The experiment was conducted in accordance with the good animal practices requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. This study was approved by the Institutional Animal Care and Use Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS (Approval No. LIHPSACUC2011-012).
Case definition of laminitis
A total of 4680 Holstein cows were selected from May to August in 2011in the Reproduction and Breeding Demonstration Center of Chinese Holstein Dairy Cow of Gansu Province, which was the member of the Chinese National technological system of dairy industry. Laminitis was identified with at least one of the following claw horn lesions: double sole, solar ulcer, solar hemorrhage, white line disease, and solar abscess . A case of cow usually has the following clinical signs: redness, heat, pain, or sensitivity to percussion, and swelling of the lamellar hoof, as well as the corresponding systemic symptoms, which are diagnosed by hoof trimmers and veterinarians. A total of 36 adult cows in lactation with acute laminitis were selected as a sick group from the dairy herds during the routine herd trimming, with at least one hoof suffering from laminitis. A total of 15 healthy dairy cows with no evident clinical signs of other diseases were included as a control group. All of the cows enrolled in the study aged 3–5 years, were around 400 kg in body weight, and had not received any drug treatment for 1 month before trials. All procedures were followed up by a veterinary assistance and according to the corresponding ethical and animal welfare guidelines.
Collection and selection of blood samples
Blood samples were withdrawn from the jugular vein by cava venepuncture using a 16-gauge needle in 10 mL tubes and immediately transferred into sealed vacutainer glass tubes that contained EDTA-K2 as an anticoagulant (for plasma). After collection, samples were placed on ice and transferred to the laboratory in 4 h. Plasma was obtained by centrifugation at 2000 g for 15 min at 4 °C. To emphasize proteomic differences between the groups while eliminating potential individual contributions, six cows were randomly selected from the same group and equal volumes of plasma were pooled from them. Two mixed plasma samples were available for the next proteomic experiment, which included comparison between sick and healthy groups.
Preparation of protein samples and depletion of abundant proteins
Plasma samples were analyzed using two commercial kits: Albumin and IgG Removal kit (GE Healthcare, NJ USA) and 2-D Clean Up kit (GE Healthcare, NJ USA). The most abundant proteins of albumin and immunoglobulin (IgG) in plasma were removed by the immune affinity-based method according to the manufacturer’s instructions. After fractionation, samples were desalted using the 2-D Clean Up kit.
The protein precipitate in samples were resuspended in a lysis buffer containing 8 M urea, 2 % 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) (w/v), 18 mM dithiothreitol (DTT), 1 % ampholytes (v/v), and bromophenol blue (silver-stained gels) or 30 mM Tris-HCl, and sonicated to completely dissolve the aggregated protein (15 s, 0.5 cycles/s, 60 % amplitude). Samples were centrifuged at 16,000 g for 15 min at 4 °C in a 5415R centrifugal machine (Eppendorf 5415R, Germany) to get suspensions, which were diluted in the lysis buffer using protease inhibitor (Complete EDTA-free Protease Inhibitor Cocktail Tablets, Roche, Spain) until the final protein concentration was 5–10 μg/μL as determined using the 2-D QUANT KIT (GE Healthcare, NJ, USA). Samples were aliquoted once the experiment was over and stored at −80 °C until 2-DE analysis.
To discover the potential new biomarkers in plasma, 2-DE was performed to screen differentially expressed proteins between healthy and sick cows. Isoelectric focusing (IEF) was run on an Ettan IPG phor II (GE Healthcare, CA, USA) using 24 cm nonlinear immobilized pH gradient strips (pH 3–10; GE Healthcare). Protein samples (150 μg) pooled with rehydration solution (8 M urea, 2 % CHAPS, 20 mM DTT, 0.5 % (v/v) immobilized pH gradient (IPG) buffer (pH 3–10), and 0.001 % bromophenol blue) were placed for 12 h at 4 °C. The linear ramping mode of the IEF voltage was applied in the focusing program as description in reference . Strips were sequentially incubated for 15 min in 10 mL equilibration solution with 2.5 % (w/v) DTT or iodoacetamide (IAA). Second-dimension electrophoresis was performed on 12.5 % sodium dodecyl sulfate gels in an Ettan DALT six apparatus (Amersham Bioscience, Uppsala, Sweden) with constant power at 5 W per gel for the first 30 min, and then at 12 W per gel for 6–7 h until the bromophenol blue line reached the bottom of the gels. Gels were treated in triplicate and silver stained according to published procedures . Gels were scanned at 300 dpi resolution (UMAX USB2100XL, Taiwan, China), and the profiles were renamed as the experiment.
Image analysis and protein identification
Differential analysis was performed using Image Master 2D platinum software (Version 5.0, GE Healthcare, CA, USA) for spot detection, quantification, matching, and comparative and statistical analyses. Data were averaged from three independent gels, and the mean and standard deviations were calculated and assessed for statistical significance by normalized intensities of spots. Finally the differentially expressed proteins were defined between the sick and healthy groups with a paired t test if P values were less than 0.05, and the average spot intensity was greater than threefold.
Protein spots of interest were excised manually from the gel, subjected to destaining and trypsin digestion according to the protocol described by Wu , and purified using ZipTip microliter plates (Millipore). MALDI-TOF MS analysis of tryptic peptides was performed on an Ultraflex TOF/TOF instrument (Bruker Daltonics). Proteins were identified by peptide mass fingerprint (PMF) using the Mascot search engine (http://www.matrixscience.com; Matrix Science Ltd., London, UK) and the Swiss-Prot 55.4 database.
The identified proteins were searched in the Uniprot database (http://www.uniprot.org/), AgBase (http://www.agbase.msstate.edu/), and published literature for their functions . According to the combined search results, these proteins were divided into different functional groups. Categorical annotation was supplied in the form of gene ontology (GO) biological process (BP), molecular function (MF), cellular component (CC), as well as participation in a Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway and membership in a protein complex as defined by the comprehensive resource of mammalian protein complexes (CORUM) .
Validation of differentially expressed protein
To add confidence to the results obtained by 2-DE, 2 of 16 differentially expressed proteins in plasma were measured in healthy and sick groups. The concentrations of haptoglobin were detected by Bovine ELISA kits (Shanghai Institute of Biological enzyme-linked, Shanghai, China). ApoA-I in plasma was detected by immunoturbidimetric method in the automatic biochemical analyzer (Mindary 420, Shenzhen, China) using commercial test kits for human (Mindary, Shenzhen, China). The standard curve was developed with the known ApoA-I concentration (0 g/L, 0.180 g/L, 0.530 g/L, 1.26 g/L, and 2.45 g/L). The procedure was performed according to the manufacturer’s instructions.
Plasma samples (1:2000 dilutions) were added in duplicate to each well in enzyme-linked immunosorbent assay (ELISA) plate precoated with monoclonal antibody (McAb) against bovine haptoglobin, and then 10 μL of biotin-labeled McAb and 50 μL of streptavidin–HRP conjugates were added to the wells. After incubation at 37 °C for 1 h, the ELISA plate was washed for three times using PBST [0.5 % (v/v) Tween-20, PBS, pH 7.4]. Coloration was developed by 3′, 5, 5′-tetramethylbenzidine solution, and the reaction was stopped with 50 μL of 2 M H2SO4. The absorbance was measured at a wavelength of 450 nm. In ELISA test, bovine haptoglobin solutions with known concentrations (800 mg/L, 400 mg/L, 200 mg/L, 100 mg/L, and 50 mg/L) were used to prepare a standard curve according to the ELISA procedure described by the manufacture. Haptoglobin concentration in plasma was calculated according to the sample absorbance and standard curve.
Assay of antioxidant ability of plasma in dairy cows
Plasma samples were analyzed for the total antioxidative capacity (T-AOC), malonaldehyde (MDA), super oxygen dehydrogenases (SOD), and glutathione peroxidase (GSH-Px) using colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), and detected by microplate reader (Spectra Max M2, Molecular Devices, CA, USA). All samples were tested in duplicate. The T-AOC concentration was determined by the reaction of phenanthroline and Fe2+ using spectrophotometer at 520 nm. MDA was measured by the thiobarbituric acid method. SOD activity was determined by inhibiting nitroblue tetrazolium reduction due to superoxide anion generation by a xanthine–xanthine oxidase system. The GSH-Px level was determined using the direct measurement of the remaining GSH after the enzyme-catalyzed reaction. All assay procedures were performed according to the manufacturer’s instructions.
Statistical analysis was performed using the SPSS statistical package v 17.0. Normality of data was tested using the one-sample Kolmogorov–Smirnov test. Data were analyzed by one-way analysis of variance, and differences between group means were evaluated with the Duncan test. Differences between groups were analyzed with independent t test (P < 0.05).