W. G. Bottje

Aflatoxicosis alters avian renal function, calcium, and vitamin d metabolism

Experiments were designed to determine the effects of aflatoxicosis on avian renal function, calcium (CA), inorganic phosphorous (Pi), and vitamin D metabolism, and to determine if the effects of aflatoxin are reversible upon discontinuation of toxin administration. Three-week-old male broiler chickens (n = 12 per treatment) received aflatoxin (AF; 2 mg/kg po) or an equal volume of corn oil, the AF carrier vehicle, for 10 consecutive days. After 10 d of treatment, half of the birds from each treatment group were anesthetized and prepared for renal function analysis, which included a 2-h phosphate loading period. Ten days after discontinuation of AF treatment, the remaining birds in each treatment group were anesthetized and prepared for renal function analysis. AF decreased plasma 25-hydroxy vitamin D [25(OH)D] and 1,25-dihydroxy vitamin D [1,25(OH)2D] levels after 5 d of treatment. After 10 d of treatment, urine flow rate (V), fractional sodium excretion (FENa), and fractional potassium excretion (FEK) were lower in AF-treated birds. In addition, total plasma Ca tended to be lower (p = .10) and fractional Ca excretion (FECa) tended to be higher (p = .10) in the AF-treated birds. Intravenous phosphate loading produced a sharp increase in urine hydrogen ion concentration ([H+]) in the AF-treated birds. Glomerular filtration rate (GFR) was reduced and plasma osmolality was increased in AF-treated birds 10 d after discontinuation of toxin administration. The results indicate that AF directly or indirectly affects Ca and Pi metabolism in avians. At the present time, the effects may be related to altered vitamin D and parathyroid hormone (PTH) metabolism. Aflatoxicosis may decrease endogenous PTH synthesis and the renal sensitivity to PTH. The AF-related increase in urine [H+] during phosphate loading is probably due to increased Na+/H+ counterport, suggesting that AF stimulates sodium reabsorption. Also, the decrease in GFR exhibited 10 d after toxin removal indicates that AF may cause prolonged alteration in renal function.

Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge.

Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10(6) to 10(8) cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, DeltaaroA-DeltahtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain DeltaaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, and DeltaaroA-DeltahtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with DeltaaroA-DeltahtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.

Protein metabolism during an acute phase response in chickens

Summary. Fractional rates of liver, muscle, plasma and acute phase portein synthesis were measured in chickens injected with saline or E. coli lipopolysaccharide (LPS). Male Single Comb White Leghorns were infused with a primed constant infusion of 15N-L-methionine and 2H5-L-phenylalanine into the portal vein for 2 h. Changes in plasma amino acid enrichment were similar for both amino acids reaching an apparent plateau by the 30 min sampling time. The enrichment of plasma protein-bound amino acid was measurable after 1 h of isotope infusion and increased linearly over 2 h. LPS injection decreased free phenylalanine enrichment in the carotid artery (50%), and reduced tissue free methionine enrichment in the liver, pectoralis, and gastrocnemius by 16, 41, and 31% respectively. Isotopic enrichment of phenyl-alanine in liver protein, plasma protein and hemopexin increased in LPS injected birds relative to control birds. Fractional rates of muscle protein synthesis were not affected by LPS injection, however, liver protein, plasma protein, and hemopexin fractional synthesis rates increased 141, 161 and 266% respectively compared with untreated animals.

Heart and breast muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus)

This study was conducted to determine function and defects in electron transport in muscle mitochondria of meat chickens (broilers) with pulmonary hypertension syndrome (PHS). The respiratory control ratio (RCR, indicative of respiratory chain coupling) was higher in the control than in PHS breast and heart muscle mitochondria, but there were no differences in the ADP/O (an index of oxidative phosphorylation). Sequential additions of ADP improved the RCR in the control breast muscle mitochondria and the ADP/O in PHS breast and heart muscle mitochondria. Basal hydrogen peroxide production, (an indicator of electron leak), was higher in PHS breast and heart muscle mitochondria than in controls and differences in electron leak in PHS mitochondria were magnified by inhibiting electron transport at Complex I and III (cyt b(562)). Complex I activity was lower in PHS heart mitochondria but there was no difference in Complex II activity. Thus, compared to controls, PHS mitochondria exhibited site-specific defects in electron transport within Complex I and III that could contribute to lower respiratory chain coupling. Additionally, it appears that healthy broilers may exhibit higher basal levels of electron leak compared to other avian species. Together, these findings provide insight into inefficient cellular use of oxygen that may contribute to the development of PHS in broilers.

Gene expression in breast muscle associated with feed efficiency in a single male broiler line using a chicken 44K microarray. II. Differentially expressed focus genes

Global RNA expression in breast muscle obtained from a male broiler line phenotyped for high or low feed efficiency (FE) was investigated. Pooled RNA samples (n = 6/phenotype) labeled with cyanine 3 or cyanine 5 fluorescent dyes to generate cRNA probes were hybridized on a 4 × 44K chicken oligo microarray. Local polynomial regression normalization was applied to background-corrected red and green intensities with a moderated t-statistic. Corresponding P-values were computed and adjusted for multiple testing by false discovery rate to identify differentially expressed genes. Microarray validation was carried out by comparing findings with quantitative reverse-transcription PCR. A 1.3-fold difference in gene expression was set as a cutoff value, which encompassed 20% (782 of 4,011) of the total number of genes that were differentially expressed between FE phenotypes. Using an online software program (Ingenuity Pathway Analysis), the top 10 upregulated genes identified by Ingenuity Pathway Analysis in the high-FE group were generally associated with anabolic processes. In contrast, 7 of the top 10 downregulated genes in the high-FE phenotype (upregulated in the low-FE phenotype) were associated with muscle fiber development, muscle function, and cytoskeletal organization, with the remaining 3 genes associated with self-recognition or stress-responding genes. The results from this study focusing on only the top differentially expressed genes suggest that the high-FE broiler phenotype is derived from the upregulation of genes associated with anabolic processes as well as a downregulation of genes associated with muscle fiber development, muscle function, cytoskeletal organization, and stress response.

Polymorphisms in Uncoupling Protein, Melanocortin 3 Receptor, Melanocortin 4 Receptor, and Pro-Opiomelanocortin Genes and Association with Production Traits in a Commercial Broiler Line

Because avian uncoupling protein (avUCP), melanocortin 3 receptor (MC3R), melanocortin 4 receptor (MC4R), and pro-opiomelanocortin (POMC) genes may be associated with production traits [e.g., BW, weight gain (WG), and feed conversion ratio (FCR)], male and female broilers from an elite broiler line were screened for polymorphisms in these genes. The PCR-restriction fragment length polymorphism (RFLP) tests were developed to type the missense polymorphisms UCPAla118Val, MC4RSer76Leu, MC3R-Met54Leu, and Gly104Ser and POMCPro61Leu. Of 39 single nucleotide polymorphisms identified in all 4 genes, 24/39 were transitions with 11 having a C to T change. Of the 23 polymorphisms in UCP, 17 represented at least 7 haplotypes in this pedigreed broiler line. The UCP Ala-118Val allele was associated with a) high feed efficiency (FE; P = 0.03) and WG (P = 0.053) in selected males, and b) high BW in selected females (P = 0.07) and unselected males (P = 0.015). The UCPVal118Val allele was found in approximately 10% of the birds that were screened. Five silent substitutions, 3 in MC3R and 2 in MC4R, were also identified. Thirteen polymorphisms were identified in the POMC gene representing at least 3 different alleles. A missense Pro61Leu heterozygote was associated with greater BW in females. The heterozygote MC3R Gly104Ser polymorphism was associated with greater FE in selected males (P = 0.03) and greater BW in unselected males (P = 0.007). The MC4R Ser76Leu heterozygote polymorphism was associated with greater BW than the Leu76 homozygote in females (P = 0.05). From these findings, we hypothesize that UCP, MC3R, MC4R and POMC genes may play important roles and could be candidate loci for production traits such as feed conversion and BW in commercial broiler breeding stock.

Aflatoxin and glutathione in domestic fowl (Gallus domesticus)—II. Effects on hepatic blood flow

1. The effect of aflatoxin on plasma aspartate aminotransferase (AST), protein, and hepatic glutathione (GSH) and hepatic blood flow (perfusion), were determined in 3-week-old male chickens. 2. Daily aflatoxin gavage (2 mg/kg body wt, in corn oil) for 5 and 10 days elevated plasma AST and hepatic GSH, and depressed plasma protein and hepatic perfusion. Also, renal GSH was elevated after 10 days of aflatoxin treatment. 3. Birds given aflatoxin for 10 days followed by a 10-day recovery period exhibited tissue GSH, plasma AST and protein levels that were not different from control, but hepatic perfusion remained depressed.

Investigation of mitochondrial protein expression and oxidation in heart muscle in low and high feed efficient male broilers in a single genetic line

The objective of this study was to assess the expression of mitochondrial proteins and oxidized proteins in heart muscle homogenate obtained from male broilers exhibiting either high or low feed efficiency (G:F) phenotypes. Tissue homogenate was prepared from ventricular tissue obtained from broilers with high (0.80+/-0.01, n=8) and low (0.62+/-0.02, n=8) FE. The levels of specific immunoreactive proteins and oxidized proteins (carbonyls) were determined using Western blot analysis. The expression of 6 electron transport chain proteins [complex II, 70S subunit (CII 70S); iron-sulfur-containing protein (ISP), cytochrome b (Cyt b), cytochrome (Cyt c1) (of complex III); and cytochrome oxidase subunit II (COX II) (of complex IV)] and adenine nucleotide translocator 1 (ANT1) were higher in low feed efficiency heart mitochondria, but 1 protein [NAD subunit 6c (NAD6c) (complex I)] was higher in high feed efficiency birds. Protein carbonyl levels, indicative of oxidized proteins, were higher in heart tissue of low compared with high feed efficiency broilers.

Improved hatchability and posthatch performance in turkey poults receiving a dextrin-iodinated casein solution in ovo

Two experiments were conducted with a commercial turkey company using a commercial egg injection system to investigate the effect of a dextrin-iodinated casein solution injected in ovo at 25 d of incubation on turkey poult hatchability, hatch weight, and growth (6 or 7 d posthatch). In experiment 1, a total of 3,900 turkey eggs (1,300 per group) were injected at 25 d of incubation with either 200 μL of a control (physiological saline) solution or a dextrin solution (18% maltodextrin and 10% potato starch dextrin) with 75 or 375 μg/mL of iodinated casein (DexIC75 or DexIC375, where Dex and IC refer to dextrin and iodinated casein, respectively). Two hundred poults from each group were neck-tagged, weighed (hatch weight), placed in a commercial turkey house within a single brooder ring, and weighed again (7 d posthatch). In experiment 2, a total of 5,200 eggs (2,600 per group) were injected with the control or DexIC75 solution. A total of 600 poults (300 per group) were neck-tagged and hatch weights were obtained, followed by placement in a single brooder ring in a commercial house and a second weighing (6 d posthatch). Eggs in experiments 1 and 2 were obtained from hen flocks that were 33 and 5 wk into the laying cycle, respectively. In experiment 1, the DexIC75 injection resulted in a 1.8% increase (P = 0.03) in hatch weight. In experiment 2, the DexIC75 treatment resulted in a 2.4% increase in hatchability (P = 0.01), a 4.3% increase in hatch weight (P < 0.001), and a 1.8% increase in 6-d poult weights (P < 0.03) compared with controls. Results of this study indicate that a solution containing dextrin and 75 μg/mL of iodinated casein injected into turkey eggs at 25 d of incubation may be used to improve early poult weights, hatchability, or both in commercial turkey production.

The hepatic extraction of plasma free amino acids and response to hepatic portal venous infusion of methionine sources in anesthetized SCWL males (Gallus domesticus).

This study was conducted to investigate the hepatic extraction of plasma free amino acids in anesthetized Single Comb White Leghorn (SCWL) males (Gallus domesticus). SCWL males were anesthetized and implanted with cannulae in the carotid artery, hepatic vein, hepatic portal vein and the left hepatic duct. Free amino acids in plasma and bile were determined before, during and after 30-min infusions of Saline (control), DL-Methionine (DL-Met) or DL-2-hydroxy-4-methylthio-butanoic acid (DL-HMB) into the hepatic portal vein. Hepatic extraction rates (HER) of amino acids were calculated based on the concentration of amino acids in plasma multiplied by estimations of blood flow in the hepatic portal vein, hepatic artery and hepatic vein. For the non-essential amino acids, alanine had the highest HER (46%). The liver also removed more than 20% of hepatic inflow of tyrosine and asparagine with substantial extraction (14-18%) of serine, glycine and glutamine, also. In contrast, less than 5% of hepatic inflow of glutamate and cystine were removed by liver. For the essential amino acids, HER for methionine, histidine and phenylalanine were 30, 14 and 17%, respectively, with less than 5% for branched-chain amino acids, lysine, arginine and threonine. Biliary secretion of amino acids represented a small percentage (<0.2%) of total hepatic extraction turnover of the amino acids. Infusion of methionine sources, DL-Met and DL-HMB, had no effect on hepatic metabolism of amino acids other than methionine. The results demonstrated for the first time, the hepatic extraction of circulating free amino acids in avian species in vivo.