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Selection Based on Indirect Genetic Effects for Growth, Environmental Enrichment and Coping Style Affect the Immune Status of Pigs

  • Inonge Reimert ,

    inonge.reimert@wur.nl

    Affiliation Wageningen University, Department of Animal Sciences, Adaptation Physiology Group, Wageningen, The Netherlands

  • T. Bas Rodenburg,

    Affiliation Wageningen University, Department of Animal Sciences, Behavioural Ecology Group, Wageningen, The Netherlands

  • Winanda W. Ursinus,

    Affiliations Wageningen University, Department of Animal Sciences, Adaptation Physiology Group, Wageningen, The Netherlands, Wageningen UR Livestock Research, Animal Behaviour & Welfare, Wageningen, The Netherlands

  • Bas Kemp,

    Affiliation Wageningen University, Department of Animal Sciences, Adaptation Physiology Group, Wageningen, The Netherlands

  • J. Elizabeth Bolhuis

    Affiliation Wageningen University, Department of Animal Sciences, Adaptation Physiology Group, Wageningen, The Netherlands

Selection Based on Indirect Genetic Effects for Growth, Environmental Enrichment and Coping Style Affect the Immune Status of Pigs

  • Inonge Reimert, 
  • T. Bas Rodenburg, 
  • Winanda W. Ursinus, 
  • Bas Kemp, 
  • J. Elizabeth Bolhuis
PLOS
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Abstract

Pigs living in intensive husbandry systems may experience both acute and chronic stress through standard management procedures and limitations in their physical and social environment, which may have implications for their immune status. Here, the effect of a new breeding method where pigs were selected on their heritable influence on their pen mates' growth, and environmental enrichment on the immune status of pigs was investigated. Hereto, 240 pigs with a relatively positive genetic effect on the growth of their pen mates (+SBV) and 240 pigs with a relatively negative genetic effect on the growth of their pen mates (−SBV) were housed in barren or straw-enriched pens from 4 to 23 weeks of age (n  =  80 pens in total). A blood sample was taken from the pigs before, three days after a 24 h regrouping test, and at week 22. In addition, effects of coping style, as assessed in a backtest, and gender were also investigated. Mainly, +SBV were found to have lower leukocyte, lymphocyte and haptoglobin concentrations than -SBV pigs. Enriched housed pigs had a lower neutrophil to lymphocyte (N:L) ratio and lower haptoglobin concentrations, but had higher antibody titers specific for Keyhole Limpet Hemocyanin (KLH) than barren housed pigs. No interactions were found between SBV class and housing. Furthermore, pigs with a proactive coping style had higher alternative complement activity and, in the enriched pens, higher antibody titers specific for KLH than pigs with a reactive coping style. Lastly, females tended to have lower leukocyte, but higher haptoglobin concentrations than castrated males. Overall, these results suggest that +SBV pigs and enriched housed pigs were less affected by stress than -SBV and barren housed pigs, respectively. Moreover, immune activation might be differently organized in individuals with different coping styles and to a lesser extent in individuals of opposite genders.

Introduction

In response to stressful situations, the HPA-axis and sympathetic nervous system are activated which subsequently results in the release of glucocorticoids and catecholamines which prepare the body to fight or flight [1]. However, it is now well-known that the experience of stress also has an effect on various components of the immune system [2], [3]. For instance, one of the best known effects of acute stress is a reduction in the number of several blood leukocyte types [4]. Generally, acute stress has been suggested to enhance and chronic stress to suppress immune activation [4], although experimental results are not always that straightforward [5], [6]. The effect of stress on the immune system is suggested to be particularly mediated by glucocorticoids and catecholamines [5], [7], [8], [9].

Pigs in intensive farming systems experience acute stress during standard management procedures such as castration, tail docking, abrupt weaning, regrouping and transport [10], [11], [12], [13], [14], [15], and at the same time they have to cope with prolonged limitations in their living environment. The absence of proper substrates for oral manipulation in most intensive farming systems [16], [17] prevents pigs from performing highly motivated behaviors such as rooting and chewing [18] and may, therefore, induce chronic stress which is reflected in changes in the HPA-axis [19], [20], cognitive impairment [20], [21], [22] and in the expression of abnormal behaviors, such as tail biting and stereotypies [23], [24], [25]. Chronic stress may also be caused by ongoing social stress [26], [27]. Both short-term and prolonged stressful situations have indeed been found to influence the immune status and immune reactivity of pigs [6], [28], [29], [30], and also have major implications for pig welfare and productivity [13], [31], [32], [33].

One solution to diminish these negative side effects of stress may be provided by genetics [34], [35], [36]. For instance, social stress may be reduced by breeding pigs that perform well in group housing and do not show harmful behavior directed towards their group mates. Direct selection for pigs that perform favorable behaviors seems, however, not feasible in commercial pig breeding [34], [37], but selection on group performance is feasible and this may, indirectly, also lead to pigs with improved behavioral skills [37]. Early work of Griffing [38] and later work of Muir [39], [40] and Bijma et al. [41], [42] has shown that a phenotypical trait of an individual that lives in a group is not only influenced by its own genes, but also by the genes of its group members. This indirect genetic effect [43] on another's phenotypical trait is also referred to as associative effect [40] or social (genetic) effect [44], [45] and can relatively easy be included as a social breeding value (SBV) for production traits in commercial breeding programs [37], [42]. Hence, via indirect selection on each other's performance, animals can perform better as a group. A series of selection experiments in which laying hens were selected by taking indirect genetic effects on performance of cage mates into account, not only showed that these laying hens indeed performed better as a group, but also suggested that these hens were less sensitive to stress compared to laying hens that were selected on individual performance only [46]. Pigs can be selected for the genetic effect on each other's growth during the finishing phase [44], [45], [47] and the first results of a one generation selection experiment indicated that pigs that were selected to have a relatively positive indirect genetic effect on the growth of their pen mates (+SBV) are somewhat less fearful [48], [49] and less sensitive to stress [50] than pigs that were selected to have a relatively negative indirect genetic effect on the growth of their pen mates (−SBV). Effects of this divergent selection on indirect genetic effects for growth on immune status are so far unknown. Besides genetics, the provision of environmental enrichment is suggested to alleviate pigs from (prolonged) stress as well [17]. Environmental enrichment has, moreover, been reported to affect certain components of the immune system [51], [52].

The aim of this study was therefore to investigate both the separate and interacting effects of this new breeding method and housing on the immune status of pigs. Furthermore, the coping style of the pigs was also taken into account, because pigs with different coping styles do not only respond differently to acute and chronic stress [53], [54], [55], [56], but have also been found to differ in immune responses [57], [58]. To that aim, a contrast in prolonged stress was created by housing +SBV and −SBV pigs from 4 to 23 weeks of age in either relatively barren or straw-enriched pens. Furthermore, at 9 weeks of age all pigs were subjected to a 24 h regrouping test to induce acute stress [13]. Effects of SBV class, housing and coping style on leukocyte subsets [59], haptoglobin [60] and innate immune components [61], [62], [63], [64] were studied by taking three blood samples, i.e. before and after the regrouping test and at 22 weeks of age. We expected the +SBV pigs to be less affected by stress than the −SBV pigs which would be, subsequently, reflected in their immune status. In addition, we expected differences in immune status between pigs in barren and enriched housing as the latter are likely to suffer less from prolonged stress than barren housed pigs.

Materials and Methods

Ethics Statement

The experiment described in this study was approved by the Animal Care and Use Committee of Wageningen University (Protocol Number: 2010055f). Blood sampling was carried out by trained assistants and done as quickly as possible to minimize suffering.

Animals and housing

The pigs in this study −480 in total, equally divided over five batches - were the same pigs as described in Reimert et al. [49]. In short, pigs were born at the experimental farm of Topigs Research Center IPG in Beilen, The Netherlands and reared in conventional farrowing pens. Pigs were weaned at four weeks of age and transported to the experimental farm ‘de Haar’ of Wageningen University in Wageningen, The Netherlands. Here, half of the pigs were housed in barren pens (∼1 m2/pig) with a partially slatted and partially concrete solid floor. Barren housed pigs received two hands of wood shavings each day from 6 weeks of age onwards. The other half of the pigs were housed in pens (∼1 m2/pig) enriched with 1.5 kg of straw and 12 kg of wood shavings. All pens were cleaned daily and afterwards 3 kg of fresh wood shavings and fresh straw (250 g at the start of the experiment and then gradually increased to 1.5 kg) were added to the enriched pens. In all pens, a metal chain with a ball and, from 8 weeks of age, a jute sack were attached to the wall of each pen. The jute sack was replaced when needed.

Each group of pigs consisted of three gilts and three barrows and at least two high-resisting (HR) and two low-resisting (LR) pigs (see section 2.2.1.) and groups diverged in indirect genetic effects for growth, i.e. the heritable effect on the growth of their group members. All pigs in a pen had either an estimated relatively positive indirect genetic effect (+SBV, average of 2.00 g/day) or an estimated relatively negative indirect genetic effect on the growth of their pen mates (−SBV, average of −1.62 g/day) during the finishing period (from app. 25 to 110 kg). During this period, the growth of a pig is theoretically affected by each of its pen mates which means that the total estimated indirect genetic effect on a pig's growth in this experiment was 10 g/day ((6−1) * 2.00) for the +SBV pigs and −8.1 g/day ((6−1) * −1.62) for the −SBV pigs. Pigs were obtained by mating Topigs-20 sows and Tempo boars with the most extreme positive or negative estimated indirect genetic effects for growth that were available for each batch. Direct breeding values for growth were kept as similar as possible for both SBV classes (for details about the (social) breeding value estimations see Camerlink et al., 2013). The study was approved by the Animal Care and Use Committee of Wageningen University.

Behavioral tests

Backtest.

Pigs were subjected to the backtest at approximately two weeks of age [48] to assess their personality or coping style [57]. In short, a piglet was put on its back for 1 min and manually restrained. During the test, the number of struggles, the latency to struggle, the number of vocalizations, and the latency to vocalize were recorded. Piglets were classified as high-resisters (HR) if they showed two struggles and produced at least 25 vocalizations, or showed at least three struggles. Low-resisting (LR) pigs were piglets that showed 0 or 1 struggle, or 2 struggles and produced less than 25 vocalizations.

Regrouping test.

At 9 weeks of age, pigs were exposed to a regrouping test [50] which is a stressful event for pigs [13]. In short, a pair of pigs was regrouped for 24 h in a new pen with two other pairs of unfamiliar pigs. Pairs of pigs were always mixed with other pairs from the same SBV class and housing condition and the new temporary group composition was balanced for gender and coping style. After the 24 h, each pair of pigs was put in its original pen and group again.

Blood collection and analyses

Blood was collected from the pigs in the week before the regrouping test at 8 weeks of age, three days after the regrouping test at 9 weeks of age, and at 22 weeks of age (Figure 1). Hereto, a pig was immobilized on its back in a crib (for the first and second collection) or fixated using a nose sling (for the third collection) and blood was taken by venipuncture from the jugular vein. Housing condition and SBV class were taken into account in the order of blood collection. Blood was collected in serum separating tubes (Greiner bio-one, Kremsmünster, Austria) which were stored at room temperature (RT) and in K3 EDTA tubes (Greiner bio-one, Kremsmünster, Austria) which were stored on ice after blood sampling.

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Figure 1. Flow diagram of the experiment.

A flow diagram of the experiment showing the different treatments, the number of pigs and pens, and the number of pigs sampled at 8, 9 and 22 weeks of age.

https://doi.org/10.1371/journal.pone.0108700.g001

In the laboratory, the serum separating tubes were incubated for one hour at 37°C after which they were centrifuged at 5251 g for 12 min at 20°C. Obtained sera were stored at −80°C until further analysis (sections 2.3.1, 2.3.2 and 2.3.4). Blood from the EDTA tubes was used directly (section 2.3.3).

Complement activity via the classical (CPW) and alternative pathway (APW).

The hemolytic activity of both CPW and APW complement was measured using the hemolytic complement assay of Demey et al. [65]. In short, for CPW complement activity, 50 µl of serum was diluted serially in a gelatin-VBS-salt buffer and incubated with hemolysin sensitized sheep red blood cells for 90 min at 37°C in 96-well microtiter plates. During incubation, plates were shaken every 30 min in a Titertrek (Flow Laboratories). After 90 min, the amount of light scattered by the red blood cells upon lysis was read at 655 nm in a microplate reader (BioRad model 3550). The readings were transformed using a log-log equation [66] and the hemolytic titer was expressed as the titer that lysed 50 % of the red blood cells (CH50 U/ml). For APW complement activity, the same assay was used except that sera were diluted serially in a gelatin-VBS-EGTA buffer and incubated with rabbit red blood cells [65].

IgG and IgM antibody titers specific for KLH.

Antibody titers of IgG and IgM specific for Keyhole Limpet Hemocyanin (KLH) [67] were determined by a two-step enzyme-linked immunosorbent assay (ELISA) similar to Bolhuis et al. [57] and Lammers et al. [67]. First, medium binding microtiter plates (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) were coated overnight at 4°C with 2 mg/ml KLH in coating buffer (0.05 M Na2CO3 × 10 H2O, pH 9.6). After washing with tap water containing 0.05% Tween 20, serial dilutions of serum were added and incubated for one hour at RT. After washing, plates were incubated for one hour at RT with a 1:20000 diluted peroxidase (PO)-conjugated goat antibody directed to swine IgGFC (GαSw-IgGFC/PO, Bethyl Laboratories, Montgomery, USA) to detect binding of IgG and with 1:20000 diluted peroxidase (PO)-conjugated goat antibody directed to swine IgMFC (GαSw-IgMFC/PO, Bethyl Laboratories, Montgomery, USA) to detect binding of IgM, respectively. After washing, tetramethylbenzidine and 0.05% H2O2 were added as a substrate and incubated for 10 min at RT. The reaction was stopped with 2.5N H2SO4 and the absorbance was measured at 450 nm with a Multiskan (Flow, Irvine, UK). Each absorbance was expressed relatively to the absorbance of a standard positive control serum and antibody titers were determined as described in Schrama et al. [58].

Leukocytes, lymphocytes and the neutrophil to lymphocyte ratio.

With 150 µl of the blood in the EDTA tubes, the concentration of leukocytes (109/l) was determined with a Sysmex F-820 (Sysmex Corporation, Kobe, Japan) and with 10 µl, a smear was made on a microscope plate. After the smears had dried, they were fixed with a methanol solution and thereafter stained using a rapid staining kit (Hemacolor staining kit, Merck KGaA, Darmstadt, Germany). Surplus staining solution was washed away with PBS and then smears were dried. The percentages of lymphocytes, neutrophils, monocytes and eosinophils (the last two were not used further in this study, because of low occurrence: monocytes: 8.4 ± 0.2 %, eosinophils: 2.0 ± 0.1 % (overall mean ± SEM)) were determined by microscopic examination of the smears and counting 100 leukocytes in total using an Assistant-Counter AC-8. From these counts, the neutrophil:lymphocyte ratio (N:L ratio) was determined. Furthermore, the concentration of lymphocytes (109/l) was determined by multiplying the percentage of lymphocytes with the leukocyte concentration.

Haptoglobin.

Haptoglobin concentrations were determined in serum using a commercial kit based on the hemoglobin-binding capacities of haptoglobin (Phase Haptoglobin, Tridelta Development Limited, Maynooth, Ireland) which has been validated for pigs (GD Animal Health Service, Deventer, The Netherlands). Briefly, 100 µl of hemoglobin was added to 7.5 µl serum and solutions were gently mixed. Thereafter, 140 µl of chromogen was added and incubated for 5 min at RT. The absorbance was read immediately at 600 nm in a microplate reader. Haptoglobin concentrations (mg/ml) were calculated by using a standard linear curve with known concentrations of haptoglobin.

Statistical analyses

SAS (SAS 9.2, SAS Institute Inc.) was used for all statistical analyses. Variables could not be obtained from all 480 pigs at each sampling period (Figure 1). This was because some pigs died during the experiment (respiratory problems, n = 3; meningitis n = 2, other causes n = 2), and some pigs were removed from the experiment because they had tail biting wounds (n = 10), were lame (n = 5), or had an umbilical cord hernia (n = 4) or rectal prolapse (n = 2). These numbers were not different for the SBV classes, housing conditions or coping styles, except that in barren housing more pigs were removed from the experiment due to a tail wound (barren vs. enriched: n = 9 vs. n = 1). In addition, missing values were present for another 20 pigs due to technical problems during blood sampling or during the laboratory assays. Moreover, for 22 other pigs the blood in the EDTA tubes had clotted after sampling which therefore became unreliable to use for further testing. Also these pigs were equally distributed across the different treatments (P ≥ 0.20). Prior to analysis, the variables CPW complement, haptoglobin, and N:L ratio were log transformed and the number of lymphocytes square root transformed to obtain normally distributed residuals. The effects of week, SBV class, housing, backtest classification and gender on the variables were assessed with a repeated linear mixed model with the fixed factors week, SBV class, housing, backtest classification, their interactions, gender, its interaction with week, and batch. Values in time of individual pens and pigs were taken as repeated measurements, i.e. SBV class, housing and batch effects were tested against the random effect of pen, and backtest classification and gender effects were tested against the random effect of pig. The order of collection within a sampling day was included as covariate.

To investigate the effect of SBV class, housing, backtest classification and gender on the variables after acute stress, the delta between week 9 and week 8 was calculated and subsequently analyzed with a linear mixed model with SBV class, housing, backtest classification, their two-way interactions, gender, and batch as fixed effects and pen, nested within SBV class, housing, and batch, as random effect. Prior to this analysis, CPW complement activity was log transformed to obtain normality of residuals.

For brevity, only significant interactions are reported. Significant interactions were further investigated with post hoc pairwise comparisons using the differences of the least square means. Results are presented as means ± SEM.

Results

Classical (CPW) and alternative (APW) complement activity

CPW complement activity was not affected by SBV class, housing or backtest classification (P ≥ 0.29). There was, however, an effect of week on CPW complement activity (P <0.001) (Figure 2A and 2B), with higher activity in week 9, three days after regrouping, than in week 8 or 22. The increase in CPW complement activity from week 8 to 9 (i.e. the delta) was not affected by SBV class, housing or backtest classification (P ≥ 0.12).

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Figure 2. Means and SEM of complement activity and antibody titers.

Compliment activity via the classical (CPW) (panels A and B) and alternative pathway (APW) (panels C and D), and IgG (panels E and F) and IgM titers (panels G and H) to Keyhole Limpet Hemocyanin (KLH) of pigs with a high-resisting (HR) and low-resisting (LR) backtest classification in barren and enriched housing measured before a 24 h regrouping test at 8 weeks of age, after the regrouping test at 9 weeks of age and at 22 weeks of age. Significance of effects of housing and backtest classification is given in the text.

https://doi.org/10.1371/journal.pone.0108700.g002

APW complement activity was affected by backtest classification (P <0.01) and week (P <0.001) (Figure 2C and 2D). HR pigs had a higher APW complement activity than LR pigs (overall: 64.3 ± 1.1 vs. 58.9 ± 1.1 CH50 U/ml). In addition, APW complement activity was lower in week 22 compared to weeks 8 and 9. APW complement activity was not affected by SBV class or housing (P ≥ 0.54). Although APW complement activity did, overall, not differ between weeks 8 and 9, the delta between weeks 8 and 9 was affected by backtest classification (P <0.05). From week 8 to 9, after regrouping, APW complement activity decreased slightly for HR pigs, but increased for LR pigs (−1.0 ± 1.7 vs. 3.4 ± 1.6 CH50 U/ml). The delta in APW complement activity was not affected by SBV class or housing (P ≥ 0.93).

IgG and IgM titers specific for KLH

KLH-IgG titers were affected by housing (P <0.001) and week (P <0.001) and by the interaction between housing and backtest classification (P <0.05) (Figure 2E and 2F). Post hoc analysis showed that enriched housed HR pigs (4.0 ± 0.1) had a higher titer than enriched housed LR pigs (3.8 ± 0.1) and that both had a higher titer than the barren housed HR and LR pigs (both: 3.5 ± 0.1). Furthermore, KLH-IgG titers increased from week 8 to 9 to 22. KLH-IgG titers were not affected by SBV class (P  =  0.43). The increase of the KLH-IgG titer from week 8 to 9 tended to be smaller for +SBV pigs than for -SBV pigs (0.37 ± 0.06 vs. 0.47 ± 0.06, P <0.1) and was larger for enriched housed pigs than for barren housed pigs (0.59 ± 0.07 vs. 0.32 ± 0.08, P <0.05), but was not affected by backtest classification (P  =  0.15).

KLH-IgM titers were affected by week (P <0.001) and the interaction between housing, backtest classification and week (P <0.001) (Figure 2G and 2H). Post hoc analysis showed that KLH-IgM titers, similar to the KLH-IgG titers, increased from week 8 to 9 to 22. Housing and backtest classification did not affect the KLH-IgM titer in weeks 8 and 9, but HR enriched housed pigs had a higher KLH-IgM titer than the other pigs in week 22. KLH-IgM titers were not affected by SBV class (P  =  0.19). The increase of the KLH-IgM titer from week 8 to 9 was not affected by SBV class, housing or backtest classification (P ≥ 0.16).

Leukocytes, lymphocytes and the ratio of neutrophils to lymphocytes (N:L ratio).

The concentration of leukocytes was affected by SBV class (P <0.05). Pigs with a +SBV had, overall, lower concentrations than −SBV pigs (17.8 ± 0.2 vs. 18.6 ± 0.2 109/l). The concentration of leukocytes was also affected by week (P <0.001) and by the interaction between housing and week (P <0.05) (Figure 3A and 3B). Post hoc analysis revealed that leukocyte concentrations of enriched housed pigs were lower in weeks 9 and 22 compared to week 8, whereas leukocyte concentrations of barren housed pigs were lower in week 9 compared to weeks 8 and 22. In addition, leukocyte concentrations did not differ between enriched and barren housed pigs in weeks 8 and 9, but enriched housed pigs had lower leukocyte concentrations than barren housed pigs in week 22 (Figure 3A and 3B). Leukocyte concentrations were not affected by backtest classification (P  =  0.71). The decrease in the concentration of leukocytes from week 8 to 9 tended to be larger for +SBV pigs than for −SBV pigs (−2.6 ± 0.4 vs. −1.7 ± 0.4 109/l, P <0.1), but was not affected by housing or backtest classification (P ≥ 0.19).

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Figure 3. Means and SEM of leukocytes, lymphocytes, N:L ratio and haptoglobin.

The concentrations of leukocytes (panels A and B), lymphocytes (panels C and D), the neutrophil to lymphocyte ratio (N:L ratio) (panels E and F), and haptoglobin concentrations (panels G and H) of pigs that have an estimated relative positive genetic effect (+SBV) or negative genetic effect (−SBV) on the growth of their pen mates in barren and enriched housing measured before a 24 h regrouping test at 8 weeks of age, after the regrouping test at 9 weeks of age and at 22 weeks of age. Significance of effects of housing and SBV is given in the text.

https://doi.org/10.1371/journal.pone.0108700.g003

The concentration of lymphocytes was also affected by SBV class (P <0.05) and week (P <0.01) (Figure 3C and 3D). Pigs with a +SBV had lower lymphocyte concentrations than −SBV pigs (8.6 ± 0.2 vs. 9.2 ± 0.2 109/l). Furthermore, lymphocyte concentrations were lower in week 22 compared to weeks 8 and 9. Lymphocyte concentrations were not affected by housing or backtest classification (P ≥ 0.22). The delta in the lymphocyte concentration between weeks 8 and 9 was also not affected by SBV class, housing or backtest classification (P ≥ 0.20).

The N:L ratio was affected by housing (P <0.05) and week (P <0.001) (Figure 3E and 3F). Enriched housed pigs had a lower N:L ratio than barren housed pigs (0.90 ± 0.03 vs. 0.96 ± 0.04). Moreover, the N:L ratio was higher in week 8 compared to weeks 9 and 22. The delta in N:L ratio between weeks 8 and 9 was not affected by housing (P  =  0.57), but was affected by the interaction between SBV class and backtest classification (P <0.05). Post hoc analysis showed, however, no differences between the groups.

Haptoglobin

Haptoglobin concentrations tended to be affected by SBV class (P <0.1) (Figure 3G and 3H). Concentrations tended to be lower for +SBV pigs than −SBV pigs (0.57 ± 0.03 vs. 0.65 ± 0.03 mg/ml). Moreover, haptoglobin concentrations were affected by housing (P <0.01) and week (P <0.001) (Figure 3G and 3H). Enriched housed pigs had lower haptoglobin concentrations than barren housed pigs (0.57 ± 0.03 vs. 0.65 ± 0.03 mg/ml) and haptoglobin concentrations were higher in week 9 compared to weeks 8 and 22. Haptoglobin concentrations were not affected by backtest classification (P  =  0.85). The increase in haptoglobin from week 8 to 9 was not affected by SBV class, housing or backtest classification (P ≥ 0.32).

Gender did not affect any of the immune variables, except that over the three sampling points, gilts tended to have lower leukocyte concentrations than barrows (17.9 ± 0.2 vs. 18.4 ± 0.2 109/l, P <0.10) and higher haptoglobin concentrations (0.62 ± 0.02 vs. 0.60 ± 0.02 mg/ml, P  =  0.1).

Discussion

In this study, we investigated the effects of divergent selection for indirect genetic effects on growth (+SBV pigs vs. −SBV pigs) and environmental enrichment on the immune status of pigs.

In response to acute stress or inflammation, the acute phase response is activated which results, amongst others, in an increase of acute phase proteins, such as haptoglobin, and in complement activation [2], [64], [68]. The increased CPW complement activity and haptoglobin concentrations found three days after the regrouping test could, thus, indicate that the pigs experienced the test as stressful, but may also have resulted from skin inflammations caused by vigorous fighting during regrouping [50]. Moreover, pigs also had lower leukocyte concentrations and a lower N:L ratio after regrouping, whereas lymphocyte concentrations did not differ between before and after regrouping. These results are not in line with the generally reported effects of acute stress on different leukocyte types, which could be due to differences in the timing of blood sampling [59], i.e. the leukocyte levels three days after regrouping in our study may have partly reflected recovery from acute stress than the effect of regrouping stress per se.

The other effects of week on the variables measured could point to an effect of age. For instance, the increase in KLH-IgG and KLH-IgM natural antibody titers from week 8 to 9 to 22 is in line with other studies reporting rising natural antibody titers with age [62], [69]. On the other hand, APW complement activity, the concentration of lymphocytes and the N:L ratio decreased over weeks in our study. A similar result was found by Blount et al. [70] for the concentration of lymphocytes, but other studies showed a different pattern with age for these three variables [62], [71]. These inconsistencies might be due to individual variation as distinct individual variation in age-related immune changes has been reported [72].

Even though the pigs in this study were all relatively healthy and were not deliberately immunologically challenged, SBV class, housing and coping style did have clear effects on the immune variables measured. These effects were mainly found for levels of the immune variables per se and only few effects on their change due to regrouping stress. It should be stated that the amount of fresh lesions counted after regrouping did not differ between enriched and barren housing and between the two SBV classes [50], and not between the two coping styles (unpublished data). Housing affected all immune variables, except the CPW and APW complement activity and lymphocyte concentrations, and SBV class mainly affected the leukocyte, lymphocyte and haptoglobin concentrations. Moreover, effects of coping style were mainly found on the innate immune variables APW complement activity and KLH-IgG and KLG-IgM natural antibody titers. Effects of gender were also found, but these effects were rather subtle. The interpretation of these results with respect to health and (chronic) stress is, for clarity reasons, discussed in the separate sections below.

Housing

Effects of housing were found on all variables measured except for complement activity and lymphocyte concentrations. Enriched housed pigs had, partly in line with other studies, overall a higher KLH-IgG titer [73], a lower N:L ratio [74] and lower haptoglobin concentrations [75], [76] than barren housed pigs. Enriched housed pigs also had lower leukocyte concentrations than barren housed pigs, in line with Manciocco et al. [77], but only at 22 weeks of age. On the other hand, Merlot et al. [74] found no effect of conventional or enriched housing on haptoglobin concentrations in a conventional pig breed and Manciocco et al. [77] actually found a higher N:L ratio and lower CPW complement activity in enriched housed pigs. This could be explained by differences in type of enrichment (e.g. straw, extra outdoor area, or toys), duration of enrichment provided, and age of the animals tested. The lower N:L ratio and lower haptoglobin concentrations in enriched housed pigs could indicate that enriched housed pigs were less stressed [59], [78], [79] which confirms our expectation and is in accordance with the well-established benefits of straw on behavior and welfare [17]. Straw bedding has been described as unfavorable for pathogen presence and hygiene [80] which could explain the higher circulating KLH-IgG in enriched housed pigs, but more research is needed to confirm this. All in all, the results of this study show that housing (i.e. relatively barren vs. deep straw bedding) has a substantial impact on variables related to both stress physiology and (innate) immunity which could, therefore, have consequences for both pig health and welfare. The biological significance of these effects needs to be demonstrated as pigs were not immunologically challenged. Future experimental studies addressing effects of enrichment on susceptibility to infectious challenges should reveal this, and it may be important to take the pigs' microbiome into account as well as straw enrichment has been suggested to affect gut health [81].

Social breeding values for growth

Most notably, +SBV pigs had overall lower concentrations of leukocytes and lymphocytes and tended to have overall lower haptoglobin concentrations than −SBV pigs. In addition, the decrease in the concentration of leukocytes from week 8 to 9 tended to be larger for +SBV pigs than for −SBV pigs, suggesting that they responded differently to the 24 h regrouping test at 9 weeks of age.

Although no differences were found in the number of skin lesions after regrouping, indicating no major differences in aggression, after reunion −SBV pigs tended to act more aggressively towards their own original group members, indicating that they differently coped with the stress of regrouping. This seems to be in line with their response to other stressful situations. Previously, it was found that these +SBV pigs behaved somewhat less fearful than the −SBV pigs in several novelty tests [48], [49]. In addition, injurious biting behavior directed at pen mates (e.g. tail and ear biting) has been reported to occur less in the +SBV pens than in the −SBV pens (Camerlink et al., in press). As all pigs within a pen either had a +SBV or −SBV, these behavioral results could indicate that +SBV pigs create a less stressful social environment for themselves than the −SBV pigs. The lower leukocyte, lymphocyte and haptoglobin concentrations in the +SBV pigs support this indication and confirms our expectation, because higher leukocyte and lymphocyte levels have been associated with higher stress levels [4], [82], [83], [84] and a higher level of haptoglobin with chronic or repeated stress [78], [79]. Some caution should be made, however, because lower leukocyte and lymphocyte levels have also been associated with more stress, probably also depending on the duration of stress (e.g. acute vs. chronic stress) [59]. The observed behavioral and immunological differences between the +SBV and −SBV pigs are likely related, but in what way is, at present, not clear. It may be speculated that a higher concentration of leukocytes, lymphocytes and haptoglobin in the −SBV pigs points to a more active immune system which, in turn, could have led to an increased need of specific amino acids (e.g. for synthesis of acute phase proteins such as haptoglobin) and, thus, a reduced availability for other systems such as growth [85]. This may have stimulated these −SBV pigs to search for food and thereby have led to more biting behavior and more stress [86]. On the other hand, as all pigs in one pen were either +SBV or −SBV pigs, more biting behavior in the −SBV pens also meant receiving more bites which, likely, resulted in more inflammations and that could have led to a more active immune system in the −SBV pigs. It should be noted, though, that significantly less biting behavior was observed in the enriched pens (Camerlink et al., in press [87]), whereas the immunological differences between the +SBV and −SBV pigs were independent of housing condition (see below), suggesting that the higher leukocyte, lymphocyte and haptoglobin levels of the −SBV pigs are not the sole cause or consequence of the injurious biting behaviors. Whether the found immunological differences between the +SBV and −SBV pigs have different implications for their health is difficult to say, because both higher and lower leukocyte concentrations have been associated with better health [59] and the haptoglobin concentrations in the current study are much lower compared to haptoglobin concentrations of pigs with health problems [88]. In our study, pigs were not deliberately immunologically challenged and only few pigs became ill during the experiment, and their number did not differ between +SBV and −SBV pigs. Further research is necessary to investigate the biological significance of the differences between +SBV and −SBV pigs under less hygienic/more challenging conditions. Moreover, it is worthwhile to further investigate the health of pigs from this selection method compared with conventional selection in which selection is based on individual performance only, because one of the consequences of conventional selection has been suggested to be a heightened susceptibility to disease [89], [90].

No interactions were found between SBV class and coping style which is in agreement with earlier findings on the behavior of these pigs [48].

Interestingly, the differences between the +SBV and −SBV pigs are comparable with the differences found between enriched and barren housing: pigs housed in a better physical environment (i.e. enriched pens) may experience less chronic stress and pigs housed in a better social environment (i.e. +SBV pens) also seem to experience less chronic stress. In addition, no interactions were found between SBV class and housing condition which is in line with behavioral results of these pigs [49], [50]. This suggests that effects of this selection method on pig behavior and physiology are independent from those of housing. This is important as it may show that only a combined effort of optimizing both the breeding program and the housing environment will yield optimal results in terms of pig welfare and health.

Coping style

In many different animal species two extremes in coping style or personality have been described: proactive and reactive [91], [92], [93]. Generally, proactive animals are aggressive, active, bold, prone to take risks and they hardly pay attention to environmental cues, whereas reactive animals are less aggressive, more cautious, avoid taking risks, but are very attentive to cues from the environment [91], [93], [94]. In pigs, similar coping styles have been distinguished based on their response in a backtest at a young age. Pigs that struggle and vocalize relatively much in this test are classified as high-resisting (HR) pigs and they resemble proactive copers, and pigs that hardly struggle and vocalize in the backtest are classified as low-resisting (LR) pigs and they resemble reactive copers [53], [54], [95], [96], [97], [98], [99], [100], [101], [102].

In this study we also found immunological differences between HR and LR pigs, even though we simply divided the population of pigs into HR or LR and did not use the extremes of the population as was done in earlier pig studies [97], [98], [103], [104], [105]. HR pigs had an overall higher APW complement activity, although the regrouping test had a more substantial effect on APW complement activity in the LR pigs. Furthermore, HR pigs also had a higher KLH-IgG titer, but only in enriched housing. At 22 weeks of age, the same result was found for the KLH-IgM titer. Together these results could indicate that on the long term HR pigs have a more active innate immune system, but that acute stress due to regrouping has a larger impact on the LR pigs. As LR or reactive pigs are more attentive to environmental cues [91], [96], a change of environment (i.e. regrouping) could indeed have more impact on them than on HR or proactive pigs. The more chronic higher innate immune activity in the HR pigs could be related to their behavior: they explore a new environment faster than LR pigs. It could be hypothesized that HR pigs are earlier exposed to pathogens than LR pigs and, perhaps, also to different pathogens. To defend themselves against these pathogens, having an active innate immune system which is the first line of defense may fit with this suggestion [106], [107]. In addition, several other pig studies investigated whether HR and LR pigs differed in specific immune responses and their results indicated that HR pigs had a stronger cell-mediated immune response, while LR pigs had a more pronounced humoral immune response [57], [58], [108]. Hessing et al. (1995) proposed that a difference in balance between T-helper 1 (Th1) and T-helper 2 (Th2) cells was underlying this difference with a shift towards Th1 in HR pigs and Th2 in LR pigs, because activation of Th1 cells leads to an inflammatory response and activation of Th2 to the production of antibodies [108], [109]. In support of this, results of an unpublished pig study by Bolhuis et al. indeed found more pro-inflammatory cytokine production in HR pigs. Furthermore, as complement activation is also associated with the activation of pro-inflammatory cytokines [110], the results of our study seem to be in line with these results. Together, these results may indicate that HR and LR pigs may have different strategies to deal with immune challenges. However, studies that investigated differences in immune responses in proactive and reactive animals are scarce and the results of these studies do not always agree with each other [111], [112], [113], [114]. In addition, in this study housing had a modulating effect on the natural antibody titers of the HR pigs, whereas Bolhuis et al. [57] found that housing had a modulating effect on specific antibody titers of the LR pigs. We propose, therefore, that more research is needed not only to be able to draw more definite conclusions about differences in immune function between proactive and reactive animals [107], but also because of the relevance of personality in actual disease susceptibility [115], [116].

Gender

Gilts were found to have lower concentrations of leukocytes, but higher haptoglobin concentrations than barrows. The barrows were surgically castrated at 3 days of age which could explain their higher concentrations of leukocytes, because Prunier et al. [117] described that surgical castration might have long-term negative effects on the health of the pigs. The higher concentrations of haptoglobin in the gilts have also been found by others [118], [119], [120] and could indicate a fundamental difference between female and male pigs. It has been suggested that females have both a more active humoral and a more active cell mediated immune system which could be reflected in a higher susceptibility to parasites and infections in males, and females being more prone to autoimmune diseases [121], [122], [123], [124]. These differences have been attributed to differences in sex steroids, but also to differences in how females and males deal with stress on both a physiological and psychological level [121], [122], [123], [124]. As the male pigs in this study were castrated, we will refrain from speculating whether sex steroids could underlie the found immunological differences in the gilts and barrows, but differences in dealing with stress is a likely possibility, because these same gilts and barrows have been found to behave very differently in various novelty tests [48], [49] and in the regrouping test [50].

Conclusions

Environmental enrichment is known to alleviate stress in animals. In this study, enriched housed pigs were found to have a lower N:L ratio and lower haptoglobin concentrations than the barren housed pigs which indeed suggests that enrichment has stress-reducing effects. Stress-reducing effects were also seen in pigs selected for a relatively positive genetic effect on the growth of their pen mates (i.e. +SBV pigs), because these pigs had lower leukocytes, lymphocyte and haptoglobin concentrations compared to pigs that were selected for a relatively negative genetic effect of the growth of their pen mates. Together these results indicate that both genetics and environmental enrichment can be used to improve the welfare of pigs and that the use of both together likely yields the best results.

Two effects of gender were found, but these effects were rather subtle. On the other hand, clear differences were found between pigs with a proactive or reactive coping style. Pigs with a proactive coping style seemed to have a more active innate immune status compared to pigs with a reactive coping style, pointing to a difference in dealing with immune challenges. The biological relevance of the results and their implications for health merit further research.

Acknowledgments

The authors are very grateful to Sophie de Graaf, Jolanda de Jong, Mike Nieuwland, Monique Ooms, Marjoke Scherpenzeel, Merel Verhoeven and Ger de Vries Reilingh for their help in the laboratory. We would also like to thank Fleur Bartels, Irene Camerlink, the animal caretakers and many students for their contribution in taking care of the pigs and blood sampling.

Author Contributions

Conceived and designed the experiments: IR WWU JEB. Performed the experiments: IR WWU. Analyzed the data: IR TBR WWU JEB BK. Wrote the paper: IR TBR WWU BK JEB.

References

  1. 1. Chrousos GP, Gold PW (1992) The concepts of stress and stress system disorders. JAMA 267: 1244–1252.
  2. 2. Campisi J, Fleshner M (2003) Role of extracellular HSP72 in acute stress-induced potentiation of innate immunity in active rats. J Appl Physiol 94: 43–52.
  3. 3. Khansari DN, Murgo AJ, Faith RE (1990) Effects of stress on the immune system. Immunol Today 11: 170–175.
  4. 4. Dhabhar FS (2002) Stress-induced augmentation of immune function - the role of stress hormones, leukocyte trafficking, and cytokines. Brain Behav Immun 16: 785–798.
  5. 5. Moynihan JA (2003) Mechanisms of stress-induced modulation of immunity. Brain Behav Immun 17: S11–S16.
  6. 6. Salak-Johnson JL, McGlone JJ (2007) Making sense of apparently conflicting data: stress and immunity in swine and cattle. J Anim Sci 85: E81–E88.
  7. 7. Sorrells SF, Sapolsky RM (2007) An inflammatory review of glucocorticoid actions in the CNS. Brain Behav Immun 21: 259–272.
  8. 8. Nance DM, Sanders VM (2007) Autonomic innervation and regulation of the immune system (1987–2007). Brain Behav Immun 21: 736–745.
  9. 9. Padgett DA, Glaser R (2003) How stress influences the immune response. Trends Immunol 24: 444–448.
  10. 10. Noonan GJ, Rand JS, Priest J, Ainscow J, Blackshaw JK (1994) Behavioural observations of piglets undergoing tail docking, teeth clipping and ear notching. Appl Anim Behav Sci 39: 203–213.
  11. 11. Dudink S, Simonse H, Marks I, De Jonge FH, Spruijt BM (2006) Announcing the arrival of enrichment increases play behaviour and reduces weaning-stress-induced behaviours of piglets directly after weaning. Appl Anim Behav Sci 101: 86–101.
  12. 12. Von Borell E (2001) The biology of stress and its application to livestock housing and transportation assessment. J Anim Sci 79: E260–E267.
  13. 13. Stookey JM, Gonyou HW (1994) The effects of regrouping on behavioral and production parameters in finishing swine. J Anim Sci 72: 2804–2811.
  14. 14. Geverink NA, Bühnemann A, Van de Burgwal JA, Lambooij E, Blokhuis HJ, et al. (1998) Responses of slaughter pigs to transport and lairage sounds. Physiol Behav 63: 667–673.
  15. 15. Rault JL, Lay DC Jr, Marchant-Forde JN (2011) Castration induced pain in pigs and other livestock. Appl Anim Behav Sci 135: 214–225.
  16. 16. Day JEL, Spoolder HAM, Burfoot A, Chamberlain HL, Edwards SA (2002) The separate and interactive effects of handling and environmental enrichment on the behaviour and welfare of growing pigs. Appl Anim Behav Sci 75: 177–192.
  17. 17. Van de Weerd HA, Day JEL (2009) A review of environmental enrichment for pigs housed in intensive housing systems. Appl Anim Behav Sci 116: 1–20.
  18. 18. Studnitz M, Jensen MB, Pedersen LJ (2007) Why do pigs root and in what will they root? A review on the exploratory behaviour of pigs in relation to environmental enrichment. Appl Anim Behav Sci 107: 183–197.
  19. 19. Beattie VE, O'Connell NE, Kilpatrick DJ, Moss BW (2000) Influence of environmental enrichment on welfare-related behavioural and physiological parameters in growing pigs. Anim Sci 70: 443–450.
  20. 20. De Jong IC, Prelle IT, Van de Burgwal JA, Lambooij E, Korte SM, et al. (2000) Effects of environmental enrichment on behavioral responses to novelty, learning, and memory, and the circadian rhythm in cortisol in growing pigs. Physiol Behav 68: 571–578.
  21. 21. Bolhuis JE, Oostindjer M, Hoeks CWF, De Haas EN, Bartels AC, et al. (2013) Working and reference memory of pigs (Sus scrofa domesticus) in a holeboard spatial discrimination task: the influence of environmental enrichment. Anim Cogn 16: 845–850.
  22. 22. Sneddon IA, Beattie VE, Dunne L, Neil W (2000) The effect of environmental enrichment on learning in pigs. Anim Welf 9: 373–383.
  23. 23. Averós X, Brossard L, Dourmad J, De Greef KH, Edge HL, et al. (2010) A meta-analysis of the combined effect of housing and environmental enrichment characteristics on the behaviour and performance of pigs. Appl Anim Behav Sci 127: 73–85.
  24. 24. Schrøder-Petersen DL, Simonsen HB (2001) Tail Biting in Pigs. Vet J 162: 196–210.
  25. 25. Beattie VE, Walker N, Sneddon IA (1995) Effects of environmental enrichment on behaviour and productivity of growing pigs. Anim Welf 4: 207–220.
  26. 26. Turner SP, Nath M, Horgan GW, Edwards SA (2013) Measuring chronic social tension in groups of growing pigs using inter-individual distances. Appl Anim Behav Sci 146: 26–36.
  27. 27. Turner S, Roehe R, D′Eath RB, Ison SH, Farish M, et al. (2009) Genetic validation of postmixing skin injuries in pigs as an indicator of aggressiveness and the relationship with injuries under more stable social conditions. J Anim Sci 87: 3076–3082.
  28. 28. Merlot E, Meunier-Salaün M, Prunier A (2004) Behavioural, endocrine and immune consequences of mixing in weaned piglets. Appl Anim Behav Sci 85: 247–257.
  29. 29. De Groot J, Ruis MAW, Scholten JW, Koolhaas JM, Boersma WJ (2001) Long-term effects of social stress on antiviral immunity in pigs. Physiol Behav 73: 145–158.
  30. 30. Kanitz E, Tuchscherer M, Puppe B, Tuchscherer A, Stabenow B (2004) Consequences of repeated early isolation in domestic piglets (Sus scrofa) on their behavioural, neuroendocrine, and immunological responses. Brain Behav Immun 18: 35–45.
  31. 31. Wiepkema PR, Koolhaas JM (1993) Stress and animal welfare. Anim Welf 2: 195–218.
  32. 32. Ekkel ED, Van Doorn CEA, Hessing MJC, Tielen MJM (1995) The Specific-Stress-Free housing system has positive effects on productivity, health, and welfare of pigs. J Anim Sci 73: 1544–1551.
  33. 33. Hyun Y, Ellis M, Riskowski G, Johnson RW (1998) Growth performance of pigs subjected to multiple concurrent environmental stressors. J Anim Sci 76: 721–727.
  34. 34. Turner SP (2011) Breeding against harmful social behaviours in pigs and chickens: state of the art and the way forward. Appl Anim Behav Sci 134: 1–9.
  35. 35. Rodenburg TB, Turner SP (2012) The role of breeding and genetics in the welfare of farm animals. Anim Front 2: 16–21.
  36. 36. Star L, Ellen ED, Uitdehaag K, Brom FWA (2008) A plea to implement robustness into a breeding goal: poultry as an example. J Agric Environ Ethics 21: 109–125.
  37. 37. Bijma P (2012) Socially affected traits, inheritance and genetic improvement. In: Meyers RA, editor. Encyclopedia of sustainability science and technology. Larkspur: Springer Science+Business Media. pp.9358–9394.
  38. 38. Griffing B (1967) Selection in reference to biological groups I. Individual and group selection applied to populations of unordered groups. Aust J Biol Sci 20: 127–140.
  39. 39. Muir WM (1996) Group selection for adaptation to multiple-hen cages: selection program and direct responses. Poult Sci 75: 447–458.
  40. 40. Muir WM (2005) Incorporation of competitive effects in forest tree or animal breeding programs. Genetics 170: 1247–1259.
  41. 41. Bijma P, Muir WM, Ellen ED, Wolf JB, Van Arendonk JAM (2007) Multilevel selection 2: Estimating the genetic parameters determining inheritance and response to selection. Genetics 175: 289–299.
  42. 42. Bijma P, Muir WM, Van Arendonk JAM (2007) Multilevel selection 1: Quantitative genetics of inheritance and response to selection. Genetics 175: 277–288.
  43. 43. Wolf JB, Brodie ED III, Cheverud JM, Moore AJ, Wade MJ (1998) Evolutionary consequences of indirect genetic effects. Trends Ecol Evol 13: 64–69.
  44. 44. Bergsma R, Kanis E, Knol EF, Bijma P (2008) The contribution of social effects to heritable variation in finishing traits of domestic pigs (Sus scrofa). Genetics 178: 1559–1570.
  45. 45. Bergsma R, Mathur PK, Kanitz E, Verstegen MWA, Knol EF, et al. (2013) Genetic correlations between lactation performance and growing-finishing traits in pigs. J Anim Sci 91: 3601–3611.
  46. 46. Rodenburg TB, Bijma P, Ellen ED, Bergsma R, De Vries S, et al. (2010) Breeding amiable animals? Improving farm animal welfare by including social effects in breeding programmes. Anim Welf 19: 77–82.
  47. 47. Chen CY, Johnson RK, Newman S, Van Vleck L (2007) A general review of competition genetic effects with an emphasis on swine breeding. Genet Mol Res 6: 594–606.
  48. 48. Reimert I, Rodenburg TB, Ursinus WW, Duijvesteijn N, Camerlink I, et al. (2013) Backtest and novelty behavior of female and castrated male piglets with diverging social breeding values for growth. J Anim Sci 91: 4589–4597.
  49. 49. Reimert I, Rodenburg TB, Ursinus WW, Kemp B, Bolhuis JE (2014) Responses to novel situations of female and castrated male pigs with divergent social breeding values and different backtest classifications in barren and straw-enriched housing. Appl Anim Behav Sci 151: 24–35.
  50. 50. Camerlink I, Turner SP, Bijma P, Bolhuis JE (2013) Indirect genetic effects and housing conditions in relation to aggressive behaviour in pigs. PLoS One 8: e65136.
  51. 51. Huff GR, Huff WE, Balog JM, Rath NC (2003) The effects of behavior and environmental enrichment on disease resistance of turkeys. Brain Behav Immun 17: 339–349.
  52. 52. Marashi V, Barnekow A, Ossendorf E, Sachser N (2003) Effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice. Horm Behav 43: 281–292.
  53. 53. Bolhuis JE, Schouten WGP, Schrama JW, Wiegant VM (2005) Behavioural development of pigs with different coping characteristics in barren and substrate-enriched housing conditions. Appl Anim Behav Sci 93: 213–228.
  54. 54. Bolhuis JE, Schouten WGP, Schrama JW, Wiegant VM (2006) Effects of rearing and housing environment on behaviour and performance of pigs with different coping characteristics. Appl Anim Behav Sci 101: 68–85.
  55. 55. Geverink NA, Schouten WGP, Gort G, Wiegant VM (2003) Individual differences in behaviour, physiology and pathology in breeding gilts housed in groups or stalls. Appl Anim Behav Sci 81: 29–41.
  56. 56. Geverink NA, Heetkamp MJW, Schouten WGP, Wiegant VM, Schrama JW (2004) Backtest type and housing condition of pigs influence energy metabolism. J Anim Sci 82: 1227–1233.
  57. 57. Bolhuis JE, Parmentier HK, Schouten WGP, Schrama JW, Wiegant VM (2003) Effects of housing and individual coping characteristics on immune responses of pigs. Physiol Behav 79: 289–296.
  58. 58. Schrama JW, Schouten JM, Swinkels JW, Gentry JL, De Vries Reilingh G, et al. (1997) Effect of hemoglobin status on humoral immune response of weanling pigs differing in coping styles. J Anim Sci 75: 2588–2596.
  59. 59. Davis AK, Maney DL, Maerz JC (2008) The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct Ecol 22: 760–772.
  60. 60. Cray C (2012) Acute phase proteins in animals. Prog Mol Biol Transl Sci 105: 113–150.
  61. 61. Oostindjer M, Priester M, Van den Brand H, Parmentier HK, De Vries Reilingh G, et al. (2013) Environment and coping with weaning affect immune parameters of piglets 25 days after weaning. In: Hötzel MJ, Filho LCPM, editors. Proceedings of the 47th International Congress of the International Society for Applied Ethology (ISAE). Florianopolis: Wageningen Academic Publishers. p. 126.
  62. 62. Star L, Frankena K, Kemp B, Nieuwland MGB, Parmentier HK (2007) Natural humoral immune competence and survival in layers. Poult Sci 86: 1090–1099.
  63. 63. Sun Y, Ellen ED, Parmentier HK, Van der Poel JJ (2013) Genetic parameters of natural antibody isotypes and survival analysis in beak-trimmed and non-beak-trimmed crossbred laying hens. Poult Sci 92: 2024–2033.
  64. 64. Ayensu WK, Pucilowski O, Mason GA, Overstreet DH, Rezvani AH, et al. (1995) Effects of chronic mild stress on serum complement activity, saccharin preference, and corticosterone levels in Flinders lines of rats. Physiol Behav 57: 165–169.
  65. 65. Demey F, Pandey V, Baelmans R, Agbede G, Verhulst A (1993) The effect of storage at low temperature on the haemolytic complement activity of chicken serum. Vet Res Commun 17: 37–40.
  66. 66. Von Krogh M (1916) Colloidal chemistry and immunology. J Infect Dis 19: 452–477.
  67. 67. Lammers A, Klomp MEV, Nieuwland MGB, Savelkoul HFJ, Parmentier HK (2004) Adoptive transfer of natural antibodies to non-immunized chickens affects subsequent antigen-specific humoral and cellular immune responses. Dev Comp Immunol 28: 51–60.
  68. 68. Cray C, Zaias J, Altman NH (2009) Acute phase response in animals: a review. Comp Med 59: 517–526.
  69. 69. Parmentier HK, Lammers A, Hoekman JJ, De Vries Reilingh G, Zaanen ITA, et al. (2004) Different levels of natural antibodies in chickens divergently selected for specific antibody responses. Dev Comp Immunol 28: 39–49.
  70. 70. Blount DG, Pritchard DI, Heaton PR (2005) Age-related alterations to immune parameters in Labrador retriever dogs. Vet Immunol Immunopathol 108: 399–407.
  71. 71. Juul-Madsen HR, Jensen KH, Nielsen J, Damgaard BM (2010) Ontogeny and characterization of blood leukocyte subsets and serum proteins in piglets before and after weaning. Vet Immunol Immunopathol 133: 95–108.
  72. 72. Lutgendorf SK, Costanzo ES (2003) Psychoneuroimmunology and health psychology: an integrative model. Brain Behav Immun 17: 225–232.
  73. 73. Ernst K, Tuchscherer M, Kanitz E, Puppe B, Manteuffel G (2006) Effects of attention and rewarded activity on immune parameters and wound healing in pigs. Physiol Behav 89: 448–456.
  74. 74. Merlot E, Vincent A, Thomas F, Meunier-Salaün M, Damon M, et al. (2012) Health and immune traits of Basque and Large White pigs housed in a conventional or enriched environment. Appl Anim Behav Sci 85: 247–257.
  75. 75. Scollo A, Di Martino G, Bonfanti L, Stefani AL, Schiavon E, et al. (2013) Tail docking and the rearing of heavy pigs: the role played by gender and the presence of straw in the control of tail biting. Blood parameters, behaviour and skin lesions. Res Vet Sci 95: 825–830.
  76. 76. Scott K, Chennells DJ, Campbell FM, Hunt B, Armstrong D, et al. (2006) The welfare of finishing pigs in two contrasting housing systems: fully-slatted versus straw-bedded accommodation. Livest Sci 103: 104–115.
  77. 77. Manciocco A, Sensi M, Moscati L, Battistacci L, Laviola G, et al. (2011) Longitudinal effects of environmental enrichment on behaviour and physiology of pigs reared on an intensive-stock farm. Ital J Anim Sci 10: 224–232.
  78. 78. Piñeiro C, Piñeiro M, Morales J, Carpintero R, Campbell F, et al. (2007) Pig acute-phase protein levels after stress induced by changes in the pattern of food administration. Animal 1: 133–139.
  79. 79. Salamano G, Mellia E, Candiani D, Ingravalle F, Bruno R, et al. (2008) Changes in haptoglobin, C-reactive protein and pig-MAP during a housing period following long distance transport in swine. Vet J 177: 110–115.
  80. 80. Tuyttens FAM (2005) The importance of straw for pig and cattle welfare: a review. Appl Anim Behav Sci 92: 261–282.
  81. 81. Oostindjer M, Bolhuis JE, Mendl M, Held S, Gerrits W, Van den Brand H, Kemp B (2010) Effects of environmental enrichment and loose housing of lactating sows on piglet performance before and after weaning. J Anim Sci 88: 3554–3562.
  82. 82. Boscarino JA, Chang J (1999) Higher abnormal leukocyte and lymphocyte counts 20 years after exposure to severe stress: research and clinical implications. Psychosom Med 61: 378–386.
  83. 83. Lewis CRG, Hulbert LE, McGlone JJ (2008) Novelty causes elevated heart rate and immune changes in pigs exposed to handling, alleys, and ramps. Livest Sc 116: 338–341.
  84. 84. Ots I, Murumägi A, Horak P (1998) Haematological health state indices of reproducing great tits: methodology and sources of natural variation. Funct Ecol 12: 700–707.
  85. 85. Van de Kampman-van de Hoek E, Gerrits WJJ, Van den Borne JJGC, Van der Peet-Schwering CMC, Van Beers H, et al. (2013) Challenge models to study the effect of immune system activation on amino acid metabolism in pigs. In: Oltjen JW, editor. Proceedings of the 4th EAAP Internations Symposium on energy and protein metabolism and nutrition. Sacramento: Wageningen Academic Publishers. pp.237–238.
  86. 86. Taylor NR, Main DCJ, Mendl M, Edwards SA (2010) Tail-biting: a new perspective. Vet J 186: 137–147.
  87. 87. Ursinus WW, Van Reenen CG, Kemp B, Bolhuis JE (2014) Tail biting behaviour and tail damage in pigs and the relationship with general behaviour: predicting the inevitable? Appl Anim Behav Sci 156: 22–36.
  88. 88. Petersen HH, Dideriksen D, Christiansen BM, Nielsen JP (2002) Serum haptoglobin concentration as a marker of clinical signs in finishing pigs. Vet Rec 151: 85–89.
  89. 89. Prunier A, Heinonen M, Quesnel H (2010) High physiological demands in intensively raised pigs: impact on health and welfare. Animal 4: 886–898.
  90. 90. Rauw WM, Kanis E, Noordhuizen-Stassen EN, Grommers FJ (1998) Undesirable side effects of selection for high production efficiency in farm animals: a review. Livest Prod Sci 56: 15–33.
  91. 91. Koolhaas JM, De Boer SF, Coppens CM, Buwalda B (2010) Neuroendocrinology of coping styles: towards understanding the biology of individual variation. Front Neuroendocrinol 31: 307–321.
  92. 92. Carere C, Caramaschi D, Fawcett TW (2010) Covariation between personalities and individual differences in coping with stress: converging evidence and hypotheses. Curr Zool 56: 728–740.
  93. 93. Coppens CM, De Boer SF, Koolhaas JM (2010) Coping styles and behavioural flexibility: towards underlying mechanisms. Philos Trans R Soc Lond B Biol Sci 365: 4021–4028.
  94. 94. Sih A, Bell AM, Johnson JC, Ziemba RE (2004) Behavioral syndromes: an integrative overview. Q Rev Biol 79: 241–277.
  95. 95. Hessing MJC, Hagelsø AM, Schouten WGP, Wiepkema PR, Van Beek JAM (1994) Individual behavioral and physiological strategies in pigs. Physiol Behav 55: 39–46.
  96. 96. Bolhuis JE, Schouten WGP, De Leeuw JA, Schrama JW, Wiegant VM (2004) Individual coping characteristics, rearing conditions and behavioural flexibility in pigs. Behav Brain Res 152: 351–360.
  97. 97. Ruis MAW, Te Brake JHA, Van de Burgwal JA, De Jong IC, Blokhuis HJ, et al. (2000) Personalities in female domesticated pigs: behavioural and physiological indications. Appl Anim Behav Sci 66: 31–47.
  98. 98. Geverink NA, Schouten WGP, Gort G, Wiegant VM (2002) Individual differences in behavioral and physiological responses to restraint stress in pigs. Physiol Behav 77: 451–457.
  99. 99. Bolhuis JE, Schouten WGP (2002) Behavioural responses in a restraint test of pigs wit different Backtest classifications. In: Koene P, editor. Proceedings of the 36th International Congress of the International Society for Applied Ethology (ISAE). Egmond aan Zee: Ponsen and Looijen. p. 172.
  100. 100. Jansen J, Bolhuis JE, Schouten WGP, Spruijt BM, Wiegant VM (2009) Spatial learning in pigs: effects of environmental enrichment and individual characteristics on behaviour and performance. Anim Cogn 12: 303–315.
  101. 101. Bolhuis JE, Schouten WGP, Schrama JW, Wiegant VM (2005) Individual coping characteristics, aggressiveness and fighting strategies in pigs. Anim Behav 69: 1085–1091.
  102. 102. Ruis MAW, Te Brake JHA, Engel B, Buist WG, Blokhuis HJ, et al. (2002) Implications of coping characteristics and social status for welfare and production of paired growing gilts. Appl Anim Behav Sci 75: 207–231.
  103. 103. Bolhuis JE, Schouten WGP, De Jong IC, Schrama JW, Cools AR, et al. (2000) Responses to apomorphine of pigs with different coping characteristics. Psychopharmacology 152: 24–30.
  104. 104. Van Erp-van der Kooij E, Kuijpers AH, Schrama JW, Ekkel ED, Tielen MJM (2000) Individual behavioural characteristics in pigs and their impact on production. Appl Anim Behav Sci 66: 171–185.
  105. 105. Hessing MJC, Hagelsø AM, Van Beek JAM, Wiepkema PR, Schouten WGP, et al. (1993) Individual behavioural characteristics in pigs. Appl Anim Ethol 37: 285–295.
  106. 106. Barber I, Dingemanse NJ (2010) Parasitism and the evolutionary ecology of animal personality. Philos Trans R Soc Lond B Biol Sci 365: 4077–4088.
  107. 107. Kortet R, Hedrick AV, Vainikka A (2010) Parasitism, predation and the evolution of animal personalities. Ecol Lett 13: 1449–1458.
  108. 108. Hessing MJC, Coenen GJ, Vaiman M, Renard C (1995) Individual differences in cell-mediated and humoral immunity in pigs. Vet Immunol Immunopathol 45: 97–113.
  109. 109. Sanders VM (2006) Epigenetic regulation of Th1 and Th2 cell development. Brain Behav Immun 20: 317–324.
  110. 110. Ricklin D, Hajishengallis G, Yang K, Lambris JD (2010) Complement - a key system for immune surveillance and homeostasis. Nat Immunol 11: 785–797.
  111. 111. Sild E, Sepp T, Hõrak P (2011) Behavioural trait covaries with immune responsiveness in a wild passerine. Brain Behav Immun 25: 1349–1354.
  112. 112. Koolhaas JM (2008) Coping style and immunity in animals: making sense of individual variation. Brain Behav Immun 22: 662–667.
  113. 113. Niemelä PT, Dingemanse NJ, Alioravainen N, Vainikka A, Kortet R (2013) Personality pace-of-life hypothesis: testing genetic associations among personality and life history. Behav Ecol 24: 935–941.
  114. 114. Geverink NA, Parmentier HK, De Vries Reilingh G, Schouten WGR, Gort G, et al. (2004) Effect of response to backtest and housing condition on cell-mediated and humoral immunity in adult pigs. Physiol Behav 80: 541–546.
  115. 115. Mehta PH, Gosling SD (2008) Bridging human and animal research: a comparative approach to studies of personality and health. Brain Behav Immun 22: 651–661.
  116. 116. Friedman HS (2008) The multiple linkages of personality and disease. Brain Behav Immun 22: 668–675.
  117. 117. Prunier A, Bonneau M, Von Borell E, Cinotti S, Gunn M, et al. (2006) A review of the welfare consequences of surgical castration in piglets and the evaluation of non-surgical methods. Anim Welf 15: 277–289.
  118. 118. Piñeiro M, Piñeiro C, Carpintero R, Morales J, Campbell FM, et al. (2007) Characterisation of the pig acute phase protein response to road transport. Vet J 173: 669–674.
  119. 119. Piñeiro C, Piñeiro M, Morales J, Andrés M, Lorenzo E, et al. (2009) Pig-MAP and haptoglobin concentration reference values in swine from commercial farms. Vet J 179: 78–84.
  120. 120. Clapperton M, Bishop SC, Cameron N, Glass E (2005) Associations of acute phase protein levels with growth performance and with selection for growth performance in Large White pigs. Anim Sci 81: 213–220.
  121. 121. Kurtz J, Wiesner A, Götz P, Sauer KP (2000) Gender differences and individual variation in the immune system of the scorpionfly Panorpa vulgaris (Insecta: Mecoptera). Dev Comp Immunol 24: 1–12.
  122. 122. Darnall BD, Suarez EC (2009) Sex and gender in psychoneuroimmunology research: past, present and future. Brain Behav Immun 23: 595–604.
  123. 123. Stefanski V, Grüner S (2006) Gender difference in basal and stress levels of peripheral blood leukocytes in laboratory rats. Brain Behav Immun 20: 369–377.
  124. 124. Baum A, Grunberg NE (1991) Gender, stress, and health. Health Psychol 10: 80–85.