Yolanda Saco

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.

Effects of trailer design on animal welfare parameters and carcass and meat quality of three Pietrain crosses being transported over a long distance.

This study aimed at evaluating the effects of trailer design on stress responses and meat quality traits of 3 different pig crosses: 50% Pietrain breeding with halothane (HAL)(Nn) (50Nn); 50% Pietrain breeding with HAL(NN) (50NN); and 25% Pietrain breeding with HAL(NN) genotype (25NN). Over a 6-wk period, pigs (120 pigs/crossbreed) were transported for 7 h in either a pot-belly (PB) or flat-deck (FD) trailer (10 pigs/crossbreed(-1)·trailer(-1)·wk(-1)). Temperature (T) and relative humidity (RH) were monitored in each trailer. Behaviors during loading and unloading, time to load and unload, and latency to rest in lairage were recorded, whereas a sub-population of pigs (4 pigs/crossbreed(-1)·trailer(-1)·wk(-1)) was equipped with gastro-intestinal tract (GIT) temperature monitors. Blood samples were collected at exsanguination for measurement of cortisol, creatine kinase (CK), lactate, haptoglobin, and Pig-MAP concentrations. Meat quality data were collected at 24 h postmortem from the LM and semimembranosus (SM) and adductor (AD) muscles of all 360 pigs. Greater T were recorded in the PB trailer during transportation (P = 0.006) and unloading (P < 0.001). Delta GIT temperature was greater (P = 0.01) in pigs unloaded from the PB. At loading, pigs tended to move backwards more (P = 0.06) when loaded on the FD than the PB trailer. At unloading, an interaction was found between trailer type and crossbreed type, with a greater (P < 0.01) frequency of overlaps in 50NN and 25NN pigs and slips/falls in 50Nn and 50NN pigs from the FD than the PB trailer. Cortisol concentrations at slaughter were greater (P = 0.02) in pigs transported in the PB than FD trailer. Greater lactate concentrations were found in 50Nn and 50NN pigs (P = 0.003) and greater CK concentrations (P < 0.001) in 50Nn pigs. As expected, 50Nn pigs produced leaner (P < 0.001) carcasses, with greater (P = 0.01) dressing percentages, as well as lower (P < 0.001) ultimate pH values and greater (P < 0.001) drip loss percentages in the LM and greater (P = 0.002) drip losses and a paler color (greater L* values, P = 0.02) in the SM than 50NN pigs. When used for long distance transportation under controlled conditions, the PB trailer produced no detrimental effects on animal welfare or pork quality. Pigs with 50% Pietrain crossbreeding appear to be more responsive to transport stress, having the potential to produce acceptable carcass and pork quality, provided pigs are free of the HAL gene.

Evaluation of serum haptoglobin and C-reactive protein in dogs with mammary tumors.

BACKGROUND In veterinary medicine, there is increasing interest in measuring acute phase proteins as a tool in the diagnosis and monitoring of neoplastic diseases. Although mammary neoplasms are the most common type of cancer in dogs, acute phase proteins have not been extensively evaluated in dogs with mammary tumors. OBJECTIVES The aim of this study was to evaluate serum haptoglobin (Hp) and C-reactive protein (CRP) concentrations in the dogs with mammary tumors and assess their potential association with malignancy. METHODS A retrospective study of dogs with mammary tumors was performed. Serum concentrations of CRP and Hp were determined in healthy control dogs (n=20) and dogs with mammary tumors before surgery (n=41). Mammary tumors were grouped as carcinomas (n=24), fibrosarcoma (n=1), malignant mixed tumors (n=7), benign mixed tumors (n=6), and adenomas (n=3). CRP and Hp concentrations were compared in dogs with different tumor types and were also compared based on tumor size, lymph node infiltration, skin ulceration, fixation to underlying tissue, and time between tumor identification and removal. RESULTS Hp concentration was significantly (P<.043) higher in dogs with mammary tumors (median 2.03 g/L, range 0.09-2.94 g/L) compared with controls (1.38 g/L, range 0.08-3.00 g/L), but the range of values overlapped considerably. CRP concentration was higher in dogs with carcinomas (4.70 mg/L, range 0.63-128.96 mg/L) vs controls (2.11 mg/L, range 0.25-6.57 mg/L) (P=.0008) and in dogs with ulcerated skin (14.8 mg/L, range 5.7-128.9 mg/L, n=3) compared with those without ulceration (2.4 mg/L, range 0.11-30.3 mg/L, n=38) (P=.048). CONCLUSIONS Serum Hp and CRP do not appear to have value in diagnosing or predicting malignancy of mammary tumors in dogs. Higher CRP concentrations in dogs with mammary carcinoma suggest a role for inflammation in this tumor type.

Plasma protein electrophoresis of Trachemys scripta and Iguana iguana

BACKGROUND Protein electrophoresis is widely applied in veterinary medicine, but is not used often in reptiles, in part because of lack of reference values. OBJECTIVE The goals of this study were to compare plasma protein profiles obtained by cellulose acetate electrophoresis (CAE) and agarose gel electrophoresis (AGE), measure precision and examine interference by sample hemolysis, and establish preliminary reference intervals for 2 reptile species. METHODS Heparinized plasma samples from healthy and diseased adult female Iguana iguana (n=40) and Trachemys scripta (n=60) were analyzed by CAE and AGE. Total protein concentration was measured by the biuret method. Electrophoresis results were compared using Bland-Altman plots and Passing-Bablok regression analysis. Precision and the effects of sample hemolysis were determined. Results from clinically healthy animals were used to determine reference intervals. RESULTS Five protein fractions were identified in both species, with bisalbuminemia observed in 23/40 iguanas. High correlation was observed between the 2 methods for all fractions, with few proportional and systematic errors. Coefficients of variation were lower using AGE vs CAE and for I. iguana vs T. scripta. Two additional bands were observed in hemolyzed samples from T. scripta; 1 additional band was observed for I. iguana. Minimum and maximum values were reported for healthy I. iguana (n=14) and T. scripta (n=22). CONCLUSIONS Although both methods are acceptable, the performance of AGE was slightly better than that of CAE for analysis of plasma from reptiles. Furthermore, reptile electrophoretic patterns should be interpreted based on the method used, the species analyzed, and the quality of the plasma sample.

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems.

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions). In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system. Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR). Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions.

Plasma iron, C-reactive protein, albumin, and plasma fibrinogen concentrations in dogs with systemic inflammatory response syndrome.

OBJECTIVE To investigate the diagnostic and prognostic value over time of plasma iron compared with the inflammatory markers albumin, C-reactive protein (CRP), and fibrinogen in dogs with systemic inflammatory response syndrome (SIRS). DESIGN Prospective observational study of sequentially enrolled dogs. SETTING ICU of a veterinary teaching hospital. ANIMALS One hundred and sixteen client-owned dogs: 54 dogs with SIRS or sepsis, 42 with focal inflammation, and 20 clinically healthy dogs. MEASUREMENTS AND MAIN RESULTS Blood samples were obtained on admission in all study groups, and then on alternate days until discharge or death in both inflammation groups. On admission, dogs with SIRS had significantly lower plasma iron (65 ± 5.8 μg/dL, P = 0.001) concentrations than dogs with focal inflammation (89.5 ± 6.2 μg/dL, P = 0.001). Plasma iron, albumin, and CRP effectively discriminated the SIRS/sepsis group from those presenting with focal inflammation with areas under the curve for the receiver operating curves of 0.679, 0.834, and 0.704, respectively. The admission values for these variables did not discriminate survivors from nonsurvivors within the SIRS/sepsis group. However, the magnitude of increase in iron concentration and the decrease in CRP concentration from admission to hospital discharge was higher in survivors than in nonsurvivors within the SIRS/septic group (22.8 vs. 2.51 μg/dL, respectively, P = 0.021 for iron; -67.1 vs. -4.1 mg/L, respectively, P = 0.002 for CRP), resulting in iron and CRP concentrations at hospital discharge for survivors similar to those in the focal inflammation group. CONCLUSION Hypoferremia is a sensitive marker of systemic inflammation in dogs. In this study, the increase in iron concentrations during the hospitalization period of SIRS/septic dogs was associated with a better prognosis, suggesting that plasma iron in combination with CRP and albumin concentrations might be used to monitor dogs with inflammatory disease processes.Objective To investigate the diagnostic and prognostic value over time of plasma iron compared with the inflammatory markers albumin, C-reactive protein (CRP), and fibrinogen in dogs with systemic inflammatory response syndrome (SIRS). Design Prospective observational study of sequentially enrolled dogs. Setting ICU of a veterinary teaching hospital. Animals One hundred and sixteen client-owned dogs: 54 dogs with SIRS or sepsis, 42 with focal inflammation, and 20 clinically healthy dogs. Measurements and Main Results Blood samples were obtained on admission in all study groups, and then on alternate days until discharge or death in both inflammation groups. On admission, dogs with SIRS had significantly lower plasma iron (65 ± 5.8 μg/dL, P = 0.001) concentrations than dogs with focal inflammation (89.5 ± 6.2 μg/dL, P = 0.001). Plasma iron, albumin, and CRP effectively discriminated the SIRS/sepsis group from those presenting with focal inflammation with areas under the curve for the receiver operating curves of 0.679, 0.834, and 0.704, respectively. The admission values for these variables did not discriminate survivors from nonsurvivors within the SIRS/sepsis group. However, the magnitude of increase in iron concentration and the decrease in CRP concentration from admission to hospital discharge was higher in survivors than in nonsurvivors within the SIRS/septic group (22.8 vs. 2.51 μg/dL, respectively, P = 0.021 for iron; −67.1 vs. −4.1 mg/L, respectively, P = 0.002 for CRP), resulting in iron and CRP concentrations at hospital discharge for survivors similar to those in the focal inflammation group. Conclusion Hypoferremia is a sensitive marker of systemic inflammation in dogs. In this study, the increase in iron concentrations during the hospitalization period of SIRS/septic dogs was associated with a better prognosis, suggesting that plasma iron in combination with CRP and albumin concentrations might be used to monitor dogs with inflammatory disease processes.

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.

Validation of an immunoturbidimetric method for determination of porcine serum C-reactive protein.

Measurement of porcine C-reactive protein (CRP) in serum is an important tool for monitoring health and welfare in pigs. In this study, an immunoturbidimetric method from Olympus System Reagent (OSR 6147) used to measure human CRP in serum that employ a human traceable calibrator has been evaluated in porcine serum samples. Intra- and inter-assay imprecision were lower than that obtained with the porcine-specific commercially available ELISA. The expected difference in serum CRP between healthy and non-healthy pigs was detected. CRP values measured by the immunoturbidimetric method showed a good correlation with those obtained by ELISA, although differences in absolute CRP values were observed. When an in-house porcine standard was used a better agreement was obtained. In conclusion, the immunoturbidimetric method of Olympus can be used with porcine samples. The easier use of this method should facilitate the implementation of CRP serum determination for diagnostic and prognostic purposes in swine medicine. The results emphasize the need to establish species-specific standard and methods to decrease inter-laboratory discrepancies.

Association between plasma metabolites and gene expression profiles in five porcine endocrine tissues

BackgroundEndocrine tissues play a fundamental role in maintaining homeostasis of plasma metabolites such as non-esterified fatty acids and glucose, the levels of which reflect the energy balance or the health status of animals. However, the relationship between the transcriptome of endocrine tissues and plasma metabolites has been poorly studied.MethodsWe determined the blood levels of 12 plasma metabolites in 27 pigs belonging to five breeds, each breed consisting of both females and males. The transcriptome of five endocrine tissues i.e. hypothalamus, adenohypophysis, thyroid gland, gonads and backfat tissues from 16 out of the 27 pigs was also determined. Sex and breed effects on the 12 plasma metabolites were investigated and associations between genes expressed in the five endocrine tissues and the 12 plasma metabolites measured were analyzed. A probeset was defined as a quantitative trait transcript (QTT) when its association with a particular metabolic trait achieved a nominal P value < 0.01.ResultsA larger than expected number of QTT was found for non-esterified fatty acids and alanine aminotransferase in at least two tissues. The associations were highly tissue-specific. The QTT within the tissues were divided into co-expression network modules enriched for genes in Kyoto Encyclopedia of Genes and Genomes or gene ontology categories that are related to the physiological functions of the corresponding tissues. We also explored a multi-tissue co-expression network using QTT for non-esterified fatty acids from the five tissues and found that a module, enriched in hypothalamus QTT, was positioned at the centre of the entire multi-tissue network.ConclusionsThese results emphasize the relationships between endocrine tissues and plasma metabolites in terms of gene expression. Highly tissue-specific association patterns suggest that candidate genes or gene pathways should be investigated in the context of specific tissues.