Raquel Peña

Identification of serum stress biomarkers in pigs housed at different stocking densities.

Eight Duroc×(Landrace×Large White) male pigs housed at a stocking rate of 0.50m(2)/pig were subjected to a higher stocking rate of 0.25m(2)/pig (higher density, HD) for two 4-day periods over 26 days. Using biochemical and proteomic techniques serum and plasma samples were examined to identify potential biomarkers for monitoring stress due to HD housing. HD housed pigs showed significant differences (P<0.001) in total cholesterol and low density lipoprotein-associated cholesterol, as well as in concentrations of the pig-major acute phase protein (Pig-MAP) (P=0.002). No differences were observed in serum cortisol or other acute phase proteins such as haptoglobin, C-reactive protein or apolipoprotein A-I. HD-individuals also showed an imbalance in redox homeostasis, detected as an increase in the level of oxidized proteins measured as the total plasma carbonyl protein content (P<0.001) with a compensatory increase in the activity of the antioxidant enzyme glutathione peroxidase (P=0.012). Comparison of the serum proteome yielded a new potential stress biomarker, identified as actin by mass spectrometry. Cluster analysis of the results indicated that individuals segregated into two groups, with different response patterns, suggesting that the stress response depended on individual susceptibility.

Establishment of the biochemical and endocrine blood profiles in the Majorera and Palmera dairy goat breeds: the effect of feed restriction

Feed restriction, and seasonal weight loss (SWL), are major setbacks for animal production in the tropics and the Mediterranean. They may be solved through the use of autochthonous breeds particularly well adapted to SWL. It is therefore of major importance to determine markers of tolerance to feed restriction of putative use in animal selection. Two indigenous breeds from the Canary Islands, Palmera and Majorera, are commonly used by dairy goat farmers and, interestingly, have different phenotype characteristics albeit with a common ancestry. Indeed, Majorera is well adapted to feed restriction whereas the Palmera is susceptible to feed restriction. In addition, regardless of their importance in dairy production, there are only a limited number of reports relating to these breeds and, to the best of our knowledge, there is no description of their blood metabolite standard values under control conditions or as affected by feed restriction. In this study we analysed the blood metabolite profiles in Majorera and Palmera goats aiming to establish the differential responses to feed restriction between the two breeds and to characterise their metabolite standard values under control conditions. We observed significant differences in creatinine, urea, non-esterified fatty acids (NEFAs), cholesterol, IGF-1 and T3 due to underfeeding. Furthermore, a PCA analysis, revealed that animals submitted to undernutrition could be distinguished from the control groups, with the formation of three separate clusters (Palmera individuals after 22 d of subnutrition (PE22); Majorera individuals after 22 d of subnutrition (ME22) and animals assigned to control conditions (MC0, MC22, PC0 and PC22)), highlighting different responses of the two breeds to undernutrition.

Biochemical and proteomic analyses of the physiological response induced by individual housing in gilts provide new potential stress markers

BackgroundThe objective assessment of animal stress and welfare requires proper laboratory biomarkers. In this work, we have analyzed the changes in serum composition in gilts after switching their housing, from pen to individual stalls, which is generally accepted to cause animal discomfort.ResultsBlood and saliva samples were collected a day before and up to four days after changing the housing system. Biochemical analyses showed adaptive changes in lipid and protein metabolism after the housing switch, whereas cortisol and muscular markers showed a large variability between animals. 2D-DIGE and iTRAQ proteomic approaches revealed variations in serum protein composition after changing housing and diet of gilts. Both techniques showed alterations in two main homeostatic mechanisms: the innate immune and redox systems. The acute phase proteins haptoglobin, apolipoprotein A-I and α1-antichymotrypsin 3, and the antioxidant enzyme peroxiredoxin 2 were found differentially expressed by 2D-DIGE. Other proteins related to the innate immune system, including lactotransferrin, protegrin 3 and galectin 1 were also identified by iTRAQ, as well as oxidative stress enzymes such as peroxiredoxin 2 and glutathione peroxidase 3. Proteomics also revealed the decrease of apolipoproteins, and the presence of intracellular proteins in serum, which may indicate physical injury to tissues.ConclusionsHousing of gilts in individual stalls and diet change increase lipid and protein catabolism, oxidative stress, activate the innate immune system and cause a certain degree of tissue damage. We propose that valuable assays for stress assessment in gilts may be based on a score composed by a combination of salivary cortisol, lipid metabolites, innate immunity and oxidative stress markers and intracellular proteins.

C-reactive protein, haptoglobin and Pig-Major acute phase protein profiles of pigs infected experimentally by different isolates of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiologic agent of PRRS, one of the most important diseases in swine worldwide. In the present work, the effects of different PRRSV strains were tested on a piglet experimental model to study the induced acute phase response. For this purpose, pigs (n=15 for each group) were intranasally inoculated with one of five PRRSV strains (isolates EU10, 12, 17, 18 from genotype 1 and isolate JA-142 from genotype 2). The acute phase response was monitored by measuring acute phase proteins (APPs). Specifically, the serum concentration of haptoglobin (Hp), C-reactive protein (CRP) and Pig-Major Acute Protein (Pig-MAP) was determined at 0, 3, 6, 9, 12, 15, 18 and 21 days p.i. Clinical signs and growth performance were also monitored during the experiment. All animals became viremic after inoculation during the study period. The APP response was heterogeneous and dependent on the strain, being strains EU10, EU 18 and JA-142 those that induced the highest response and the strongest clinical signs. In general, Hp was the most sensitive biomarker for PRRSV infection, CRP behaved as moderate and Pig-MAP was the less responsive during the course of PRRSV experimental infection. Hp and CRP were significantly discriminatory between infected and control pigs, but not Pig-MAP.

A new automated turbidimetric immunoassay for the measurement of canine C‐reactive protein

BACKGROUND In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. OBJECTIVES The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. METHODS A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). RESULTS The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. CONCLUSIONS The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use.

Effects on pig immunophysiology, PBMC proteome and brain neurotransmitters caused by group mixing stress and human-animal relationship

Peripheral blood mononuclear cells (PBMC) are an interesting sample for searching for biomarkers with proteomic techniques because they are easy to obtain and do not contain highly abundant, potentially masking proteins. Two groups of pigs (n = 56) were subjected to mixing under farm conditions and afterwards subjected to different management treatments: negative handling (NH) and positive handling (PH). Serum and PBMC samples were collected at the beginning of the experiment one week after mixing (t0) and after two months of different handling (t2). Brain areas were collected after slaughter and neurotransmitters quantified by HPLC. Hair cortisol and serum acute phase proteins decreased and serum glutathione peroxidase increased at t2, indicating a lower degree of stress at t2 after adaptation to the farm. Differential gel electrophoresis (DIGE) was applied to study the effects of time and treatment on the PBMC proteome. A total of 54 differentially expressed proteins were identified, which were involved in immune system modulation, cell adhesion and motility, gene expression, splicing and translation, protein degradation and folding, oxidative stress and metabolism. Thirty-seven protein spots were up-regulated at t2 versus t0 whereas 27 were down-regulated. Many of the identified proteins share the characteristic of being potentially up or down-regulated by cortisol, indicating that changes in protein abundance between t0 and t2 are, at least in part, consequence of lower stress upon adaptation to the farm conditions after group mixing. Only slight changes in brain neurotransmitters and PBMC oxidative stress markers were observed. In conclusion, the variation in hair cortisol and serum APPs as well as the careful analysis of the identified proteins indicate that changes in protein composition in PBMC throughout time is mainly due to a decrease in the stress status of the individuals, following accommodation to the farm and the new group.

Effect of handling on neurotransmitter profile in pig brain according to fear related behaviour

Chemical neurotransmitters (NT) are principal actors in all neuronal networks of animals. The central nervous system plays an important role in stress susceptibility and organizes the response to a stressful situation through the interaction of the dopaminergic and the serotonergic pathways, leading to the activation of the hypothalamus-pituitary-adrenal axis (HPA). This study was designed to investigate: a) the effects of stressful handling of pigs at the slaughterhouse on the neurotransmitter profile in four brain areas: amygdala, prefrontal cortex (PFC), hippocampus and hypothalamus, and b) whether the alterations in the brain NT profile after stressful handling were associated with fear, determined by the tonic immobility (TI) test. In the first place, the characterization of the NT profile allowed to distinguish the four brain areas in a principal component analysis. The most crucial pathway involved in the reaction of pigs to a stressful handling was the serotonergic system, and changes were observed in the amygdala with a decrease in serotonin (5-HT) and total indoleamines, and in the hippocampus, where this pathway was activated. Fearful and non-fearful pigs did not show significant differences in their NT profile in control conditions, but when subjected to a stressful handling in the slaughterhouse, fearful animals showed a significant variation in the serotonin pathway and, in a lesser extent, the dopamine (DA) pathway. In conclusion, the existence of an underlying biological trait - possibly fearfulness - may be involved in the pigs response toward stressful challenges, and the serotonergic system seems to play a central role in this response.

Pig cognitive bias affects the conversion of muscle into meat by antioxidant and autophagy mechanisms.

Slaughter is a crucial step in the meat production chain that could induce psychological stress on each animal, resulting in a physiological response that can differ among individuals. The aim of this study was to investigate the relationship between an animals emotional state, the subsequent psychological stress at slaughter and the cellular damage as an effect. In all, 36 entire male pigs were reared at an experimental farm and a cognitive bias test was used to classify them into positive bias (PB) or negative bias (NB) groups depending on their decision-making capabilities. Half of the animals, slaughtered in the same batch, were used for a complete study of biomarkers of stress, including brain neurotransmitters and some muscle biomarkers of oxidative stress. After slaughter, specific brain areas were excised and the levels of catecholamines (noradrenaline (NA) and dopamine (DA)) and indoleamines (5-hydroxyindoleacetic acid and serotonin (5HT)) were analyzed. In addition, muscle proteasome activity (20S), antioxidant defence (total antioxidant activity (TAA)), oxidative damage (lipid peroxidation (LPO)) and autophagy biomarkers (Beclin-1, microtubule-associated protein I light chain 3 (LC3-I) and LC3-II) were monitored during early postmortem maturation (0 to 24 h). Compared with PB animals, NB pigs were more susceptible to stress, showing higher 5HT levels (P<0.01) in the hippocampus and lower DA (P<0.001) in the pre-frontal cortex. Furthermore, NB pigs had more intense proteolytic processes and triggered primary muscle cell survival mechanisms immediately after slaughter (0 h postmortem), thus showing higher TAA (P<0.001) and earlier proteasome activity (P<0.001) and autophagy (Beclin-1, P<0.05; LC3-II/LC3-I, P<0.001) than PB pigs, in order to counteract the induced increase in oxidative stress, that was significantly higher in the muscle of NB pigs at 0 h postmortem (LPO, P<0.001). Our study is the first to demonstrate that pigs cognitive bias influences the animals susceptibility to stress and has important effects on the postmortem muscle metabolism, particularly on the cell antioxidant defences and the autophagy onset. These results expand the current knowledge regarding biomarkers of animal welfare and highlight the potential use of biomarkers of the proteasome, the autophagy (Beclin-1, LC3-II/LC3-I ratio) and the muscle antioxidant defence (TAA, LPO) for detection of peri-slaughter stress.

Neurotransmitter levels and proteomic approach in pig brain: pre-slaughter handling stress and cognitive biases

Stress can be defined as a brain-body reaction towards stimuli arising from the environment or from internal cues that are interpreted as a disruption of homeostasis. The organization of the response to a stressful situation involves the activity of several areas of the limbic system through neurotransmitters synthesis (Mora et al., 2012). Changes in a neurotransmitter’s concentrations are also related to the activation and modulation of behavioral processes and autonomic response (Herman et al. 2005).

Oxidative stress and acute phase response associated to changes in housing from pen to individual stalls in reproductive sows

Under field conditions, gilts are usually housed in pens of 10–15 sows by group, when they arrive into the production facility from the multiplication unit. After spending there the quarantine period, they are moved into the mating room, where they are housed in small and individual stalls where the mating process takes place. This individual housing system could be stressful for the animals [1]. The aims of our study were twofold. The first one is to confirm that this change of housing from pen to stalls is really stressful for gilts and the second one is to find new welfare/chronic stress markers if it were possible in this experimental setting.