Effects of N-acetyl-l-cysteine on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens.

The purpose of this study was to discuss the effects of N-acetyl-l-cysteine (NAC) on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens in different periods. A total of 120 Hy-Line variety brown laying hens (12 weeks old) were randomly assigned to 4 groups with 6 replicates. The control group (C group) (22 ± 1 °C) received a basal diet, the NAC-treated group (N group) (22 ± 1 °C) received a basal diet with 1000 mg/kg NAC, and 2 heat-stressed groups (36 ± 1 °C for 10 h per day and 22 ± 1 °C for the remaining time) were fed a basal diet (HS group) or a basal diet with 1000 mg/kg NAC (HS + N group) for 21 consecutive days. The influence of NAC on histologic changes, oxidative stress and proinflammatory cytokine production was measured and analysed in hens with heat stress-induced hypothalamic changes. NAC effectively alleviated the hypothalamic morphological changes induced by heat stress. In addition, NAC attenuated the activity of the Nf-κB pathway activated by heat stress and decreased the expression of the proinflammatory cytokines IL-6, IL-18, TNF-α, IKK, and IFN-γ. In addition, NAC treatment regulated the expression of HO-1, GSH, SOD2 and PRDX3 by regulating the activity of Nrf2 at different time points to resist oxidative stress caused by heat exposure. In summary, dietary NAC may be an effective candidate for the treatment and prevention of heat stress-induced hypothalamus injury by preventing Nf-κB activation and controlling the Nrf2 pathway.

Transversal gene expression panel to evaluate intestinal health in broiler chickens in different challenging conditions.

There is a high interest on gut health in poultry with special focus on consequences of the intestinal diseases, such as coccidiosis and C. perfringens-induced necrotic enteritis (NE). We developed a custom gene expression panel, which could provide a snapshot of gene expression variation under challenging conditions. Ileum gene expression studies were performed through high throughput reverse transcription quantitative real-time polymerase chain reaction. A deep review on the bibliography was done and genes related to intestinal health were selected for barrier function, immune response, oxidation, digestive hormones, nutrient transport, and metabolism. The panel was firstly tested by using a nutritional/Clostridium perfringens model of intestinal barrier failure (induced using commercial reused litter and wheat-based diets without exogenous supplementation of enzymes) and the consistency of results was evaluated by another experiment under a coccidiosis challenge (orally gavaged with a commercial coccidiosis vaccine, 90× vaccine dose). Growth traits and intestinal morphological analysis were performed to check the gut barrier failure occurrence. Results of ileum gene expression showed a higher expression in genes involved in barrier function and nutrient transport in chickens raised in healthy conditions, while genes involved in immune response presented higher expression in C.perfringens-challenged birds. On the other hand, the Eimeria challenge also altered the expression of genes related to barrier function and metabolism, and increased the expression of genes related to immune response and oxidative stress. The panel developed in the current study gives us an overview of genes and pathways involved in broiler response to pathogen challenge. It also allows us to deep into the study of differences in gene expression pattern and magnitude of responses under either a coccidial vaccine or a NE.

Dietary taurine supplementation ameliorates muscle loss in chronic heat stressed broilers via suppressing the perk signaling and reversing endoplasmic reticulum-stress-induced apoptosis.

BACKGROUND: Heat stress seriously affects animal health and induces enormous financial losses in poultry production. Exploring the appropriate means for ameliorating unfavorable effects caused by heat stress is essential. We investigated whether taurine supplementation could attenuate breast muscle loss in chronic heat-stressed broilers, as well as its mechanism. We designed three groups: a normal control group (22 °C), a heat stress group (32 °C) and a taurine treatment group (32 °C, basal diet + 5 g·kg-1 taurine). RESULTS: We found that taurine significantly moderated the decreases of breast muscle mass and yield, as well as the increases of serum aspartate aminotransferase activity and serum urine acid level in chronic heat-stressed broilers. Additionally, supplementary taurine significantly alleviated elevations of the cytoplasm Ca2+ concentration, protein expressions of GRP78 and p-PERK, mRNA expressions of Ca2+ channels (RyR1, IP3R3) and endoplasmic reticulum (ER) stress factors (GRP78, GRP94, PERK, EIF2α, ATF4, IRE1, XBP1, ATF6 and CHOP), apoptosis (Caspase-3 and TUNEL), protein catabolism, and the reduction of taurine transporter (TauT) mRNA expression in the breast muscle induced by chronic heat stress. CONCLUSION: Supplementary taurine could attenuate chronic heat stress-induced breast muscle loss via reversing ER stress-induced apoptosis and suppressing protein catabolism. © 2020 Society of Chemical Industry.

Effects of Amino Acid-Bound Zinc and Manganese Feed Additives on MHC Haplotype Chickens Challenged with Infectious Bronchitis Coronavirus.

Major histocompatibility complex (MHC) congenic chicken lines have been used as a model to study infectious bronchitis virus (IBV) immune responses in chickens. Zinc (Zn) and manganese (Mn) are trace minerals that act as enzyme cofactors in cellular reactions. In addition, Zn is an important modulator of immune responses, especially in the respiratory tract. Zinc and Zn + Mn amino acid complex supplements were tested to alleviate the effects of an IBV challenge using relatively resistant and susceptible MHC congenic chicken lines. Prior to the challenge with IBV, the amino acid-bound supplements induced better weight gain in the IBV-resistant chicken line (331/B2) compared to the birds fed with the sulfate-delivered supplements. No body weight differences were detected between IBV-challenged and unchallenged 331/B2 birds supplemented with Zn in amino acid complex. A reduction of respiratory signs was observed in 335/B19 birds fed with the diet supplemented with Zn in amino acid complexes at 4 dpi. Compared to the sulfate-bound trace minerals, 331/B2 chickens fed with the amino acid-bound supplements presented milder clinical sign trends at 6 dpi and less severe airsacculitis at 14 dpi. The total antibody response in serum in 331/B2 birds fed with the amino acid-bound Zn ration was the highest among all groups tested. Both amino acid-delivered trace mineral supplements induced a slightly higher antibody response than the sulfate-bound ration in both chicken lines. This experiment provides insights into the effect of Zn and Mn on the immunity of chickens with known different susceptibilities to IBV.

Selenium Yeast Alleviates Ochratoxin A-Induced Hepatotoxicity via Modulation of the PI3K/AKT and Nrf2/Keap1 Signaling Pathways in Chickens.

The aim of this study was to investigate the protective effects of selenium yeast (Se-Y) against hepatotoxicity induced by ochratoxin A (OTA). The OTA-induced liver injury model was established in chickens by daily oral gavage of 50 µg/kg OTA for 21 days. Serum biochemistry analysis, antioxidant analysis, as well as the qRT-PCR and Western blot (WB) analyses were then used to evaluate oxidative damage and apoptosis in chicken liver tissue. The results showed that Se-Y significantly increased liver coefficient induced by OTA (P < 0.05). OTA + Se-Y treated group revealed that Se-Y reduced the OTA-induced increase in glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and malonaldehyde (MDA) content, and reversed the decrease in antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) (P < 0.05). In this study, we found that OTA is involved in the mRNA expression levels about Nrf2/Keap1 and PI3K/AKT signaling pathways, such as oxidative stress-related genes (Nrf2, GSH-Px, GLRX2 and Keap1) and apoptosis-related genes (Bax, Caspase3, P53, AKT, PI3K and Bcl-2). Besides, significant downregulations of protein expression of HO-1, MnSOD, Nrf2 and Bcl-2, as well as a significant upregulation of Caspase3 and Bax levels were observed after contaminated with OTA (P < 0.05). Notably, OTA-induced apoptosis and oxidative damage in the liver of chickens were reverted back to normal level in the OTA + Se-Y group. Our findings indicate that pretreatment with Se-Y effectively ameliorates OTA-induced hepatotoxicity.

Selenium-mediated gga-miR-29a-3p regulates LMH cell proliferation, invasion, and migration by targeting COL4A2.

Selenium (Se) is an essential trace element that has several functions in cellular processes related to cancer prevention. While the cancericidal effect of Se has been reported in liver cancer, the mechanism has not been clarified. MiR-29a has widely been reported as a tumor suppressor; however, it also acts as a carcinogenic agent by increasing cell invasion in human epithelial cancer cells and hepatoma cells. In a previous study, we found that miR-29a-3p is a Se-sensitive miRNA. However, its effect in the chicken hepatocellular carcinoma cell line (LMH) is still unknown. In the present study, we found that the expression of miR-29a-3p in LMH cells was decreased by Se supplementation and increased under Se-deficient conditions. Flow cytometry and CCK-8 results suggested that Se decreased LMH cell proliferation induced by miR-29a-3p overexpression. Transwell and gap-closure assays implied that Se mediated LMH cell invasion and migration by downregulating miR-29a-3p. Quantitative real-time polymerase chain reaction and Western blotting results suggested that Se mitigated miR-29a-3p overexpression-induced LMH cell proliferation by downregulating CDK2, cyclin-D1, CDK6, and cyclin-E1. We further demonstrated that collagen type IV alpha 2 (COL4A2) is a target gene of miR-29a-3p. COL4A2 activates the RhoA/ROCK pathway to promote LMH cell invasion and migration. In conclusion, Se mediated miR-29a-3p overexpression induced LMH cell invasion and migration by targeting COL4A2 to inactivate the RhoA/ROCK pathway.

Dietary taurine supplementation decreases fat synthesis by suppressing the liver X receptor α pathway and alleviates lipid accumulation in the liver of chronic heat-stressed broilers.

BACKGROUND: Chronic heat stress can enhance fat synthesis and result in lipid accumulation in the liver of broilers. To investigate the effects and molecular mechanisms of dietary taurine supplementation on fat synthesis and lipid accumulation in the liver of chronic heat-stressed broilers, 144 28 day-old chickens (Arbor Acres) were randomly distributed to normal control (NC, 22 °C, basal diet), heat stress (HS, consistent 32 °C, basal diet), or heat stress plus taurine (HS + T, consistent 32 °C, basal diet +5.00 g kg-1 taurine) groups for a 14-day feeding trial. RESULTS: Compared with those of the HS group, dietary taurine supplementation significantly decreased the level of very-low-density lipoprotein and the activity of aspartate aminotransferase in plasma and the relative weight of liver in the HS + T group. In addition, dietary taurine supplementation also significantly decreased the levels of triglyceride, acyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and suppressed the mRNA expression levels of liver X receptor α (LXRα), sterol response element-binding protein 1c, ACC and FAS in the liver of chronic heat-stressed broilers. Meanwhile, dietary taurine supplementation effectively alleviated lipid accumulation in the liver of broilers exposed to chronic heat stress. CONCLUSION: Chronic heat stress significantly increased fat synthesis and resulted in excess lipid deposition in the liver of broilers. Dietary taurine supplementation can effectively decrease fat synthesis by suppressing the LXRα pathway and alleviate lipid accumulation in the liver of chronic heat-stressed broilers. © 2019 Society of Chemical Industry.

The regulatory effects of miR-138-5p on selenium deficiency-induced chondrocyte apoptosis are mediated by targeting SelM.

Apoptosis is a common paradigm of cell death and plays a key role in cartilage damage and selenium (Se) deficiency. Selenoproteins play major roles in determining the biological effects of Se, and are potentially involved in the pathophysiological processes in bone tissue. MicroRNAs (miRNAs) play important roles in cell proliferation, differentiation, apoptosis and tumorigenesis. Based on the preliminary results, the expression of selenoprotein M (SelM) was significantly decreased (69%) in chicken cartilage tissues with Se deficiency, and we subsequently screened and verified that SelM is one of the target genes of miR-138-5p in chicken cartilage using a dual luciferase reporter assay and real-time quantitative PCR (qRT-PCR). The expression of miR-138-5p was increased in response to Se deficiency, and the overexpression of miR-138-5p increased caspase-3, caspase-9, BAX and BAK levels, while the BCL-2 level was decreased, suggesting that miR-138-5p induced apoptosis via the mitochondrial pathway in vivo and in vitro. We explored whether oxidative stress, mitochondrial fission and fusion, and energy metabolism might trigger apoptosis to obtain an understanding of the mechanisms underlying the effects of miR-138-5p on Se deficiency-induced apoptosis in cartilage. The levels of indicators of oxidative stress, mitochondrial dynamics and energy metabolism were changed as well. This study confirmed that SelM is one of the target genes of miR-138-5p, and the overexpression of miR-138-5p induced by Se deficiency triggered oxidative stress, an imbalance in mitochondrial fission and fusion, and energy metabolism dysfunction. Therefore, miR-138-5p is involved in the mitochondrial apoptosis pathway via targeting SelM in chicken chondrocytes.

Dietary enzymatically treated Artemisia annua L. supplementation alleviates liver oxidative injury of broilers reared under high ambient temperature.

Heat stress induced by high ambient temperature is a major concern in commercial broiler production. To evaluate the effects of dietary enzymatically treated Artemisia annua L. (EA) supplementation on growth performance and liver oxidative injury of broilers reared under heat stress, a total of 320 22-day-old male broilers were randomly allotted into five groups with eight replicates of eight birds each. Broilers in the control group were housed at 22 ± 1 °C and fed the basal diet. Broilers in the HS, HS-EA1, HS-EA2, and HS-EA3 groups were fed basal diet supplemented with 0, 0.75, 1.00, and 1.25 g/kg EA, respectively, and reared under cyclic high temperature (34 ± 1 °C for 8 h/day and 22 ± 1 °C for 16 h/day). Broilers fed EA diets had higher final body weight, average daily body weight gain, and average daily feed intake, as well as liver concentration of reduced glutathione, activities of antioxidant enzymes, abilities to inhibit hydroxyl radical and superoxide radical (HS-EA2 and HS-EA3), and lower liver concentrations of reactive oxygen metabolites, malondialdehyde, and protein carbonyl (HS-EA1, HS-EA2, and HS-EA3) than HS group (P < 0.05). EA treatment downregulated the mRNA levels of heat shock proteins 70 and 90, upregulated the mRNA levels of nuclear factor erythroid 2-related factor 2 (HS-EA1, HS-EA2, and HS-EA3) and heme oxygenase 1 (HS-EA2 and HS-EA3) in liver of heat-treated broilers (P < 0.05). In conclusion, EA alleviated heat stress-induced growth depression and liver oxidative injury in broilers, possibly through improving the antioxidant capacity and regulating the pertinent mRNA expression. The appropriate inclusion level of EA in broiler diet is 1.00-1.25 g/kg.

Cloning, characterization, and expression analysis of LGP2 cDNA from goose, Anser cygnoides.

Laboratory of genetics and physiology 2 ( LGP2: ) is a homologue of the retinoic acid inducible gene-I and melanoma differentiation associated gene 5 that lacks the caspase activation and recruitment domain required for signaling. It plays a pivotal role in host immune response. In this study, we cloned and characterized the full-length open reading frame ( ORF: ) sequence of LGP2 in the Qingyuan goose (Anser cygnoides) and evaluated the mRNA expression of this gene post infection with an H5N1 highly pathogenic avian influenza virus ( HPAIV: ). The full-length goose LGP2 ORF (2,028 bp) encoded a polypeptide of 675 amino acids. The deduced amino acid sequence contained 5 main overlapping structural domains-2 DEAD/DEAH box helicase domains, one conserved restriction domain of bacterial type III restriction enzyme, one helicase superfamily C-terminal domain and one C-terminal regulatory domain. Quantitative real-time PCR analysis indicated that goose LGP2 was constitutively expressed in all 19 investigated tissues, but the expression level was different among them. It was high expressed in the trachea, jejunum, bursa, kidney and heart, but low in the glandular stomach, lung, liver, spleen, crop and muscular stomach. A significant increase in the transcription of LGP2 was detected in the brain, spleen and lungs of geese post infection with H5N1 HPAIV versus uninfected tissues. These findings indicated that goose LGP2 was an important receptor that is involved in the host antiviral innate immune defense to H5N1 HPAIV in geese.

Uncovering molecular events associated with the chemosuppressive effects of flaxseed: a microarray analysis of the laying hen model of ovarian cancer.

BACKGROUND: The laying hen model of spontaneous epithelial ovarian cancer (EOC) is unique in that it is the only model that enables observations of early events in disease progression and is therefore also uniquely suited for chemoprevention trials. Previous studies on the effect of dietary flaxseed in laying hens have revealed the potential for both amelioration and prevention of ovarian cancer. The objective of this study was to assess the effect of flaxseed on genes and pathways that are dysregulated in tumors. We have used a bioinformatics approach to identify these genes, followed by qPCR validation, immunohistochemical localization, and in situ hybridization to visualize expression in normal ovaries and tumors from animals fed a control diet or a diet containing 10% flaxseed. RESULTS: Bioinformatic analysis of ovarian tumors in hens led to the identification of a group of highly up-regulated genes that are involved in the embryonic process of branching morphogenesis. Expression of these genes coincides with expression of E-cadherin in the tumor epithelium. Levels of expression of these genes in tumors from flax-fed animals are reduced 40-60%. E-cadherin and miR200 are both up-regulated in tumors from control-fed hens, whereas their expression is decreased 60-75% in tumors from flax-fed hens. This does not appear to be due to an increase in ZEB1 as mRNA levels are increased five-fold in tumors, with no significant difference between control-fed and flax-fed hens. CONCLUSIONS: We suggest that nutritional intervention with flaxseed targets the pathways regulating branching morphogenesis and thereby alters the progression of ovarian cancer.

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-ß promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1ß and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

Influence of bacteriophage P22 on the inflammatory mediator gene expression in chicken macrophage HD11 cells infected with Salmonella Typhimurium.

This study was designed to evaluate the effects of bacteriophage on the intracellular survival and immune mediator gene expression in chicken macrophage-like HD11 cells. The invasive ability and intracellular survival of Salmonella Typhimurium (ST(P22-) ) and lysogenic S. Typhimurium (ST(P22+) ) in HD11 cells were evaluated at 37 °C for 24 h postinfection (hpi). The expression of inflammatory mediator genes was determined in ST(P22-) - and ST(P22+) -infected HD11 cells treated with and without bacteriophage P22 at 1 and 24 hpi using quantitative RT-PCR. The ability of ST(P22-) and ST(P22+) to invade HD11 cells was significantly decreased by bacteriophage P22 at 1 hpi. The numbers of intracellular ST(P22-) and ST(P22+) were significantly decreased from 2.39 to 1.62 CFU cm(-2) and from 3.40 to 1.72 CFU cm(-2) in HD11 cells treated with bacteriophage P22, respectively, at 24 hpi. The enhanced expression of inflammatory mediators was observed in ST(P22-) - and ST(P22+) -infected HD11 cells treated with and without bacteriophage P22. These results suggest that the application of bacteriophage could be an effective way to control the intracellular infection.

Use of Artemisia annua as a natural coccidiostat in free-range broilers and its effects on infection dynamics and performance.

This work investigated the preventive effect of Artemisia annua L. dried leaves supplied as a botanical coccidiostat to two broiler genotypes reared in a Danish free-range system in a factorial experiment (two genotypes and ± supplement of dried A. annua leaves). The genotypes White Bresse L40, a pure slow-growing line, and Kosmos 8 Ross, a hybrid genotype with medium growing characteristics, were used. Broilers were raised indoor until 29-days-old and kept free of parasites. Twelve groups of 30 randomly selected broilers were placed in the range forming three replicates for each treatment combination. The paddocks were cultivated with a mix of grass and clover. A separate group of broilers was naturally infected with Eimeria spp. oocysts and five animals nominated as "seeders" were introduced to the above mentioned 12 groups, 10 days after its formation, with each group consisting of 35 animals per plot. This infection strategy was meant to imitate the transmission pathway observed at farm level. Ten individual birds from each of the 12 groups, in total 120 animals of mixed sex, were monitored twice weekly for 30 days for oocysts excretion. PCR of pooled faecal samples, oocyst morphology and localization upon necropsy were used to identify the Eimeria species involved in the infection. In general, broilers from both genotypes in the range coped well with a coccidia infection caused by Eimeria acervulina and Eimeria maxima as no clinical symptoms, or deaths, were reported during the experiment. In general, broilers supplemented with A. annua dried leaves showed a significantly (p<0.05) reduced number of excreted oocysts during the infection with no interaction to genotype. Females generally had a significantly higher shedding of oocysts than males (p<0.05). The overall body weight gain and the daily weight gain when infection was subdued showed a three-way interaction among genotype, sex and treatment - accounted mainly for the fact that Kosmos females responded positively to the Artemisia treatment while Kosmos males responded negatively, and only minor differences were found between sexes for the White Bresse genotype. In conclusion, supply of A. annua dried leaves as a botanical coccidiostat significantly reduced oocyst output in free ranged broilers and thus may form part of a strategy to prevent commercial losses.

Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction.

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.

Proteomics in the chicken: tools for understanding immune responses to avian diseases.

The entire chicken genome sequence will be available by the time this review is in press. Chickens will be the first production animal species to enter the "postgenomic era." This fundamental structural genomics achievement allows, for the first time, complete functional genomics approaches for understanding the molecular basis of chicken normo- and pathophysiology. The functional genomics paradigm, which contrasts with classical functional genetic investigations of one gene (or few) in isolation, is the systematic holistic genetic analyses of biological systems in defined contexts. Context-dependent gene interactions are the fundamental mechanics of all life. Functional genomics uses high-throughput large-scale experimental methods combined with statistical and computational analyses. Projects with expressed sequence tags in chickens have already allowed the creation of cDNA microarrays for large-scale context-dependant mRNA analysis (transcriptomics). However, proteins are the functional units of almost all biological processes, and protein expression very often bears no correlation to mRNA expression. Proteomics, a discipline within functional genomics, is the context-defined analysis of complete complements of proteins. Proteomics bridges the "sequence-to-phenotype gap;" it complements structural and other functional genomics approaches. Proteomics requires high capital investment but has ubiquitous biological applications. Although currently the fastest-growing human biomedical discipline, new paradigms may need to be established for production animal proteomics research. The prospective promise and potential pitfalls of using proteomics approaches to improve poultry pathogen control will be specifically highlighted. The first stage of our recently established proteomics program is global protein profiling to identify differentially expressed proteins in the context of the commercially important pathogens. Our trials and tribulations in establishing our proteomics program, as well some of our initial data to understand chicken immune system function, will be discussed.

The expression of calbindin in chicks that are divergently selected for low or high incidence of tibial dyschondroplasia.

Three experiments were conducted with broiler chicks that were divergently selected for low or high incidence of tibial dyschondroplasia (LTD and HTD, respectively) to determine if the expression of intestinal calbindin-28 kD mRNA and protein differed between the 2 strains. In addition, levels of intestinal vitamin D receptor mRNA and plasma thyroid hormone concentrations were also examined. In experiment 1, LTD and HTD chicks were fed a corn-soybean meal diet that was adequate in all nutrients except cholecalciferol (D3), which was titrated to 5 or 40 microg/kg diet in a completely randomized 2 x 2 factorial arrangement. At 4 and 8 d of age, HTD chicks fed 5 microg of D3/kg of diet had a lower (P < 0.05) expression level of calbindin-28 kD mRNA than the LTD chicks fed the same diet. At 4 and 8 d of age, HTD chicks fed 5 microg of D3 had the lowest intestinal expression of calbindin-28 kD protein. Expression of vitamin D receptor mRNA did not differ for broiler strains at either level of D3 supplementation. In experiment 2, there was no significant difference in the expression of calbindin-28 kD mRNA or vitamin D receptor mRNA between day-of-hatch LTD, HTD, and commercial broiler chicks. Experiment 3 was similar in design to the first experiment except that the birds were fed for 18 d. Calbindin-28 kD and vitamin D receptor mRNA expression levels at 18 d were similar to those observed in experiment 1. Plasma triiodothyronine and free-triiodothyronine concentrations were greater for LTD chicks, regardless of dietary D3 supplementation levels. These results suggest that divergent selection of broilers for LTD or HTD alters the physiological response to nutritionally inadequate levels of dietary D3.

Influence of dietary phytase supplementation on incidence and severity in broilers divergently selected for tibial dyschondroplasia.

A study was conducted to determine the effect of dietary supplementation of phytase on the incidence and severity of tibial dyschondroplasia (TD) in chickens selected for high (HTD) and low (LTD) incidences of TD for 11 generations. By feeding a phosphorus-deficient diet (0.1% nonphytate phosphorous; nPP), HTD and LTD chickens were further identified as high-sensitivity birds (HS) and low-sensitivity birds (LS) to phosphorus deficiency based on mortality. Two hundred forty 1-d-old chicks from HTD and LTD lines (five replications of four birds per treatment) were randomly assigned to a control diet with 0.5% nPP and two treatment diets (0.1% nPP) with and without 600 phytase units (FTU) Natuphos phytase/kg. Feed consumption and growth rate were measured for 3 wk, and both tibiae were scored for TD incidence, average TD score, and total number of TD lesions with the most severe form of the abnormality (lesions that were scored 3). The addition of phytase had no influence on TD incidence and lesion scores of 3 in HTD chicks. However, a nonsignificant reduction in TD incidence (P = 0.07), TD score, and no. 3 lesions (P < or = 0.01) were observed in LTD chicks. Interactions between sensitivity (to P deficiency) and phytase (P < or = 0.01) and sensitivity and nPP (P < or = 0.01) were observed for no. 3 scores in LTD chicks. These results indicate that phytase was effective in reducing TD incidence and severity in LTD chicks but not in HTD chicks.

The effects of dietary protein independent of essential amino acids on growth and body composition in genetically lean and fat chickens.

1. Growth performance between 28 and 49 d of age and carcase composition at 49 d in genetically lean (LL) and fat (FL) broilers fed on diets varying in non-essential amino acid (NEAA) concentrations were compared in 2 experiments. In experiment 1, 3 crude protein (CP) contents (133, 155, and 178 g/kg) were compared. In experiment 2, 4 CP levels (131, 150, 170 and 189 g/kg) were compared. All diets were supplemented with synthetic amino acids to cover the EAA requirement of the LL birds. 2. Weight gains of FL chickens were not affected by dietary treatments, while those of LL increased when CP level increased. 3. Reducing CP content always increased body lipids, abdominal fat and food conversion ratio in both lines in both experiments; however, the effect on abdominal fat was more pronounced in the FL birds. 4. Reducing CP concentration always decreased breast muscle proportion in both lines in both experiments, even when growth rate was not affected by CP. 5. It is concluded that LL chickens require diets more concentrated in NEAA than fat chickens and that there seems to be an effect of NEAA on breast muscle development.

Effects of divergent selection for incidence of tibial dyschondroplasia (TD) on purebred and crossbred performance. 1. TD incidence and calcium and phosphorus plasma concentrations.

1. The effect of divergent selection for high (H) or low (L) incidence of tibial dyschondroplasia (TD) for 7 generations on blood calcium (Ca) and phosphorus (P) concentrations was studied. 2. The chicks used in this experiment were obtained from diallel crosses between H and L lines to obtain HH, HL, LH, and LL lines. A random-bred control (CC) line was also used. 3. The chicks were reared under standard management conditions. Body weight and plasma Ca and P concentrations were measured at 2, 4, and 7 weeks of age. Incidence of TD was recorded at 4 and 7 weeks. 4. Although HH birds had lower body weights than the other lines, there was no significant effect attributable to line selection for body weight at 2, 4, and 7 weeks of age. 5. The LL line birds had no incidence of TD at 4 weeks of age; however, the incidence of TD in LL line birds was 5.3% at 7 weeks of age. The incidence of TD was higher in HH line birds than the other line at 4 and 7 weeks of age. 6. Plasma Ca and P concentrations and Ca:P ratios increased with age. These results showed that HH line birds had higher plasma Ca, lower plasma P and higher Ca:P ratios than birds of the LL line.