Source: UNIVERSITY OF ARKANSAS submitted to
ROLE OF ANTIOXIDANTS IN HEALTH AND DISEASE IN POULTRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0171676
Grant No.
(N/A)
Project No.
ARK01664
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2002
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Project Director
Bottje, W. G.
Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703
Performing Department
POULTRY SCIENCE
Non Technical Summary
Mitochondrial function is essential for the production of energy for the cell. Distruptions or inefficiences in mitochondrial function can therefore have profound effects on growth and development. This project will examine mitochondrial function and biochemistry in health and disease in poultry that are being studied.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3033220103020%
3053220102080%
Goals / Objectives
1.To investigate the relationships between mitochondrial function and biochemistry, antioxidant status, and free radical biochemistry in health and disease in various tissues in poultry. 2. To interrelate observations of mitochondrial function and biochemistry, antioxidant status and free radical biochemistry to enhancement of productivity in poultry.
Project Methods
This project will investigate the role of mitochondrial function and biochemistry in healthy broilers fed various nutrients (e.g. feed additives or probiotics), as well as in broilers that may be experiencing abnormal health (e.g. metabolic disease such as pulmonary hypertension syndrome, different populations of bacteria in the g.i. tract). Considerable effort will be devoted towards understanding relationships between mitochondrial function and feed efficiency in healthy broilers. Mitochondria will be isolated from various tissues for functional and biochemical analyses. Functional analysis may include oxgen consumption, determination of respiratory control ration, ADP:O ratio, and site-specific defects in mitochondrial electron transport chain activity. Biochemical analyses may include assessment of antioxidants, respiratory chain complex activity, expression of mitochondrial proteins, generation of hydrogen peroxide. By understanding how changes in biochemical indices occur in mitochondria, and how such changes affect mitochondrial function and the relationship of these changes to the overall health of the animal, these studies will provide a better understanding of the role that mitochondria play in health and disease in poultry.

Progress 10/01/02 to 09/30/08

Outputs
OUTPUTS: Research in the role that mitochondrial function plays in the phenotypic expression of feed efficiency (i.e. in animal health) indicated that compared to broilers exhibiting high feed efficiency (FE), broilers with low FE had site specific defects in electron transport (observed in muscle, and duodenal tissue) that resulted in higher oxygen radical production and higher protein oxidation. These observations are profoundly important to understanding feed efficiency and to our knowledge were unique; i.e. never previously reported. Higher protein oxidation was consistently observed in tissues obtained from animals with low FE - this was observed in breast and leg muscle, liver, duodenum, heart and lymphocytes and indicates that a significant role of redox chemistry may be involved in the phenotypic expression of feed efficiency. Oxidative stress was also observed in duodenal tissues due to the presence of higher oxidized glutathione, and a higher oxidized to reduced glutatione ratio. Besides protein oxidation, higher levels of ubiquitin expression were also observed. As ubiquitin is instrumental in targeting damaged proteins for recycling of amino acids and protein repair, a very energetically expensive cellular process, this is additional evidence that oxidative stress contributes to low feed efficiency in broilers. It would appear that the mitochondria are at the center of this oxidative stress due to the higher rate of oxygen radical production found in broilers with low feed efficiency. It is interesting to note that steers with low FE also exhibited higher protein oxidation compared to those with higher feed efficiency. Thus, the increased oxidation observed with low FE may occur in other animal species besides poultry. Research was also conducted on proton leak kinetics to determine if differences exist in proton leak that could have an impact on mitochondrial oxygen radical production. It was determined that under a wide range of chemical manipulations, proton leak in mitochondria from broilers with high FE was always less than or equal to, but never greater than proton leak in low FE broilers. The results suggest that there are subtle differences in membrane characteristics (e.g. lipids. integral membrane proteins) that affect proton conductance in broiler muscle mitochondria that may in turn play a role in the phenotypic expression of feed efficiency PARTICIPANTS: Dr. Carol Ojano-Dirain received her Ph.D. on this project and served as a technician (program associate) that was critical to the overall success of the project. After completing her Ph.D., she was hired as a post doctoral research associate in the Mitochondrial Disease Laboratory, College of Medicine, University of Florida. She has recently been promoted to a research assistant professor on a permanent basis. Kentu Lassiter received an M.S. degree for his research on lymphocytes and feed efficiency. He currently is a program associate working in the Center of Excellence for Poultry Science, and plans to begin a Ph.D. with the P.I. this fall. Two visiting scientists contributed to the project on proton leak kinetics; Dr. Martin Brand (Buck Institute for Age Research, Novato, CA) and Dr. Masaaki Toyomizu (Graduate School of Agricultural Science, Tohoku University, Sendai Japan). TARGET AUDIENCES: The major target audience would be the scientific community primarily in animal agriculture. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
One of the most significant impacts our research appears to have had was to influence other research groups to investigate cellular mechanisms associated with feed efficiency using a similar paradigm; i.e. using animals from a same genetic line and provided the same feed. This research was carried out in angus steers by two independent groups from the University of Missouri (Dept.'s of Animal Science and Veterinary Pathobiology) and from Texas A&M University (Dept. of Animal Science).

Publications

  • Bottje, W. G., and G. Carstens, 2009. Association of mitochondria with feed efficiency in livestock and poultry. J. Anim. Sci. 87:E48-E63.
  • Bottje, W., M. D. Brand, C. Ojano-Dirain, K. Lassiter, M. Toyomizu, and T. Wing. 2009. Mitochondrial proton leak kinetics and relationship with feed efficiency within a single genetic line of male broilers. Poultry Sci. (in press)


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Increased hydrogen peroxide (H2O2) production was observed in duodenal mitochondria obtained from broiler chickens with low feed efficiency (FE). As a decrease in mitochondrial membrane potential (MMP) due to mild uncoupling of oxidative phosphorylation reduces reactive oxygen species production, this study was conducted to evaluate the effect of uncoupling on MMP and H2O2 production in duodenal mitochondria isolated from broilers with low (0.48 +/- 0.02) and high (0.68+/- 0.01) FE. Membrane potential and H2O2 production were measured fluorometrically and in the presence of different levels of an uncoupler, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The Dym was higher (P < 0.05) in high FE mitochondria at 0 to 600 nM FCCP. A decrease in Dym was observed at 600 and 1,000 nM FCCP in the low and high FE groups, respectively. H2O2 generation was higher in the low FE mitochondria at all FCCP levels except at 200 nM. Adding 200 to 800 nM FCCP decreased H2O2 production in low but not in high FE mitochondria. These results showed that FCCP-induced uncoupling lowered H2O2 production in low FE but not in high FE duodenal mitochondria and suggest that mitochondrial membrane potential may influence H2O2 production in low FE mitochondria. PARTICIPANTS: Ahmad Mujahid (Tohuku University, Sendai Japan) conducted studies in our lab as part of his PhD program. Masaaki Toyomizu (Professor, Animal Nutrition and Life Sciences, Tohuku University) was A. Mujahid's advisor and helped with mRNA studies that were ongoing in the lab. TARGET AUDIENCES: Target audiences would be the greater poultry science community as well as commercial poultry breeding companies.

Impacts
The results of this study provide insight into the mitochondrial dysfunction associated with the phenotypic expression of low feed efficiency within a specific line of male broiler breeders. Higher hydrogen peroxide production in mitochondria of broilers with low feed efficiency may require additional energy input in terms of repair and recycling of proteins and amino acids and additional protein synthesis which requires considerable energy input. An additional study was conducted to investigate oxygen radical and lipid peroxidation in heat-stressed broilers. The results of this study indicate that mitochondrial oxygen radical production may be responsible for increased oxidative damage to lipids and proteins in broilers.

Publications

  • Ojano-Dirain, C., N. B. Tinsley, T. Wing, M. Cooper, and W. Bottje, 2007. Membrane potential and hydrogen peroxide production in duodenal mitochondria in broilers chickens (Gallus gallus) with low and high feed efficiency. Comp. Biochem Physiol. 147:934-941
  • Mujahid, A., N. R. Pumford, W. Bottje, K. Nakagawa, T. Miyazawa, Y. Akiba, and M. Toyomizu, 2007. Mitochondrial oxidative damage in chicken skeletal muscle induced by acute heat stress. J. Poultry Sci. 44:439-445.
  • Ojano-Dirain, C.P, N.R. Pumford, M. Toyomizu and W. G. Bottje, 2007. Association of mitochondrial function and feed efficiency. J. Poultry Sci. 44:221-237.


Progress 01/01/06 to 12/31/06

Outputs
Previous studies have revealed differences in protein expression and mitochondrial function in broilers with low and high phenotypic expression of feed efficiency (FE) as well as higher oxidative stress in tissues obtained from broilers with low feed efficiency. These studies examined mRNA expression of mitochondrial transcription factors that affect mitochondrial biogenesis and energy metabolism as well as protein oxidation in breast muscle and duodenal tissue in broilers with low and high feed efficiency. Total RNA was extracted from snap-frozen tissues from male broilers with low (0.55 +/- 0.01) and high (0.72 +/- 0.01) FE (n = 8 per group). Total RNA was reverse-transcribed using oligo (dT) and/or random primers followed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Protein oxidation, measured as protein carbonyls, was also evaluated in duodenal mucosa. Protein carbonyls were higher in low FE mucosa in both tissue homogenate and the mitochondrial fraction. The mRNA expression of inducible nitric oxide synthase iNOS and and peroxisome proliferators activated receptor gamma (PPAR-g) in the duodenum was lower in the low FE broilers, with no differences in avian adenine nucleotide transporter (avANT), cytochrome oxidase III (COX III) and PPAR-g co-activator 1-alpha (PGC1-a). In contrast, mRNA expression of avANT and COX III in breast muscle was lower in low FE broilers with no differences in iNOS, PPAR-g, and PGC1-a. The avUCP in breast muscle was higher (P = 0.068) in low FE broilers. These results indicate that there are differences in the expression of mRNA encoding for mitochondrial transcription factors and/or proteins in breast muscle and duodenal tissue between low and high FE birds. The differences that were observed may also reflect inherent metabolic and gene regulation differences between tissues.

Impacts
Feed costs represent up to 70% of the total cost of raising an animal to market weight. Our studies are helping to elucidate basic cellular mechanisms associated with feed efficiency. In addition, we are working on identify one or more biomarkers, that could be used in conjunction with a biosensor to provide real-time detection of feed efficiency with a blood sample. Such a tool would help commercial companies in their selection program for replacement stock, and increase the progress made in selecting broilers with improved feed efficiency.

Publications

  • Bottje, W., N. Pumford, C. Ojano-Dirain, M. Iqbal, and K. Lassiter, 2006. feed efficiency and mitochondrial function. Poult. Sci. 85:8-14.
  • K. Lassiter, M. Iqbal, N. R. Pumford, C. Ojano-Dirain, N. Tinsley, T. Wing, M. Cooper, and W. Bottje, 2006. Differential expression of mitochondrial and extra-mitochondrial proteins in lymphocytes of low and high feed efficient male broilers. Poultry Sci. 85:2251-2259.


Progress 01/01/05 to 12/31/05

Outputs
Increased hydrogen peroxide (H2O2) production was observed in duodenal mitochondria obtained from broilers with low feed efficiency (FE). As a decrease in mitochondrial membrane potential (MMP) due to stimulation of uncoupling of oxidative phosphorylation reduces reactive oxygen species (ROS, e.g., H2O2) production, a study was conducted to evaluate the effect of uncoupling on MMP and H2O2 production in duodenal mitochondria from broiler breeder males with low and high FE. Duodenal mitochondria were isolated from broilers with low (0.48 +/- 0.02, n = 8) and high (0.68 +/- 0.01, n = 7) FE. Membrane potential and H2O2 production were measured fluorometrically in the presence of different levels (0, 200, 400, 600, 800 and 1,000 nM) of an uncoupler, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). The MMP was higher in the high FE mitochondria at 0 to 600 nM FCCP. A decrease in MMP was observed at 600 and 1,000 nM FCCP for the low and high FE groups, respectively. H2O2 generation was higher in the low FE mitochondria at all FCCP levels except at 200 nM. Adding 200 to 800 nM FCCP caused a decrease in H2O2 production in low but not in high FE mitochondria. The results indicate that FCCP-induced uncoupling lowered H2O2 production in low FE but not in high FE duodenal mitochondria and indicate that MMP may influence ROS production in broilers with low FE.

Impacts
In commercial poultry production, feed costs represent up to 70% of the total cost of raising an animal to market weight. Our studies are helping to elucidate basic cellular mechanisms associated with feed efficiency. In addition, we are trying to identify one or more biomarkers, that could be used in conjunction with a biosensor to provide real-time detection of feed efficiency with a blood sample. Such a tool would help commercial companies in their selection program for replacement stock, and increase the progress made in selecting broilers with improved feed efficiency.

Publications

  • Ojano-Dirain, C., W. Bottje, T. Wing and M. Cooper, 2005. Glutathione and respiratory chain complex activities in duodenal mitochondria from broilers with low and high feed efficiency. Poultry Sci. 84:782-788.
  • Iqbal, M., Pumford, N., Lassiter, K., Tang, Z. X., Wing, T., M. Cooper, W. G. Bottje, 2005. Compromised Liver Mitochondrial Function and Complex Activity in Low Feed Efficient Broilers Within a Single Genetic Line Associated With Higher Oxidative Stress and Differential Protein Expression. Poult. Sci. 84:933-941.
  • Ojano-Dirain, C., N.R. Pumford, M. Iqbal, T. Wing, and M. Cooper, W. G. Bottje, 2005. Biochemical evaluation of mitochondrial respiratory chain in duodenum of low and high feed efficient broilers, 2005. Poult. Sci. 84: 1926-1934.


Progress 01/01/04 to 12/30/04

Outputs
We are continuing to investigate relationships between mitochondrial function, biochemistry, antioxidant status and free radical biochemistry in health and disease in various tissues in poultry. We have determined that there is higher mitochondrial oxgyen radical production and/or protein oxidation in muscle, liver, intestines and lymphocytes observed in broiler breeder males with low feed efficiency (FE) compared to those with high FE. In duodenal mitochondria, although there were no differences in glutathione (GSH) peroxidase or GSH reductase activities, or in individual complex activities between low and high FE broilers, GSH levels were higher (P = 0.075) in broilers with high FE. Regression analysis revealed significant correlations (P < 0.05) between mitochondrial GSH and activities of Complex II, IV and V. These data suggest that reduced GSH may be important in maintaining or enhancing the activity of certain respiratory chain complexes, and could be important in the phenotypic expression of feed efficiency in broilers. We are continuing to investigate both mitochondrial and extramitochondrial protein expression in broilers with low and high FE with the ultimate goal of developing an assay or biomarker system that could be used to help in selection programs. Finally, additional publications were finalized in the area of pulmonary hypertension syndrome, a metabolic disease in broilers that we have shown to be characterized by oxidative stress. A study was also published on the role of vitamin E in phagocytic function of macrophages in broilers.

Impacts
We are rapidly gaining greater understanding of cellular mechanisms associated with the phenotypic expression of feed efficiency. We hope that these studies will lead towards understanding the proteins and genes controlling feed efficiency.

Publications

  • Bottje, W. G., M. Iqbal, C. Ojano-Dirain, N. R. Pumford, and K. Lassiter, 2004. Role of mitochondria in the phenotypic expression of feed efficiency. J. Appl. Poultry Res. 13:94-105.
  • Iqbal, M., N. R. Pumford, Z. X. Tang, K. Lassiter, T. Wing, M. Cooper, and W. Bottje, 2004. Low Feed Efficient Broilers Within a Single Genetic Line Exhibit Higher Oxidative Stress and Protein Expression in Breast Muscle With Lower Mitochondrial Complex Activity. Poultry Sci. 83: 474-484.
  • Ojano-Dirain, C., M. Iqbal, D. Cawthon, S. Swonger, T. Wing, M. Cooper, and W. Bottje, 2004. Site specific effects in electron transport in duodenal mtiochondria is associated with low feed efficiency in broiler breeder males. Poultry Sci. 83:1394-1403.
  • Konjufca, V.K., W. G. Bottje, and G. F. Erf, 2004. Influence of dietary vitamin E on phagocytic function of macrophages in broilers. Poultry Sci. 83: 1530-34.
  • Iqbal, MJ. N.R. Pumford, K. Lassiter, N. Tinsley, C. Ojano-Dirain, W. Bottje, T. Wing, and M. Cooper, 2004. Differences in protein banding patterns in several tissues in broiler breeder males with low and high feed efficiency. Southern Poultry Science Society, Jan. 26-27, Atlanta GA p. 49. (Abstract)
  • Higgins, J, N.R. Pumford, M. Iqbal, T. Wing, M. Cooper, and W. G. Bottje, 2004. Evidence of protein oxidation in mitochondrial respiratory complexes in broilers with high and low feed efficiency. Poultry Sci. 83(1): 110. (Abstract)
  • Sharma, P., W. Bottje, and R. Okimoto, 2004. Allele frequencies of genes associated with feed efficiency traits in selected and unselected population in a commercial broiler line. 83(1): 262 (Abstract) esistant and susceptible lines. Poultry Sci. 83:1420-1426.
  • Pumford, N.R., M. Iqbal, W. Bottje, J. O. Lay, T. Wing and M. Cooper, 2004. Plasma proteins were differentially expressed from broilers with High and Low Feed Efficiency. Poult. Sci. 83(1):189. (Abstract)
  • Tinsley, N., N. Pumford, M. Iqbal*, K. Lassiter*, T. Wing+, M. Cooper,+ and W.Bottje, 2004 Expression of proteins in cardiac tissue in broilers with low and high feed efficiency. Poultry Sci. 83(1):188. (Abstract)
  • Lassiter, K., M. Iqbal, C. Ojano-Dirain, N. Tinsley, W. Bottje, T. Wing and M. Cooper, 2004. Mitochondrial Biochemistry in Lymphocytes Associated with the Phenotypic Expression of Feed Efficiency in Broilers. Poultry Sci. 83(1): 188. (Abstract)
  • Iqbal, M., N.R. Pumford, Z. X. Tang, K. Lassiter, W. Bottje, T. Wing and M. Cooper, 2004. Compromised Liver Mitochondrial Function and Complex Activity in Low Feed Efficient Broilers Associated With Higher Oxidative Stress and Differential Expression of Proteins Within a Single Male Line. Poultry Sci. 83(1):110. (Abstract)
  • Ojano-Dirain, C., N. Tinsley, T. Wing, M. Cooper, and W. Bottje, 2004. Membrane Potential and Hydrogen Peroxide Production in Duodenal Mitochondria in Broilers with Low and High Feed Efficiency. Poultry Sci. 83(1): 110. (Abstract)


Progress 01/01/03 to 12/31/03

Outputs
Research is currently focused on the role of mitochondria in the phenotypic expression of feed efficiency. We have now determined that in broilers with low feed efficiency, there is a higher amount of oxygen radical production and higher protein oxidation present than in mitochondria from broilers with high feed efficiency. Furthermore, an correlation between protein carbonyls and complex V (ATP synthase) activity in intestinal mitochondria was observed suggesting that protein oxidation. There was also higher protein oxidation occuring in Complex III from muscle mitochondria in broilers with low feed efficiency. Thus, lower activities of respiratory complexes may be related to the higher protein oxidation in low feed efficiency mitochondria.

Impacts
We are rapidly gaining greater understanding of cellular mechanisms associated with the phenotypic expression of feed efficiency. We hope that these studies will lead towards understanding the genes controlling feed efficiency.

Publications

  • M. Iqbal, N. R. Pumford, Z. X. Tang, K. Lassiter, T. Wing, M. Cooper, and W. Bottje, 2004. Low Feed Efficient Broilers Within a Single Genetic Line Exhibit Higher Oxidative Stress and Protein Expression in Breast Muscle With Lower Mitochondrial Complex Activity. Poultry Sci. 83:474-484.


Progress 01/01/02 to 12/31/02

Outputs
Focus of research on this project is shifting toward the role of mitochondrial function and antioxidants in feed efficiency and health less on disease aspects at this time. Previous studies indicate that breast muscle mitochondria obtained from broilers with low feed efficiency (FE) generate more oxygen radicals than do mitochondria from broiliers with high feed efficiency. Antioxidant analyses reveal that low FE muscle tissue had lower activities of glutathione peroxidase, but there were no differences in activities of glutathione reductase, superoxide dismustase, or gamma-glutamyl cysteine synthetase. Findings suggest that oxidation may be confined to the mitochondrial compartment of cells. Additional studies indicate that intestinal mitochondria of low FE broilers have higher oxygen radical production (similar to muscle) but that sites of electron leak appear to be at respiratory complexes I and II as opposed to I and III in muscle. The results of these studies indicate that oxidation processes are critical to the phenotypic expression of feed efficiency in broilers.

Impacts
Results of these studies will help in understanding the cellular basis of feed efficiency. It is hoped that methods will be developed from these studies that will help in identifying animals with superior feed efficiency.

Publications

  • Iqbal M., J.D. Freiburger, G.F. Erf, and W.G. Bottje, 2002. Immunohistochemical Evidence of Cytochrome c Oxidase Subunit II Involvement in Pulmonary Hypertension Syndrome (PHS) in Broilers. Poultry Sci. 81:1231-1235.
  • Iqbal, M., N. Pumford, K. Lassiter, Z. X. Tang, T. Wing, M. Cooper and W. G. Bottje, 2003. Association of mitochondrial dysfunction with feed efficiency in male broilers within a single genetic line: A proteomics approach. SPSS January, Atlanta GA p. 13.
  • Ojano-Dirain, C., M. Iqbal, D. Cawthon, S. Swonger, T. Wing, M. Cooper, and W. Bottje, 2003. Site-specific defects in electron transport in duodenal mitochondria is associated with low feed efficiency in broiler breeder males. SPSS January, Atlanta GA p. 11


Progress 01/01/01 to 12/31/01

Outputs
Studies are continuing to investigate the role and relationships of mitochondrial function in feed efficiency and in pulmonary hypertension syndrome (PHS) in broilers. Studies now indicate that in addition to better mitochondrial function with less radical production in muscle mitochondria of broilers with higher feed efficiency, there is also lower amounts of protein carbonyls; an indicator of oxidized or damaged proteins in muscle mitochondria of broilers with high feed efficiency. There was also higher activity of glutathione peroxidase in breast muscle tissue of broilers with high feed efficiency. Broilers with low feed efficiency appeared to have higher expression of a protein (47 kDa) as determined by gel electrophoresis that was inversely correlated with feed efficiency. Immunohistochemistry techniques have revealed that there may be higher levels of expression of cytochrome oxidase (subunit II, COX II) in broilers with PHS compared to healthy broilers. The results of these combined studies help in understanding the role of mitochondria and antioxidants in health and disease in poultry.

Impacts
Results of studies being conducted may help lead to methods of identifying broilers with greater feed efficiency, or broilers that are resistant or susceptible to developing pulmonary hypertension. The overall goal of such studies is to improve productive efficiency in poultry.

Publications

  • Wang, S., W.G. Bottje, Z. Song, K. Beers, M. Vazquez-Anon, and J. J. Dibner, 2001. Uptake of DL 2-hydroxy-4-methylthio-butanoic acid (DL-HMB) in the broiler liver in vivo. Poultry Sci. 80:1619-1624.
  • Song, Z., K. Beers, J. J. Dibner, M. Vazquez-Anon, R. McNew, and W. Bottje, 2001. The hepatic extraction of plasma free amino acids and response to hepatic portal venous infusion of methionine sources in anesthetized SCWL males (Gallus domesticus). Comp. Biochem. Physiol. (B) 130:237-250.
  • Iqbal, M., D. Cawthon, K. Beers, and W. Bottje, 2001. Antioxidant enzyme activities and mitochondrial fatty acids in pulmonary hypertension syndrome (PHS) in broilers. Poultry Sci. (in press).
  • Barnes, D. M., Z. Song, K. C. Klasing, and W. Bottje, 2001. Protein metabolism during an acute phase response in chickens. Amino Acids (in press).
  • Bottje, W., Z. Tang, M. Iqbal, D. Cawthon, R. Okimoto, T. Wing, M. Cooper, 2001. Association of mitochondrial function with feed efficiency within a single genetic line of male broilers. Poultry Sci. (in press).
  • Tang, Z., M. Iqbal, D. Cawthon, and W. G. Bottje, 2001. Heart and muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus) Comp. Biochem. Physiol. (in press).


Progress 01/01/00 to 12/31/00

Outputs
Feed represents 60% of the cost of raising an animal to market weight. Studies have been initiated to investigate the role of mitochondrial function in feed efficiency. Preliminary findings indicate that muscle mitochondria isolated from broilers with low feed efficiency are less tightly coupled (lower RCR), exhibit greater electron leak and ROS formation, and lower Complex I and II activities compared to mitochondria from broilers with high feed efficiency. There were also positive correlations between mitochondrial function and complex activity with feed efficiency. These findings suggest that mitochondrial function may be linked to the phenotypic expression of feed efficiency in an animal. We also observed that whereas Complex I activity was lower in heart mitochondria from broilers with pulmonary hypertension syndrome (PHS), that there were no differences in Complex II activity between groups. Studies are being initiated to determine the differences in Complex activities between broilers with and without PHS, and in broilers with different feed efficiencies.

Impacts
The research provides new information on the role that mitochondria play in determining feed efficiency of an animal. The research on mitochondrial function in pulmonary hypertension syndrome give specific clues at the cellular/molecular level that relate to the defect that leads birds to develop pulmonary hypertension syndrome. Such information may be relevant to research on familial pulmonary hypertension in humans as well.

Publications

  • Song, Z., D. Cawthon, K. Beers, and W. G. Bottje, 2000. Hepatic and extra-hepatic stimulation of glutathione release by norepinephrine in vivo. Poultry Sci 79:1632-1639.
  • Song, Z., W. G. Bottje, D. Cawthon, and K.W. Beers, 2000. Biliary glutathione (GSH) secretion in male SCWL chickens after inhibition of g-glutamyl transpeptidase. Poultry Sci. 79:1829-1832.
  • Cawthon, D., K. Beers, and W.G. Bottje, 2000. Electron transport chain defect and inefficient respiration may both underlie pulmonary hypertension syndrome (PHS)-associated mitochondrial dysfunction in broilers. Poultry Sci. (in press).
  • Iqbal, M., D. Cawthon, R.F. Wideman, Jr., and W. G. Bottje, 2000. Lung mitochondrial dysfunction in pulmonary hypertension syndrome. I. Site specific defects in electron transport chain. Poultry Sci. (in press).
  • Iqbal, M., D. Cawthon, R.F. Wideman, Jr., K.W. Beers, and W.G. Bottje, 2000. Lung mitochondrial dysfunction in pulmonary hypertension syndrome. II. Oxidative stress and inability to improve function with repeated additions of ADP. Poultry Sci. (in press).


Progress 01/01/99 to 12/31/99

Outputs
Studies have been conducted that clearly indicated site-specific defects in the electron transport chain of mitochondria obtained from broilers with pulmonary hypertension syndrome (PHS). Using a series of chemical inhibitors of respiratory chain activity, it was determined that defects in electron transport were present in Complex I and III of PHS lung mitochondria and Complex II of liver mitochondria. These defects likely result in increased leak of electrons from the respiratory chain that react with oxygen to form oxygen radicals. Furthermore, these defects may have a genetic basis; i.e. birds that are susceptible to PHS may have an inherently greater electron leak and oxygen radical production than do birds that are resistant to PHS. These defects could therefore contribute to both the inefficiency of oxygen use and oxidative stress that characterizes this metabolic disease in broilers. Mitochondrial oxidative stress was apparent due to the diminished levels of vitamin E and glutathione in PHS mitochondria. An unexpected finding of these studies was that sequential additions of ADP to isolated mitochondria improved function in Control mitochondria to a much greater extent than in PHS mitochondria. These findings may be important for the understanding of this metabolic disease, as well as increasing the basic understanding of mitochondrial physiology. Other studies using a method of obtaining blood samples from pre- and post-hepatic blood vessels in vivo have revealed that the stress hormone causes the release of glutathione, an important endogenous antioxidant, into plasma from both hepatic and extra-hepatic tissue. This research indicates that glutathione may be mobilized from tissue during periods of physiological stress.

Impacts
The findings of these studies reveal important insight into the cellular basis of pulmonary hypertension syndrome. The findinss also suggest that there is a genetic basis for mitochondrial dysfunction, and that the dysfunction does not occur only as a secondary response to the onset of disease symptoms.

Publications

  • Cawthon, D., K.W. Beers, R. McNew and W.G. Bottje, 1999. Evidence of hepatic mitochondrial dysfunction in broilers with pulmonary hypertension syndrome (ascites): Effect of t-butyl hydroperoxide on mitochondrial function and glutathione. Poultry Sci. 78:114-124.
  • Wideman RF Jr, P Maynard, and W.G. Bottje, 1999. Thromboxane mimics the pulmonary but not systemic vascular responses to bolus HCl injections in broiler chickens. Poultry Sci. 78:714-721.
  • Wideman, R.F. Jr, P. Maynard, and W. G. Bottje, 1999. Venous blood pressure in broilers during acute inhalation of 5 pct carbon dioxide or unilateral pulmonary artery occlusion. Poultry Sci. 78:1443-1451
  • Iqbal, M., D. Cawthon, R.F. Wideman, Jr. and W. Bottje, 1999. Site Specific H2O2 Production in Lung Mitochondria of Broilers with Pulmonary Hypertension Syndrome (PHS). Free Rad. Biol. Med. 27(1):S27.
  • Cawthon, D., and W. Bottje, 1999. Hepatic mitochondrial dysfunction and electron leak in avian pulmonary hypertension syndrome (PHS). Free Rad. Biol. Med. 27(1):S243
  • Song, Z., K. Beers, and W. Bottje, 1999. Evidence of hepatic and extra-hepatic stimulation of glutathione release into plasma by norepinephrine. Poultry Sci. 78 (1):88.


Progress 01/01/98 to 12/31/98

Outputs
Glutathione (GSH) is an important antioxidant found in high levels in all tissues with particularly high levels in the liver. The liver functions to synthesize and export GSH into the plasma where it is then taken up by extrahepatic cells. We have now documented the existence of an interorgan circulation of glutathione (GSH) in birds. Another major route for GSH turnover is through excretion of GSH, oxidized GSH (GSSG) and/or GSH conjugates into the bile. Gamma-glutamyl transpeptidase (gGT) is an enzyme found in high concentrations in the bile duct and biliary tree of mammals. However, the amount of biliary secretion of GSH and the importance of gGT activity in the bile duct of domestic birds has not been previously documented. By infusing AT-125, a specific inhibitor of gGT activity, we have recently determined that domestic chickens have a high level of gGT activity and that hepatic export of GSH accounts for approximately 40% of the total export of GSH from the liver of domestic birds. During the coming year we plan to investigate hormonal regulation of sinusoidal and biliary secretion of GSH in avian liver. We have also been investigating hepatic metabolism of 4-hydroxy-methyl butanoic acid (HMB, a methionine precursor). Results of these studies indicate that the liver has a tremendous capacity for clearance of HMB. (Methionine, via the cystathionine pathway, can be converted to cysteine which is the rate-limiting amino acid in GSH synthesis.)

Impacts
(N/A)

Publications

  • Bottje W.G., S. Wang, F.J. Kelly, C. Dunster, A. Williams, and I. Mudway, 1998. Antioxidant defenses in lung lining fluid of domestic fowl: Impact of poor ventilation conditions. Poultry Sci. 77:516-522.
  • Wang, S., D. Cawthon, and W.G. Bottje, 1998. Age-related changes of plasma glutathione and cysteine in broilers. Effect of dithiothreitol reduction in vitro on free and bound pools. Poultry Sci. 77:1234-1240
  • Wang, S, W.G. Bottje, D. Cawthon, C. Evenson, K. Beers, and R. McNew, 1998. Hepatic export of glutathione and uptake of constituent amino acids, glutamate and cysteine, in broilers in vivo. Poultry Sci. 77:1556-1564.
  • Bottje, W. G., S. Wang, K. W. Beers, and D. Cawthon, 1998. Lung lining fluid antioxidants in broilers: Age related changes under thermoneutral and cold temperature conditions. Poultry Sci. 77:1905-1912.
  • Bottje, W.G., S. Wang, K. Beers, M. Vazquez-Anon, and P. McCullough. 1998. Clearance of DL-2-Hydroxy-4-(Methylthiobutanoic Acid) HMB by broiler liver in vivo. Poultry Sci. 77(1):31.
  • Bottje, W., S. Wang, K. Beers, and D. Cawthon, 1998. Age-related changes in lung epithelial lining fluid epithelial lining fluid (ELF) antioxidants in broilers under cold temperature conditions. Poultry Sci. 77(1):127
  • Fritts, C.A., W. G. Bottje, T.K. Bersi, and G. F. Erf, 1998. Altered immune cell profiles in broilers with pulmonary hypertension syndrome. Poultry Sci. 77(1):132.


Progress 01/01/97 to 12/31/97

Outputs
In mammals, the presence of antioxidants in epithelial lining fluid (ELF) of the lung is instrumental in protecting the lung from oxidant stress caused by inhalation of air pollutants. Previously, we determined that broilers had ELF antioxidants similar to levels in mammals, and that high levels of dust and ammonia produced an oxidative stress on ELF fluid. As these birds were also exposed to cold temperatures, a second experiment was conducted to determine if cold temperatures alone would induce an oxidative stress, and to document age-related changes in ELF antioxidants in broilers. The results of the study indicate that there were age-related decreases in alpha-tocopherol, uric acid and glutathione (GSH), but not ascorbic acid in ELF. Cold temperatures produced an increase in protein and in the GSSG/GSH ratio (an indicator of oxidative stress) at 7 weeks, and a decrease in ELF ascorbic acid at 7 week. These results indicate that ELF antioxidant protection appears to decrease with age, and that cold temperatures induce an oxidative stress in ELF. The increase in ELF protein may impart some antioxidant protection, but may have resulted from damage to lung cells in birds maintained in the cold environment.

Impacts
(N/A)

Publications

  • WANG SY, WG BOTTJE, P MAYNARD, J DIBNER, AND W SCHERMER, 1997. EFFECT OF SANTOQUIN AND OXIDIZED FAT ON LIVER AND INTESTINAL GLUTATHIONE IN BROILERS. POULTRY SCI. 76:961-967.
  • BOTTJE, WG, GF ERF, TK BERSI, S WANG, D BARNES, AND K. W. BEERS, 1997. EFFECT OF DL- -TOCOPHEROL AND PULMONARY HYPERTENSION SYNDROME (ASCITES) IN BROILERS. POULTRY SCI. 76:1506-1512.
  • BEAUPRE C, CJ TRESSLER, WG BOTTJE, SJ BEAUPRE, AND JD KIRBY, 1997. THE TESTIS OF DOMESTIC FOWL IS MAINTAINED AT CORE BODY TEMPERATURE.
  • BOTTJE WG, S WANG, FJ KELLY, C DUNSTER, A WILLIAMS, AND I. MUDWAY, 1997. ANTIOXIDANT DEFENSES IN LUNG LINING FLUID OF DOMESTIC FOWL: IMPACT OF POOR VENTILATION CONDITIONS. POULTRY SCI. . (IN PRESS).
  • ERF GF, WG BOTTJE, AND TK BERSI, 1996. CD4, CD8 AND TCR DEFINED T CELL SUBSETS IN BROILER CHICKENS: AGE ASSOCIATED CHANGES IN THE PROPORTIONS OF RARE AND COMMON T CELL POPULATIONS. VET. IMMUNOL. (IN


Progress 01/01/96 to 12/30/96

Outputs
An experiment was conducted to determine the effect of high dietary vitamin E (VE) on lung and liver tissue VE, on immune function and on pulmonary hypertension syndrome (PHS) mortality. The results of the study indicated that lung and liver alpha-tocopherol levels increased in response to dietary VE and with age. Cumulative PHS during the study was 21% and was unaffected by level of dietary VE. Interestingly, considerable new information was obtained relative to various populations of T lymphocytes (T cells) in broilers. Various populations of T cells in thymus, spleen and blood were ascertained at 2, 5 and 7 weeks of age by flow cytometry. There was a general increase in the proportion of CD8+ (cytotoxic T cells) with age. However, high levels of dietary VE caused a shift in T cells populations resulting in an increase in CD4+ (T helper cells) and a decrease in CD8+ cells at 7 weeks of age. Data from this experiment is still being analyzed and additional information on the effect of VE on immune T cells populations is anticipated. An experiment was also conducted to determine the effect of a feed grade antioxidant (Santoquin, S) on intestinal and liver glutathione (GSH) and on the incidence of PHS in birds fed normal or oxidized fat. The results indicate that S had no affect on lowering the incidence of PHS, but S did cause an elevation in intestinal GSH. Thus, S appears to promote intestinal tissue antioxidant protection.

Impacts
(N/A)

Publications

  • Erf GE, and WG Bottje, 1996. Nutrition and Immune Function in Chickens: Benefits of dietary Vitamin E supplementation. Arkansas Nutrition Conference (Sept. 10-12, Fayetteville AR.
  • Bottje, W.G. and S. Wang, 1996. Lowering Oxidative stress with Santoquin: Antioxidant sparing activity, growth, and possible importance to ascites. Novus International Technical Symposium. Rio De Jinero Brazil (Oct 11 and 12).
  • Bottje, W.G., 1996. Oxidative Stress and Antioxidant Protection in Ascites Syndrome. BASF Canada, Technical Symposia, (Nov. 4-6).
  • Wang SY, WG Bottje, P Maynard, J Dibner, and W Schermer, 1996. Effect of santoquin and oxidized fat on liver and intestinal glutathione in broilers. Poultry Sci. (in press).
  • Beaupre C, CJ Tressler, WG Bottje, SJ Beaupre, and JD Kirby, 1996. The testis of domestic fowl is maintained at core body temperature. Poultry Sci. 75(1):48. PB Wang S, W Bottje, B Drake, C Evenson, K Emmerson, and J Dibner, 1996. Age-related changes in tissue glutathione (GSH) in broilers fed .2% or .5% Alimet. Poultry Sci 75(1):150.
  • Wang SY, W Bottje, K Emmerson, C Evenson, and R Moore, 1996. Time.