, Prostaglandin-H2 D-isomerase, Retinolbinding protein 4, and Salivary lipocalin. The odorant-binding protein (OBP) is found in the nasal epithelium and it binds a wide variety of volatile hydrophobic molecules [22]. The OBP is assumed to be directly involved in chemical communication and in the pre-mating recognition process [23]. It has been reported that it is expressed in both sexes in contrast to the analogues, urinary, and salivary lipocalins [24]. Our results demonstrated the presence of nasal,
The lipocalins constitute a heterogeneous family of small secreted proteins with a variety of different functions, including roles in retinol transport, cryptic coloration, olfaction, pheromone transport, and enzyme synthesis of prostaglandins. Moreover, the lipocalins have also been implicated in the regulation of immune response and the mediation of cell homeostasis [21]. Eight proteins have been correctly reviewed from this protein family in the pig UniProtKB database: Apolipoprotein M, Protein AMBP, Lipocalin-1, Beta-lactoglobulin-1A/1C, 5
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Fig. 4. Discriminant analysis of the proteins that appeared differentially regulated in 2DE analysis. The plot shows tight clustering of each group, female (blue data) and male (green data) pigs. The centroids (red) show mean discriminant scores for each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
proteins related to social and sexual communication. Surprisingly, the significant differences were found in males in comparison to female pigs, with a diminished expression in the males. Further experiments are needed to evaluate the functional impact of the altered proteins and their associated pathways after a proper validation of the identified proteins.
salivary, and tear lipocalins, which appeared differentially regulated, in the saliva of male and female pigs. The salivary lipocalin (SAL 1) binds the pheromones, released from the saliva of males, and affect the sexual behaviour of females. It has been localized in the submaxillary salivary glands of mature male pigs, but virtually absent in the females, with an apparent molecular mass of 20 kDa [25]. According to this study, it could not be concluded that the SAL 1 is absent in the submaxillary gland of porcine females, since the SDS-PAGE technique is not an optimal tool for protein quantification, and only one biological replicate was used in the mentioned study. In contrast, we have obtained higher values of SAL 1 in female than in male pigs in several spots identified as SAL 1. Moreover, it has been reported that the level of lipocalins and, consequently, the perception of pheromones is differently interpreted according to the age and the endocrinal status of the animals [26] and their disease condition [17]; so, several factors could have influenced our contradictory results. The lipocalin 1 (LCN1) is secreted in large amounts by the pig lachrymal glands and von Ebner's glands (in tongue) [27]. In humans, the LCN1 is supposed to act as a protection factor for the epithelia, with the ability to play a role in the nonimmunological defence against microorganisms and viruses, and the control of inflammatory processes [28]. This result is in concordance with the previous studies in which the LCN1 was proposed as the biomarker of health status in male pigs [17]. A close relation between the three lipocalins identified and the immune status has been previously reported in literature. The high level of lipocalin observed in females, in comparison to male pigs, is in concordance with the literature in which higher levels of immune markers have been reported in the females [7], and also with the results of the salivary biomarkers of immune status quantified in the present study, with higher values of Hp, CRP and ADA in females in comparison to the male pigs. The Hp and CRP are well-known acute phase proteins whose levels increase after a homeostasis disturbance [29], and the ADA is involved in the activation and maintenance of the immune system via lymphocyte development [30]. Thus, the possible future use of lipocalins as biomarkers of immune status in porcine saliva would be a great tool that adds a new perspective of immune characterization in pigs. However, taking into account that our present results showed higher OBP, LCN 1 and SAL 1 values in females in comparison to males, the normal range values of these lipocalins should be established in both sexes prior to its possible use as biomarker of immune status.
Competing interest Competing interests do not compromise this study. We agree to its publication, providing it is considered suitable, and we accept the rules set out in authors consent to publication (copyright). Acknowledgment The authors wish to thank Karin Hummel, Katharina Nöbauer and Ebrahim Razzazi Fazeli (VetCore Facility for Research, University of Veterinary Medicine Vienna, Austria), for the provision of MS data and the excellent technical assistance. References [1] C.R. Pierozan, P.S. Agostini, J. Gasa, A.K. Novais, C.P. Dias, R.S.K. Santos, M. Pereira Jr., J.G. Nagi, J.B. Alves, C.A. Silva, Factors affecting the daily feed intake and feed conversion ratio of pigs in grow-finishing units: the case of a company, Porcine Health Manag. 2 (2016) 7. [2] H.H. Petersen, A.K. Ersboll, C.S. Jensen, J.P. Nielsen, Serum-haptoglobin concentration in Danish slaughter pigs of different health status, Prev. Vet. Med. 54 (2002) 325–335. [3] M. Clapperton, S.C. Bishop, N.D. Cameron, E.J. Glass, Associations of acute phase protein levels with growth performance and with selection for growth performance in Large White pigs, Anim. Sci. 81 (2005) 213–220. [4] A.M. Gutiérrez, S. Martínez-Subiela, L. Soler, F.J. Pallarés, J.J. Cerón, Use of saliva for haptoglobin and C-reactive protein quantifications in porcine respiratory and reproductive syndrome affected pigs in field conditions, Vet. Immunol. Immunopathol. 132 (2009) 218–223. [5] M.A. Ruis, J.H. Te Brake, B. Engel, E.D. Ekkel, W.G. Buist, H.J. Blokhuis, J.M. Koolhaas, The circadian rhythm of salivary cortisol in growing pigs: effects of age, gender, and stress, Physiol. Behav. 62 (1997) 623–630. [6] D. Escribano, A.M. Gutiérrez, M. Fuentes-Rubio, J.J. Cerón, Saliva chromogranin A in growing pigs: a study of circadian patterns during daytime and stability under different storage conditions, Vet. J. 199 (2014) 355–359. [7] H.A. Hakimov, S. Walters, T.C. Wright, R.G. Meidinger, C.P. Verschoor, M. Gadish, D.K. Chiu, M.V. Strömvik, C.W. Forsberg, S.P. Golovan, Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization, Proteomics 9 (2009) 4000–4016. [8] S.P. Golovan, H.A. Hakimov, C.P. Verschoor, S. Walters, M. Gadish, C. Elsik, F. Schenkel, D.K. Chiu, C.W. Forsberg, Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders, and conventional and genetically enhanced lines, Comp. Biochem. Physiol. D Genomics Proteome 3 (2008) 234–242. [9] E. Kaufman, I.B. Lamster, The diagnostic applications of saliva—a review, Crit. Rev. Oral Biol. Med. 13 (2002) 197–212. [10] J.R. Prickett, J.J. Zimmerman, The development of oral fluid-based diagnostics and applications in veterinary medicine, Anim. Health Res. Rev. 11 (2010) 207–216. [11] A.M. Gutiérrez, D. Escribano, M. Fuentes, J.J. Cerón, Circadian pattern of acute phase proteins in the saliva of growing pigs, Vet. J. 196 (2013 May) 167–170. [12] A.M. Gutiérrez, E. De La Cruz-Sánchez, A. Montes, J. Sotillo, C. Gutiérrez-Panizo,
5. Conclusion The two-dimensional proteome analysis proved to be an effective tool to detect saliva proteins with altered expression in male or female pigs. Three lipocalin proteins showing differential expression profiles in the saliva of finishing pigs have been highlighted. It could be postulated that the sexual hormones are directly influencing these results, since the most relevant differences between the genders were observed in 6
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[13]
[14] [15] [16]
[17]
[18] [19] [20]
[21]
(1996) 1–14. [22] P. Pelosi, Odorant-binding proteins, Crit. Rev. Biochem. Mol. Biol. 29 (1994) 199–228. [23] C. Meslin, F. Brimau, P. Nagnan-Le Meillour, I. Callebaut, G. Pascal, P. Monget, The evolutionary history of the SAL1 gene family in eutherian mammals, BMC Evol. Biol. 11 (2011) 148. [24] M. Tegoni, P. Pelosi, F. Vincent, S. Spinelli, V. Campanacci, S. Grolli, R. Ramoni, C. Cambillau, Mammalian odorant binding proteins, Biochim. Biophys. Acta 1482 (2000) 229–240. [25] S. Marchese, D. Pes, A. Scaloni, V. Carbone, P. Pelosi, Lipocalins of boar salivary glands binding odours and pheromones, Eur. J. Biochem. 252 (1998) 563–568. [26] P. Nagnan-Le Meillour, A.S. Vercoutter-Edouart, F. Hilliou, C. Le Danvic, F. Lévy, Proteomic analysis of pig (Sus scrofa) olfactory soluble proteome reveals O-linkedN-acetylglucosaminylation of secreted odorant-binding proteins, Front. Endocrinol. (Lausanne) 5 (2014) 202. [27] M. Garibotti, H. Christiansen, H. Schmale, P. Pelosi, Porcine VEG proteins and tear prealbumins, Chem. Senses 20 (1995) 69–76. [28] P. Holzfeind, P. Merschak, P. Wojnar, B. Redl, Structure and organization of the porcine LCN1 gene encoding Tear lipocalin/von Ebner's gland protein, Gene 202 (1997) 61–67. [29] C. Gabay, I. Kushner, Acute-phase proteins and other systemic responses to inflammation, N. Engl. J. Med. 340 (1999) 448–454. [30] A.K. Rai, C.P. Thakur, T. Velpandian, S.K. Sharma, B. Ghosh, D.K. Mitra, High concentration of adenosine in human visceral leishmaniasis despite increased ADA and decreased CD73, Parasite Immunol. 33 (2011) 632–636.
P. Fuentes, P.L. Tornel, J. Cabezas-Herrera, Easy and non-invasive disease detection in pigs by adenosine deaminase activity determinations in saliva, PLoS One 12 (2017) e0179299. M.M. Bradford, Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 222 (1970) 680–685. I. Miller, J. Crawford, E. Gianazza, Protein stains for proteomic applications: which, when, why? Proteomics 6 (2006) 5385–5408. A.M. Gutiérrez, I. Miller, D. Kolarich, K. Hummel, K. Nöbauer, E. Razzazi-Fazeli, Detection and first characterization of an uncommon haptoglobin in porcine saliva of pigs with rectal prolapse by using boronic acid sample enrichment, Animal 11 (2017) 845–853. A.M. Gutiérrez, K. Nöbauer, L. Soler, E. Razzazi-Fazeli, M. Gemeiner, J.J. Cerón, I. Miller, Detection of potential markers for systemic disease in saliva of pigs by proteomics: a pilot study, Vet. Immunol. Immunopathol. 151 (2013) 73–82. V. Miller, M. Hay, Principles of Sex-Based Differences in Physiology, Academic Press/Elsevier Inc, Amsterdam, 2004. A.H. Schuurs, H.A. Verheul, Effects of gender and sex steroids on the immune response, J. Steroid Biochem. 35 (1990) 157–172. A. Ramirez, C. Wang, J.R. Prickett, R. Pogranichniy, K.J. Yoon, R. Main, J.K. Johnson, C. Rademacher, M. Hoogland, P. Hoffmann, A. Kurtz, E. Kurtz, J. Zimmerman, Efficient surveillance of pig populations using oral fluids, Prev. Vet. Med. 104 (2012) 292–300. D.R. Flower, The lipocalin protein family: structure and function, Biochem. J. 318
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Contents lists available at ScienceDirect
Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot
Gender influence on the salivary protein profile of finishing pigs ⁎
Ana M. Gutiérreza, , Ana Montesa, Cándido Gutiérrez-Panizoa, Pablo Fuentesc, Ernesto De La Cruz-Sánchezb a b c
Department of Animal Medicine and Surgery, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, Espinardo, Murcia, Spain Department of Physical Activity and Sport, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, San Javier, Murcia, Spain Cefu S.A., Alhama de Murcia, Murcia, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Gender Saliva Lipocalin Pig Proteome
A study on gender differences in the normal range of biomarkers in porcine saliva samples has the scope for further attention. In the present study, the salivary protein profiles of age-matched healthy male and female finishing pigs were compared. The levels of salivary adenosine deaminase (ADA) activity, haptoglobin (Hp) and C-reactive protein (CRP) have been quantified in 32 male and 32 female pigs to ensure the presence of gender effect on the median levels of salivary biomarkers. Moreover, the total salivary protein content was quantified and compared. The overall salivary protein distribution was compared with SDS-PAGE in 14 male and 14 female pigs and the possible gender influence in the salivary protein profile was analysed by 2DE in 6 male and 6 female pigs. Statistically significant differences were observed in the median values of Hp, CRP, and ADA between male and female pigs (p < 0.005). Although the total salivary protein content was not different between the sexes, the salivary protein distribution and profile showed specific gender differences in three proteins of the lipocalin family: the odorant-binding protein, salivary lipocalin and lipocalin 1. These proteins have been related to animal immune status and should be further explored as possible porcine salivary biomarkers. Significance: The biological relevance of the reported research is based on the possible gender influence on the discovery of salivary biomarkers in porcine production. As differences have been reported in the salivary protein distribution in male pigs in comparison to that of female pigs, the normal-range values, according to gender, of the newly discovered biomarkers should be explored and defined prior to its application in the porcine production system. A hormonal sexual influence is highly hypothesized.
1. Introduction Gender influence, which has been widely reported in porcine production, is varied from its effects on the performance and dietary feed intake values, with better parameters in sows than barrows [1], to differences in the levels of several biomarkers of health status. As an example, the normal-range levels of acute phase proteins (APP) have been reported to be higher in females than males in the serum [2,3] and saliva samples [4]. Moreover, the effects of gender have been postulated for a long time on the stress markers such as salivary cortisol [5], with higher average basal concentrations in barrows in comparison to gilts. However, no gender difference has been reported in other stress biomarkers, such as Chromogranin A [6]. The importance of gender annotations in research studies is widely discussed in order to provide homogeneous data or to reduce any possible gender effect. Although, the explanations about gender influence on the levels of salivary biomarkers or on the protein distribution
⁎
in porcine saliva have been studied less. Unlike the relative stability of genome, the proteome is a dynamic entity that is affected by environmental, genetic, and epigenetic factors. Therefore, in order to catalogue the range of proteins in a specific tissue or biological fluid, it is desirable to analyse the variations caused by sexually dimorphic gene expression (male/female), as it has been reported in porcine muscle proteins [7]. Moreover, it has been published that 5.6% of liver proteins are differentially expressed between the males and females when the liver proteome was analysed by iTRAQ [8]. Taking into account that saliva is composed of a mixture of fluids from different origins - oral fluids, expectorated secretions, serum and blood derivates, bacteria, fungi and viruses and food debris [9] - differences in the salivary protein profile should be also expected. The saliva is a body fluid that the field of swine herd health management is increasingly interested in. The oral fluid testing was postulated as the optimal sample collection procedure to overcome the shortage of timely information on the circulation of pathogens, since it
Corresponding author. E-mail address: [email protected] (A.M. Gutiérrez).
https://doi.org/10.1016/j.jprot.2017.11.023 Received 31 July 2017; Received in revised form 20 November 2017; Accepted 25 November 2017 1874-3919/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Gutierrez, A., Journal of Proteomics (2017), https://doi.org/10.1016/j.jprot.2017.11.023
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2.3. Sampling procedure
offers an opportunity to easily collect group-level disease data [10]. Moreover, it has been postulated that the measurement of several salivary biomarkers in individual saliva samples has high diagnostic value for disease detection [11]. Thus, detailed information of porcine saliva at the molecular level, including gene and proteome composition, seems to be of great value. The salivary proteomic analyses reported until now in pigs have been performed exclusively in males, avoiding the possible gender influence on the analysis. However, no evidence about the possible variation of salivary protein profiles in female pigs in comparison to their male counterparts has been published. The present study is focused on the analysis of possible gender differences in the salivary protein profiles of pigs at the last stage of the production system. The overall salivary protein distribution and profile have been compared in agematched male and female pigs using one- and two- dimensional gel electrophoresis. Moreover, the proteins that appeared differentially regulated in one gender have been identified. The study could serve as a basic data for future studies on salivary proteomics.
The saliva samples were obtained prior to the veterinary clinical examination by introducing a sponge, of approximately 1 cm × 1 cm in size, clipped to a thin metal rod, in the mouth of the pigs individually for 1–2 min. The sponges were placed in specific tubes (Salivette tubes, Sarstedt, Nümbrecht, Germany) and stored on ice until processing at the laboratory, for not > 2 h. The saliva was processed by centrifugation of the tubes at 3000 × g for 10 min at the laboratory, and was stored in 2 identical aliquots, for biomarker quantification and proteomics analysis respectively, at − 80 °C until analysis. Two experienced veterinarians collected the saliva samples from all the animals at 2 consecutive days, at approximately 10:00 h a.m., in the beginning of February. The average ambient temperature in the finishing porcine unit at the time of sampling was 19.08 °C. 2.4. Salivary protein measurements The salivary levels of two acute phase proteins, Hp and CRP, were quantified in all the saliva samples for two purposes. First, to obtain an overall objective inflammatory-infection condition assessment and to identify any possible subclinical disorder, and second, to ensure the gender effect observed in previous studies [11]. For Hp and CRP quantification, previously validated homemade time-resolved immunofluorometric assays were used [4]. In addition, the activity of ADA in the saliva samples was quantified as a novel biomarker of immune status, by using an adaptation of a commercially available enzymatic assay, as recently published [12]. Moreover, the total salivary protein content of all the samples was determined according to Bradford [13]. To test if the values came from a Gaussian distribution, the Kolmogorov-Smirnov normality test was performed for each parameter quantified. As the results did not meet the normal distribution criteria, the median values obtained in males and females were compared with a non-parametric test, the Mann-Whitney test, with a specific statistical software (Graph Pad Prism 5, Graph Pad Software Inc. La Jolla, United States). The spearmen correlation coefficients were calculated among Hp, CRP, ADA and the total protein measurements in the saliva samples.
2. Material and methods 2.1. Sample size estimation and power analysis The sample size of the study has been estimated, according to the guidelines of the Ethical Committee for Animal Research in the University of Murcia, using specific statistical tools (http://bit.ly/ 2eco822). For an expected mean statistical power of 80% and an alpha level of 0.05, the analysis suggested a sample size of 29 animals/ group. Taking into account any possible experimental contingencies, an extra 10% has been added, making the total sample size of 64 animals (32 animals/group). 2.2. Animal characterization A total of 64 Duroc × Large White commercial pigs, from the same farm located in the southeast of Spain, were included in the study. The inclusion criteria were as follows: - Pigs should be at the last stage of the finishing process. - Animals should belong to animal-units with similar ages. - Randomly selection of animals until 32 male and 32 female animals were sampled. - All animals with any abnormal behaviour or any clinical sign of disease after veterinary inspection were excluded.
2.5. Salivary protein distribution analysis Afterwards, the saliva samples from 14 male and 14 female pigs were randomly selected and subjected to the SDS-PAGE analysis according to the method of Laemmli [14]. Briefly, 5 μg of the total saliva samples was reduced with dithiothreitol (DTT) at 95 °C and separated with 140 × 140 × 1.5 mm gradient gels (10–15% T, 2.7% C) in a vertical electrophoresis chamber (GE Healthcare, Life Sciences, Munich, Germany). The gels were silver-stained, to visualise the overall protein distribution [15], digitalized with an ImageScanner II (GE Healthcare Life Sciences, Uppsala, Sweden) and analysed by using a specific software (Image Quant TL, GE Healthcare Life Sciences, Uppsala, Sweden). The relative % volume of the different bands in the gel was used for protein distribution comparison as the normalized value. To evaluate the band % volume differences between the sexes, a non-parametric Mann-Whitney test was used with specific statistical software (Graph Pad Prism 5, Graph Pad Software Inc. La Jolla, United States) since the results did not meet the normal distribution criteria after the Kolmogorov-Smirnov normality test.
The animals were given ad libitum access to a nutritionally balanced commercial diet (3300 kcal ED/kg, 3.1% fibre, 16% protein, and 5% fat). Water was constantly available. The pigs were housed in unit-pens in groups of 10 with a minimum of 0.65 square meters per animal (Directive 2001/88/CE). Each unit was composed of 10 pens. All the procedures involving animals were approved by the Murcia University Ethics Committee and followed the recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Council of Europe, ETS no. 123). All the methods were performed in accordance with the relevant ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and regulations. All animals were subjected to a general clinical veterinary examination at the farm. The parameters taken into account during the examination includes the detailed observation of the patient pig and the other pigs in the group and their environment, the general aspect of the individual animals and the annotation of any clinical sign of disease or abnormal behaviour. If any alteration was observed in a pig, it was directly excluded from the analysis. Only one male pig was excluded in the experimental phase of the study, since a mild rectal prolapse was observed during its physical examination.
2.6. 2-DE analysis The saliva samples from 6 male and 6 female pigs were randomly selected from those subjected to the SDS-PAGE to perform a 2DE analysis. From the selected samples, 30 μg of the total freeze-dried saliva proteins were dissolved in rehydration buffer (8M Urea, 2% CHAPS, 2
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and 362.6 U/L (with 233.8 and 455.1 as the 25th and 95th percentiles respectively) respectively in males, and 24.67 ng/mL (with 9.17 and 39.80 as the 25th and 95th percentiles respectively) and 614.9 U/L (with 428.3 and 739.6 as the 25th and 95th percentiles respectively) respectively in females. No differences were found in the level of total salivary protein between the male and female pigs. The median values oscillated between 1.23 mg/mL (with 0.93 and 1.70 as the 25th and 95th percentiles respectively) in males and 1.35 mg/mL (with 0.94 and 1.75 as the 25th and 95th percentiles respectively) in females. A positive correlation was reported between the different markers analysed (Table 1). The highest coefficients observed among the ADA, Hp and CRP measurements were 0.596 and 0.462 respectively.
20 mM DTT and 0.5% IPG buffer pH 3–11 NL (GE Healthcare Life Sciences, Munich, Germany)) and swollen into immobilized pH gradient strips with 11 cm long nonlinear gradients pH 3–11 (GE Healthcare Life Sciences, Munich, Germany) as reported before [16]. In brief, the first-dimensional separation was carried out under reducing and denaturing conditions in a Multiphor II electrophoresis chamber (GE Healthcare, Life Sciences, Munich, Germany) using a multistep gradient protocol with a maximum of 3500 V until 12 kVh at 20 °C was reached. For the second dimension, the strips were equilibrated with 2% DTT solution, followed by 2.5% of iodoacetamide solution, and the SDS-PAGE was carried out at 15 °C and 25 mA/gel for 4–5 h in homemade gradient gels. After silverstaining, the 2DE gel images were digitalized and evaluated by using specific software (Image Master 2D Platinum 7.0, GE Healthcare Life Sciences, Uppsala, Sweden). The analysis included spot detection, landmarking, and spot matching of protein patterns of all the gels in the set. Two sets were performed, male and female. The relative % of spot volumes from each set was statistically compared by using a ttest with the software mentioned above. To explore the similarity of the salivary 2D protein profile in males and females, a scatter plots analysis for matched spots was performed, including linear regression and correlation coefficient calculations, by using the 2D software detailed above. Moreover, a multiple discriminant analysis with a stepwise method was employed in order to obtain the discriminant coefficients between the genders of each differentially expressed spot, deriving an equation for the linear combination of the modelled variables that have enabled the discrimination. A stepwise method (Wilk's lambda) was used for the analysis. At each step, the variable that minimized the overall Wilk's lambda was entered. The SPSS statistical package was employed to perform the analysis (SPSS 19.0). The differentially abundant spots that discriminate between genders with a high efficacy have already been identified in 2D pig saliva gels in a previous study [17]. In brief, the protein identification was performed by searching the peptide mass fingerprints (PMF) and MS/MS spectra against the Swiss Prot database with “Sus scrofa” as the taxonomy. A mass spectrometer (Bruker Ultraflex II mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany)), based on matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF-TOF) mass spectrometry (MS), was used for spectra acquisition in the MS and MS/MS modes. For database searches, the processed MS/MS spectra was searched in the Swiss-Prot database or in the NCBInr using the following search parameters - no limitation in taxonomy, global modifications: carbamido-methylation on cysteine, variable modifications: oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid, MS tolerance 100 ppm, MS/MS tolerance 1 Da, one missed cleavage allowed. The identifications were considered statistically significant when p < 0.05 and scores ≥ 60. To retrieve additional function information on proteins, a publicly available website was used - the UniProtKB database (http://www. uniprot.org/) for “Sus scrofa” proteins.
3.2. Salivary protein distribution and 2D pattern analysis
3. Results
Slight differences in the overall protein distribution of porcine saliva were observed between the male and female finishing pigs by SDSPAGE (Fig. 2). Only a band, at approximately 14.4 kDa, was differentially regulated according to gender, with higher values in males than in females. A further 2D analysis was performed in order to search for any possible difference that could be masked in the SDS-PAGE. First, spots were detected and analysed in the 2D gels (6 from saliva of females and 6 from saliva of male pigs) and a total of 77 matched spots were recorded. To the analysed gel similarities, the scatter plots for matched spots were performed. A high level of similarity in the salivary 2D protein pattern was obtained within groups in both female and male pigs, with coefficients of correlation of 0.90 and 0.95 respectively. The overall salivary matched spots data of the 12 animals (6 female and 6 male pigs) included in the 2D analysis were statistically filtered and resulted in 15 regulated protein spots (Fig. 3) out of the 77 matched spots analysed. The regulated protein spots were defined as those spots which appeared statistically differentially expressed in males in comparison to females (Table 2). A discriminant analysis of the differentially regulated spots showed 4 spots that differentiate the porcine gender with a confidence of 100% when analysed together. The results of the stepwise method are summarized in Table 3. The eigenvalue was 948.826 (> 1) and the canonical correlation was rc = 0.999 (> 0.35). The Wilk's lambda is 0.001, p < 0.000. Thus, the following function explains correctly the variation: F = 2.832 (spot 21) + 11.693 (spot 27) + 16.279 (spot 81) − 14.759 (spot 82). The cut-off for discriminant classification was 27.551 (Fig. 4). 100% of the original grouped cases have been correctly classified, and 100% of the cross-validated grouped cases have also been correctly classified, with 100% sensitivity and specificity. The predictive accuracy of positive and negative values is 100%. The affected spots of the discriminant analysis were identified by MS as the odorant-binding protein, salivary lipocalin and lipocalin 1 [17]. According to the database, these three unique identified proteins, that appeared differentially regulated in the saliva of male and female pigs, belong to the lipocalin family.
3.1. Salivary protein measurements
4. Discussion
The overall results of all the salivary measurements performed can be seen in Fig. 1. Statistically significant differences were observed for Hp, CRP and ADA measurements between males and females, with higher values in females. The biggest differences were observed in the CRP and ADA values. The median values of salivary Hp were 0.53 μg/mL (with 0.32 and 1.06 as the 25th and 95th percentiles respectively) in males and 0.93 μg/mL (with 0.56 and 1.96 as the 25th and 95th percentiles respectively) in females. The salivary CRP and ADA values were 3.63 ng/ mL (with 1.76 and 11.85 as the 25th and 95th percentiles respectively)
The differences in physiology between males and females extend far beyond the differences in reproductive functions [18]. Elevated immune responses and the higher incidence of autoimmune diseases in female humans and animals have been known for a long time [19]. In the last decade, the use of salivary markers of immune response is of great importance in porcine production. Moreover, saliva has been considered as a powerful tool for porcine surveillance programs [20]. However, the possible gender influence on porcine saliva as a biological fluid is minimal. We have studied the salivary proteomic profile in male and female 3
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Fig. 1. Concentrations of CRP (a), Hp (b), ADA (c) and total protein content (d) in the saliva samples of clinically healthy male and female pigs. The plot shows the median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) and outliers (°). The asterisk represents the statistically significant differences from each group. *level of significance p < 0.05. **level of significance p < 0.01.
in comparison to the female pigs by 2DE. The resulting data were treated by a stepwise discriminant analysis in order to ascertain the efficacy of these spots in classifying the animals according to their gender. Four of the differentiated expressed spots, 21, 27, 81 and 82, can discriminate between groups; therefore, the proposed model correctly describes group differences and it can be used for determining the animal gender. The MS analysis in a previous study identified those four spots as three unique proteins: the odorant-binding protein, lipocalin-1, and salivary lipocalin, which belong to the lipocalin family. Since no additional protein validation technique has been performed, it should be desirable to validate the protein identifications with appropriate methods such as western blot or ELISA in further studies, and could be considered as one of the main limitations of our study.
Table 1 Spearman correlation coefficients between the different salivary measurements performed: Hp, CRP, ADA and total protein content*: statistically significant (p < 0.01).
Total protein content Hp ADA
Hp
ADA
CRP
0.458*
0.327* 0.596*
0.141 0.354* 0.462*
pigs for the first time in a group of healthy finishing pigs with differences in the concentration of salivary immune markers, specifically Hp, CRP and ADA. After the 2DE-MS approach, a total of 9 spots were successfully identified as differentially regulated in the saliva of males
Fig. 2. Detailed view of the salivary protein distribution in male and female pigs after SDS-PAGE.
4
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Fig. 3. Detailed views of the spots differentially regulated from the 2DE patterns obtained in a healthy female (a1) and male (a2) pig. Protein spots successfully identified by MALDI-TOF/ TOF are marked with red circles (numbering corresponds with the data in Table 2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Detailed list of the spots differentially expressed in male or female pigs after 2DE. The statistical data obtained using a 2DE software (ImageMaster™ 2D Platinum 7.0, GE Healthcare Life Sciences, Munich, Germany) and a statistical software (SPSS 19.0). Mean: mean value (% spot volume). The list was sorted according to the p values. Acc. no. = accession number. PMF = identified as peptide mass fingerprint. MSMS = identified as tandem mass spectrometry. *Identification according to previously reported [17]. Match spot no
p value
*Protein Identification
Mean ♀
Mean ♂
Difference (SE)
t ratio
68 27 17 18 87 92 21 81 62 123 80 7 82 56 12
0.0002 0.0006 0.0045 0.0060 0.0100 0.0114 0.0117 0.0157 0.020 0.029 0.0304 0.032 0.0424 0.043 0.046
No identification Odorant-binding protein Lipocalin 1 Lipocalin 1 Salivary lipocalin Salivary lipocalin Lipocalin 1 Salivary lipocalin No identification No identification Salivary lipocalin No identification Salivary lipocalin No identification No identification
0.48 1.48 1.66 2.59 1.56 1.25 1.28 0.60 0.49 1.46 0.75 0.73 1.69 0.65 0.82
0.18 0.55 0.80 1.00 0.52 0.32 0.49 0.20 0.16 0.50 0.39 0.32 0.80 0.21 0.37
0.30 0.93 0.86 1.59 1.04 0.92 0.79 0.39 0.32 0.95 0.37 0.40 0.89 0.44 0.44
6.11 5.46 3.91 3.70 3.35 3.26 3.25 3.05 2.88 2.65 2.63 2.58 2.41 2.40 2.35
(0.04) (0.17) (0.22) (0.43) (0.31) (0.28) (0.24) (1.3) (0.11) (0.36) (0.14) (0.15) (0.37) (0.18) (0.19)
Table 3 The spots selected by stepwise discriminant analysis. There is only one discriminant function to determine the groups. λ Wilks = Wilks's lambda; df = degree freedom; p = level of significance. Step
Entered variable
Removed variable
λ Wilks Statistic
1 2 3 4 5 6
Spot Spot Spot Spot
68 27 82 81 Spot 68
Spot 21
0.176 0.101 0.059 0.005 0.005 0.001
df1
1 2 3 4 3 4
df2
1 1 1 1 1 1
df3
8 8 8 8 8 8
Exact F Statistic
df1
df2
p
37.450 31.132 31.629 243.614 382.097 1186.033
1 2 3 4 3 4
8 7 6 5 6 5
0.000 0.000 0.000 0.000 0.000 0.000
Odorant-binding protein, Prostaglandin-H2 D-isomerase, Retinolbinding protein 4, and Salivary lipocalin. The odorant-binding protein (OBP) is found in the nasal epithelium and it binds a wide variety of volatile hydrophobic molecules [22]. The OBP is assumed to be directly involved in chemical communication and in the pre-mating recognition process [23]. It has been reported that it is expressed in both sexes in contrast to the analogues, urinary, and salivary lipocalins [24]. Our results demonstrated the presence of nasal,
The lipocalins constitute a heterogeneous family of small secreted proteins with a variety of different functions, including roles in retinol transport, cryptic coloration, olfaction, pheromone transport, and enzyme synthesis of prostaglandins. Moreover, the lipocalins have also been implicated in the regulation of immune response and the mediation of cell homeostasis [21]. Eight proteins have been correctly reviewed from this protein family in the pig UniProtKB database: Apolipoprotein M, Protein AMBP, Lipocalin-1, Beta-lactoglobulin-1A/1C, 5
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Fig. 4. Discriminant analysis of the proteins that appeared differentially regulated in 2DE analysis. The plot shows tight clustering of each group, female (blue data) and male (green data) pigs. The centroids (red) show mean discriminant scores for each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
proteins related to social and sexual communication. Surprisingly, the significant differences were found in males in comparison to female pigs, with a diminished expression in the males. Further experiments are needed to evaluate the functional impact of the altered proteins and their associated pathways after a proper validation of the identified proteins.
salivary, and tear lipocalins, which appeared differentially regulated, in the saliva of male and female pigs. The salivary lipocalin (SAL 1) binds the pheromones, released from the saliva of males, and affect the sexual behaviour of females. It has been localized in the submaxillary salivary glands of mature male pigs, but virtually absent in the females, with an apparent molecular mass of 20 kDa [25]. According to this study, it could not be concluded that the SAL 1 is absent in the submaxillary gland of porcine females, since the SDS-PAGE technique is not an optimal tool for protein quantification, and only one biological replicate was used in the mentioned study. In contrast, we have obtained higher values of SAL 1 in female than in male pigs in several spots identified as SAL 1. Moreover, it has been reported that the level of lipocalins and, consequently, the perception of pheromones is differently interpreted according to the age and the endocrinal status of the animals [26] and their disease condition [17]; so, several factors could have influenced our contradictory results. The lipocalin 1 (LCN1) is secreted in large amounts by the pig lachrymal glands and von Ebner's glands (in tongue) [27]. In humans, the LCN1 is supposed to act as a protection factor for the epithelia, with the ability to play a role in the nonimmunological defence against microorganisms and viruses, and the control of inflammatory processes [28]. This result is in concordance with the previous studies in which the LCN1 was proposed as the biomarker of health status in male pigs [17]. A close relation between the three lipocalins identified and the immune status has been previously reported in literature. The high level of lipocalin observed in females, in comparison to male pigs, is in concordance with the literature in which higher levels of immune markers have been reported in the females [7], and also with the results of the salivary biomarkers of immune status quantified in the present study, with higher values of Hp, CRP and ADA in females in comparison to the male pigs. The Hp and CRP are well-known acute phase proteins whose levels increase after a homeostasis disturbance [29], and the ADA is involved in the activation and maintenance of the immune system via lymphocyte development [30]. Thus, the possible future use of lipocalins as biomarkers of immune status in porcine saliva would be a great tool that adds a new perspective of immune characterization in pigs. However, taking into account that our present results showed higher OBP, LCN 1 and SAL 1 values in females in comparison to males, the normal range values of these lipocalins should be established in both sexes prior to its possible use as biomarker of immune status.
Competing interest Competing interests do not compromise this study. We agree to its publication, providing it is considered suitable, and we accept the rules set out in authors consent to publication (copyright). Acknowledgment The authors wish to thank Karin Hummel, Katharina Nöbauer and Ebrahim Razzazi Fazeli (VetCore Facility for Research, University of Veterinary Medicine Vienna, Austria), for the provision of MS data and the excellent technical assistance. References [1] C.R. Pierozan, P.S. Agostini, J. Gasa, A.K. Novais, C.P. Dias, R.S.K. Santos, M. Pereira Jr., J.G. Nagi, J.B. Alves, C.A. Silva, Factors affecting the daily feed intake and feed conversion ratio of pigs in grow-finishing units: the case of a company, Porcine Health Manag. 2 (2016) 7. [2] H.H. Petersen, A.K. Ersboll, C.S. Jensen, J.P. Nielsen, Serum-haptoglobin concentration in Danish slaughter pigs of different health status, Prev. Vet. Med. 54 (2002) 325–335. [3] M. Clapperton, S.C. Bishop, N.D. Cameron, E.J. Glass, Associations of acute phase protein levels with growth performance and with selection for growth performance in Large White pigs, Anim. Sci. 81 (2005) 213–220. [4] A.M. Gutiérrez, S. Martínez-Subiela, L. Soler, F.J. Pallarés, J.J. Cerón, Use of saliva for haptoglobin and C-reactive protein quantifications in porcine respiratory and reproductive syndrome affected pigs in field conditions, Vet. Immunol. Immunopathol. 132 (2009) 218–223. [5] M.A. Ruis, J.H. Te Brake, B. Engel, E.D. Ekkel, W.G. Buist, H.J. Blokhuis, J.M. Koolhaas, The circadian rhythm of salivary cortisol in growing pigs: effects of age, gender, and stress, Physiol. Behav. 62 (1997) 623–630. [6] D. Escribano, A.M. Gutiérrez, M. Fuentes-Rubio, J.J. Cerón, Saliva chromogranin A in growing pigs: a study of circadian patterns during daytime and stability under different storage conditions, Vet. J. 199 (2014) 355–359. [7] H.A. Hakimov, S. Walters, T.C. Wright, R.G. Meidinger, C.P. Verschoor, M. Gadish, D.K. Chiu, M.V. Strömvik, C.W. Forsberg, S.P. Golovan, Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization, Proteomics 9 (2009) 4000–4016. [8] S.P. Golovan, H.A. Hakimov, C.P. Verschoor, S. Walters, M. Gadish, C. Elsik, F. Schenkel, D.K. Chiu, C.W. Forsberg, Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders, and conventional and genetically enhanced lines, Comp. Biochem. Physiol. D Genomics Proteome 3 (2008) 234–242. [9] E. Kaufman, I.B. Lamster, The diagnostic applications of saliva—a review, Crit. Rev. Oral Biol. Med. 13 (2002) 197–212. [10] J.R. Prickett, J.J. Zimmerman, The development of oral fluid-based diagnostics and applications in veterinary medicine, Anim. Health Res. Rev. 11 (2010) 207–216. [11] A.M. Gutiérrez, D. Escribano, M. Fuentes, J.J. Cerón, Circadian pattern of acute phase proteins in the saliva of growing pigs, Vet. J. 196 (2013 May) 167–170. [12] A.M. Gutiérrez, E. De La Cruz-Sánchez, A. Montes, J. Sotillo, C. Gutiérrez-Panizo,
5. Conclusion The two-dimensional proteome analysis proved to be an effective tool to detect saliva proteins with altered expression in male or female pigs. Three lipocalin proteins showing differential expression profiles in the saliva of finishing pigs have been highlighted. It could be postulated that the sexual hormones are directly influencing these results, since the most relevant differences between the genders were observed in 6
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