antioxidants Article Trace Elements and Ferritin in Pig Saliva: Variations during Fattening, Time of Sampling, Effect of Dirtiness and Stability under Different Storage Conditions Alba Ortín-Bustillo 1 , Damián Escribano 1,2 , Silvia Martínez-Subiela 1 , Asta Tvarijonaviciute 1 , Alberto Muñoz-Prieto 1 , Marina López-Arjona 3 , José J. Cerón 1 and Fernando Tecles 1, * 1 Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, Campus de Espinardo s/n, 30100 Murcia, Spain 2 Department of Animal Production, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, Campus de Espinardo s/n, 30100 Murcia, Spain 3 Department of Animal and Food Science, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain * Correspondence: ftecles@um.es; Tel.: +34-868-887-082 Abstract: The objective of this study was to evaluate the possible changes of zinc (Zn), copper (Cu), iron (Fe) and ferritin during the entire productive cycle in fattening pigs and at different diurnal sampling times. Moreover, the possible effects of the presence of pen contaminants and storage stability at different temperature conditions were assessed. The analytes changed along the different phases of the fattening productive cycle, showing, in general, higher values at the initial phases. In addition, statistically significant variations were found in Zn and Cu measurements at different sampling times of the day. In the spectrophotometric assays, the values of all analytes significantly increased after adding high concentrations of feces or feed. However, when low concentrations of feces or feed were added, only Cu showed a significant increase. Overall, the salivary levels of Zn, Cu, Fe and ferritin in pigs can change during different fattening phases and the different hours of the Citation: Ortín-Bustillo, A.; day. These analytes were more stable at −80 ◦ C and, if saliva is contaminated with feces or feed, it Escribano, D.; Martínez-Subiela, S.; can lead to an increase in these analytes. Tvarijonaviciute, A.; Muñoz-Prieto, A.; López-Arjona, M.; Cerón, J.J.; Keywords: saliva; pig; fattening cycle; diurnal; variations; dirtiness; stability; biomarkers; sampling Tecles, F. Trace Elements and Ferritin in Pig Saliva: Variations during Fattening, Time of Sampling, Effect of Dirtiness and Stability under 1. Introduction Different Storage Conditions. Antioxidants 2023, 12, 649. https:// The application of the saliva as a non-invasive sample for analysis has grown more doi.org/10.3390/antiox12030649 popular in recent years [1,2]. Saliva has a wide spectrum of components [3], with potential as biomarkers to assess health status [4]. Collecting saliva samples is a non-invasive and Academic Editor: Evangelos Zoidis easy way to perform sampling without the need to acquire specialized skills [5,6]. In Received: 20 February 2023 veterinary science, the use of saliva implies an easier collection in less time and, therefore, Revised: 1 March 2023 more efficient sampling of a higher number of animals [7]. The use of saliva is particularly Accepted: 3 March 2023 convenient in porcine species, where blood sampling is stressful for the animal and tech- Published: 5 March 2023 nically complicated due to its thick layer of fat body, lack of easily accessible superficial vessels and susceptibility to develop a state of stress [8]. Sialochemistry is a term that defines a series of laboratory tests performed on saliva and includes biomarkers that provide diverse information on the health status of the Copyright: © 2023 by the authors. animals [2]. Trace elements such as iron (Fe), zinc (Zn) and copper (Cu) are related to Licensee MDPI, Basel, Switzerland. iron deficiency [9–11], inflammation and activation of the immune response among other This article is an open access article physiological pathways [12–15]; they can be measured in porcine saliva by automated distributed under the terms and commercial assays [16]. Ferritin is a biomarker related to Fe that participates in several conditions of the Creative Commons cellular functions [17] and it has a role in inflammation [18]. Attribution (CC BY) license (https:// In a previous report, an analytical validation was performed for Fe, Zn, Cu and creativecommons.org/licenses/by/ 4.0/). ferritin measurement in porcine saliva. These analytes were measured in saliva of Fe Antioxidants 2023, 12, 649. https://doi.org/10.3390/antiox12030649 https://www.mdpi.com/journal/antioxidants Antioxidants 2023, 12, 649 2 of 10 supplemented and non-supplemented piglets, with the supplemented group having higher values of ferritin and Zn in saliva [16]. Although the measurement of these analytes in the saliva of pigs is still not widely used, it could be of interest for the evaluation of different physiological processes in which these analytes are involved, for example, to evaluate the addition of different Cu levels to diet [19], the effect of Zn in the stress and immune response [20] or Fe in intestinal inflammation [21]. The aim of this study was to gain knowledge about the measurements of Zn, Cu, Fe and ferritin in the saliva of pigs. For this purpose, we evaluated whether these analytes could change under different physiological conditions, such as the entire productive cycle in fattening pigs and different sample times during the day. In addition, the effect of pen feces and feed contamination, and also the stability of the storage of these salivary analytes, were assessed in order to evaluate possible variations in the measurements due to these factors. 2. Materials and Methods 2.1. Animals and Biomedical Ethics Saliva samples from pigs (Sus Scrofa domesticus, Large-White) located at the Veterinary Teaching Farm of the University of Murcia (Guadalupe, Murcia, Spain), which is free of the porcine respiratory virus and reproductive syndrome, were used. Animals were accommodated under general commercial housing and conditions conforming to European Union guidelines (Directive 2010/63/EU1). 2.2. Sampling Saliva collection tubes (Salivette, Sarstedt, Aktiengesellschaft and Co., Nümbrecht, Germany) and fragments (5 × 2 × 2 cm) of polypropylene sponges (Esponja Marina, La Griega E. Koronis, Madrid, Spain) were used for sampling as previously reported [22]. Animals were allowed to chew a fragment of sponge attached to a thin, flexible metal rod for 1–2 min or until thoroughly moistened. Then, the sponge was introduced in a Salivette tube and transported refrigerated to the laboratory (which took around 2 h) where they were centrifuged (3000× g, 10 min, 4 ◦ C). The obtained volume of saliva was transferred to 1.5 mL tubes (Eppendorf Ibérica, Spain) to be stored at −80 ◦ C until analysis (less than a month in all cases). During sampling, animals that were drinking or eating were avoided. Dirty sponges or oral fluid with color were discarded. 2.3. Analysis of Zn, Cu, Fe and Ferritin Fe, Zn and Cu were analyzed using colorimetric methods from BioSystems (Iron- Ferrozine, 11,509, BioSystems S.A., Barcelona, Spain) and Randox (Zinc, ZN2341, and Copper, CU2340, Randox Laboratories Ltd., Crumlin, UK). Ferritin was measured using an automated immunoturbidimetric assay (Ferritin latex, 31,935, Biosystems S.A., Barcelona, Spain). Chemistry analyzers (Olympus AU 400 for Zn, Cu and ferritin and Olympus AU 600 for Fe, Olympus Diagnostica GmbH1, Hamburg, Germany) were used for the analysis. The four methods were previously analytically validated for pig saliva samples, showing adequate precision and accuracy in a simple and fast manner, without using deproteinization pre-treatments in case of trace elements [16]. In that report, the intra- and inter-assay imprecision was <12% in average for all the assays, and the variation percentage compared with the values expected in the recovery tests was <9% [16]. 2.4. Evaluation of Changes of Zn, Cu, Fe and Ferritin in Fattening Pigs during a Complete Productive Cycle This was made in surplus samples from Large White pigs (24 males and 21 females) from 5 litters (average litter size of 10.0 ± 0.7) from a previous experiment [23]. Sampling was performed in the sucking phase at 24 days of life (T1); in the nursery phase one week after weaning (T2); at the end of nursery (T3); at the beginning of the growing-finishing phase after one week for acclimatization (T4); and at the end of the phase (T5). Antioxidants 2023, 12, 649 3 of 10 2.5. Evaluation of Changes of Zn, Cu, Fe and Ferritin According to the Time of Sampling (Diurnal Variations Study) This was made in surplus samples from a previous experiment [24]. A total of 40 Large White pigs (20 males and 20 females) were randomly chosen for the experimental study with an age around 5 months approximately, and they had ad libitum access to water and a commercial suitable diet. Saliva was sampled from the pigs at 0800 h, 1200 h, 1600 h and 2000 h within a same day. Pen temperature oscillated from 19 ◦ C in the morning to 24 ◦ C at midday. 2.6. Influence of Fecal and Feed Contamination of Saliva Samples in the Measurements of Zn, Cu and Fe The experimental assay was performed based on a previous study made to evaluate the effect of pen feces and feed in salivary biomarker assays [25]. Clean saliva samples collected from apparently healthy finishing pigs (around 110 kg body weight and 5 months of age) were mixed to obtain pools (n = 16) of 12 mL. Pen feces and animals feed were also collected in falcon tubes (Eurotubo® , sterilized conic tubes 15 mL and 50 mL, Deltalab S.L, Barcelona, Spain). Feed was a commercial finisher diet (Cefusa, Murcia, Spain) integrated by corn, wheat, sunflower, barley, rapeseed and soybeans, nutritionally balanced (15.5% of crude protein -CP-; 0.79% standardized ileal digestible lysine, %SID Lys; and 13.5 MJ of metabolizable energy per kg -ME/kg-). It was given ad libitum in pellets with 400 µm particle size. After that, five aliquots of each saliva pools were prepared and treated as follows: (1) control group (C group, n = 16) with 3.5 mL of saliva without any treatment; (2) effect of high pen feces contamination (HD group, n = 8) by adding 875 mg of pen feces, previously collected in a Falcon tube from the farm of the study, to 3.5 mL of clean pig saliva; (3) effect of low pen feces (LD group, n = 8), by adding 87.5 mg of pen feces to 3.5 mL of clean saliva; (4) effect of high feed contamination (HF group, n = 8) by adding 875 mg of commercial porcine feed, used at the farm of this study, to a 3.5 mL of clean saliva; (5) effect of low feed contamination (LF group, n = 8) by adding 87.5 mg of feed to 3.5 mL of clean pig saliva. For preparing those mixes, the pen feces or feed inside falcon tubes were crushed and mixed with the pools of saliva during 1 min and incubated at 38 ◦ C for 5 min. Then, sponges were introduced in each falcon to absorb the mixture, placed in saliva collection tubes and centrifuged to obtain the pools. The trace elements concentration of those mixtures was analyzed by the colorimetric methods, and also with ICP-MS (Agilent 7900 Inductively Coupled Plasma-Mass Spec- trometry), to assess the possible effect of the method of measurement in the interferences. ICP-MS is a highly sensitive and accurate technique [26], previously validated for trace elements measurement in saliva [27], which requires a previous treatment of the samples with HNO3 65% (500 µL of acid added to 500 µL of sample). As additional controls, the trace elements concentration of 87.5 mg of feces and of 87.5 mg of feed diluted in 3.5 mL of distilled water was previously measured by ICP. Concentrations were 245.55 ppb of Fe, 304.83 ppb of Cu and 723.82 ppb of Zn for the feces dilution; and 175.17 ppb of Fe, 731.67 ppb of Cu and 743.79 ppb of Zn for feed dilution. Therefore, the concentrations of each element were 10.07 µg of Fe, 15.24 µg of Cu and 29.68 µg of Zn per mg of feces; and 8.76 µg of Fe, 29.99 µg of Cu and 30.50 µg of Zn per mg of feed. 2.7. Storage Stability The effect of different storage temperature conditions through time was evaluated for Zn, Cu, Fe and ferritin measurement in saliva of pigs. Ten saliva samples were collected from the Teaching Farm of the University of Murcia and were measured after collection (T0) and then, each sample was aliquoted in three subsamples: one was refrigerated at 4 ◦ C, one frozen at −20 ◦ C and one frozen at −80 ◦ C. The samples were measured before storage (T0) and after 1 week (TW) and 6 months (T6). Percentages of loss and recovery of markers after each point of measurements were calculated as percentages from initial analysis (T0 Antioxidants 2023, 12, 649 4 of 10 as 100% of each analyte concentration of saliva sample). Measurements were calculated according to the following formula: (T − T0) × 100/T0 [28]. 2.8. Statistical Analysis All data were assessed for normality by the Shapiro–Wilk method. Since most of the data showed non-normal distribution, continuous variables were naturally log transformed prior to analysis by applying the formula ln (x + 1) for further analysis [29]. For the longitudinal and diurnal variation studies, a Mixed Linear Model of repeated measures was used in which time and sex were fixed factors and the individual was a random factor. Univariate analysis and Bonferroni post hoc test were further performed for any of those fixed factors considered as significant. For assessing the effect of the presence of dirt in the saliva samples, the results ob- tained after the addition of feces or feed to the sample were analyzed by a General Linear Model (GLM) or repeated measures, followed by a Bonferroni post hoc test for pairwise comparisons. For stability storage evaluation, the imprecision of the assay, calculated as the intra- assay coefficient of variation (Intra-CV), was used to assess stability. Thus, the biomarkers of this study were considered stable when changes observed in the stored samples did not exceed the significant change limit (SCL) acceptable for the assay, which was defined as SCL = 100% ± 2 × Intra-CV [30]. A two-way ANOVA test of repeated measures, followed by Dunnett’s multiple comparison test, was used to assess whether the percentage of change observed in analytes’ levels over time was statistically significant at different temperatures. Changes out of SCL and with significant differences from T0 were considered to have unacceptable stability for that storage conditions. The calculations were made using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) and SPSS (IBM SPSS Statistics for Windows, Version 28.0.1. IBM Corp, NY, USA) statistical packages, and the significant level was set at p < 0.05. 3. Results 3.1. Longitudinal Study The results of the salivary analytes obtained at the different phases are shown in Table 1. Time effect was significant for all analytes. Table 1. Results obtained of salivary zinc, copper, iron and ferritin in 45 Large White pigs (21 females, 24 males) at suckling phase (T1); beginning (T2) and end (T3) of nursery phase; beginning (T4) and end (T5) of growing phase. Median (interquartile range) are expressed. Sampling Time Biomarkers T1 T2 T3 T4 T5 Fixed Factors p Value Zn (µg/dL) a 452.6 (453.7) b 2154.6 (3104.3) c 186.0 (212.1) c 170.3 (258.1) d 59.9 (65.4) Time <0.001 Female 517.4 a (681.2) 2340.3 b (2040.7) 261.0 c (251.9) 172.5 c (208.2) 79.3 d (82.3) Sex 0.102 Male 444.2 a (382.4) 1841.7 b (3328.4) 166.5 c (160.0) 170.0 c (288.4) 46.1 d * (64.4) Time * sex 0.189 Cu (µg/dL) 23.2 a (26.6) 194.8 b (185.8) 57.2 c (39.3) 73.6 c (94.5) 71.3 c (85.4) Time <0.001 Female 27.3 a (28.4) 182.2 b (248.5) 67.2 c (64.6) 81.9 c (95.6) 112.2 c (90.2) Sex 0.038 Male 18.0 a (27.7) 195.7 b (151.9) 47.7 c * (27.4) 64.1 c (99.6) 65.8 c * (29.5) Time * sex 0.203 Fe (µg/dL) 183.2 a (112.6) 133.2 ab (127.9) 46.7 d (30.7) 59.5 bc (128.0) 57.8 cd (50.5) Time <0.001 Female 160.8 a (130.2) 144.8 ab (139.8) 57.6 c (38.0) 58.5 bc (139.3) 74.2 bc (91.3) Sex 0.046 Male 186.2 a (104.0) 98.2 ab (115.0) 46.4 c * (31.6) 63.7 b (114.9) 49.6 c * (38.9) Time * sex 0.079 Ferritin (µg/L) 79.1 a (144.1) 8.3 b (15.6) 4.2 c (5.3) 3.0 c (5.0) 3.4 c (4.0) Time <0.001 Female 97.8 a (219.1) 16.1 b (25.7) 5.6 c (4.7) 2.9 c (5.4) 4.2 c (5.6) Sex <0.001 Male 60.0 a (101.4) 6.5 b *** (8.8) 2.9 c ** (2.8) 3.1 bc (5.1) 1.6 c * (3.1) Time * sex 0.200 Zn: zinc; Cu: copper; Fe: iron; ferritin; BW: birth weight. Statistical analysis: a different letter indicates significant differences between sampling times; asterisks indicate significant differences between sexes (*: p < 0.05; **: p < 0.01; ***: p < 0.001). Antioxidants 2023, 12, 649 5 of 10 Zn concentrations reached the highest values at the beginning of nursery. Then a progressive decrease was observed until they reached the lower values at the end of the growing phase. Cu concentrations in saliva were lower at lactation and then significantly increased, reaching its peak at the beginning of nursery. Then Cu decreased in the growing phase. A significant gender effect was observed, with Cu concentrations being significantly higher in females than in males at the end of the nursery and growing phases. Fe salivary concentrations were higher at lactation and the beginning of nursery, then they decreased by the end of nursery and values remained stable since then. Significantly higher Fe concentrations were detected in females than in males at the end of the nursery and growing periods. Ferritin concentrations in saliva were higher at lactation, showing a strong decrease at the beginning of nursery. By the end of nursery, the ferritin concentrations achieved stable values until the end of the growing phase. Sex effect was significant since females showed higher values than males at nursery and by the end of growing phase. In spite of the gender differences observed, the evolution of salivary biomarker con- centrations was similar in both sexes since time–gender interaction was not significant in any case. 3.2. Diurnal Variations Study The results of this section are shown in Figure 1. Time effect was significant for Zn and Cu salivary concentrations. Significantly lower Zn concentrations were observed at 1600 h compared with 1200 h. Cu concentrations were significantly lower at 1600 h and Antioxidants 2023, 12, x FOR PEER REVIEW 2000 h compared with 0800 h. No gender effect was observed in Zn and Cu throughout 6 of 11 the hours of the day. Fe and ferritin salivary concentrations remained stable along the day, with no significant changes due to time or gender. Figure 1. Results for the zinc (Zn), copper (Cu), iron (Fe) and ferritin concentrations obtained in Figure 1. Results saliva from for the 40 healthy zinc (Zn), fattening pigscopper (Cu), 20 (20 females, iron (Fe) at males) and ferritinhours different concentrations of the day.obtained in pB : p value saliva from 40 healthy fattening pigs of the Bonferroni post hoc analysis. (20 females, 20 males) at different hours of the day. p B: p value of the Bonferroni post hoc analysis. 3.3. Influence of Fecal and Feed Contamination The results of this section appear in Figure 2. The addition of high concentrations of feces or feed to the saliva samples significantly increased Zn, Cu and Fe values in both colorimetric and ICP methods (GLM p < 0.001 in all cases). No statistically significant Antioxidants 2023, 12, 649 6 of 10 3.3. Influence of Fecal and Feed Contamination The results of this section appear in Figure 2. The addition of high concentrations of feces or feed to the saliva samples significantly increased Zn, Cu and Fe values in both colorimetric and ICP methods (GLM p < 0.001 in all cases). No statistically significant changes were found in the colorimetric assays for Zn and Fe after the addition of Antioxidants 2023, 12, x FOR PEER REVIEW low 7 of 11 concentrations of feces or feed, whereas Cu was affected when the concentration of dirtiness was low. In the case of ICP assays, the presence of feces or feed at any concentration significantly increased the results in all analytes. (a) (b) Figure 2. Figure 2. Results Results for for the the zinc zinc (Zn), (Zn), copper copper (Cu) (Cu) and and iron iron (Fe) (Fe) after after adding adding different different quantities quantities ofof pen pen feces (a) or feed (b) in pig saliva. Concentrations obtained in saliva pools without any treatment feces (a) or feed (b) in pig saliva. Concentrations obtained in saliva pools without any treatment (UN -untreated)), after adding low quantity of pen feces or feed (FcL) and high quantity of pen feces (UN—untreated)), after adding low quantity of pen feces or feed (FcL) and high quantity of pen feces or feed (FcH) by automated colorimetric assays and by ICP-MS method. pB: p value of the Bonfer- or feed roni (FcH) post hoc by automated analysis. colorimetric Asterisks indicateassays and by significant ICP-MS method. differences pBC from the : pgroup value (***: of thep