Skip to main content

Influence of the circadian cycle, sex and production stage on the reference values of parameters related to stress and pathology in porcine saliva

Abstract

Background

The concentration of biomarkers in saliva could be influenced by several factors not related to the specific condition under analyses, which should be considered for proper clinical interpretation. In the present study, the circadian rhythm of C-reactive protein (CRP), haptoglobin (Hp), Pig-MAP, S100A12, Cu, Zn, Adenosine deaminase (ADA), total protein (TP), total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI), cortisol and α-amylase in saliva of 20 female and 20 male pigs was investigated. Moreover, the influence of sex and production phase (post-weaning, fattening and finishing) on the concentrations of biomarkers in a total of 414 healthy pigs was studied and the reference intervals for all salivary biomarkers were calculated accordingly.

Results

All parameters except Pig-MAP, OSI and α-amylase varied significantly along the daytime, and most of them peak around early afternoon (13–15 h). The cosinor analysis described the temporal dynamics of circadian rhythms for all parameters. The range values showed differences between male and female pigs in 8 out of the 13 biomarkers, with higher concentrations in females in comparison to male pigs. The influence of the production phase on the salivary concentrations was observed for all the biomarkers. The highest concentrations were observed for Pig-MAP, S100A12 and α-amylase in post-weaning animals, for TP in growing pigs and for OSI in finishing animals. Most of the sex-influenced biomarkers showed the highest concentrations at growing stages with some exceptions such as ADA or Hp that showed the peak at finishing and post-weaning stages respectively.

Conclusions

It is necessary to establish the optimal daytime for routine saliva sampling to avoid circadian variations and for that end, the time interval between 10:00 a.m. to 12:00 a.m. is highly recommended. The factors sex and production phase influence the concentration of biomarkers and should be considered for proper biomarker interpretation. The reference intervals presented here for each salivary biomarker will help to correctly interpret the results of these analytes and contribute to the use of saliva as a non-invasive sample for the diagnosis and monitoring of the health status of swine farms.

Background

Over the years, saliva has become an important sample to assess the health status in pigs, due to its non-invasive character and easy collection that does not need experienced staff. Components of saliva mirror in many cases those from plasma and they may provide a good indication of the health status of the individual [1, 2]. Moreover, saliva shows proteins originated from the different salivary sources that could also be of interest for health assessment and monitoring [1]. Several compounds related to stress, oxidation, inflammation, immune activation and other causes have already been assayed in pig saliva. One of the first analytes to be measured was the stress hormone cortisol since the collection of saliva may be performed without altering the animal, being a more adequate indicator of stress compared to the plasma hormone [3]. Furthermore, it is known that only free cortisol is found in saliva and that there is an equilibrium between the concentration of cortisol in saliva and free cortisol in plasma, although concentration in saliva is much lower than in plasma (5–10% of the free plasma cortisol concentration) [4]. Besides cortisol, many other analytes related to stress and pathology have been determined in pig saliva including acute phase proteins (APPs) [5,6,7], α-amylase [8, 9], carbonic anhydrase VI [10], interleukin (IL)-18 [11], immunoglobulins (Ig) A, G and M [12, 13], adenosine deaminase (ADA) [14], chromogranin A [15], markers of the oxidative status [16] and trace elements [17].

Variations in the pig saliva concentration of one or more of these parameters are indicative of stress [2, 11, 12, 18,19,20,21,22] and several pathologies [2, 6, 14, 16, 23, 24], increasing the interest on these biomarkers and this type of sample for the diagnosis and monitoring of the health status of swine farms.

On this regard, it is of the uttermost importance to analyze in detail all the potential sources of results variation, due to the conditions of the individual and/or differences in the preanalytical phase of the procedure. It has to be considered that the saliva flux is variable, in contrast to the plasma volume, and it may be affected by the circadian cycle. Likewise, it is essential to establish reference intervals (RI) not only for each animal species, but also taking into account variables like age and management conditions. In fact, the effect of age, breed and productive stage has been already described by our group for salivary ADA, total antioxidant capacity (TAC) and the APPs haptoglobin (Hp) and C-reactive protein (CRP) [16], as well as the influence of the circadian rhythm on salivary cortisol [25, 26] and Hp and CRP [27].

Thus, the objective of the present study is to define the optimal daytime for routine saliva sampling and to establish actual reference range values for salivary biomarkers of health and welfare status in commercial pigs, specifically for CRP, Hp, Pig-MAP, S100A12, Cu, Zn, ADA, total protein (TP), TAC, total oxidant status (TOS), oxidative stress index (OSI), cortisol and α-amylase.

Results

Circadian rhythm trial

Statistically significant differences in the concentrations of salivary biomarkers during the daytime were observed for all parameters except Pig-MAP, OSI and α-amylase (Fig. 1). The highest values of biomarkers were reported in the saliva samples collected at 15:00 p.m. except for trace elements in which the highest values were reported at 11:00 a.m. No differences were observed in the daytime variations of salivary parameters between male and female (p value > 0.05 for interaction between sex and time). The power of the differences found in the concentration of salivary parameters between timepoints were higher to 90% for all analyses (Table 1).

Fig. 1
figure 1

Concentration of salivary analytes studied at different timepoints in healthy pigs. Pig-MAP (ng/mL) (A), CRP (ng/mL) (B), Hp (µg/mL) (C), ADA (U/mL) (D), TP (mg/mL) (E), S100A12 (µg/mL) (F), Cu (µg/mL) (G), Zn (µg/mL) (H), TAC (µM/L Trolox equivalents) (I), TOS (µM/L peroxidase equivalents) (J), OSI (TOS/TAC ratio) (K), cortisol (µg/dL) (L) and salivary α-amylase (U/L) (M) concentrations at different timepoints (7:00 a.m., 11:00 a.m., 15:00 p.m. and 19 p.m.). Graph showing the distribution of the population, the median (central horizontal line), 25th and 75th percentiles (non-central horizontal lines within the plot), maximum and minimum (edges of the figure). Statistical differences are indicated by *, **, ***, and **** for p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively

Table 1 Achieved power for the analysis of variations in the concentration of salivary biomarkers during the daytime and the effect size of the differences observed between timepoints

The cosinor model showed the circadian curves for all parameters (Fig. 2). Overall, the concentration of salivary biomarkers peaks between 13:00–15:00 p.m. for most of the parameters. For OSI, the highest concentrations were obtained at midnight while α-amylase showed different peaks for male (17:00 p.m.) and females (3:00 a.m.). Statistically significant differences between the mean midline-estimating statistics of rhythm (MESOR), or acrophase by sex were observed for ADA and Hp (MESOR) and for OSI (acrophase) (Table 2).

Fig. 2
figure 2

Fitted cosine curves of the different salivary analytes studied in female (n = 20) and male (n = 20) pigs in the circadian study in which animals were sampled at 4 h interval in a 12 h period between 7:00 a.m. and 19:00 p.m. Pig-MAP (ng/mL) (A), CRP (ng/mL) (B), Hp (µg/mL) (C), ADA (U/mL) (D), TP (mg/mL) (E), S100A12 (µg/mL) (F), Cu (µg/mL) (G), Zn (µg/mL) (H), TAC (µM/L Trolox equivalents) (I), TOS (µM/L peroxidase equivalents) (J), OSI (TOS/TAC ratio) (K), cortisol (µg/dL) (L) and salivary α-amylase (U/L) (M)

Table 2 Extracted parameters from the cosinor method analysis in female (n = 20) and male (n = 20) pigs

Reference interval trial

The concentrations of 5 biomarkers (Pig-MAP, S100A12, α-amylase, TP and OSI) were significantly different in pigs at different production stages but similar in male and female pigs, while the other 8 biomarkers showed statistically significant differences in both factors (production stage and sex).

Three RIs were established for those biomarkers that showed an influence of production phase in the salivary levels (Table 3), specifically RI for post-weaning, growing, and finishing pigs. The highest levels of salivary biomarkers were observed in post-weaning animals for Pig-MAP, S100A12 and α-amylase, in growing pigs for TP and in finishing animals for OSI (Fig. 3).

Table 3 Reference range intervals in commercial pigs at different production stages for salivary biomarkers not sex-influenced
Fig. 3
figure 3

Concentration of salivary analytes in pigs at different stages of the production system. Pig-MAP (ng/mL) (A), TP (mg/mL) (B), S100A12 (µg/mL) (C), OSI (TOS/TAC ratio) (D) and salivary α-amylase (U/L) (E). Graph showing the distribution of the population, the median (central horizontal line), 25th and 75th percentiles (non-central horizontal lines within the plot), maximum and minimum (edges of the figure). Statistical differences are indicated by **** for p < 0.0001

For the biomarkers that showed an influence of sex and production stage in their salivary concentrations, six RIs were defined (Table 4). Overall, for all salivary biomarkers higher concentrations were recorded in female pigs in comparison to male counterparts. Moreover, the concentration of most biomarkers was higher at growing stages in males and females, except for ADA that showed the highest values at finishing and Hp and CRP that showed highest values at growing in males while the highest values were recorded at post-weaning state in females (Fig. 4).

Table 4 Reference range intervals in male and female pigs at different production stages for salivary biomarkers
Fig. 4
figure 4

Concentration of salivary analytes in pigs of different sexes and stages of the production system. CRP (ng/mL) (A), Hp (µg/mL) (B), ADA (U/mL) (C), TAC (µM/L Trolox equivalents (D), TOS (µM/L peroxidase equivalents) (E), cortisol (µg/dL) (F), Cu (µg/mL) (G), and Zn (µg/mL) (H). Graph showing the distribution of the population, the median (central horizontal line), 25th and 75th percentiles (non-central horizontal lines within the plot), maximum and minimum (edges of the figure). Statistical differences are indicated by *, **, ***, and **** for p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively

Discussion

To use saliva as a routine sample type to assess pig health it is mandatory to acknowledge and characterize the potential sources of variation that may affect the analytical results, like it is well accepted for plasma parameters [28]. Furthermore, it is necessary to extend the experimental studies on the calculation of RIs to provide a robust set of data to be used by the clinician.

Amongst the sources of variation, the influence of the circadian rhythm is important, as demonstrated by the well-known example of salivary cortisol [25, 26]. To describe the circadian rhythms of the different tested parameters, we have used the cosinor method described previously by Ekkel et al. to assess the circadian rhythm of salivary cortisol in pigs [18, 25]. In this method, point and 95% confidence interval estimates of the following rhythm characteristics are obtained: the MESOR (the rhythm-adjusted mean), the circadian amplitudes (measures of the extent of the predictable change within one cycle), and the circadian acrophases (measures of the timing of overall high values recurring in each cycle). Using this chronobiological approach, not only the average cortisol level is found but the amplitude of the circadian rhythm is also analysed [25, 29, 30]. In the present study, the salivary cortisol rhythmicity is similar to other reports, peaking around noon or early afternoon [31,32,33]. Pigs in the present study were 120 days old and were sampled in spring. Although the circadian rhythms may be influenced due to environmental conditions [31], gender or age [32] being less marked in very young animals, it has been described that around 20 weeks of age they reach an adult and stable profile [32]. The similarities between the fitted curve of our 12-hour study, 4-hourly values, and the one reported before for 24-hours covering [32], showed the usefulness of cosinor model for rhythmicity parameters comparison when sampling frequency are restricted to few times along the scale of a given rhythm as reported before [29].

Besides cortisol, the influence of the circadian rhythm on other salivary parameters has been studied by us in the present work. Thus, the influence on CRP was found to be significant with the lowest concentrations at 19:00 p.m., and a fitted cosinor curve similar to the one observed for cortisol in this study and in previous reports [34] with peak concentrations in the late morning. Hp is higher in the afternoon than in the morning, with no additional variations between males and females but with higher MESOR values in females than in males. This difference in the MESOR between males and females was also observed for ADA concentrations and could be explained by the sex influence in the levels of salivary biomarkers as reported before [33, 35]. Moreover, our peak around 15:00 p.m. in ADA levels also agree with the results reported for the peaks observed in ADA isoforms [33]. Although Pig-MAP showed no variations in the mean concentrations during the daytime, the cosinor analysis revealed a circadian curve with higher concentrations in the early afternoon. Regarding the other parameters, the majority of them (TP, S100A12, Cu, Zn, TAC and TOS) are affected by the daytime except OSI and α-amylase which are not affected. However, the cosinor curves indicated that most of the parameters peak around early afternoon (13:00-15:00 p.m.) with the exception of Cu and Zn in males (peak between 10:00 a.m. and 11:00 a.m.). The curves of females and males behave similarly for most of the parameters studied, although the amplitude, which measure the extent of rhythmic change, is clearly higher in females for Pig-MAP, Cu, Zn, TAC and TOS, whereas the amplitude is clearly higher in males for S100A12. The curves for OSI and α-amylase are different in shape between females and males, showing different peaks for the concentrations in females and males. Our results in male pigs agree with previous studies in which a peak of α-amylase was observed at around 16:00 p.m. [33]. However, we observed differences in the TAC levels over the daytime peaking at 15:00 p.m., with a statistical power of 90.95%, which is contradictory to the results of the latest study. It has been described that circadian rhythms have a significant impact on oxidative stress, but there is still an incomplete understanding of the molecular mechanisms linking circadian rhythms and oxidative stress [36] that should be further studied.

Nevertheless, the variations observed in all parameters fall within the normal range and they would not have any clinical significance as reported before for APPs [27].

It is difficult to compare these results to other animal species since there is very little or no information about the influence of the sampling time in salivary biomarker concentrations. In humans, the influence of daytime was analysed for salivary α-amylase which peaks in the afternoon (similar to our results in male pigs) [37], and for salivary antioxidant mechanisms which show several regular peaks along the daytime [38]. Our results show that TAC and TOS peak in early afternoon, but it is difficult to compare the results since the analytical techniques were different.

In general, it is clear that daytime may affect the concentration of many components in saliva [34], as studies performed in humans have already shown for melatonin [39], androgen derivatives [40], some steroid hormones and IgA [41], peptide hormones such as oxytocin and arginine-vasopressin [42], serotonin [43] and minerals such as Ca, P, Na and K [44]. None of these compounds were assayed in the present work.

In other animal species, some research has been carried out in goats but referred only to electrolytes and urea and found diurnal acrophases for most of the tested parameters [45].

We recommend establishing a time interval for routine saliva sampling to avoid misinterpretations due to circadian variations. The optimal time interval should be compatible with farm and laboratory timetable to guarantee optimal biomarker analysis and/or sample storage, so the time interval between 10:00 a.m. and 12:00 a.m. is highly recommended.

Each of the salivary parameters studied has its own clinical significance and the information from one could not be replaced by other, but compatible. Those biomarkers without circadian influence in its levels, specifically Pig-MAP, α-amylase and OSI, would be preferable than others, however, for biomarker’s recommendation other factor should also be considered such as the specific conditions to be evaluated, since not all the conditions alter the level of biomarkers in a similar way [19, 63].

On the other hand, RIs must be set for each animal species, and the knowledge of the influence of age and management conditions has been recognized as an essential condition to the correct use of these RI [28, 46]. Several reports have analysed the effects of age, breed or management conditions on plasma/serum parameters including APPs and metabolic compounds [47,48,49,50,51,52,53] and found relevant differences in RI values depending on the conditions. Thus, the plasma antioxidant potential, hydroperoxides, the oxidative stress index (OSI) and vitamin A and E differed in the young piglets depending on the weaning status [47]. Sex and age have an influence of serum acute phase proteins [48, 50,51,52,53,54], and plasma cytokines [51, 52]. Many of the most important differences occurs in young ages and around weaning, but they occur also in adult ages and depending on the conditions of the farm [53].

Despite the amount of information on serum or plasma analytes, scarce information is available on saliva. Our group has previously described the influence of sex, breed (Iberian or Large White x Duroc) and the production phase (post-weaning, nursery, fattening, and finishing) on ADA, CRP, Hp and TAC [16]. The results presented in this work extend this information to other salivary parameters. However, our study is limited to commercial entire male and female pigs and additional studies should be performed to extent the analysis to castrated or even immunocastrated pigs. Moreover, our results are limited to some commercial standardized genetics and further studies should be performed to cover other hybrids of other genetic lines.

Our results indicate that significant differences exist in several parameters depending on the production stage but were not affected by sex: Pig-MAP, S100A12 and α-amylase present higher values in post-weaning animals, whereas TP is higher in growing pigs and OSI in finishing pigs. Furthermore, on the other tested parameters, female animals showed higher RIs than males, and most of them higher values in the growing phase except for ADA, Hp and CRP. Our results are in concordance with previous ones in which higher levels of immune markers have been reported in saliva samples of female in comparison to age-matched male pigs [35]. Regarding CRP, Hp, ADA and TAC, our results were also similar to our previous study [16] taking into account the technical and biological differences between the animals included in each study.

The interest of the results described here on the influence of the sampling time and the calculation of reference intervals, using a high number of individuals, resides on the previously reported study by us showing that these parameters measured in saliva may provide a diagnostic tool more adequate than serum to evaluate the health status of the herds [1]. In that work, the diagnostic power of saliva biomarkers to detect disease conditions in pigs was analysed in a multi-herd experimental approach, under field commercial conditions using two animal groups, healthy and diseased, and matching by breed, gender and age. The present study goes further on the full characterization of these analytes to ensure that saliva could be considered as an alternative specimen to serum for detection of disease in pigs. Furthermore, these results will help to find the best combination of biomarkers to develop an optimal algorithm, for its possible implementation for disease monitoring and/or health status assessment in the field, as suggested previously [17]. However, further studies should be performed to characterize the behaviour of each biomarker under different health conditions to define optimal analytical models for detection of homeostasis dysregulation’s in field conditions.

Conclusions

The concentration of the studied salivary biomarkers of health and stress status showed variations during the daytime, so it is necessary to establish a time interval for routine saliva sampling for proper interpretation. The time interval between 10:00 a.m. and 12:00 a.m. is highly recommended since is compatible to both farm and laboratory labour. Sex and production stage influence the concentration of salivary biomarkers in healthy animals and both factors should be considered for reference range values calculation. We have established actual reference intervals for male and female pigs at post-weaning, growing and finishing states for 13 salivary biomarkers that will contribute to the use of saliva as a non-invasive sample for the diagnosis and monitoring of the health and stress status of swine farms.

Materials and methods

Aim, design and setting of the study

The aim of the study was to analyse the possible influence of different factors on the concentration of salivary biomarkers under healthy conditions that should be considered for proper clinical interpretation. Specifically, the factors to be studied were the circadian rhythm variations, and the possible differences according to sex and production phase of the pigs. To overcome the objective of the study, two separated trials were performed: circadian rhythm trial and RI trial.

For the first trial, a group of 40 animals around 120 days of life were sampled at different daytimes (07:00, 11:00, 15:00 and 19:00 h) on May 25th, 2022. The room temperature oscillated a maximum of 2.4ºC between samplings (from a minimum medium temperature of 22 ºC at early morning to a maximum medium temperature of 24.1 ºC in the afternoon). Four experimented veterinary researchers performed the clinical examination, to discard any clinical sign of disease, and the sampling of animals at each timepoint (1 researcher/10 animals) to reduce the duration of the experimental procedure.

The RI trial was performed in a total of 6 days between June 1st and 28th, 2022. Three different production phases were included in the study: post-weaning (pigs with a median weight of 21.6 kg), fattening (pigs with a median weight of 63.4 kg) and finishing (pigs with a median weight of 98.7 kg). Three farms per production phase were included in the study, in which a minimum of 20 males and 20 females were sampled at the same time of the day (between 10 and 12 h), after discarding any clinical signs of disease during proper clinical examination. A minimum of 120 animals (60 males and 60 females) were selected per production phase which gave a minimum total sample size of 360 pigs (Table 5).

Table 5 Characteristics of the animals from the reference interval analysis

Animals and housing conditions

For circadian rhythm trial, one commercial farm from the southeast of Spain was selected while for reference range interval trial, nine commercial farms from the Southeast of Spain were selected from the same commercial company to obtain data over different environments.

The vaccination of animals was the same for all farms within each production phase. The vaccination program consisted of the administration of a first dose against porcine circovirus and mycoplasma before weaning at 21 days of life, the vaccination against enzootic pneumonia at 10 and 14 weeks of life and the vaccination against Aujezsky disease at 11 and 15 weeks of life.

All pigs were housed in pen groups with 0.65 m2/pig following the official standards [54] with ad libitum access to balanced dry food and water.

The sample size used for the circadian rhythm trial was 40 (20 males and 20 females) as reported before [27]. To ensure a proper statistical significance of the analysis, the optimal power for daytime variations was established as 90%.

For the establishment of RIs, a minimal sample size of 60 per condition was used following the general guidelines for the determination of reference intervals in veterinary species [46].

Saliva sampling procedure

Saliva samples were collected individually, without animal restrain, by using 1 × 1 × 1 cm sponges clipped to a thin metal rod. Pigs were allowed to chew the sponge for 1–2 min. Afterwards, sponges were included in specifically designed tubes for saliva collection (Salivette tubes, Sarstedt, Nümbrecht, Germany), individually labelled and stored in boxes with cold accumulators until transported to the laboratory within 4 h after collection. Saliva collection tubes were centrifuged at 3000 g for 10 min to obtain the clear whole saliva from the sponges and remove food or cell debris. Saliva samples were stored at -80ºC until analysis within 1 week.

Acute phase proteins determination: CRP, Hp and Pig-MAP

The measurement of CRP and Hp was carried out using previously in-house developed time-resolved immunofluorometric assays (TR-IFMA) which were validated for the optimal quantification of CRP [55] and Hp [56] in porcine saliva samples. The concentrations of Pig-MAP were quantified using an in-house TR-IFMA, recently optimized and validated for proper saliva quantification from a previous ELISA [57]. In summary, the new developed assay showed good intra-assay and inter-assay precision with coefficient of variation (CV) lower than 10.56% and 13.73% respectively, good accuracy investigated by linearity under dilution with coefficient of correlation of 0.99 and a limit of detection of 4.09 ng/mL. The fluorometric signals were quantified in a multilabel counter (Victor 1420, Perkin Elmer, Turku, Finland). For saliva Pig-MAP determinations, saliva samples were analysed undiluted, and the calibration curve used covered a range between 7.8 and 2000 ng/mL.

S100A12 quantifications

S100A12 levels were quantified by an in-house sandwich enzyme-linked immunosorbent assay (ELISA) recently developed and validated for porcine saliva determinations [58]. The assay consisted in a sandwich ELISA with good analytical parameters. In summary, the assay showed a high precision, with coefficients of variations lower than 7% for intra and inter-assay precision, good accuracy with coefficient of correlation of 0.98, when linearity under dilution was evaluated, and a limit of detection of 3.19 ng/mL. For saliva determinations, samples were diluted 1:1000 and the calibration curve used was constructed using porcine recombinant protein (1.95 to 125 ng/mL) (GenScript Biotech, Leiden, Netherlands).

Measurement of Cu and Zn levels

For the measurements of Cu and Zn levels, samples were subjected to acid digestion as reported before [17] followed by atomic absorption spectrometry (Varian model SpectrAA 55B spectrometer, Palo Alto, CA, USA). The contents of Cu and Zn in the saliva samples were calculated by the interpolation of the signal in a standard curve (from 1 to 4 µg/mL of certified standard solutions for Cu and Zn (Agilent Technologies Spain, Madrid, Spain)), with standard concentration on the x-axis and signal on the y-axis drawing the best-fit curve through the standard points and taking into account the dilution of the saliva according to the digestion protocol used (0.5mL of saliva in a final volume of 2.5 mL).

ADA activity determination

The procedure is an adaptation to microtitration plates of a commercial automatized assay (BioSystems S.A., Barcelona, Spain) based on Slaats et al. [59]. The assay was previously optimized and validated for porcine saliva samples with good precision, accuracy and limit of detection [14]. The levels of total ADA activity are calculated in U/mL according to manufacturer’s instructions taking into account the dilution factor.

Total protein content determination

The total protein content was determined according to Bradford protocol [60]. Saliva samples were diluted (1:40) for its proper quantification using an albumin calibration curve (from 5 to 100 ng/mL).

TAC, TOS and OSI determinations

TAC was measured through the ferric reducing antioxidant power (FRAP) assay [61]. The assay was validated for porcine saliva samples with good analytical performance [16]. The levels of TAC are calculated using a standard curve of Trolox (a water-soluble analogue of vitamin E employed as a control antioxidant agent for assay calibration), ranging from 1.5 to 100 µM Trolox equivalents/L.

The procedure for Total Oxidant Status (TOS) measurement is an in-house adaptation of a commercially available assay (Pierce Quantitative Peroxide Assay Kit, aqueous-compatible formulation) based on Nourooz-Zadeh et al. [62]. The assay has been previously validated for porcine saliva determinations with high precision, accuracy and good limit of detection [63]. The levels of TOS are calculated using a standard curve of Hydrogen peroxide (0.97 to 31.25 µM Peroxidase equivalents/L).

The Oxidative Stress Index (OSI) was calculated as the ratio TOS/TAC according to previous studies [64].

Cortisol and salivary alpha-amylase measurements

The cortisol content was measured using an optimized commercial competitive ELISA (Extended range high sensitivity salivary cortisol Enzyme immunoassay kit, Salimetrics, USA). Cortisol concentration in µg/dL was calculated according to the manufacturer’s instructions by interpolation of the signal in a standard curve (0.012 µg/dL − 3 µg/dL) using porcine saliva without previous dilution.

Salivary alpha-amylase was quantified using an adaptation of the commercial kinetic enzyme assay (Salivary alpha-amylase kinetic enzyme assay kit, Salimetrics, USA). In the adaptation, 8 µL of saliva sample or control were incubated with 320 µL of amylase substrate during 5 min at 37ºC. The increase in absorbance from minute 1 to 5 was recorder for alpha-amylase activity calculation.

Statistical analysis

Prior to statistical analysis, all groups of data were subjected to Shapiro-Wilk normality test followed by Fligner-Killeen test of homogeneity of variances to check the normality and homoscedasticity of the data and select the appropriate statistical tool. Data from both the circadian rhythm and RI trials passed the normality and homoscedasticity criteria so mixed ANOVA and two-way ANOVA test were used (for detailed results see supplementary Tables 1 and 2).

To show if differences exist between the timepoints during the daytime, results from the circadian rhythm trial were analysed by mixed ANOVA in which Maulchly’s test was used to check for sphericity using Greenhouse-Geisser or Huynh-Feldt sphericity corrections. When statistically significant differences were detected in the mixed ANOVA, a post-hoc multiple comparisons test was performed with Tukey’s correction and the size effect was calculated as the coefficient Cohen’s d. The achieved power for the mixed ANOVA test in the sample size studied was calculated for each biomarker. Afterwards, time series were analysed by the mixed-effects cosinor model to obtain a 24 h fitted cosine curve for each parameter and to estimate the effects of biological sex on each parameter curve (waveform). The estimated mean and confidence intervals for the means were calculated for the MESOR, amplitude and acrophase by sex and pairwise contrast by sex for each component were estimated [18, 25].

For the calculation of the RIs, histograms were prepared to illustrate the distribution of data and highlight possible outliers. Horn’s algorithm was used for outlier detection and elimination. Two-way ANOVA test were used considering sex and production phase as factors. When differences in the biomarker’s concentration due to sex or production phase were observed, partitioning criteria were used for more refined RI calculation within subgroups. A minimum of 120 reference animals were defined as the limit for the determination of reference limits by nonparametric methods. Bootstrapping was used to determine 90% confidence intervals for groups of less than 120 animals, following general recommendations [46].

All statistics were performed using R software version 4.0.3. The level of significance was set at p < 0.05.

Data Availability

The datasets generated and analyzed during the current study are available in the Digitum repository, http://hdl.handle.net/10201/130522.

References

  1. Sánchez J, Fuentes N, Ibáñez-López FJ, López-García I, Gutiérrez AM. A multi-herd study shows that saliva is more than a reflection of serum biomarkers in pigs. Animal. 2021. https://doi.org/10.1016/J.ANIMAL.2021.100413.

    Article  PubMed  Google Scholar 

  2. Cerón JJ, Contreras-Aguilar MD, Escribano D, Martínez-Miró S, López-Martínez MJ, Ortín-Bustillo A, et al. Basics for the potential use of saliva to evaluate stress, inflammation, immune system, and redox homeostasis in pigs. BMC Vet Res. 2022. https://doi.org/10.1186/S12917-022-03176-W.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Bushong DM, Friend TH, Knabe DA. Salivary and plasma cortisol response to adrenocorticotropin administration in pigs. Lab Anim. 2000. https://doi.org/10.1258/002367700780457482.

    Article  PubMed  Google Scholar 

  4. Gallagher NL, Giles LR, Wynn PC. The development of a circadian pattern of salivary cortisol secretion in the neonatal piglet. Biol Neonate. 2002. https://doi.org/10.1159/000047195.

    Article  PubMed  Google Scholar 

  5. Gutiérrez AM, Martínez-Subiela S, Eckersall PD, Cerón JJ. C-reactive protein quantification in porcine saliva: a minimally invasive test for pig health monitoring. Vet J. 2009. https://doi.org/10.1016/j.tvjl.2008.03.021.

    Article  PubMed  Google Scholar 

  6. Gutierrez AM, Martinez-Subiela S, Soler L, Pallares FJ, Ceron JJ. Use of saliva for haptoglobin and C-reactive protein quantifications in porcine respiratory and reproductive syndrome affected pigs in field conditions. Vet Immunol Immunopathol. 2009;132:218–23.

  7. Soler L, Gutiérrez A, Martínez-Subiela S, Cerón JJ. Development and validation of a novel competitive ELISA for the detection of serum amyloid A in pigs. Vet J. 2011. https://doi.org/10.1016/J.TVJL.2011.02.016.

    Article  PubMed  Google Scholar 

  8. Gutiérrez AM, Cerón JJ, Fuentes P, Montes A, Martínez-Subiela S. Longitudinal analysis of acute-phase proteins in saliva in pig farms with different health status. Animal. 2012. https://doi.org/10.1017/S1751731111001662.

    Article  PubMed  Google Scholar 

  9. Fuentes M, Tecles F, Gutiérrez A, Otal J, Martínez-Subiela S, Cerón JJ. Validation of an automated method for salivary alpha-amylase measurements in pigs (Sus scrofa domesticus) and its application as a stress biomarker. J Vet Diagn Invest. 2011. https://doi.org/10.1177/104063871102300213.

    Article  PubMed  Google Scholar 

  10. Nishita T, Itoh S, Arai S, Ichihara N, Arishima K. Measurement of carbonic anhydrase isozyme VI (CA-VI) in swine sera, colostrums, saliva, bile, seminal plasma and tissues. Anim Sci J. 2011. https://doi.org/10.1111/J.1740-0929.2011.00888.X.

    Article  PubMed  Google Scholar 

  11. Muneta Y, Minagawa Y, Nakane T, Shibahara T, Yoshikawa T, Omata Y. Interleukin-18 expression in pig salivary glands and salivary content changes during acute immobilization stress. Stress. 2011. https://doi.org/10.3109/10253890.2011.565392.

    Article  PubMed  Google Scholar 

  12. Muneta Y, Yoshikawa T, Minagawa Y, Shibahara T, Maeda R, Omata Y. Salivary IgA as a useful non-invasive marker for restraint stress in pigs. J Vet Med Sci. 2010. https://doi.org/10.1292/JVMS.10-0009.

    Article  PubMed  Google Scholar 

  13. Escribano D, Gutiérrez AM, Martínez Subiela S, Tecles F, Cerón JJ. Validation of three commercially available immunoassays for quantification of IgA, IgG, and IgM in porcine saliva samples. Res Vet Sci. 2012. https://doi.org/10.1016/J.RVSC.2011.09.018.

    Article  PubMed  Google Scholar 

  14. Gutiérrez AM, De La Cruz-Sánchez E, Montes A, Sotillo J, Gutiérrez-Panizo C, Fuentes P, et al. Easy and non-invasive disease detection in pigs by adenosine deaminase activity determinations in saliva. PLoS ONE. 2017. https://doi.org/10.1371/JOURNAL.PONE.0179299.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Escribano D, Soler L, Gutiérrez AM, Martínez-Subiela S, Cerón JJ. 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. Animal. 2013. https://doi.org/10.1017/S1751731112002005.

    Article  PubMed  Google Scholar 

  16. Sánchez J, García A, Ruiz JM, Montes AM, Cabezas-Herrera J, Ros-Lara S, et al. Porcine breed, sex, and production stage influence the levels of health status biomarkers in saliva samples. Front Vet Sci. 2019. https://doi.org/10.3389/FVETS.2019.00032.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Sánchez J, Montilla M, Gutiérrez-Panizo C, Sotillo J, Fuentes P, et al. Analytical characterization of trace elements (zinc, copper, cadmium, lead and selenium) in saliva of pigs under common pathological conditions in the field: a pilot study. BMC Vet Res. 2020. https://doi.org/10.1186/S12917-020-2245-6.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Ekkel ED, Savenije B, Schouten WGP, Wiegant VM, Tielen MJM. The effects of mixing on behavior and circadian parameters of salivary cortisol in pigs. Physiol Behav. 1997. https://doi.org/10.1016/S0031-9384(97)00037-1.

    Article  PubMed  Google Scholar 

  19. Ott S, Soler L, Moons CPH, Kashiha MA, Bahr C, Vandermeulen J, et al. Different stressors elicit different responses in the salivary biomarkers cortisol, haptoglobin, and chromogranin A in pigs. Res Vet Sci. 2014;97:124–8.

    Article  CAS  PubMed  Google Scholar 

  20. Huang Y, Liu Z, Liu W, Yin C, Ci L, Zhao R, et al. Short communication: salivary haptoglobin and chromogranin A as non-invasive markers during restraint stress in pigs. Res Vet Sci. 2017. https://doi.org/10.1016/j.rvsc.2017.02.023.

    Article  PubMed  Google Scholar 

  21. Soler L, Gutiérrez A, Escribano D, Fuentes M, Cerón JJ. Response of salivary haptoglobin and serum amyloid A to social isolation and short road transport stress in pigs. Res Vet Sci. 2013. https://doi.org/10.1016/J.RVSC.2013.03.007.

    Article  PubMed  Google Scholar 

  22. Escribano D, Gutiérrez AM, Tecles F, Cerón JJ. Changes in saliva biomarkers of stress and immunity in domestic pigs exposed to a psychosocial stressor. Res Vet Sci. 2015. https://doi.org/10.1016/j.rvsc.2015.07.013.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Soler L, Gutiérrez A, Müllebner A, Cerón J, Duvigneau J. Towards a better understanding of salivary and meat juice acute phase proteins determination in pigs: an expression study. Vet Immunol Immunopathol. 2013. https://doi.org/10.1016/J.VETIMM.2013.09.018.

    Article  PubMed  Google Scholar 

  24. Gomez-Laguna J, Gutierrez A, Pallares FJ, Salguero FJ, Ceron JJ, Carrasco L. Haptoglobin and C-reactive protein as biomarkers in the serum, saliva and meat juice of pigs experimentally infected with porcine reproductive and respiratory syndrome virus. Vet J. 2010;185:83–7.

    Article  CAS  PubMed  Google Scholar 

  25. Ekkel ED, Dieleman SJ, Schouten WGP, Portela A, Cornélissen G, Tielen MJM, et al. The circadian rhythm of cortisol in the saliva of young pigs. Physiol Behav. 1996. https://doi.org/10.1016/0031-9384(96)00107-2.

    Article  PubMed  Google Scholar 

  26. Gatti R, Antonelli G, Prearo M, Spinella P, Cappellin E, De Palo EF. Cortisol assays and diagnostic laboratory procedures in human biological fluids. Clin Biochem. 2009;42:1205–17.

    Article  CAS  PubMed  Google Scholar 

  27. Gutiérrez AM, Escribano D, Fuentes M, Cerón JJ. Circadian pattern of acute phase proteins in the saliva of growing pigs. Vet J. 2013. https://doi.org/10.1016/j.tvjl.2012.10.003.

    Article  PubMed  Google Scholar 

  28. Geffre A, Friedrichs K, Harr K, Concordet D, Trumel C, Braun JP. Reference values: a review. Vet Clin Pathol. 2009;38:288–98.

    Article  PubMed  Google Scholar 

  29. Bingham C, Arbogast B, Guillaume G, Lee JK. Inferential statistical methods for estimating and comparing cosinor parameters. Chronobiology. 1982;9:397–439.

    CAS  Google Scholar 

  30. Halberg F, Chronobiology. Annu Rev Physiol. 1969. https://doi.org/10.1146/ANNUREV.PH.31.030169.003331.

    Article  PubMed  Google Scholar 

  31. de Jong IC, Prelle IT, van de Burgwal JA, Lambooij E, Korte SM, Blokhuis HJ, et al. Effects of environmental enrichment on behavioral responses to novelty, learning, and memory, and the circadian rhythm in cortisol in growing pigs. Physiol Behav. 2000;68:571–8.

    Article  PubMed  Google Scholar 

  32. Ruis MAW, Te Brake JHA, Engel B, Ekkel ED, Buist WG, Blokhuis HJ, et al. The circadian rhythm of salivary cortisol in growing pigs: effects of age, gender, and stress. Physiol Behav. 1997. https://doi.org/10.1016/S0031-9384(97)00177-7.

    Article  PubMed  Google Scholar 

  33. Ortín-Bustillo A, Contreras-Aguilar MD, Rubio CP, Botia M, Cerón JJ, López-Arjona M, et al. Evaluation of the Effect of Sampling Time on biomarkers of stress, Immune System, Redox Status and other Biochemistry Analytes in Saliva of Finishing Pigs. Animals. 2022. https://doi.org/10.3390/ani12162127.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Wisser H, Breuer H. Circadian changes of clinical chemical and endocrinological parameters. Clin Chem Lab Med. 1981;19:323–38.

    CAS  Google Scholar 

  35. Gutiérrez AM, Montes A, Gutiérrez-Panizo C, Fuentes P, De La Cruz-Sánchez E. Gender influence on the salivary protein profile of finishing pigs. J Proteom. 2018. https://doi.org/10.1016/j.jprot.2017.11.023.

    Article  Google Scholar 

  36. Wilking M, Ndiaye M, Mukhtar H, Ahmad N. Circadian rhythm connections to oxidative stress: implications for human health. Antioxid Redox Signal. 2013. https://doi.org/10.1089/ars.2012.4889.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Rohleder N, Nater UM. Determinants of salivary alpha-amylase in humans and methodological considerations. Psychoneuroendocrinology. 2009. https://doi.org/10.1016/J.PSYNEUEN.2008.12.004.

    Article  PubMed  Google Scholar 

  38. Atsumi T, Tonosaki K, Fujisawa S. Salivary free radical-scavenging activity is affected by physical and mental activities. Oral Dis. 2008;14:490–6.

    Article  CAS  PubMed  Google Scholar 

  39. Kennaway DJ. A critical review of melatonin assays: past and present. J Pineal Res. 2019. https://doi.org/10.1111/JPI.12572.

    Article  PubMed  Google Scholar 

  40. Nowotny HF, Auer MK, Lottspeich C, Schmidt H, Dubinski I, Bidlingmaier M, et al. Salivary profiles of 11-oxygenated androgens follow a diurnal rhythm in patients with congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2021. https://doi.org/10.1210/CLINEM/DGAB446.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Rapson A, Collman E, Faustini S, Yonel Z, Chapple IL, Drayson MT, et al. Free light chains as an emerging biomarker in saliva: Biological variability and comparisons with salivary IgA and steroid hormones. Brain Behav Immun. 2020;83:78–86.

    Article  CAS  PubMed  Google Scholar 

  42. Kagerbauer SM, Debus JM, Martin J, Gempt J, Jungwirth B, Hapfelmeier A, et al. Absence of a diurnal rhythm of oxytocin and arginine-vasopressin in human cerebrospinal fluid, blood and saliva. Neuropeptides. 2019;78:101977.

    Article  CAS  PubMed  Google Scholar 

  43. Tan ZL, Bao AM, Tao M, Liu YJ, Zhou JN. Circadian rhythm of salivary serotonin in patients with major depressive disorder. Neuroendocrinol Lett. 2007;28:395–400.

    CAS  PubMed  Google Scholar 

  44. Bel’skaya LV, Kosenok VK, Sarf EA. Chronophysiological features of the normal mineral composition of human saliva. Arch Oral Biol. 2017;82:286–92.

    Article  PubMed  Google Scholar 

  45. Assenza A, Fazio F, Marcenò G, Piccione G, Caola G. Daily rhythms of serum and salivary parameters in goats. Aust Vet J. 2009. https://doi.org/10.1111/J.1751-0813.2009.00480.X.

    Article  PubMed  Google Scholar 

  46. Friedrichs KR, Harr KE, Freeman KP, Szladovits B, Walton RM, Barnhart KF et al. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. 2012. https://doi.org/10.1111/vcp.12006.

  47. Buchet A, Belloc C, Leblanc-Maridor M, Merlot E. Effects of age and weaning conditions on blood indicators of oxidative status in pigs. PLoS ONE. 2017. https://doi.org/10.1371/JOURNAL.PONE.0178487.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Christoffersen B, Jensen SJ, Ludvigsen TP, Nilsson SK, Grossi AB, Heegaard PMH. Age- and Sex-Associated Effects on Acute-Phase Proteins in Göttingen Minipigs. Comp Med. 2015;65:333.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Clapperton M, Bishop SC, Glass EJ. Innate immune traits differ between Meishan and large White pigs. Vet Immunol Immunopathol. 2005;104:131–44.

    Article  CAS  PubMed  Google Scholar 

  50. Itoh H, Tamura K, Izumi M, Motoi Y, Kidoguchi K, Funayama Y. The influence of age and health status on the serum alpha 1-acid glycoprotein level of conventional and specific pathogen-free pigs. Can J Vet Res. 1993;57:74.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Llamas Moya S, Boyle LA, Lynch PB, Arkins S. Age-related changes in pro-inflammatory cytokines, acute phase proteins and cortisol concentrations in neonatal piglets. Neonatology. 2007. https://doi.org/10.1159/000096970.

    Article  PubMed  Google Scholar 

  52. Yu K, Canalias F, Solà-Oriol D, Arroyo L, Pato R, Saco Y, et al. Age-Related serum biochemical reference intervals established for Unweaned Calves and Piglets in the post-weaning period. Front Vet Sci. 2019. https://doi.org/10.3389/fvets.2019.00123.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Pineiro C, Pineiro M, Morales J, Andres M, Lorenzo E, Pozo MD, et al. Pig-MAP and haptoglobin concentration reference values in swine from commercial farms. Vet J. 2009;179:78–84.

  54. Council Directive. 2008/120/EC of 18 December 2008 laying down minimum standards for the protection of pigs. https://eur-lex.europa.eu/eli/dir/2008/120/oj. Accessed 22 February 2022.

  55. Gutiérrez AM, Nöbauer K, Soler L, Razzazi-Fazeli E, Gemeiner M, Cerón JJ, et al. Detection of potential markers for systemic disease in saliva of pigs by proteomics: a pilot study. Vet Immunol Immunopathol. 2013. https://doi.org/10.1016/j.vetimm.2012.10.007.

    Article  PubMed  Google Scholar 

  56. Gutierrez AM, Martinez-Subiela S, Ceron JJ. Evaluation of an immunoassay for determination of haptoglobin concentration in various biological specimens from swine. Am J Vet Res. 2009;70:691–6.

  57. Piñeiro M, Lampreave F, Alava MA. Development and validation of an ELISA for the quantification of pig major acute phase protein (Pig-MAP). Vet Immunol Immunopathol. 2009. https://doi.org/10.1016/j.vetimm.2008.10.318.

    Article  PubMed  Google Scholar 

  58. Gutiérrez AM, Matas-Quintanilla M, Piñeiro M, Sánchez J, Fuentes P. S100A12 protein as a porcine health status biomarker when quantified in saliva samples. 202X. YTVJL-D-23_00131-3.

  59. Slaats EH, Keimpema ARJ, Kruijswijk H. A continuous method for the estimation of adenosine deaminase catalytic concentration in pleural effusions with a Hitachi 705 discrete analyser. J Clin Chem Clin Biochem. 1985. https://doi.org/10.1515/CCLM.1985.23.10.677.

    Article  PubMed  Google Scholar 

  60. Bradford MA. Rapid and Sensitive Method for the quantification of Microgram quantities of protein utilizing the Principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

    Article  CAS  PubMed  Google Scholar 

  61. Benzie I, Strain J. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal Biochem. 1996;15:70–6.

    Article  Google Scholar 

  62. Nourooz-Zadeh J, Tajaddini-Sarmadi J, Wolff S. Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine. Anal Biochem. 1994;220:403–9.

    Article  CAS  PubMed  Google Scholar 

  63. Sánchez J, Matas M, Ibáñez-López F, Hernández I, Sotillo J, Gutiérrez A. The connection between stress and Immune Status in Pigs: a First Salivary Analytical Panel for Disease differentiation. Front Vet Sci. 2022. https://doi.org/10.3389/fvets.2022.881435.

    Article  PubMed  PubMed Central  Google Scholar 

  64. Wang D, Feng J, Zeng P, Yang Y, Luo J, Yang Y. Total oxidant/antioxidant status in sera of patients with thyroid cancers. Endocrinol Relat Cancers. 2011;18:773–82.

    Article  CAS  Google Scholar 

  65. Directive 2010/63/EU of the European Parliament and of the Council of 22 September. 2010 on the protection of animals used for scientific purposes. https://eur-lex.europa.eu/eli/dir/2010/63/oj. Accessed 22 February 2022.

  66. Real Decreto 53/. 2013, de 1 de febrero, por el que se establecen las normas básicas aplicables para la protección de los animales utilizados en experimentación y otros fines científicos, incluyendo la docencia. https://www.boe.es/diario_boe/txt.php?id=BOE-A-2013-1337. Accessed 22 February 2022.

Download references

Acknowledgements

Authors wish to express their gratitude to the student Irene Lozano, the veterinarians Jorge Sánchez and Aida Miralles for its involvement on animal sampling. We also thanks Dr. Cándido Gutiérrez and Dr. Ana María Montes for its support on animal sampling and for useful discussions. We also thanks Susana Ros for its technical assistance in salivary biomarker quantifications.

Funding

This study has been supported by a Grant PID2020-116310RB-I00 funded by MCIN/AEI/10.13039/501100011033.

Author information

Authors and Affiliations

Authors

Contributions

Y.S. and R.P. drafted the first version of the manuscript. M.M. was involved on sample collection in the circadian rhythm trial and analyzed TAC and TOS salivary content. F.J.I. performed the statistical analysis of the data and helped on data interpretation. M.P. contributed to Pig-MAP salivary concentrations analysis. J.S. was involved on sample collection in both trials and prepare samples for Cu and Zn quantifications. A.B. was a major contributor in writing the manuscript. A.M.G. designed the study and was involved on sample collection in both trials, salivary biomarkers analysis and data interpretation. All authors read and approved the final manuscript.

Corresponding author

Correspondence to A. M. Gutiérrez.

Ethics declarations

Competing interests

M Piñeiro was employed by Acuvet Biotech. None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper.

List of abbreviations

ADA, Adenosine deaminase.

APPs, acute phase proteins.

CRP, C-reactive protein.

ELISA, Enzyme-linked immunosorbent assay.

Hp, Haptoglobin.

Ig, Immunoglobulins.

IL, Interleukin.

MESOR, Midline-estimating statistics of rhythm.

RI, Reference intervals.

TAC, Total antioxidant capacity.

TOS, Total oxidant status.

TP, Total protein.

TR-IFMA, Time-resolved immunofluorometric assay.

OSI, Oxidative stress index.

Ethics approval

All procedures involving animals were approved by the Ethics Committee of the University of Murcia (approval number 561/2019, 14th of November of 2019) and followed the European [65] and the Spanish regulation [66] on the protection of animals used for scientific purposes.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Saco, Y., Peña, R., Matas-Quintanilla, M. et al. Influence of the circadian cycle, sex and production stage on the reference values of parameters related to stress and pathology in porcine saliva. Porc Health Manag 9, 42 (2023). https://doi.org/10.1186/s40813-023-00337-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40813-023-00337-7

Keywords