Cell bioenergetics in Leghorn male hepatoma cells and immortalized chicken liver cells in response to 4-hydroxy 2-nonenal-induced oxidative stress.

The major objectives of this study were to compare cell bioenergetics in 2 avian liver cell lines under control conditions and in response to oxidative stress imposed by 4-hydroxy 2-nonenal (4-HNE). Cells in this study were from a chemically immortalized Leghorn male hepatoma (LMH) cell line and a spontaneously immortalized chicken liver (CELi) cell line. Oxygen consumption rate (OCR) was monitored in specialized microtiter plates using an XF24 Flux Analyzer (Seahorse Bioscience, Billerica, MA). Cell bioenergetics was assessed by sequential additions of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin-A that enables the determination of a) OCR linked to adenosine triphosphate (ATP) synthase activity, b) mitochondrial oxygen reserve capacity, c) proton leak, and d) nonmitochondrial cytochrome c oxidase activity. Under control (unchallenged) conditions, LMH cells exhibited higher basal OCR and higher OCR attributed to each of the bioenergetic components listed above compared with CELi cells. When expressed as a percentage of maximal OCR (following uncoupling with FCCP), LMH cells exhibited higher OCR due to ATP synthase and proton leak activity, but lower mitochondrial oxygen reserve capacity compared with CELi cells; there were no differences in OCR associated with nonmitochondrial cytochrome c oxidase activity. Whereas the LMH cells exhibited robust ATP synthase activity up to 50 µM 4-HNE, CELi cells exhibited a progressive decline in ATP synthase activity with 10, 20, and 30 µM 4-HNE. The CELi cells exhibited higher mitochondrial oxygen reserve capacity compared with LMH cells with 0 and 20 µM 4-HNE but not with 30 µM 4-HNE. Both cell lines exhibited inducible proton leak in response to increasing levels of 4-HNE that was evident with 30 µM 4-HNE for CELi cells and with 40 and 50 µM 4-HNE in LMH cells. The results of these studies demonstrate fundamental differences in cell bioenergetics in 2 avian liver-derived cell lines under control conditions and in response to oxidative challenge due to 4-HNE.

Influence of dietary vitamin E on phagocytic functions of macrophages in broilers.

Vitamin E (VE) is known for its antioxidant properties and has been shown to modulate immune system functions in various species. This study examined the influence of different levels of dietary VE (alpha-tocopherol acetate) on phagocytic functions of macrophages (abdominal exudate cells) in broiler chickens at 3, 5, and 7 wk. Birds were fed commercial diets containing 16 (control), 110, or 220 mg of VE/kg of feed. Macrophages were elicited into the abdominal cavity by injecting a 3% Sephadex solution prepared in PBS (G50-50, 1 mL/100 g of BW) 42 h prior to harvest. The percentage of phagocytically active macrophages and the number of SRBC phagocytosed per macrophage for unopsonized and antibody-opsonized SRBC were determined. These aspects of macrophage function were assessed based on 900 macrophages per sample. When unopsonized SRBC were used, dietary VE supplementation above control level did not affect phagocytic function of macrophages at wk 3, 5, or 7. With antibody-opsonized SRBC, the percentage of phagocytically active macrophages and the number of SRBC phagocytosed per macrophage were higher (P = 0.08 and P = 0.01, respectively) in 3-wk-old birds fed 110 and 220 mg of VE/kg of feed compared with age-matched controls. This enhancing effect of VE supplementation on macrophage function was not observed in 5- and 7-wk-old broilers. It appears from this study that supplemental VE enhances Fc-receptor-mediated macrophage phagocytic activity at early stages of broiler growth.

Uptake of DL-2-hydroxy-4-methylthio-butanoic acid (DL-HMB) in the broiler liver in vivo.

The methionine source DL-2-hydroxy-4methylthio-butanoic acid (DL-HMB; Alimet feed supplement) is widely used in the poultry industry. The purpose of this study was to determine the capacity of the broiler liver to remove DL-HMB from the circulation. Cannulae were implanted in the carotid artery and hepatic and hepatic portal veins in anesthetized male broilers (3.33 +/- 0.13 kg BW). In Experiment 1, birds (n = 5) were infused with DL-HMB solutions (diluted in saline, pH 7.2 to 7.4) into the hepatic portal vein at rates ranging from 4.4 to 22 mg/min per kg BW, whereas in Experiment 2, birds (n = 6) were infused with DL-HMB at rates ranging from 2.2 to 4.4 mg/min per kg BW. Plasma samples from each vessel were obtained before and after each 10-min DL-HMB infusion period with a 10-min clearance period allowed between each DL-HMB infusion. Regression analysis revealed a highly significant correlation in the amount of DL-HMB entering the liver via afferent vessels (afferent DL-HMB) and DL-HMB removed by the liver (y = 0.86(x) - 173, r2 = 0.98). The slope of this regression indicates that 86% of DL-HMB entering in afferent blood (i.e. from both the hepatic artery and hepatic portal vein) was removed or that the liver apparently metabolized 86% of the DL-HMB that entered the liver. The results indicate that the broiler liver has the capacity to remove DL-HMB from the circulation far in excess of that needed to metabolize DL-HMB that would enter the liver following gastrointestinal absorption in birds fed a conventional poultry diet. In addition, present results implicate the liver as a major site of removal from circulation and further metabolism of DL-HMB in chickens.

Effects of dietary vitamin E on the immune system in broilers: altered proportions of CD4 T cells in the thymus and spleen.

To gain insight into the immunomodulatory effects of vitamin E (VE), immune cell population analyses were conducted using thymus and spleen from male broilers fed diets with various levels of VE supplementation (0, 17, 46, and 87 mg dl-alpha-tocopherol acetate/kg of feed). At 2 and 7 wk of age, the percentages of B cells, macrophages, and T cell subsets, delineated by the expression of CD4, CD8, and T cell receptor (TCR) isotype, in thymus and spleen were determined by flow cytometry. The percentages of thymic and splenic B cells and macrophages from 2- and 7-wk-old chickens, as well as the percentage of thymic T cells in 2-wk-old chickens, were unaffected by VE treatment. However, 7-wk-old broilers maintained on 87 mg VE/kg feed had a higher percentage of CD4+CD8- thymocytes, a higher CD4+CD8- to CD4-CD8+ thymocyte ratio, and a lower percentage of CD4+CD8+ thymocytes than chickens receiving no dietary VE supplementation. The VE-induced increase in the percentage of CD4+CD8- thymocytes was due to an increase in the TCR2+CD4+CD8- thymocyte subset, whereas the decrease in the percentage of CD4+CD8+ thymocytes involved all TCR defined T cell subsets. In the spleen, the percentage of CD4+CD8- T cells was lower in 2-wk-old chickens and higher in 7-wk-old chickens maintained on 87 mg/kg feed than in chickens receiving no dietary VE supplementation. The decrease in CD4+CD8- splenocytes at 2 wk of age was due to a decline in the percentage of TCR2+CD4+CD8- splenocytes, whereas the increase in CD4+CD8- splenocytes in 7-wk-old chicks was due to an increase in the percentages of all TCR defined CD4+CD8- T cell subsets. These data support an immunomodulatory effect of VE on CD4+CD8- T cells.

Lung lining fluid antioxidants in male broilers: age-related changes under thermoneutral and cold temperature conditions.

The purpose of this study was to determine age-related changes in lung lining fluid antioxidants in broilers reared under thermoneutral or cold temperature conditions. Male broilers (Cobb 500) were placed in floor pens within environmental chambers and fed a standard commercial starter diet. The thermoneutral Control chamber was maintained at 32, 30, 27, and 22 to 25 C for Weeks 1, 2, 3, and 4 to 7, respectively, whereas temperature in the Cold chamber was lowered to 18 C during Week 3 and maintained between 15 and 18 C for the rest of the study. At 2, 4, and 7 wk, four to six birds per chamber were selected randomly. The lungs were lavaged with heparinized saline (2 mL/g lung) to obtain lung lining fluid. Antioxidants [reduced (GSH), oxidized (GSSG), and total (TGSH) glutathione, uric acid, ascorbic acid, and alpha- and gamma-tocopherol] in lung lining fluid were determined by HPLC; protein was determined colorimetrically. In Controls, levels of alpha- and -gamma-tocopherol, uric acid, and GSH in lung lining fluid decreased between 2 and 7 wk of age. Birds in the Cold chamber exhibited higher protein, a higher GSSG:TGSH ratio, and a decrease in ascorbic acid (7 wk) in lung lining fluid relative to Controls. Lung lining fluid antioxidants were not correlated with antioxidants in plasma. To determine the effect of vitamin E supplementation on lung lining fluid antioxidants, birds were given a supplement of 200 IU alpha-tocopherol per day for 7 d. Alpha-tocopherol supplementation elevated alpha-tocopherol levels in lung lining fluid, but lowered ascorbic acid, GSH, and GSSG and had no effect on uric acid in lung lining fluid. The results of this study suggest that antioxidant protection in lung lining fluid may diminish with age, that cold conditions in this study produced an oxidative stress in lung lining fluid in broilers, and that oral supplementation of alpha-tocopherol elevated lung lining fluid alpha-tocopherol.

Effect of dietary dl-alpha-tocopherol on tissue alpha- and gamma-tocopherol and pulmonary hypertension syndrome (ascites) in broilers.

The objectives of this experiment were to determine the effects of high dietary levels of vitamin E on growth performance and pulmonary hypertension syndrome (PHS) mortality. Male broiler chicks (Cobb 500) were randomly assigned to one of four dietary treatments consisting of standard starter and grower diets supplemented with 0, 17, 46, and 87 mg dl-alpha-tocopherol acetate/kg. To encourage the development of PHS, air temperature in the house was 32 and 28 C for Weeks 1 and 2, dropped to 18 C during Week 3, and kept between 10 and 15 C during Weeks 4 through 7. Also, chicks were placed in floor pens on litter used for five previous flocks and ventilation reduced to increase dust and ammonia in the house. Ammonia levels increased from an initial 18 to 36 ppm on Day 42 with the increase in ammonia corresponding to an obvious increase in dust in the air. Lung and liver tissue obtained at 2, 5, and 7 wk of age were analyzed for tissue alpha- and gamma-tocopherol by liquid chromatography. Dietary vitamin E had no effect on body weight, feed intake, or feed efficiency. Cumulative PHS mortality through 7 wk of age was 21% and was also unaffected by dietary treatment. Liver and lung alpha-tocopherol concentrations exhibited a dose-response increase to dietary tocopherol and there was a high correlation between lung and liver tissue alpha-tocopherol (r = 0.72, P < 0.05). Whereas gamma-tocopherol concentrations in lung and liver were unaffected by dietary treatment, liver and lung exhibited age-dependent increases in both alpha- and gamma-tocopherol. Despite dose-dependent increases in tissue alpha-tocopherol, supplementation of diets with up to 87 mg dl-alpha-tocopherol acetate had no effect on growth performance or PHS mortality in broilers under the conditions used in this study.

Influence of diethyl maleate and cysteine on tissue glutathione and growth in broiler chickens.

The objectives of this study were to determine the effects of diethyl maleate (DEM) and l-cysteine (L-Cys) on tissue glutathione (GSH) and growth in male broiler chickens. In Experiment 1, broilers were treated with DEM (0, 1.5, 3, 6, or 12.0 mmol/kg BW, i.p.). After 1 h, maximum GSH depletions were to 9, 24, 20, 19, and 35% of control (0 mmol DEM/kg) for liver, lung, kidney, heart, and brain, respectively. In Experiment 2, time-course changes following 1.5 mmol DEM/kg (i.p.) were determined; time-controls received an equal amount of corn oil (CO, .25 mL/kg BW). Levels of GSH in all tissues were low at 1 and 2 h after DEM in comparison to time-control values. Tissue GSH concentrations returned to values that were not different from controls by 5 h in liver and kidney, by 12 h in heart, and by 24 h in brain and lung. In Experiment 3, the effects of feeding a control diet (0% L-Cys) or one supplemented with 1% L-Cys from 3 to 7 wk of age with weekly i.p. injections (at 3,4,5, and 6 wk of age) of DEM (1.5 mmol/kg BW) or CO (.25 mL/kg BW) on growth rate and tissue GSH were determined. There were no differences in BW among treatment groups between 3 and 6 wk of age. Although there were no differences in 7-wk BW between controls (0% L-Cys/CO) and birds treated with DEM fed either diet, the 1% L-Cys/CO group was heavier (P < .05) than either the 0% or 1% L-Cys/DEM groups, and heavier (P = .066) than controls at 7 wk of age. At 5 wk of age, 1% L-Cys raised GSH concentrations in liver, kidney, lung, and duodenum, but had no effect on heart GSH in birds treated with either CO or DEM. Control hepatic GSH concentrations were higher at 7 than at 5 wk of age. With the exception of duodenal GSH in CO birds, 1% L-Cys had no effect on tissue GSH concentrations in 7-wk-old birds. The results of this study provide an initial characterization of GSH metabolism in commercial male broilers and indicate that DEM produced dose- and time-dependent changes in GSH similar to reported changes in mammals. Results of this study also indicate that increased tissue GSH may be beneficial for growth.

Supplemental L-arginine attenuates pulmonary hypertension syndrome (ascites) in broilers.

The incidence of pulmonary hypertension syndrome (PHS; ascites) was evaluated in two experiments using broiler breeder male by-product chicks exposed after 3 wk of age to cool environmental temperatures (10 to 15 C). In Experiment 1, 3- to 6-wk-old birds were fed a grower diet to which 0 (Control), .25, .5, or 1% supplemental L-arginine HCl had been added. During Weeks 7 to 8, all groups in Experiment 1 were fed a finisher diet containing no supplemental arginine. In Experiment 2, the Control group received no supplemental arginine, a second group was fed a grower diet supplemented with 1% L-arginine HCl (Weeks 3 to 6), and a third group was fed grower and finisher diets supplemented with 1% L-arginine HCl (Weeks 3 to 8). Cumulative PHS mortality was significantly reduced by 1% L-arginine HCl on Days 34 to 46 in Experiment 1. When data from all birds fed grower or finisher diets supplemented with 1% L-arginine HCl were pooled in Experiment 2, cumulative PHS mortality was marginally lower (P = .065) than for the Control group. Supplemental L-arginine HCl had no effect on final body weights, weight gain, or feed conversion in either experiment. Neither body weight on Day 1 or 21 nor net weight gain from Days 1 to 21 determined susceptibility to PHS during the subsequent grower and finisher intervals in either experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Altered renal function in broilers during aflatoxicosis.

Experiments were conducted to determine the effects of aflatoxicosis on acid-base balance, urine flow rate (V), glomerular filtration rate (GFR), clearance of para-aminohippuric acid (CPAH), plasma osmolality, and the renal handling of Na, K, Ca, and P. Three-week-old broilers were gavaged with aflatoxin at a dose of 2 mg/kg of BW per day for 10 consecutive days. Control birds received an equal volume of corn oil, the aflatoxin carrier vehicle. On the eleventh day, the birds were anesthetized and prepared for renal function analysis. A solution containing inulin, para-aminohippuric acid, and mannitol was infused at a low infusion rate (.1 mL/kg of BW per min) and a high infusion rate (.4 mL/kg of BW per min) to determine if aflatoxin affects the renal response to an acute volume load. Aflatoxicosis decreased the fractional excretion of phosphorous (FEP) and plasma Ca concentration but did not significantly alter any other renal function or acid-base variables. The decrease in FEP and plasma Ca may be a direct result of renal tubular damage, decreased Ca absorption from the gut, or a result of altered circulating levels of parathyroid hormone (PTH), and possibly decreased renal sensitivity to PTH.

Effect of carbonated water on growth performance of cockerels subjected to constant and cyclic heat stress temperatures.

Three growth trials were performed to determine the effect of carbonated water on growth performance of cockerels subjected to heat stress temperatures. In Trial 1, a 2 X 2 X 2 factorial design was used to test growth performance of Columbian crossbred cockerels between 8 and 11 weeks of age. The birds were subjected to either cyclic (day-night) heat stress (H) temperature (29 to 34 C) or cyclic (day-night) moderate (M) temperature (25 to 29 C), fed either a corn soy grower (G) diet or a 20% alfalfa diet (A), and provided with tap (TW) or carbonated (CW) drinking water. In Trial 2, a 2 X 4 X 2 factorial was used to access the effect of CW on growth performance of Hubbard cockerels between 4 and 7 weeks of age. Birds were grown in the M or H cyclic thermal environments with dietary treatments consisting of A, G, A plus 1% sodium bicarbonate (AB) and G plus 1% sodium bicarbonate (GB). In Trial 3, a 2 X 2 factorial was used to test the effect of CW on growth performance of Hubbard cockerels fed the G diet and subjected to either constant heat stress (33 C) or thermoneutral (25 C) temperatures. A 24-hr photoperiod was used in each experiment. In all three trials, heat stress reduced (P less than .05) average daily gain (ADG), feed intake, and feed efficiency (G/F).(ABSTRACT TRUNCATED AT 250 WORDS)