Progress 10/01/09 to 09/30/13
Outputs
Target Audience: The target audience includes individuals working in the following fields: animal agriculture, food science, food biochemistry, nutritional sciences, metabolomics, biochemistry, biophysical chemistry, and the meat industry. Specific industries with interest in this project include all sectors of the poultry industry, National Pork Board, American Meat Institute, National Cattlemen’s Beef Association, aquaculture, aquatic, and seafood industry sectors. Knowledge from this project will be delivered via a graduate level Food Lipids course that is taught on the UW-Madison campus. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The Principal Investigator and PhD student that conducted this project have gained valuable experience in i) measuring gene expression differences between turkeys and chickens, ii) understanding biochemical pathways that contribute to tocopherol trafficking and tocopherol metabolism in birds, iii) working with a national research institute, another land-grant university, and a major animal agricultural ingredient company to conduct joint research, and iv) relating tocopherol levels and other compositional characteristics of avian tissues to oxidative rancidity in poultry meat. How have the results been disseminated to communities of interest? Research finding have been presented at a poultry research workshop held by a major poultry producer in August 2011, 2012, and 2013. Findings were also presented at the Reciprocal Meats Conference in 2011, 2012, and 2013. The graduate student working on this Hatch grant, Dale Perez, won a research award at the 2012 RMC meeting. Collaborative research has taken place with Archer Daniels Midland (ADM), Maple Leaf Farms, Pacific Northwest National Laboratories (PNNL), a bioinformatics specialist at UW-Madison, and with Cornell University. An invited presentation describing this research project for the Midwest American Society of Animal Science meeting was prepared during the reporting period and will be delivered in March 2014. Results will also be reported in 2014 at the Reciprocal Meats Conference. Three manuscripts for publication in peer-reviewed journals are in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In line with Objective 1, we have improved our understanding regarding the mechanism of deficient tocopherol deposition in turkey muscle. We have shown for the first time that tocopherol metabolites were higher in turkey bile compared to chicken bile. The implication is that a cytochrome P450 hydroxylase is up-regulated in turkey compared to chicken which explains the increased tocopherol metabolites in the turkey bile. Four cytochrome P450s were up-regulated in turkey liver based on RNA Seq results. This provides direction to further elucidate the mechanism of poor tocopherol deposition in turkey muscle. We also explored tocopherol absorption in the intestine as a candidate pathway by which tocopherol accumulation in turkey muscle can be compromised. Increasing the tocopherol level 10-fold in the diets of turkeys and chickens similarly increased the ‘relative’ tocopherol levels in blood and muscle, albeit lower in turkey at the high and low level. This suggested the absorption pathway was not deficient in turkeys. It should be noted that cholesterol was found to be higher in turkey bile compared to chicken bile. This has the potential to block tocopherol uptake by intestinal cells since at least one receptor in the intestine (e.g. SRB1) is known to bind both cholesterol and tocopherol. Excess cholesterol may saturate SRB1 so that less tocopherol is transported into intestinal cells. The act of eating dumps bile into the intestinal lumen. Thus, excess cholesterol in the bile will likely block SRB1 receptors of the intestinal cells at the crucial moments when dietary tocopherol is traveling through the intestinal tract during the post-prandial period. Gene expression levels of SRB1 in turkey and chicken intestinal tissue is underway. Further, our RNA Seq results in turkey and chicken liver indicated that a cytochrome P450 which is responsible for cholesterol synthesis was elevated in turkey. Thus it is a possibility that cholesterol accumulation in turkey bile limits tocopherol absorption by intestinal cells. A cytochrome P450 responsible for bile acid synthesis was up-regulated in turkey liver. An elevated concentration of bile salts in turkey liver may act as a vehicle to export cholesterol and tocopherol from the liver to the bile. Elevated gamma tocopherol in turkey compared to chicken bile was observed in one trial. Total bile salt concentrations were measured in turkey and chicken bile yet differences were not observed. Thus, the hypothesis that elevated bile salt synthesis in turkey liver will direct cholesterol to the bile and divert dietary tocopherol from muscle tissue to bile appears to be unlikely. Our RNA Seq results helped to reveal that there are six amino acid differences in the alpha tocopherol transfer protein (ATTP) when comparing turkey and chicken livers. Amino acid substitution at one of these sites has been described to decrease tocopherol transfer in human ATTP. The differing amino acid at this site in turkey compared to chicken suggests a deficient ATTP in turkey. Computational modeling and biochemical assays can be used to explore if turkey ATTP is defective compared to chicken ATTP. A defective ATTP in turkey may cause more tocopherol to be available for metabolism or transfer to the bile, rather than available to muscle. Gene expression of UDP-glucuronyl transferase was up-regulated 3.3-fold in turkey liver which presents an additional mechanism that may limit vitamin E accumulation in turkey muscle. Glucuronidation of tocopherol will prevent incorporation of tocopherol into muscle. In line with Objective 2, dietary strategies to decrease tocopherol metabolism through the tocopherol hydroxylase pathway in turkeys have been identified. Black pepper contains piperine which can inhibit hydroxylase enzymes that metabolize tocopherols. Our preliminary trial in turkeys using dietary black pepper did not improve oxidative stability in the stored muscle. Piperonyl butoxide and ground sesame seeds were also examined in related projects as hydroxylase inhibitors. Neither inhibitor improved tocopherol status in turkeys. Thus, other dietary strategies need to be examined with cost considerations in mind. In line with Objective 3, we showed that, in general, alpha tocopherol levels were lower in turkey compared to chicken erythrocytes. Low levels of alpha tocopherol have been shown to increase hemolysis, and it has been established that release of hemoglobin due to hemolysis accelerates lipid oxidation in muscle systems. Thus, part of the low oxidative stability of turkey muscle may be due to increased hemolysis due to low levels of alpha tocopherol. We demonstrated that detergent-induced hemolysis was greater in turkey compared to chicken blood, yet in this comparison tocopherol concentrations were not elevated in the chicken erythrocytes for some unknown reason. It should be noted that turkey erythrocytes promoted lipid oxidation in washed muscle more slowly compared to chicken erythrocytes. Collectively these results suggest that some component in chicken plasma is particularly protective in regards to hemolysis. Fatty acid composition of turkey and chicken erythrocytes was similar as well as catalase activity. This suggested that fatty acid composition and catalase activity could not explain the hemolytic and oxidative differences observed between chicken and turkey erythrocytes. In line with Objective 4, we have shown that fast growing turkeys deposit dietary tocopherols into muscle tissue less effectively compared to slow growing turkeys. This suggests that selecting for growth has negative consequences related to vitamin E deposition in turkey muscle. Future selection strategies to mutually optimize growth and dietary uptake of vitamin E in the muscle may be possible.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Activities included analyzing results from various experiments that were comprised of a multitude of variables and analytical techniques. One output of this work has been presenting findings at a poultry research summit in August 2012 held by a major poultry producer. Findings were also presented at the Reciprocal Meats Conference in 2012. The graduate student working on this project, Dale Perez, won a research award at the 2012 RMC meeting. Collaborative research has taken place with Archer Daniels Midland (ADM), Maple Leaf Farms, Pacific Northwest National Laboratories (PNNL), and Cornell University. PARTICIPANTS: Mark Richards (PD) directed the work of the graduate student on this project. Richards also worked with data analysis, interpretation, and manuscript preparation. Dale Perez (graduate student) conducted all experiments, gathered data, data analysis, interpretation and preparation of a manuscript. Dr. Robert Parker at Cornell University conducted bile analyses on comparative chicken, turkey, and duck samples. Dr. Mark Berres converted raw output from RNA Seq work done on chicken and turkey livers at the UW-Biotech Center to normalized results that we are in the process of mining. Dr. Aaron Wright at the Pacific Northwest National Laboratories is in the process of assessing cytochrome P450s in liver slices from comparative turkey and chicken samples. TARGET AUDIENCES: The target audience includes individuals working in the following fields: animal agriculture, food science, food biochemistry, nutritional sciences, metabolomics, biochemistry, biophysical chemistry, and the meat industry. Specific industries with interest in this project include all sectors of the poultry industry, National Pork Board, American Meat Institute, National Cattlemen's Beef Association, aquaculture, aquatic, and seafood industry sectors. Knowledge from this project will be delivered via a graduate level Food Lipids course that is taught on the UW-Madison campus. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One major outcome is advancement in our understanding regarding mechanisms of deficient tocopherol deposition in turkey muscle. Cholesterol was found to be higher in turkey bile compared to chicken bile. This may block tocopherol uptake by intestinal cells since at least one receptor in the intestine (SRB1) is known to bind both cholesterol and tocopherol. Excess cholesterol will saturate SRB1 so that less tocopherol is transported into intestinal cells. The act of eating dumps bile into the intestinal lumen. Thus, excess cholesterol in the bile will likely block SRB1 receptors of the intestinal cells at the crucial moments when dietary tocopherol is traveling through the intestinal tract during the post-prandial period. Feces output of tocopherol was higher in turkey which further suggests poor absorption of dietary tocopherol in the intestine. Our RNA Seq results in turkey and chicken liver indicated that a cytochrome P450 which is responsible for cholesterol synthesis was elevated in turkey. Thus, we are beginning to characterize a pathway that may lead to cholesterol accumulation in turkey bile that limits tocopherol uptake by intestinal cells. Another cytochrome P450 responsible for bile acid synthesis was upregulated in turkey liver. An elevated concentration of bile salts in turkey liver may act as a vehicle to export cholesterol from the liver to the bile. Tocopherol metabolites were higher in turkey bile compared to chicken bile. This suggests an additional mechanism that limits deposition of tocopherol into turkey muscle. The mechanism implicates a cytochrome P450 hydroxylase that metabolizes tocopherol is upregulated in turkey compared to chicken. Four cytochrome P450s were upregulated in turkey liver based on RNA Seq results. Future work will attempt determine which CYP or CYPs are responsible for the tocopherol metabolism. The RNA Seq data set from liver tissue of chickens and turkeys is expansive and will continue to be mined to further elucidate pathways that cause poor deposition of tocopherol into turkey muscle. We have also characterized the ability of chickens, turkeys, and ducks to deposit natural, dietary vitamin E into muscle compared to synthetic vitamin E in the diet. This is useful to producers who can take advantage of providing the natural source of vitamin E. Natural vitamin E deposited approximately twice as effectively into muscle compared to synthetic vitamin E when concentrations were normalized. The ability of Nat E in the diet to increase alpha tocopherol levels in muscle was greater in ducks and turkeys compared to chickens. Nat E in the diet decreased lipid oxidation in minced and salted chicken thigh during frozen storage when compared to Syn E in the diet. Nine genes were differentially expressed in livers from turkeys receiving Natural E compared to synthetic E (some of which may affect appetite and growth). Dietary Nat E decreased vit E metabolic products in bile from duck but not turkey and chicken as compared to dietary Syn E. There were also trends that natural vitamin E in the diet increased fat content and pH in duck thigh which can increase tenderness and palatability.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities included conducting and analyzing results from a multitude of experiments. One output of this work has been presenting findings at a poultry research summit in August 2011 held by a major poultry producer. Findings were also presented at the Reciprocal Meats Conference in 2011. This has led to interactions with industry and academia. Collaborative research has taken place with Archer Daniels Midland (ADM), Pacific Northwest National Laboratories (PNNL), and Cornell University. PARTICIPANTS: Mark Richards (PD) directed the work of the graduate student on this project. Richards also worked with data analysis, interpretation, and manuscript preparation. Dale Perez (Graduate student) conducted all experiments, gathered data, data analysis, interpretation and preparation of a manuscript. Dr. Robert Parker at Cornell University conducted some analyses. Dr. Mamduh Sifri facilitated the comparative feeding studies. Dr. Aaron Wright provided results regarding cytochrome P450 levels in liver samples. TARGET AUDIENCES: The target audience includes individuals working in the following fields: animal agriculture, food science, food biochemistry, nutritional sciences, metabolomics, biochemistry, biophysical chemistry, and the meat industry. Specific industries with interest in this project include all sectors of the poultry industry, National Pork Board, American Meat Institute, National Cattlemen's Beef Association, aquaculture, aquatic, and seafood industry sectors. Knowledge from this project will be delivered via a graduate level Food Lipids course that is taught on the UW-Madison campus. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A large feeding study was organized in which turkeys, chickens, ducks were grown for 6 weeks in a single facility to assess comparative vitamin E metabolism. The vitamin E levels in the feeds were standardized and a natural source of added vitamin E (alpha tocopherol acetate) was compared to synthetic vitamin E. A diet containing a low level of added vitamin E (basal diet) was also assessed. Feed rations used during the grow-out period were analyzed and it was confirmed that standardized levels of vitamin E were fed. The erythrocytes were isolated and tocopherol contents were determined. Chickens had the highest concentration of erythrocyte vitamin E (specifically alpha tocopherol), ducks were intermediate, and turkeys were lowest. Erythrocyte Vitamin E was higher in birds receiving the natural vitamin E diet compared to the synthetic. The ability of the isolated erythrocytes to promote lipid oxidation in washed muscle was also assessed. Chicken erythrocytes promoted lipid oxidation most effectively, duck was intermediate and turkey erythrocytes were least effective. This raises the possibility that elevated concentrations of tocopherol destabilized the erythrocytes, facilitating lipid oxidation in washed muscle. It is more likely that a low level of some antioxidant (e.g. catalase) in chicken erythrocytes explains the higher ability of chicken erythrocytes to promote lipid oxidation. The polyunsaturation index was not different when comparing turkey, chicken, and duck erythrocytes. There were some differences in individual fatty acids when comparing the different erythrocytes. Bile, plasma, duodenal sections, liver, breast muscle, and thigh muscle were also harvested. Dietary alpha tocopherol deposited most effectively into chicken thigh muscle compared to turkey and duck. Natural vitamin E deposited into thigh muscle more effectively compared to synthetic vitamin E. Dietary alpha tocopherol deposited most effectively into chicken liver, duck was intermediate and turkey liver was lowest. RNA was isolated from chicken and turkey liver samples from birds receiving the natural and synthetic dietary source of vitamin E. The RNA has been submitted for RNA SEQ analysis. This will provide gene expression levels in the liver related to tocopherol metabolism. Cytochrome P450 protein levels were also determined in livers from chickens, turkeys and ducks from industry sources (not equivalent vitamin E levels in the feeds). P450 enzymes can metabolize vitamin E. Turkey liver showed higher P450 levels compared to the duck samples when using one of the two different P450 detecting probes. Overall, these studies provide new knowledge in areas of tocopherol metabolism in avian species and oxidative stability in the muscle tissue.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities included conducting and analyzing a multitude of experiments. One output of this work has been presenting preliminary findings at a poultry research summit in August 2010 held by a major poultry producer. This has lead to interactions with industry leaders and discussions on pursuing collaborative research efforts. Collaborative efforts are also underway with Cornell University and Michigan State University. PARTICIPANTS: Mark Richards (PD) directed the work of the graduate student on this project. Richards also worked with data analysis, interpretation, and manuscript preparation. Dale Perez (Graduate student) conducted all experiments, gathered data, data analysis, interpretation and preparation of manuscript. Dr. Robert Parker at Cornell University assisted with some analyses. Dr. Gale Strasburg at Michigan State University facilitated delivery of unique turkey breast samples. TARGET AUDIENCES: The target audience includes individuals working in the following fields: animal agriculture, food science, food biochemistry, nutritional sciences, metabolomics, biochemistry, biophysical chemistry, and the meat industry. Specific industries with interest in this project include all sectors of the poultry industry, National Pork Board, American Meat Institute, National Cattlemen's Beef Association, aquaculture, aquatic, and seafood industry sectors. Knowledge from this project will be delivered via a graduate level Food Lipids course that is taught on the UW-Madison campus. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We are in the first year of this product so the impact is limited. Substantial progress has been made in assessing tocopherol isomer levels in various tissues from chicken, turkey, and duck (e.g. breast muscle, thigh muscle, liver, plasma, bile, and erythrocytes). New knowledge has been generated related to the relative levels of tocopherol isomers in turkey and duck erythrocytes (e.g. red blood cells) and how those levels relate the structural integrity of the erythrocytes in a muscle tissue matrix. The concentration of tocopherols in duck, chicken, and turkey feeds has also been assessed which is an important part of understanding the transfer of dietary tocopherol to different tissues of the birds. Tocopherol levels were also assessed in breast muscle from a commercial line of turkeys (rapid growth) compared to a line of turkeys that exhibit slow growth. These studies suggested that selecting for growth decreases tocopherol deposition into the breast muscle which can compromise meat quality. Overall, these studies should lead to new knowledge in areas of tocopherol metabolism in avian species and oxidative stability in the muscle tissue.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: There are currently no outputs since the project was initiated Oct 1, 2009. A graduate student has been identified. The student has completed one semester of class work. PARTICIPANTS: A graduate student has been identified to work on this project. The student has completed one semester of class work. The other participant is the Principal Investigator, Mark Richards. No partners have contributed to this project yet. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
There are currently no outcomes since the project was initiated Oct 1, 2009. A graduate student has been identified. The student has completed one semester of class work.
Publications
- No publications reported this period
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