Yifeng Deng

N-acetyl-cysteine protects chicken growth plate chondrocytes from T-2 toxin-induced oxidative stress.

T‐2 toxin is now considered to be related to bone malformation such as incomplete ossification, absence of bones and fused bones. In this study, primary cultures of chicken tibial growth plate chondrocytes (GPCs) were treated with various concentrations of T‐2 toxin (5, 50, and 500 n m) in the absence and presence of N‐acetyl‐cysteine (NAC) to investigate the effects of the antioxidant NAC on T‐2 toxin‐induced toxicity. Our results showed that T‐2 toxin markedly decreased cell viability, alkaline phosphatase activity and glutathione content (P < 0.05). In addition, T‐2 toxin significantly increased reactive oxygen species levels and malondialdehyde in a dose‐dependent manner. However, the T‐2 toxin‐induced cytotoxicity was reversed, in part, by the antioxidant NAC (P < 0.05). These results suggest that T‐2 toxin inhibits the proliferation and differentiation of GPCs in vitro by altering cellular homeostasis and NAC can protect GPCs against T‐2 toxin cytotoxicity by reducing the T‐2 toxin‐induced oxidative stress. Copyright

Multi-mycotoxin analysis of animal feed and animal-derived food using LC–MS/MS system with timed and highly selective reaction monitoring

AbstractMycotoxins have the potential to enter the human food chain through carry-over of contaminants from feed into animal-derived products. The objective of the study was to develop a reliable and sensitive method for the analysis of 30 mycotoxins in animal feed and animal-derived food (meat, edible animal tissues, and milk) using liquid chromatography–tandem mass spectrometry (LC–MS/MS). In the study, three extraction procedures, as well as various cleanup procedures, were evaluated to select the most suitable sample preparation procedure for different sample matrices. In addition, timed and highly selective reaction monitoring on LC–MS/MS was used to filter out isobaric matrix interferences. The performance characteristics (linearity, sensitivity, recovery, precision, and specificity) of the method were determined according to Commission Decision 2002/657/EC and 401/2006/EC. The established method was successfully applied to screening of mycotoxins in animal feed and animal-derived food. The results indicated that mycotoxin contamination in feed directly influenced the presence of mycotoxin in animal-derived food. Graphical abstractMulti-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS

Effects of Gushukang, a Chinese herbal medicine, on bone characteristics and osteoporosis in laying hens

In this study, we evaluated the effects of the herb medicine formula Gushukang (GSK) on bone characteristics and osteoporosis in end-of-lay hens. One thousand 55-wk-old ISA caged layers were allotted randomly to 2 groups. The control group was given the basal diet, and the GSK group was given the basal diet supplemented with additional GSK (1 g/kg) for 10 wk. Egg production, shell quality, bone radiographic density, and biochemical markers of bone turnover were determined. The results showed that GSK significantly increased the egg laying rate and decreased the percentage of cracked eggs (P < 0.05).The serum calcium, phosphate, and alkaline phosphatase were decreased (P < 0.05) in the GSK-treated group compared with the control group, whereas bone characteristics were significantly improved (P < 0.05). The results suggested that GSK can improve egg production and prevent bone loss by inhibiting bone turnover.

Letrozole inhibits the osteogenesis of medullary bone in prelay pullets

This study was performed to investigate the effect of letrozole, an aromatase inhibitor, on osteogenesis of medullary bone in prelay pullets. Three hundred fifteen 95-d-old ISA prelay pullets were used. After 10 d of adaptation in the cages, 15 pullets were selected randomly to collect the serum and bone samples and the rest were randomly assigned to 2 groups with 3 replicates each. One group was control and the other was letrozole-treated, fed 0.5 mg of letrozole per prelay pullet per day for 18 d. The serum and bone samples from these birds were collected during the experiment. Estradiol and testosterone in serum were assayed using commercial RIA kits. The serum alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), Ca, and inorganic P were measured by an automatic biochemistry analyzer with commercial kits. The periosteum perimeter, endosteum perimeter, cortical bone index, cortical width, cortical bone area, and cortical area ratios of tibia were measured by transmitted scanner and a computer-assisted image analyzer. Our results showed that relative to the control-fed pullet, letrozole-fed pullets had reduced serum estrogen (57.5%), Ca (33.2%), ALP (33.6%), and TRAP (24.2%) and that values of serum estrogen, Ca, estrogen receptor expression, tibia radiographic density, serum ALP, and TRAP were all reduced (P < 0.05) and the serum P had a degressive trend in letrozole-treated groups. By contrast, the serum androgen and the tibia cortical bone index values were higher in the letrozole-treated group (P < 0.05). No differences were observed in the periosteum perimeter, endosteum perimeter, cortical width, and cortical area ratios of tibia between the 2 groups. The results showed that letrozole can inhibit the development of bone and medullary osteogenesis by inhibiting the synthesis of estrogen and its receptor in prelay pullets.

Determination of trichothecenes A (T-2 toxin, HT-2 toxin, and diacetoxyscirpenol) in the tissues of broilers using liquid chromatography coupled to tandem mass spectrometry.

A stable and sensitive method has been developed for use in food and livestock product safety for the detection of mycotoxins. This newly developed method allows for the determination of T-2 toxin, HT-2 toxin and diacetoxyscirpenol (DAS) in heart, liver, spleen, lung, kidney, Glandular stomach, muscular stomach, small intestine, muscle, bone and brain samples from broilers using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The samples were initially extracted with ethyl acetate before being filtered through a 0.22μm nylon syringe filter and subjected to chromatographic separation on a reversed-phase C18 (50×2.1mm, 3μm) column. A mobile phase composed of 0.1% acetic acid and 10mM ammonium acetate in methanol and water was used in an assay of the levels of T-2 toxin, HT-2 toxin and DAS. For the analysis of the target compounds, the mass spectrometer was operated under positive electrospray ionization conditions in the selected reaction monitoring mode. The limit of detection was in the range of 0.02-0.05ng/g, whereas the limit of quantification was in the range of 0.08-0.15ng/g. The extraction recoveries of spiked samples from the high, intermediate and low levels ranged from 58.5% to 110.5%, and the relative standard deviation (RSD (%)) values were less than 17.0%. The results of inter- and intra-day precision (RSD (%)) were within 14.7%. The results revealed that the present method could be successfully applied to the analysis of T-2 toxin, HT-2 toxin and DAS in the real samples.

Expression of TRPV6 and CaBP-D28k in the egg shell gland (uterus) during the oviposition cycle of the laying hen

1. The aim of this study was to investigate the localisation of the transient receptor potential vanilloid channel type 6 (TRPV6) in egg shell gland (ESG) and examine the dynamic expression of TRPV6 and Calbindin-d28k (CaBP-D28k), as well as the changes in concentration of total calcium (Ca), total inorganic phosphorus (P), alkaline phosphatase (ALP), parathyroid hormone (PTH) and calcitonin (CT) in plasma during the oviposition cycle. 2. The plasma ALP activity was notably increased at 8 h. In addition, plasma CT was highest at 0 h and significantly lower at 8 h. The change of plasma PTH concentration increased slightly post-oviposition and reached a maximum at 16 h. 3. Immunohistochemical analysis indicated that TRPV6 was strongly localised to the apical luminal epithelium of the mucosa. The mRNA levels of TRPV6 and CaBP-D28k in the ESG remained very low from 0 to 4.5 h, but were significantly increased at 16 h. Furthermore, Western blotting analysis showed that the expression of TRPV6 and CaBP-D28k also reached a maximum at 16 h and was different from the concentration of CaBP-D28k. 4. In conclusion, the epithelial Ca2+ channel TRPV6 is strongly expressed in the epithelial cells of the eggshell gland, and the increase of TRPV6 and CaBP-D28k mRNA and protein expression during eggshell formation suggests that active Ca2+ transcellular transport exerts significant effects in delivering active calcium in the ESG.

Expression and identification of recombinant chicken vascular endothelial growth factor in Pichia pastoris and its role in the pathogenesis of tibial dyschondroplasia

Vascular endothelial growth factor (VEGF) is an essential mediator of angiogenesis and endochondral ossification. To explore the role of VEGF in avian diseases such as tibial dyschondroplasia (TD), a typical disorder of endochondral ossification, we expressed and identified recombinant chicken VEGF (chVEGF) protein in Pichia pastoris and evaluated its effects on thiram-induced TD in broiler chickens. The SDS-PAGE showed that 2 recombinant proteins, with molecular weights of ~46 and ~70 kDa, were obtained. Western blot analysis indicated that the 2 proteins were recognized by rabbit anti-chicken and goat anti-human VEGF polyclonal antibodies. Moreover, the mixture of the proteins significantly stimulated angiogenesis in the chick chorioallantoic membrane. In 21-d-old broilers that had been fed a thiram-enriched diet (100 mg/kg of thiram for 2 d at 8 d old) to induce TD, intramuscular injection of the chVEGF proteins (at a dosage of 10 or 30 μg/kg) significantly reduced the severity of TD but had no effect on TD incidence or BW; decreased serum Ca and P concentrations and tartrate-resistant acid phosphatase activity and elevated serum alkaline phosphatase activity; enhanced the total antioxidant capacity, superoxide dismutase, and glutathione peroxidase activities in the liver and kidney; upregulated the expression of type X collagen, matrix metalloproteinase (MMP)-13, and Runx2; and downregulated the Bcl-2 expression in the growth plates. In thiram-treated broilers at 15 d old, the chVEGF proteins upregulated the expression of MMP-13 and Runx2, and had different effects on type X collagen and Bcl-2 expression at different dosages. Our results indicate that exogenous chVEGF proteins promoted the recovery of TD-affected growth plates by improving the antioxidant capacity in the liver and kidney and by regulating differential expression of genes relating to endochondral ossification at different stages of TD development; VEGF deficiency in the growth plates was involved in the pathogenesis of TD.

Toxicity induced by F. poae-contaminated feed and the protective effect of Montmorillonite supplementation in broilers

The T-2 and HT-2 toxins, the main metabolites of Fusarium poae, induce toxicity in broilers and accumulate in tissues. Consequently, during the breeding process of broilers, diets are frequently supplemented with physical adsorbents to protect birds against the toxicity induced by mycotoxins. In the present research, T-2 and HT-2 were produced in maize inoculated with F. poae. Mont, the strongest adsorbent based on in vitro adsorption ratios, was added to the contaminated diet. One-day-old chickens were randomly and equally divided into the following four groups: control diet group, Mont supplemented diet group, contaminated diet group and detoxification diet group. The experiment lasted for 42 days. Compared to the control group, the contaminated group showed significant decrease in body weight, feed intake and TP (P < 0.05), and marked increase in FCR, ALP, AST and ALT activity, T-2/HT-2 residues in the tissues and the relative expressions of apoptosis-related mRNAs (P < 0.05). Mont supplementation provided protection for the treated broilers in terms of performance, blood biochemistry, hepatic function, T-2/HT-2 residue of tissues and apoptosis. Therefore, Mont may be suitable as a detoxification agent for T-2/HT-2 in feed for broilers.

A new preparative method for simultaneous purification of ochratoxin A and ochratoxin B from wheat culture inoculated with Aspergillus ochraceus

Ochratoxins are a mycotoxin family frequently found in agricultural commodities worldwide and pose a potential health risk to humans and animals. To obtain large amounts of high-purity ochratoxins for food safety monitoring and toxicological research, a novel and effective method was established for simultaneous purification of ochratoxin A (OTA) and ochratoxin B (OTB) from a wheat culture inoculated with an ochratoxin-producing Aspergillus strain. The inoculated wheat culture was first extracted with methanol:water (80:20, v/v), followed by one or two cleanup procedures involving acid-assisted liquid-liquid extraction and gel permeation chromatography. Subsequently, target analytes were separated and collected using preparative high performance liquid chromatography. Finally, a combined approach of ultra-high performance liquid chromatography, ultraviolet spectrophotometry and mass spectrometry was applied for purity analysis and structural identification of the obtained toxins. As a result, 100 g of an inoculated wheat culture yielded 69 mg of OTA and 6 mg of OTB with purities greater than 98%. This proposed method might serve as a valuable reference to obtain expensive ochratoxin standards. To the best of our knowledge, this is the first report on simultaneous preparation of OTA and OTB from artificially inoculated wheat culture.

Effect of transient receptor potential vanilloid 6 gene silencing on the expression of calcium transport genes in chicken osteoblasts

Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of transient receptor potential vanilloid 6 in chickens. In this study, we evaluated the effects of transient receptor potential vanilloid 6 gene interference on the expression of calbindin-D28K, Na+/Ca2+ exchangers, and plasma membrane Ca2+ ATPase 1b to investigate the mechanism underlying the regulation of transient receptor potential vanilloid 6. Three hairpin siRNA expression vectors targeting transient receptor potential vanilloid 6 (pSIREN- transient receptor potential vanilloid 6) and a negative control (pSIREN-control) were constructed and transfected into chicken osteoblasts. The mRNA and protein expression levels were evaluated by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. The mRNA expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 45.7% (P<0.01) and 27.9% (P<0.01), respectively, 48 h after transfection with one of the three constructs (pSIREN- transient receptor potential vanilloid 6-3) compared with the level obtained in the untreated group. There was no significant difference in the mRNA expression levels of Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPase 1b. The protein expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 40.2% (P<0.01) and 29.8% (P<0.01), respectively, 48 h after transfection with pSIREN-transient receptor potential vanilloid 6-3 compared with the level obtained in the untreated group. In conclusion, the vector-based transient receptor potential vanilloid 6-shRNA can efficiently suppress the mRNA and protein expression of transient receptor potential vanilloid 6 in chicken osteoblasts, and transient receptor potential vanilloid 6 regulates the expression of calbindin-D28K during Ca2+ transport.