Skip to main content
Download PDF

Physiology, gene expression, and behavior as potential indicators of oxidative stress in piglets

BMC Veterinary Research volume 20, Article number: 471 (2024) Cite this article

Abstract

The goal of the current study was to develop a pig model to investigate oxidative stress with a low negative impact on piglet welfare. Four independent trials (A, B, C, and D) were performed using a single intraperitoneal shot of lipopolysaccharide (LPS) as an immune challenge, aiming to assess the minimal LPS dose for piglets of different age to trigger a measurable acute oxidative stress response in healthy animals. In trial A, piglets received an LPS dose of 25 µg/KgBW at 41 days post-weaning (p.w.). In trial B, piglets received 25 µg/KgBW of LPS at 28 days p.w., in trials C And D, piglets were injected with 50 µg/KgBW of LPS at 21 days p.w., respectively. Piglets were randomly allocated either to the T1) Control group with saline solution (Ctrl), or T2) LPS challenge (LPS). The oxidative stress response was measured through the enzymatic activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT), in both plasma and intestinal tissues. Intestinal gene expression of oxidative stress and inflammatory markers was assessed. Discomfort behaviors (panting, prostration, trembling, and vomits) were also recorded. Plasmatic and intestinal oxidative stress response was inconsistent across the four trials even when the dose and pig age were similar, possibly due to individual variability. Relative gene expression differences of anti-inflammatory cytokines (IL10), oxidation precursor (iNOS), and antioxidant markers (GPx4, MnSOD, and CAT) were detected between Ctrl and LPS treatment (P < 0.05) when assessed. Behavioral observations were sensitive to the LPS dose relative to Ctrl (P < 0.05) in all four trials. These results suggest that behavioral observations can be used as a non-invasive methodology to detect the presence of oxidative stress in pigs in challenging conditions. Behavioral observations were more sensitive than other indicators (i.e., biomarkers and gene expression) in the current study. However, a sensitivity scale system needs to be developed to qualify and rank the impact of oxidative stress in pigs.

Peer Review reports

Introduction

Oxidative stress occurs when the generation of free radicals overcomes the body’s antioxidant capacity [1]. This oxidative imbalance leads to the accumulation of Reactive Oxygen Species (ROS) with the consequent damage to lipids, proteins, and DNA, impairing the physiological condition of the organism. The accumulation of ROS in the intestinal tract is especially relevant and is associated with compromised intestinal function, decreased nutrient transport, and increased susceptibility to infections [2, 3]. Such oxidative threat provokes physiological reactions to control the potential harm to tissues. Physiological responses range from activation of antioxidant proteins [i.e., glutathione peroxidase (GPx), catalase (CAT), or superoxide dismutase(SOD)] and cytokines (i.e., TNF-α, IL-6, IL-8, and IL-1b) [4, 5], to changes in behavior [6].

Oxidative stress responses are variable depending on the intensity of the stressor. Naito et al. [4] described an organism’s different physiological steps to face an oxidative stressor. Oxidative stressors generate ROS, increasing the oxidation of molecules such as lipids, proteins, enzymes, and DNA. If the oxidative threat persists, free radical-trapping antioxidants such as vitamins C and E, carotenoids, coenzyme Q10, and bilirubin are activated. Therefore, the oxidative indicators or biomarkers to assess the oxidative status of pigs are antioxidant enzymes such as the GPx, which is recognized as an antioxidant enzyme, that reduces hydrogen peroxide and organic hydroperoxides, assisting in the control of ROS [5, 7, 8]. GPx activity has been used as an indicator of the antioxidant status and potential [7]. SOD is an important enzyme that catalyzes the dismutation of super peroxide into oxygen and hydrogen peroxide [8, 9]. CAT catalyzes the transformation of hydrogen peroxide into water and oxygen [8, 9]. Finally, GST is a detoxification enzyme that protects cells from unhealthy chemical species [10]. GPx, SOD, and CAT are commonly used as anti-oxidative biomarkers as they are the first response to oppose an oxidative challenge, conforming a preventive type of antioxidant network system [4, 8].

Behavioral responses are considered adaptative responses to cope with stimuli [11,12,13]. Two major physiological pathways influence animal behavior: the sympathetic-adrenal-medullary axis (epinephrine and norepinephrine, related to the “fight or flight” reaction) and the hypothalamic-pituitary-adrenal axis. The latter controls glucocorticoids to increase protein or fat catabolism, thereby increasing glucose availability [14]. Broom [11] has stated that behavior is related to immunological activity, particularly regarding the action of cytokines, interleukins, enzymes, and other endocrinological agents. This association allows veterinarians and medical doctors to diagnose pathologies accurately [11]. Additionally, behavioral assessment has advantages over the assessment of biomarkers as it can be performed remotely without disturbing the integrity of the animals [15]. Although behavior as a stress indicator has pros such as feasible utilization and the possibility of being automatized, it is not as specific as common biomarker analyses. Nevertheless, through systematic and validated observation protocols, behavioral observations can provide significant information about the status of the animal [11].

In pig production, oxidative stress can be generated by several factors such as nutrition imbalances, negative social interactions due to excessive aggression receptions, or environmental factors that may harm the welfare of animals and their production level [2, 3, 15]. At weaning, piglets are subject to several of these stressors simultaneously, such as separation from their mother, the sudden dietary change from liquid to solid feed, the mixing with new counterparts, and a new environment [16, 17]. Thus, it is necessary to study the physiological responses triggered by these challenges and the possible indicators that might help the pig producers and researchers monitor the stress response.

Experimental stress models help pig production stakeholders to objectively evaluate mitigation strategies to preserve animal welfare and performance. Commonly, current models to study the effects of oxidative stress through immune stimulation with lipopolysaccharide (LPS), which is a component of the gram-negative bacteria membrane and is one of the most potent immune system stimulators [18] commonly used to trigger oxidative stress in experimental conditions [15]. The LPS interacts with the antigen CD14 initiating an inflammatory response including activation of free radical-generating enzymes that provoke host lipid peroxidation [19] increasing the risk of oxidative damage. However, LPS challenges in pigs are not standardized (challenge type, age, dose, etc.) and may severely impact the physiological condition and welfare of the animals [20]. Also, the heterogeneity of experimental conditions might produce a variety of results, making it difficult to elucidate the effectiveness of tested mitigation strategies to deal with oxidative damage. Moreover, the suitable period to measure the oxidative response in piglets remains unclear. Different immune challenges have been reported (e.g. 25–100 µg LPS/Kg BW) to unbalance the oxidative status of animals at different ages (from 10 to 46 days post-weaning) days and diverse housing management [21,22,23,24]. These irregular conditions across studies may generate inconsistent results, besides the intense discomfort caused by the challenge to the animals. Hou et al. [24] and Li et al. [25] observed antioxidant activity with lower doses compared to the doses reported in the literature [60–100 µg/kg BW [20,21,22, 26,27,28,29,30,31]]. Therefore, it is reasonable to subject the piglets to a mild oxidative challenge in order to measure the oxidative response while minimizing the negative impacts on their welfare.

The main goal of this research was to develop a model capable of triggering a measurable oxidative stress response aiming to provoke minimal harm to piglets and avoid clinical illness. This study assessed four trials describing the piglets’ physiological response toward an immune stimulator to trigger a physiological oxidative challenge. Additionally, the present study aims to assess behavior as a more sensitive oxidative stress indicator compared to other measurement methods used in the study. The present study will contribute to identifying different less or non-invasive biomarkers and improve the Refinement (three Rs: Replacement, Reduction, and Refinement) [32] of experimental protocols for pig oxidative stress research.

Materials and methods

Animals, housing and diets

All procedures were approved by the Laboratory Animal Care Advisory Committee of the Universitat Autònoma de Barcelona (CEA/9310/P1). Four consecutive trials were performed at an Experimental Swine Unit farm “El Castell” (Sant Aniol de Finestres, Girona, Spain) following the same housing and management routines.

All crossbred piglets [(Largewhite × Landrace) × Pietrain] were purchased, weaned, and transported to the nursery unit from the farrowing farm of origin (“La Sala”, Sant Bartomeu del Grau, Barcelona, Spain) on the same day. Healthy piglets, weighing between 3.5 and 6.5 kg of body weight (BW), were carefully chosen from sows with the most uniform litters. They were checked for clean skin, healthy joints, and no lesions or injuries. Weaning ages and weights at each trial are shown in Table 1. At arrival, piglets were housed in four rooms with 12 pens (6 piglets/pen) in each room. Except for trial A, where only two piglets were housed together in a pen with a division allowing visual contact, to increase the number of experimental units in the trial. Therefore, the piglets in trial A were considered individually housed because they did not have physical contact with the piglets beside them. Each pen (2.64 m2) had two nipple drinkers covered with a mobile metal lid to prevent external contamination (feces and urine) and to decrease water waste. Pens had one feeder per pen, with four separations per feeder covered with a metal lid. All experimental pens were equipped with completely slatted plastic floors. Piglets were randomly distributed into pens according to their initial BW and sex to homogenize the animals in each treatment group.

Table 1 Summary of the weaning age and weights of the piglets in each trial
Full size table

All pigs were fed a standard commercial diet in mash form formulated to meet or exceed the National Research Council [33] nutrient requirements (Table 2). Feed and water were offered ad libitum throughout the trials.

Table 2 Summary of the nutrient composition of the diets supplied during the phases of the trials. Creep feed diet (from day 0 to day 7 post-weaning, p.w.), pre-starter diet (from day 7 to day 21 p.w.), and starter diet (from day 21 to day 41 p.w.)
Full size table

Study design

Oxidative stress was induced by the intraperitoneal (i.p.) administration (in the upper left area of the intraperitoneal cavity) of Escherichia coli LPS (serotype 055: B5 Sigma Chemical, San Luis, USA) to healthy animals based on the experimental oxidative stress model from Hou et al., [24]. The LPS was selected as an immune stimulator because it is commonly and widely used as an oxidative challenge [15]. The i.p. administration was intended to enhance the effectiveness of the oxidative challenge in the organism, especially in the intestinal tissues. The four trials in the current study included two experimental treatment groups (i) control (Ctrl); and (ii) LPS challenge (LPS). The experimental conditions (LPS dose administered, and the age of the piglets) for each trial were adjusted based on the results of the previous one (Table 3). Pigs were randomly allocated to one of the two treatments from weaning until the challenge day. All the treatment groups were homogenized by sex and body weight. LPS doses of 25 and 50 µg/kg BW were chosen to determine if it was possible to induce an acute oxidative stress response with a milder challenge. Pigs from the LPS challenge group received a single intraperitoneal dose of an LPS agent, whereas Ctrl pigs received an equivalent intraperitoneal injection of saline solution. The LPS dose and final injection volume were determined based on the pigs’ body weight (LPS µg/KgBW). The goal of each subsequent trial was to refine the oxidative stress model by adjusting the conditions of the LPS challenge (such as the housing type, the dose of LPS, and the age of piglets) based on the piglets’ response from the previous trial. This aimed to achieve a measurable response with less impact on the piglets’ health. Similarly, the sampling procedures (i.e., blood sampling and collection of intestinal tissue) varied across the trials to identify measurement methodologies with higher sensitivity to the piglets’ oxidative response. The efficiency of the oxidative model in each trial was assessed by comparing the differences in oxidative response between the challenged piglets and non-challenged piglets.

Table 3 Summary of the four trials conditions to assess the pig oxidative stress model
Full size table

Sample collection

Sampling protocols were the same in all the trials of the current study. Piglets were in their pens during the blood sampling. Blood samples were collected from the cava cranialis vein pre-sacrifice (4 h post LPS i.p injection) using a vacutainer into pre-labeled tubes with EDTA anticoagulant. The blood tubes were immediately centrifuged (1200 g x 10 min, 4 °C) and the supernatant was separated and frozen immediately in dry ice. After blood sampling, 4 h post-LPS i.p. injection, piglets were individually moved to the sacrifice room. LPS and Ctrl pigs (10 piglets/treatment in trial A; 12 piglets/treatment in trials B, C, and D) were euthanized to collect the intestinal tissues. Piglets were stunned by a captive bolt shot in the piglet’s front and then sacrificed exsanguinating by severing the jugular and carotid veins. After cessation of any sign of consciousness (i.e., no pain and corneal reflexes, and absence of rhythmic breathing) the abdomen was sectioned to extract and dissect the intestine into jejunum, ileum, and colon. Sections of the mid jejunum, ileum, and colon were washed with 0.9% saline to remove content and longitudinally opened to collect mucosa. All samples were flash-frozen (in liquid N2 or dry ice) and stored at -80 °C.

Biological parameters

The activity of antioxidant enzymes in intestinal mucosa and plasma was measured using commercial ELISA kits following manufacturers’ protocol (Cayman Chemical, Ann Arbor, MI). The activity of antioxidant enzymes in intestinal mucosa was measured in Trials A41/25 and B28/25 but not in Trials C21/50 and D21/50.

Total RNA was isolated from 100 mg mucosal samples automatically in a KingFisher Duo Prime (ThermoFisher Scientific, Waltham, USA) with the MagMax™ mirVana™ Total RNA Isolation kit (ThermoFisher Scientific), following manufacturers’ instructions. Final RNA concentration and quality were spectrophotometrically assessed in a NanoDrop system (ThermoFisher Scientific), and cDNA synthesis was performed with the High-Capacity cDNA Reverse Transcription Kit. RT-PCR was finally performed on a StepOne Plus system (ThermoFisher Scientific Inc.) with the Fast SYBR® Green Master Mix and the primers sequences detailed in Table 4. Specific product amplification was checked by the melting curve analysis. Gene efficiencies were evaluated by generating standard curves using cDNA from a pool of samples and calculated according to the equation E = 10 (− 1/slope). Relative expression was calculated according to the 2−ΔΔCt method using TBP and β-actin as housekeeping genes. Relative expression was only measured in Trials C21/50, and D21/50.

Table 4 Sequence of primers used in the RNA isolation
Full size table

Behaviors indicating discomfort triggered by the immune challenge (Table 5) were counted through direct observation at four time points: 60, 120, 180, and 240 min after intraperitoneal injection on Trials C21/50 and D21/50. Behavioral observations were performed by the same observer, counting the number of piglets in each pen displaying the selected behaviors at each time point.

Table 5 Ethogram and definitions of the behaviors recorded to assess the impact of oxidative challenge on piglets in different trials
Full size table

A summary of the samples collected and measurements performed in the different trials is presented in Table 6.

Table 6 Samples and measurements for the trials
Full size table

Statistical analyses

All statistical analyses were performed with the statistical software SAS (version 9.4, SAS Institute Inc., Cary, NC). Oxidative markers activity and genetic expression analyses were performed by T-test (PROC TTEST) with treatment as the main effect without any adjustment for multiple comparisons. LSMEANS compared the main effect treatment group. Behavioral observations were analyzed through a Logistic model (PROC LOGISTIC) within each observation time (60, 120, 180, and 240 min after intraperitoneal injection) comparing the frequency of each behavior among the treatments. In all the parameters measured, the pen was considered the experimental unit, and the piglet was considered the observational unit. Significant differences were declared at P ≤ 0.05 whereas near-significant trends were considered at 0.05 < P ≤ 0.10.

Results

Antioxidant enzyme activity

Plasma

Table 7 summarizes the plasmatic activities of the antioxidant enzymes. No differences in the activity of GPx (P = 0.3138), SOD (P = 0.4713), GST (P = 0.2456), and CAT (P = 0.2936) were observed for Trial A41/25. In trial B28/25, the GST (P = 0.0122) and GPx (P = 0.0476) activity were increased in the LPS group relative to the Ctrl. SOD (P = 0.5166), and CAT (P = 0.6452) activities remained unchanged between groups.

Table 7 Antioxidant enzyme activities in plasma from trials A41/25, B28/25, C21/50, and D21/50
Full size table

No differences in the activity of GPx (P = 0.7964), GST (P = 0.5475), and SOD (P = 0.1696) were observed for Trial C21/50, but CAT activity of the LPS piglets tended to increase (P = 0.0611). In Trial D21/50, with piglets of the same age at the same LPS dose of Trial C, a significant increase in plasmatic SOD activity was detected in the LPS group, compared to the Ctrl group (P = 0.0060). In contrast, GPx (P = 0.2783), GST (P = 0.4039), and CAT (P = 0.2252) activities remained unaffected.

Intestinal tissues

Table 8 summarizes the activities of the antioxidant enzymes measured in the mucosa of the different tissue sections (jejunum, ileum, and colon). Oxidative markers measured in trial A41/25 were not different between Ctrl and LPS treatments in any of the intestinal tissues analyzed (P ≥ 0.1626). In trial B28/25, piglets in the LPS group presented higher SOD activity in the ileum (P < 0.05). Trial B28/25 oxidative markers in other intestinal tissues were not different between Ctrl and LPS groups (P ≥ 0.33). Antioxidant activity in the intestinal mucosa was not measured in trials C21/50 and D21/50.

Table 8 Activity of the antioxidant enzymes in intestinal tissues (jejunum, ileum, and colon) from the trials A41/25, and B28/25
Full size table

Gene expression results

Piglets on trial C21/50 from the LPS group showed a higher jejunum gene expression of the anti-inflammatory cytokine IL-10 (149.09%) the defense protein iNOS (384.67%) and the antioxidant enzymes GPx4 (22.54%), MnSOD (73.58%), and CAT (152.42%), compared to Ctrl (P < 0.05, Fig. 1A). On the other hand, the jejunal gene expression of LPS piglets in trial D21/50, using the same LPS dose and piglets age, only showed significant increases of the oxidative markers iNOS (227.60%), MnSOD (147.05%), and CAT (107.84%), (Fig. 1B) compared to Ctrl (P < 0.05). Gene expression was not performed on trials A41/25 and B28/25.

Fig. 1
figure 1

Gene expression in A) trial C21/50, and B) trial D21/50 of antioxidant markers: IL10, iNOS, GPx4, MnSOD, and CAT. *Indicate significant differences (P < 0.05)

Full size image

Other inflammatory and antioxidant markers such as IL-8 (P ≥ 0.4796), IL-1b (P ≥ 0.9090), TNFα (P ≥ 0.1832), and GPx1 (P ≥ 0.6648) were not different between Ctrl and LPS treatment groups trial C21/50. In trial D21/50 Ctrl and LPS IL-10 (P ≥ 0.6489), IL-1b (P ≥ 0.1353), TNFα (P ≥ 0.0831), and GPx1 (P ≥ 0.5917) did not differ.

Behavioral observations

Figure 2 presents discomfort behaviors’ frequency (panting, prostration, trembling, and vomits) in trials C21/50 and D21/50. In general, Ctrl animals showed less frequency of illness-related behaviors than the LPS treatment group (P < 0.05) in both trials. Panting and vomiting behaviors were only displayed in LPS piglets in both trials. LPS pigs displayed more prostration and trembling than Ctrl pigs.

Fig. 2
figure 2

Frequency of discomfort behaviors after the oxidative challenge, including (panting, prostration, trembling, and vomits frequency presented is the sum of each behavior category. A) Trial C21/50; B) Trial D21/50. *Indicate significant differences (P < 0.05). The absence of mean bars means the value is zero or close to zero

Full size image

Discussion

There is a need for simple, accurate, and less aggressive experimental setups to evaluate nutritional or husbandry strategies to mitigate oxidative stress in pigs under commercial production conditions. The ultimate goal of the current research project was to develop an experimental oxidative stress model for pigs minimizing the negative impact on the welfare of the animals and without compromising the accuracy of the results obtained. The trials performed in this study attempted to detect oxidative stress indicator changes triggered by a mild LPS injection. Each independent trial attempted to refine the oxidative stress model based on the results of the previous trial. In trials A41/25 and B28/25, the goal was to trigger a measurable antioxidant response using an LPS dose of 25 µg/KgBW. Different age groups (28- and 41-days post-weaning) of pigs were tested to see when they would be most responsive to the LPS challenge. However, since the antioxidant response couldn’t be detected through the proposed methods (assessment of the antioxidant activity in plasma and intestinal mucosa), trials C21/50 and D21/50 were adjusted. These trials used a higher LPS dose compared to trials A41/25 and B28/25 and were conducted on younger pigs (21 days post-weaning). In addition, we conducted gene expression analyses and behavioral assessments to improve the likelihood of detecting changes resulting from the LPS challenge. However, we did not measure antioxidant activity in the intestinal mucosa in Trials C21/50 and D21/50 due to its lack of sensitivity.

Overall, the trials in the current study, using different LPS doses and in different piglet ages, were not able to detect any physiological responses toward the mild oxidative challenge. Antioxidant responses such as changes in the activity of the antioxidant enzymes were expected as Hou et al. [24], and Hu et al. [34] reported with mild LPS doses. Piglets’ variability (e.g. individual immune system maturity) or the mild severity of the proposed challenges might be some of the reasons for the inconsistent oxidative stress responses detected among the different trials. When assessing the physiological response of the piglets in trial A41/25, no significant differences were detected in plasmatic markers between treatment groups with a mild LPS dose. Whereas in trial B28/25, only a significant increase in the GST plasmatic activity was detected when pigs received the LPS challenge. The maturity of the animals is an important factor to consider in the lack of detected responses on trial A41/25. Immune system resilience increases when the animal grows, becoming more robust or primed towards immune challenges [35]. In trials, C21/50 and D21/50, LPS dose and age were modified (higher dose in younger animals) aiming to detect the acute oxidative response, but again the oxidative response obtained was not consistent. No GPx, SOD, and GST activity changes were observed in trial C21/50, but in trial D21/50, only a significant increase in SOD plasmatic activity in the LPS group was detected.

Contrary to the results of the current study, other authors, such as Hu et al. [34] applied an LPS dose of 10 µg/KgBW concentration, and Hou et al. [24] used an LPS dose of 25 µg/KgBW able to trigger and detect plasmatic antioxidant responses through mild LPS doses. Even though Hu et al. [34] reported Antioxidant Capacity (AOC) differences in the LPS group relative to treatment groups with nutritional additives. The samples for the AOC analyses were collected two hours after the LPS injection, which might increase the chance of detecting a response to the oxidative threat. On the other hand, Hou et al. [24] compared the effect of the LPS injection against a saline solution after six hours of the intraperitoneal injection; the differences are presented as total AOC. Thus, it is worth considering that antioxidant responses are easier to detect when the results are presented as AOC, which is a measurement of the cumulative action of all the antioxidant components in plasma [36], instead of the individual activity of antioxidant enzymes. Additionally, as reported by Hu et al. [34] an earlier sampling might increase the likelihood to detect antioxidant enzyme activity changes. In the present study, AOC capacity was not estimated because the focus was on observing how the different antioxidant enzymes reacted to the LPS challenge.

On the other hand, antioxidant activity on the intestinal tissues measured in trials B28/25, only detected an increase in the SOD activity in the ileum. Other authors have reported changes in the antioxidant activity markers, either in plasma or intestinal tissues, when a higher dose was applied. For instance, Sun et al. [31] reported significant reductions of the GPx activity in different tissues (serum, liver, spleen, thymus, and lymph node) of pigs challenged with an LPS dose of 100 µg/kg BW relative to the control pigs. Chen et al. [21] registered a notorious decline of the plasmatic GPx of the LPS group compared to the saline group, but no differences were detected in the SOD activity in plasma with an LPS dose of 100 µg/kg. Also, the antioxidant capacity of intestinal tissues (jejunum and ileum) was reduced after the oxidative challenge, but no differences were reported in SOD activity at the jejunum, ileum, or colon. Cao et al. [5] noticed a decrease in the GPx and SOD activity in the jejunum mucosa with the 100 µg/kg BW LPS dose. However, not all the markers are sufficiently sensitive to measure oxidative stress at this high LPS concentration. For example, studies from Kang et al. [27], Yi et al. [26], and Cai et al. [30] were not able to detect differences in CAT activity with an LPS dose of 100 µg/kg BW. Hence, a high dose on an LPS challenge does not guarantee a higher expression of the oxidative markers, but it induces acute behavioral responses (i.e. trembling, vomiting, anorexia, among others) in the animal that deteriorates the animal welfare of the subjects challenged, as observed in the studies mentioned as an example.

The absence of differences among the oxidative markers in the present study could be also related to the breed. The piglets in current trials were crossbred (Large White x Landrace) x Pietrain from the same original farm. Other studies used crossbred Duroc × Landrace × Yorkshire, in which they were able to observe differences among the antioxidant markers [5, 21, 24, 25]. Thus, the breed factor might be a possible cause of the oxidative stress sensibility [13, 37]. Nguyen and McPhee [38] have reported Large White phenotypic advantages that might reduce the susceptibility towards oxidative damage, as Large White pigs are more resilient to high environmental temperatures. Also, Camara et al., [39] have reported that the inclusion of Pietran genetics might increase the growth performances of pig genetic lines, which might have an impact on the resilience to immune challenges. However, the mechanism underlying the breed-specific response towards stress is not fully understood and requires further studies [40]. Another point to consider when comparing the antioxidant results from other authors is the differences in the management and the facilities where the trials were performed, as environmental, sampling protocols, or other unmeasurable factors could affect the physiology of the piglets [14].

Literature has reported that higher doses of LPS (> 60 µg/Kg BW) do not guarantee a measurable change in physiological parameters [26, 27, 30]. Furthermore, some antioxidant markers such as the CAT might reduce its own activity as Kang et al. [27], Yi et al. [26], and Cai et al. [30] observed when a high LPS dose was used (100 µg/kg BW). Large concentrations of LPS as an oxidative stressor would result in an amount of oxidative damage that constrains the antioxidant enzymes’ energy [4] to respond with a higher capacity to control the oxidative menace [4, 41]. The activity of the antioxidant enzymes might be higher at relatively lower levels of the stressor.

The jejunum gene expression in the animals challenged with LPS in trials C21/50 and D21/50 detected the increase of the inflammatory or oxidative activity caused by the oxidative stimuli. Gene expression is a sensible method to detect responses towards oxidative challenges, either with mild oxidative stimuli (10 µg/kg BW on day 14 and 21 p.w.) [42], or severe doses of the oxidative challenge (100 µg/kg BW on day 14 and 21 p.w.) [43]. However, the practical use of this methodology is constrained due to the relatively elevated price of the analyses and the need for euthanasia to obtain the tissues.

On the other hand, behavioral responses were detected in both trials C21/50 and D21/50, LPS piglets displayed panting, prostration, tremors, and vomiting at a higher frequency than Ctrl piglets. Contrarily, Ctrl piglets in both trials C21/50 and D21/50 displayed significantly less of these discomfort behaviors. Animal behavior changes have been pointed out as an adaptative strategy to cope with the effects of a stressor and recover homeostasis [11, 44]. Thus, considering behavior as a physiological reflection of the state of an organism, it is plausible to propose the incidence of discomfort behaviors as a non-invasive oxidative stress indicator. Remarkably, the development of remote and continuous measurement methodologies to monitor animal behavior and welfare is urgently required by animal production stakeholders to effectively oversee the health and fitness of the animals [15, 45].

In the present study, the mild challenges resulted in discomfort in the pigs, as evidenced by their behaviors. However, there was no measurable antioxidant response in plasma or the intestinal mucosa. More detailed assessment methods, such as gene expression analysis, would be necessary to detect the changes caused by the mild challenges. Nevertheless, conducting such analyses would require sacrificing the pigs and collecting tissue samples.

The combination measurement methodologies have the potential to provide an accurate approach to the assessment of oxidative stress [15]. However, behavioral observation methodologies are not able yet to quantify the magnitude of the impact of the oxidative challenge. A sensitivity scale of the behaviors related to stress responses can be a versatile tool for pig production stakeholders to assess the impact of stressors.

Finally, confronting the results of the current study with other results from the literature, severe LPS doses (> 60 µg/Kg BW) might be considered physiological challenges as those can affect the physiological homeostasis of the pigs [5, 21,22,23, 25, 26, 31], reflected in the changes in antioxidant activity (gene expression or protein abundance). Contrarily, mild LPS doses, as the ones tested in the current trials (up to 50 µg/Kg BW), might be studied as physical discomfort that might not alter severely the physiological balance and is mainly reflected in the behavior of the animal. Important to consider is that piglets’ resilience toward the LPS challenge was not measured (i.e. time to recover homeostasis after the intraperitoneal LPS injection) as sampling protocols were done aiming to detect the highest point of the physiological response. Also, if such a resilience test was done, more piglets would be exposed to the LPS challenge and handling protocol which would produce unnecessary discomfort for piglets and would increase the cost of the study. However, antioxidant results obtained in the current trials can suggest that the LPS doses did not trigger severe pathophysiological responses for the piglets and support the proposal of a mild oxidative stress model.

Conclusions

Based on the empirical results obtained, it is possible to assume that the magnitude of the physiological response may be associated with the intensity of the oxidative challenge (LPS dose) and the maturity of the animal’s immune system (age). The presence of oxidative stress was verified through the measurement of gene expression markers in intestinal tissues but not in the plasma. On the other hand, our study might elucidate the possibility that behavior may be used as an indicator for continuous and non-invasive monitoring of piglets, which is sensitive enough for early detection of physiological responses to stressors able to induce oxidative stress. Nevertheless, behavior sensitivity and specificity have not been defined and require further research.

Animal models for research must guarantee the best possible welfare conditions, as well as researchers and producers’ expectations in terms of accuracy, feasibility, and reproducibility of the results obtained. For this reason, the optimization and development of less intense stress experimental models would fulfill the Refinement concept from the three R’s principle (Replacement, Reduction, and Refinement).

Data availability

Data is provided within the manuscript.

References

  1. Jiang S, Fan HG, Lu DZ, Hou JL, Song XD, Wang Y, et al. Effects of the tiletamine/zolazepam-xylazine-tramadol combination on plasma oxidative status and haematological indicators in miniature pigs. Acta Vet Brno. 2014;83(2):145–9.

    Article  CAS  Google Scholar 

  2. Liu F, Celi P, Chauhan SS, Cottrell JJ, Leury BJ, Dunshea FR. A short-term supranutritional vitamin E supplementation alleviated respiratory alkalosis but did not reduce oxidative stress in heat stressed pigs. Asian-Australasian J Anim Sci. 2018;31(2):263–9.

    Article  CAS  Google Scholar 

  3. Orengo J, Hernández F, Martínez-Miró S, Sánchez CJ, Peres Rubio C, Madrid J. Effects of commercial antioxidants in feed on growth performance and oxidative stress status of weaned piglets. Animals. 2021;11(2):1–13.

    Article  Google Scholar 

  4. Naito Y, Lee M-C, Kato Y, Nagai R, Yonei Y. Oxidative Stress Markers. ANTI-AGING Med [Internet]. 2010;7(5):36–44. https://www.jstage.jst.go.jp/article/jaam/7/5/7_5_36/_article

  5. Cao S, Zhang Q, Wang CC, Wu H, Jiao L, Hong Q, et al. LPS challenge increased intestinal permeability, disrupted mitochondrial function and triggered mitophagy of piglets. Innate Immun. 2018;24(4):221–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lay DC, Kattesh HG, Cunnick JE, Daniels MJ, Kranendonk G, McMunn KA, et al. Effect of prenatal stress on subsequent response to mixing stress and a lipopolysaccharide challenge in pigs. J Anim Sci. 2011;89(6):1787–94.

    Article  CAS  PubMed  Google Scholar 

  7. Dalto DB, Lapointe J, Matte JJ. Assessment of antioxidative and selenium status by seleno-dependent glutathione peroxidase activity in different blood fractions using a pig model: issues for clinical nutrition and research. J Anim Physiol Anim Nutr (Berl). 2018;102(1):184–93.

    Article  CAS  PubMed  Google Scholar 

  8. Vives-Bauza C, Starkov A, Garcia‐Arumi E. Measurements of the antioxidant enzyme activities of superoxide dismutase, catalase, and glutathione peroxidase. Methods Cell Biol [Internet]. 2007:379–93. https://linkinghub.elsevier.com/retrieve/pii/S0091679X06800191

  9. Marrocco I, Altieri F, Peluso I. Measurement and clinical significance of biomarkers of oxidative stress in humans. Oxid Med Cell Longev. 2017;2017.

  10. Liu G, Mo W, Cao W, Wu X, Jia G, Zhao H, et al. Effects of spermine on ileal physical barrier, antioxidant capacity, metabolic profile and large intestinal bacteria in piglets. RSC Adv. 2020;10(45):26709–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Broom DM. Behaviour and welfare in relation to pathology. Appl Anim Behav Sci. 2006;97(1):73–83.

    Article  Google Scholar 

  12. Temple D, Manteca X, Velarde A, Dalmau A. Assessment of animal welfare through behavioural parameters in Iberian pigs in intensive and extensive conditions. Appl Anim Behav Sci. 2011;131(1–2):29–39.

    Article  Google Scholar 

  13. Terlouw C. Stress reactions at slaughter and meat quality in pigs: genetic background and prior experience: a brief review of recent findings. Livest Prod Sci. 2005;94(1–2):125–35.

    Article  Google Scholar 

  14. Martínez-Miró S, Tecles F, Ramón M, Escribano D, Hernández F, Madrid J et al. Causes, consequences and biomarkers of stress in swine: An update. BMC Vet Res [Internet]. 2016;12(1):1–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-016-0791-8

  15. Guevara RD, Pastor JJ, Manteca X, Tedo G, Llonch P. Systematic review of animal-based indicators to measure thermal, social, and immune-related stress in pigs. Óvilo C, editor. PLoS One [Internet]. 2022;17(5):e0266524. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0266524

  16. Cao S, Wu H, Wang C, Zhang Q, Jiao L, Lin F, et al. Diquat-induced oxidative stress increases intestinal permeability, impairs mitochondrial function, and triggers mitophagy in piglets. J Anim Sci. 2018;96(5):1795–805.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Blavi L, Solà-Oriol D, Llonch P, López-Vergé S, Martín-Orúe SM, Pérez JF. Management and feeding strategies in early life to increase piglet performance and welfare around weaning: a review. Animals [Internet]. 2021;11(2):302. https://www.mdpi.com/2076-2615/11/2/302

  18. Yao M, Gao W, Tao H, Yang J, Huang T. The regulation effects of danofloxacin on pig immune stress induced by LPS. Res Vet Sci [Internet]. 2017;110:65–71. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.rvsc.2016.11.005

  19. Kadiiska MB, Peddada S, Herbert RA, Basu S, Hensley K, Jones DP et al. Biomarkers of oxidative stress study VI. Endogenous plasma antioxidants fail as useful biomarkers of endotoxin-induced oxidative stress. Free Radic Biol Med [Internet]. 2015;81:100–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.freeradbiomed.2015.01.006

  20. Ahasan ASMLSML, Invernizzi G, Farina G, Pilotto A, Barbé F, Bontempo V, et al. The effects of superoxide dismutase-rich melon pulp concentrate on inflammation, antioxidant status and growth performance of challenged post-weaning piglets. Animal. 2019;13(1):136–43.

    Article  CAS  PubMed  Google Scholar 

  21. Chen F, Wang H, Chen J, Liu Y, Wen W, Li Y et al. Lactobacillus delbrueckii ameliorates intestinal integrity and Antioxidant Ability in Weaned Piglets after a Lipopolysaccharide Challenge. Oxid Med Cell Longev. 2020;2020.

  22. Luo W, Xu W, Zhang J, Yao J, Xu J. The maternal diet with fish oil might decrease the oxidative stress and inflammatory response in sows, but increase the susceptibility to inflammatory stimulation in their offspring. Animals. 2020;10(9):1–14.

    Article  CAS  Google Scholar 

  23. Zhang Y, Xu BY, Zhao L, Zhu LY, Batonon-Alavo D, Jachacz J et al. Increased consumption of sulfur amino acids by both sows and piglets enhances the ability of the progeny to adverse effects induced by lipopolysaccharide. Animals. 2019;9(12).

  24. Hou X, Zhang J, Ahmad H, Zhang H, Xu Z, Wang T. Evaluation of antioxidant activities of ampelopsin and its protective effect in lipopolysaccharide-induced oxidative stress piglets. PLoS ONE. 2014;9(9).

  25. Li Y, Zhao X, Jiang X, Chen L, Hong L, Zhuo Y et al. Effects of dietary supplementation with exogenous catalase on growth performance, oxidative stress, and hepatic apoptosis in weaned piglets challenged with lipopolysaccharide. J Anim Sci. 2020;98(3).

  26. Yi D, Hou Y, Wang L, Ding B, Yang Z, Li J, et al. Dietary N-acetylcysteine supplementation alleviates liver injury in lipopolysaccharide-challenged piglets. Br J Nutr. 2014;111(1):46–54.

    Article  CAS  PubMed  Google Scholar 

  27. Kang P, Zhang L, Hou Y, Ding B, Yi D, Wang L, et al. Effects of l-proline on the growth performance, and blood parameters in weaned lipopolysaccharide (LPS)-challenged pigs. Asian-Australasian J Anim Sci. 2014;27(8):1150–6.

    Article  CAS  Google Scholar 

  28. Wang L, Hou Y, Yi D, Li Y, Ding B, Zhu H et al. Dietary supplementation with glutamate precursor α-ketoglutarate attenuates lipopolysaccharide-induced liver injury in young pigs. Amino Acids [Internet]. 2015;47(7):1309–18. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00726-015-1966-5

  29. Cao ST, Wang CC, Wu H, Zhang QH, Jiao LF, Hu CH. Weaning disrupts intestinal antioxidant status, impairs intestinal barrier and mitochondrial function, and triggers mitophagy in piglets. J Anim Sci. 2018;96(3):1073–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cai L, Li YP, Wei ZX, Li XL, Jiang XR. Effects of dietary gallic acid on growth performance, diarrhea incidence, intestinal morphology, plasma antioxidant indices, and immune response in weaned piglets. Anim Feed Sci Technol. 2020;261(January).

  31. Sun LH, Pi DA, Zhao L, Wang XY, Zhu LY, Qi DS, et al. Response of Selenium and Selenogenome in Immune tissues to LPS-Induced inflammatory reactions in pigs. Biol Trace Elem Res. 2017;177(1):90–6.

    Article  CAS  PubMed  Google Scholar 

  32. Russell, W. M. S., and Burch, R. L. (1960). The Principles of Humane Experimental Technique. Med. J. Aust. 1, 500–500. https://doiorg.publicaciones.saludcastillayleon.es/10.5694/j.1326-5377.1960.tb73127.x.

  33. National Research Council. Nutrient requirements of swine. Nutrient requirements of Swine. National Academies; 2012.

  34. Hu R, He Z, Liu M, Tan J, Zhang H, Hou DX et al. Dietary protocatechuic acid ameliorates inflammation and up-regulates intestinal tight junction proteins by modulating gut microbiota in LPS-challenged piglets. J Anim Sci Biotechnol. 2020;11(1).

  35. Islam MA, Uddin MJ, Tholen E, Tesfaye D, Looft C, Schellander K et al. Age-associated differential production of IFN-γ, IL-10 and GM-CSF by porcine alveolar macrophages in response to lipopolysaccharide. Vet J [Internet]. 2013;198(1):245–51. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.tvjl.2013.07.026

  36. Sies H. Total Antioxidant capacity: appraisal of a concept. J Nutr [Internet]. 2007;137(June):1493–1495. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jn/137.6.1493

  37. Foury A, Geverink NA, Gil M, Gispert M, Hortós M, Font I, Furnols M, et al. Stress neuroendocrine profiles in five pig breeding lines and the relationship with carcass composition. Animal. 2007;1(7):973–82.

    Article  CAS  PubMed  Google Scholar 

  38. Nguyen HN, McPhee CP. Selection for efficient lean growth on restricted feeding in large white pigs [Internet]. Ho Chi Minh City; 2001. https://era.daf.qld.gov.au/id/eprint/252/

  39. Cámara L, Berrocoso JD, Coma J, López-Bote CJ, Mateos GG. Growth performance and carcass quality of crossbreds pigs from two Pietrain sire lines fed isoproteic diets varying in energy concentration. Meat Sci [Internet]. 2016;114:69–74. https://linkinghub.elsevier.com/retrieve/pii/S0309174015301509

  40. Wei S, Xia D, Li L, Xiao J, Bao J, Parvizi N et al. Breed-specific expression of hippocampal 11β-hydroxysteroid dehydrogenases and glucocorticoid receptors in Erhualian and Pietrain pigs exhibiting distinct stress coping characteristics. Livest Sci [Internet]. 2010;131(2–3):240–4. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.livsci.2010.04.007

  41. Brubacher JL, DeWitte-Orr SJ, Zorzitto JR, Playle RC, Bols NC. Redox-active metals in commercial preparations of lipopolysaccharide: implications for studies of cellular responses to bacterial products. Cell Microbiol. 2003;5(4):233–43.

    Article  CAS  PubMed  Google Scholar 

  42. Hu R, He Z, Liu M, Tan J, Zhang H, Hou DX, et al. Dietary protocatechuic acid ameliorates inflammation and up-regulates intestinal tight junction proteins by modulating gut microbiota in LPS-challenged piglets. J Anim Sci Biotechnol. 2020;11(1):1–12.

    Article  Google Scholar 

  43. Koo B, Choi J, Holanda DM, Yang C, Nyachoti CM. Comparative effects of dietary methionine and cysteine supplementation on redox status and intestinal integrity in immunologically challenged-weaned pigs. Amino Acids. 2023;55(1):139–52.

    Article  CAS  PubMed  Google Scholar 

  44. Špinka M. How important is natural behaviour in animal farming systems? Appl Anim Behav Sci. 2006;100(1–2):117–28.

    Article  Google Scholar 

  45. Stygar AH, Gómez Y, Berteselli GV, Dalla Costa E, Canali E, Niemi JK, et al. A systematic review on commercially available and Validated Sensor Technologies for Welfare Assessment of Dairy Cattle. Front Vet Sci. 2021;8(March):1–15.

    Google Scholar 

Download references

Acknowledgements

The current study and experimental trials expenses were covered by Lucta’s Animal Science Innovation Unit. The authors would like to thank the farm staff, animal caretakers, and veterinarians who performed the study at the experimental farm led by Carles Colom. Also, to acknowledge and reward Lucta’s laboratory staff, Almudena Martínez, and Marc Vujadinovic, who oversaw running all laboratory analyses required to obtain the results of the trials presented in the current manuscript.

Funding

The study was supported by Lucta SA. PL received funding from the Ramon y Cajal program of the Spanish Ministry of Science (Spain; Grant Ref. RYC2020-029067-I).

Author information

Authors and Affiliations

  1. Animal Welfare Education Centre (AWEC) Advisors S.L., , Research Park UAB, Campus UAB, Cerdanyola del Valles, 08193, Spain

    Raúl David Guevara

  2. Department of Animal and Food Science, Universitat Autònoma de Barcelona, Edifici V, Travessera dels Turons, Cerdanyola del Valles, 08193, Spain

    Raúl David Guevara, Xavier Manteca & Pol Llonch

  3. Animal Science Innovation Division, Lucta, Cerdanyola del Vallès, Spain

    Jose J. Pastor, Sergi López-Vergé & Gemma Tedo

Authors
  1. Raúl David Guevara
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Jose J. Pastor
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Sergi López-Vergé
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Xavier Manteca
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Gemma Tedo
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Pol Llonch
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

RDG, JJP, SLV, XM, GT, and PL contributed to the conception and design of the study. RDG, JJP and SLV performed the study. RDG, JJP and SLV organized the database and performed the statistical analysis. RDG wrote the first draft of the manuscript (including Figures and tables). All authors contributed to the article and approved the submitted version.

Corresponding author

Correspondence to Raúl David Guevara.

Ethics declarations

Ethics approval and consent to participate

All procedures were approved by the Laboratory Animal Care Advisory Committee of the Universitat Autònoma de Barcelona (CEA/9310/P1).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

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

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guevara, R.D., Pastor, J.J., López-Vergé, S. et al. Physiology, gene expression, and behavior as potential indicators of oxidative stress in piglets. BMC Vet Res 20, 471 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-024-04320-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-024-04320-4

Keywords

BMC Veterinary Research

ISSN: 1746-6148

Contact us