Wenjun Yang

Screening and determination of melamine residues in tissue and body fluid samples

Melamine is a chemical product that was sporadically mixed into animal feeds to boost protein content. Excessive melamine in animal feed can induce renal failure and even death in animals. The residue of melamine in edible animal products also threatens human health. Currently, there is no real-time and high throughput method to detect residual melamine in animal tissues. Successful development of such methods is very important for fast and on-site screening of melamine residue in animal tissues to eliminate the potential threat to human health. Here we demonstrate the detection of residual melamine from swine and chicken tissues and body fluids using indirect competitive enzyme-linked immunosorbent assay (ELISA) method. A detection sensitivity of 0.5 microg mL(-1) and a limit of detection of 0.05 microg mL(-1) were achieved with this method. A gas chromatography-mass spectrometry (GC-MS) method was also developed to act as a confirmatory and quantitative procedure for the ELISA results. The limits of quantitation (LOQ) of were 0.01 microg g(-1) and 0.005 microg mL(-1) for tissues and body fluids, respectively. The two methods showed good agreement (r(2)>0.992). The method developed was performed on samples of tissues from chickens fed with melamine-spiked feed.

The effects of dietary fiber level on nutrient digestibility in growing pigs

The objective of this study was to investigate the effects of total dietary fiber level on nutrient digestibility and the relationship between apparent total tract digestibility of total dietary fiber, and soluble dietary fiber, insoluble dietary fiber and available energy. Sugar beet pulp was as the only fiber source. The experiment was designed as a 6 × 6 Latin square with an adaptation period of 7 d followed by a 5-d total collection of feces and urine. Feed intake tended to decrease (P = 0.10) as total dietary fiber level increased. The apparent total tract digestibility of dry matter, crude protein and gross energy decreased (P < 0.01) when total dietary fiber increased but the digestibility of soluble dietary fiber and insoluble dietary fiber increased (P < 0.01). The digestible energy and metabolizable energy content of diets decreased (P < 0.01) as the total dietary fiber increased.

Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk

In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk.

Deposition of Melamine in Eggs from Laying Hens Exposed to Melamine Contaminated Feed

The deposition profile of melamine was studied in eggs obtained from laying hens fed melamine contaminated feed. A total of 180 laying hens were divided into five groups and were fed diets spiked with 0, 5, 25, 50, or 100 mg of melamine per kg of feed. Eggs collected on days 1, 3, 5, 7, 9, 11, 13, and 15 were analyzed by a gas chromatography-mass spectrometry method, which was fully validated for melamine analysis prior to use. For each treatment group, the melamine level in the eggs was similar from day 1 to day 15 (P > 0.05), suggesting that laying hens did not accumulate melamine for later deposition in eggs. The average melamine concentrations in eggs were 0.00 (below limit of detection), 0.16, 0.47, 0.84, and 1.48 mg/kg for the 0, 5, 25, 50, and 100 mg/kg treatment groups, respectively, which demonstrated an apparent dose-response relationship, and a safety threshold of 164 mg/kg melamine in the feed of laying hen was estimated when a maximum tolerance level of 2.5 mg/kg melamine in egg was adopted. These results provide a scientific basis for the risk assessment of melamine in feeds fed to laying hens.

Determination of Kanamycin A in Animal Feeds by Solid Phase Extraction and High Performance Liquid Chromatography with Pre‐Column Derivatization and Fluorescence Detection

Abstract A selective method was developed for the determination of kanamycin A in medicated animal feeds by reversed‐phase high performance liquid chromatography (HPLC) with precolumn derivatization. Samples were extracted with 0.1 mol/L hydrochloric acid solution and cleanup was achieved with MCX solid phase extraction (SPE). The purified extract was derivatized with O‐phthaladehyde (OPA), separated on a reversed‐phase C18 column and the fluorescence detection was performed at the excitation and emission wavelengths of 230 and 389 nm, respectively. The run time was approximately 14 min, at the flow rate of 0.8 mL/min. This method provides the average recoveries 98.4%–106.0% in feeds spiked in the range of 10–2000 g/ton, with the coefficients of variation 1.17%–9.78%. The limit of detection in feeds was 5 g/ton and the limit of quantification in feeds was 10 g/ton, which are well below the effective dose.

Effects of Dietary Coconut Oil as a Medium-chain Fatty Acid Source on Performance, Carcass Composition and Serum Lipids in Male Broilers

This study was conducted to investigate the effects of dietary coconut oil as a medium-chain fatty acid (MCFA) source on performance, carcass composition and serum lipids in male broilers. A total of 540, one-day-old, male Arbor Acres broilers were randomly allotted to 1 of 5 treatments with each treatment being applied to 6 replicates of 18 chicks. The basal diet (i.e., R0) was based on corn and soybean meal and was supplemented with 1.5% soybean oil during the starter phase (d 0 to 21) and 3.0% soybean oil during the grower phase (d 22 to 42). Four experimental diets were formulated by replacing 25%, 50%, 75%, or 100% of the soybean oil with coconut oil (i.e., R25, R50, R75, and R100). Soybean oil and coconut oil were used as sources of long-chain fatty acid and MCFA, respectively. The feeding trial showed that dietary coconut oil had no effect on weight gain, feed intake or feed conversion. On d 42, serum levels of total cholesterol, low-density lipoprotein cholesterol, and low-density lipoprotein/high-density lipoprotein cholesterol were linearly decreased as the coconut oil level increased (p<0.01). Lipoprotein lipase, hepatic lipase, and total lipase activities were linearly increased as the coconut oil level increased (p<0.01). Abdominal fat weight/eviscerated weight (p = 0.05), intermuscular fat width (p<0.01) and subcutaneous fat thickness (p<0.01) showed a significant quadratic relationship, with the lowest value at R75. These results indicated that replacement of 75% of the soybean oil in diets with coconut oil is the optimum level to reduce fat deposition and favorably affect lipid profiles without impairing performance in broilers.

Evaluation of Energy Digestibility and Prediction of Digestible and Metabolizable Energy from Chemical Composition of Different Cottonseed Meal Sources Fed to Growing Pigs

The present experiment was conducted to determine the digestible energy (DE), metabolizable energy (ME) content, and the apparent total tract digestibility (ATTD) of energy in growing pigs fed diets containing one of ten cottonseed meals (CSM) collected from different provinces of China and to develop in vitro prediction equations for DE and ME content from chemical composition of the CSM samples. Twelve growing barrows with an initial body weight of 35.2±1.7 kg were allotted to two 6×6 Latin square designs, with six barrows and six periods and six diets for each. A corn-dehulled soybean meal diet was used as the basal diet, and the other ten diets were formulated with corn, dehulled soybean meal and 19.20% CSM. The DE, ME and ATTD of gross energy among different CSM sources varied largely and ranged from 1,856 to 2,730 kcal/kg dry matter (DM), 1,778 to 2,534 kcal/kg DM, and 42.08 to 60.47%, respectively. Several chemical parameters were identified to predict the DE and ME values of CSM, and the accuracy of prediction models were also tested. The best fit equations were: DE, kcal/kg DM = 670.14+31.12 CP+659.15 EE with R2 = 0.82, RSD = 172.02, p<0.05; and ME, kcal/kg DM = 843.98+25.03 CP+673.97 EE with R2 = 0.84, RSD = 144.79, p<0.05. These results indicate that DE, ME values and ATTD of gross energy varied substantially among different CSM sources, and that some prediction equations can be applied to predict DE and ME in CSM with an acceptable accuracy.

HPLC–MS analysis of iodotyrosines produced by sample hydrolysis: A simple method for monitoring iodinated casein in feed premixes

A new and simple HPLC-MS method was developed for monitoring iodinated casein in feed premixes. In this method, feed premixes were hydrolyzed, and the iodotyrosines thus released were analyzed. Sample pretreatment included precipitation of transition metals ions with Na(2)S, hydrolysis with sodium hydroxide, and cleaning up with an Oasis SAX cartridge. Gradient elution was carried out on a C(18) column with water (containing 0.1% formic acid) and acetonitrile. Ion detection was performed using ESI positive SIM at m/z 262, 308, 388, and 434. Iodinated casein levels were monitored by qualitative analysis of the iodotyrosines released upon sample hydrolysis and by quantifying the 3,5-diiodotyrosine released. The validation data demonstrated that the method was selective and sensitive (<or=0.2mgg(-1)) for iodinated casein and had acceptable accuracy (recoveries: 81.3-106.7%) and precision (RSD: 1.7-16.0%).

Use of near-infrared reflectance spectroscopy for the rapid determination of the digestible energy and metabolizable energy content of corn fed to growing pigs

BackgroundThe ability of near-infrared reflectance spectroscopy (NIRS) to determine the digestible energy (DE) and metabolizable energy (ME) content of corn fed to growing pigs was tested. One hundred and seventeen corn samples, comprising different planting regions and varieties were collected from all over China in a three-year period. The samples were randomly split into a calibration set (n = 88) and a validation set (n = 29). The actual and calculated DE and ME content of the corn samples was determined by digestion-metabolism experiments and the prediction equations of Noblet and Perez (J Anim Sci. 71:3389–98,1993). The samples were then subjected to NIRS scanning and calibrations were performed by the modified partial least square (MPLS) regression method based on 77 different spectral pre-treatments. The NIRS equations based on the actually determined and calculated DE and ME were built separately and then validated using validation samples.ResultsThe NIRS equations obtained from actually determined DE, the coefficient of determination for calibration (RSQcal), cross-validation (R2CV), and validation (RSQv) were 0.89, 0.87 and 0.86, and these values for determined ME were 0.87, 0.86 and 0.86. For the NIRS equations built from calculated DE, the RSQcal, R2CV, and RSQv values were 0.88, 0.85 and 0.84, and these values for calculated ME were 0.86, 0.84 and 0.82. Except for the equation based on calculated ME (RPDv = 2.38, < 2.50), the other three equations built from actually determined energy and calculated DE produced good prediction performance (RPDv ranging from 2.53 to 2.69, > 2.50) when applied to validation samples.ConclusionThese results indicate that NIRS can be used as a quantitative method for the rapid determination of the available energy in corn fed to growing pigs, and the NIRS equations based on the actually determined energy produced better predictive performance than those built from calculated energy values.

Quantitative determination of chromium picolinate in animal feeds by solid phase extraction and liquid chromatography-tandem mass spectrometry

Chromium picolinate is one of the important Cr3+ resources and is widely used in animal production. A convenient, reliable and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative determination of chromium picolinate in animal feeds. Feed samples were extracted with acetonitrile and subsequently cleaned up by solid phase extraction cartridges Supelclean™ LC-18. Chromium picolinate was efficiently separated with a Waters ACQUITY UPLC® BEH C18 column, ionized with electrospray ion source in positive mode (ESI+), and quantitatively determined by tandem mass spectrometry in multiple reaction monitoring mode. Standard calibration curve of chromium picolinate in the concentration range from 0.5 to 1000ng/mL was obtained with good linearity correlation coefficient (R2=0.9982). Average recoveries ranged from 95.37%∼105.54%, as detected by spiking 0.02∼640mg/kg of chromium picolinate in complete feed, concentrated feed and premix. Intra-day and inter-day coefficient of variation were 0.59%∼6.67% and 2.36%∼6.97%, respectively. The limits of quantitation were 0.02mg/kg, 0.025mg/kg, and 2mg/kg for complete feed, concentrated feed, and premix, respectively. Actual sample analysis indicated that the developed method can be an effective tool to monitoring CrPic content in animal feed.