| This study was aimed to screen and validate the existing and potential stress-related protein biomarkers in the swine serum and liver, to help avoid stresses and improve animal welfare in the animal husbandry production. Healthy weaning DLW (DurocxLandrace×Large White) and Huai-breed pigs as well as Large White and Erhualian pigs were selected to do the breed comparison of the physiological and biochemical indexes, serum acute phase proteins(APP) expression, serum cortisol and TNF-a concentrations. APPs’gene and protein expression levels, serum cortisol and TNF-a concentrations were also analyzed to further understand their changes under the animal stress models induced by ACTH or LPS injection. Meanwhile, we adopted2-DE/MALDI-TOF mass spectrometry method to explore the potential biomarkers may exist in the porcine liver and serum under these models.1Comparison of APP and other stress-related indexes between different breedsLiver and serum samples were taken from weaned (25d) Large White and Erhualian pigs as well as DLW and Huai-breed pigs. APPs’gene and protein expression, serum cortisol and TNF-a concentrations and routine blood indexes were detected using relative quantitative real time RT-PCR, Enzyme linked immunosorbent assay(Elisa), Radioimmunoassay(RIA) and automatic biochemical analyzer(Olympus AU2700), respectively. The results showed that a higher cortisol and much lower levels TNF-a (P<0.01) were seen in Chinese local breeds than imported pigs. HP gene expression has a higher tendency (P=0.093) in EHL than in LW pigs. At protein expression level, HP exhibits both breeds difference and growing stage difference (P<0.05), Pig-MAP showed a difference between two growing stages (P<0.05). EHL pigs’HP>Pig-MAP and TTR plasma concentrations showed significant higher than LW pigs (P<0.05). RBC, HCT and MCH showed significant differences between two breeds and growing stages, lymphocyte and neutrophil counts varied between different breeds (P<0.01), and Platelet differenced between two growing stages (P<0.05), all of which showed a significantly higher level in Huai-breed pigs. HDL-C of Huai-breed pigs was significantly higher than that of DLW pigs, while low density lipoprotein cholesterol(LDL-C) show a converse trend which is also higher in growth stage (P<0.01). Concerning the growing stages, the content of serum GSH-PX and SOD were significantly higher in growing period than that in finishing period (P<0.01). In addition, correlation analysis showed that CRP, HP and Pig-MAP had a significant correlation between each other and Pig-MAP was strongly correlated with HDL-C (P<0.05). RBC and HCT counts were strongly correlated with each other (P<0.05) and still with cortisol concentration (r=0.38,0.47, P<0.05). The above results revealed Chinese local breed pigs will suffer a much less stress than imported breeds under the same kind of breeding conditions and have their advantages in the substances’metabolism like cholesterol, et al. Otherwise, the data demonstrated the practicality of indexes such as cortisol, TNF-a, HP and Pig-MAP, provided new theory evidences for the further study of physiological characteristics of different varieties of pigs.2Detection of stress-related indexes and hepatic proteomics study of pigs injected with ACTHSixteen DLW pigs of15-20kg were divided into two groups, experimental group pigs were im. injected with1IU/kg BW ACTH at9:00am and16:00pm each day for one week long and control group were dealt with same volume of saline. Samples were collected on day8post the final injection. APPs’gene and protein expression, serum cortisol and TNF-a concentrations analyzed by using RT-PCR, Elisa and RIA techniques. The data revealed that APPs’gene expression level coupled with serum cortisol and TNF-a concentrations stay peaceful after the stimulation, and no serum APP concentration except HP (P<0.01) was changed post ACTH injection.2-DE and MALDI-TOF techniques were employed to further analysis the protein expression profile of porcine liver.32out of33discrepant protein spots were positively identified, they represented27different proteins which were involved in material metabolism or transportation, cells’function and growth cycle regulation and immune defenses, etc. Revealing the happening of a series of physiological changes under the long-term chronic stress, provided new biological indicators for the animals’welfare and health monitoring and the data support for the follow-up scientific studies.3Proteomics study of porcine serum after LPS infectionTwelve weaned male DLW piglets (28d) were employed in the present study, control group pigs were i.m. injected with2mL of saline and LPS group was suffered to2mL of saline containing15μg/kg BW of E.Coli serotype lipopolysaccharide.6hour after infection, pigs were sacrificed for sampling. APPs’gene and protein expression as well as serum cortisol and TNF-a concentrations were analyzed by RT-PCR, Elisa and RIA. The results showed that gene expressions of CRP, HP and Pig-MAP were significantly up-regulated and TTR was down-regulated (P<0.001), serum concentrations of CRP, HP, TNF-a and cortisol raised notablely (P<0.05) post LPS infection. Meanwhile, body surface temperature and serum creatinine content elevated markedly (P<0.05), yet seralbumin showed a decreased tendency (P=0.076). These indexes demonstrated severe physiological reactions were induced by the LPS stimulation. Protein expression profiles were gained through2-DE/MALDI-TOF analysis,10different proteins were identified from18protein spots differed between two groups. Most of them belong to the classical APP definitive category. In addition, a new stress-related biomarker, ZAG, was reported for the first time, whose concentration will raise by more than2-fold with the LPS induction, hinted that ZAG can be regarded as another potential biomarker under stress conditions, but more further researches are needed to get new insight into the ZAG regulated mechanism. |
PDF Full Text Request