Duration: 10/01/2007 to 09/30/2012

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need as indicated by the stakeholders: Dietary recommendations have been made for n-3 fatty acids as a part of Dietary Reference Intakes (DRIs) by the Institute of Medicine of the National Academy of Sciences (NAS) (1,2). The Adequate Intake (AI) for n-3 fatty acids has been set, based on the observed median n-3 intakes in the US, at 1.6 g/day alpha linolenic acid (ALA) for men and 1.1 g/day ALA for women (1). While the NAS acknowledges that up to ten percent of the ALA recommendation can be met from long chain n-3 fatty acids such as eicosapentaenoic and docosahexaenoic acids (EPA and DHA), the committee chose to base n-3 recommendations on current n-3 intakes of ALA. In contrast to this approach, the American Heart Association (AHA) Diet and Lifestyle Recommendations Revision 2006 has reemphasized the importance of EPA and DHA consumption by recommending the consumption of fish, especially oily fish, at least twice weekly (3). Furthermore, for the first time the AHA has recommended increased EPA and DHA consumption either from fish or fish oil supplements - for those with established cardiovascular disease and obesity-induced hyperlipidemia (3).
The problem: Not only are Americans unlikely to achieve the enhanced AHA recommendation for long-chain polyunsaturated (PUFA) n-3 fatty acid intakes by increasing fish consumption, but Americans are likely to be confused by the competing recommendations from the NAS and the AHA. Basing n-3 recommendations on ALA, the NAS approach, suggests to consumers that the benefits of ALA consumption are equal to those from long-chain n-3 sources such as EPA and DHA and alternative agricultural sources of n-3 fatty acids. Members of the NC-1167 Committee have been at the forefront in showing that the benefits of long-chain n-3 PUFA are quite unique and distinct from those of the parent fatty acid, ALA. Because of concerns regarding the sustainability of fish stocks and the contamination of fish by both mercury and pesticides agricultural-based sources of n-3 PUFA must be investigated as an alternative means of achieving equivalent health promotion and disease prevention benefits. N-3 PUFA comprise a heterogeneous group of fatty acid with diverse and unique health benefits. The overall goal of this application is to determine the effectiveness of both agricultural-based n-3 PUFA and marine-based n-3 PUFA, and to determine both the form and the amount of n-3 PUFA, necessary to promote health and prevent disease.

This project addresses the goals of the ESCOP Science Roadmap Challenge 7: We can ensure improved food safety and health through agricultural and food systems, by improving the nutritional value of foods and discovering better educational methods to help individuals make informed food choices.

The project also addresses CSREES/GPRA Goal 3, Healthy, well-nourished children, youth and families by: 1. reduced disease prevalence and enhanced quality of life by defining the relationship between diet and the risk of chronic disease; 2. improving the scientific basis for more effective Federal food assistance programs by better defining nutrient requirements &.. and analyzing the effects of nutrition information on food choices and diets; 3. generate a more nutritious food supply through research to modify the health-promoting properties of plant and animal foods and 4. enhance public understanding of diets role in lifelong health through nutrition education.
This project addresses three Food and Nutrition Crosscutting Research Areas and Objectives of the NCRA identified in Appendix A1 of the NCRA Manual. The three crosscutting objectives to be addressed by NC-1167 are:
Research Objective 1: Expand our understanding of the relationship between diet, health and disease prevention with particular focus on dietary lipids
Research Objective 2: Elucidate the health benefits associated with the functional or phytochemical properties of food constituents.
Dietary guidelines for fat intakes were originally based on the formulae that predicted the impact of polyunsaturated fatty acids (PUFA), saturated fatty acids (SFA) and monounsaturated fatty acid (MUFA) on serum cholesterol levels as the cardinal biomarker of cardiovascular disease (CVD) risk. There has been an evolution in the dietary guidelines over the 1996-2006 period, which recognizes that while PUFA are associated with a reduction in serum cholesterol and CVD risk, not all PUFA are created equal with regards to their beneficial effects and excessive consumption of n-6 PUFA is not without detrimental effects. The 2006 Revision of the American Heart Association (AHA) dietary guidelines (3) has highlighted the abandonment of the nutrient-based PUFA recommendation in favor of food-based recommendations of unsaturated fat consumption from fish, vegetables, legumes and nuts. The 2006 AHA revision is the first guideline to recommend enhanced long-chain n-3 consumption in the form of EPA and DHA, either from fish or fish-oil supplements, for those with heart disease and hypertriglyceridemia. These new guidelines emphasize that n-3 PUFA confer benefits over and above those attributable to improved serum lipid profiles- for example in reducing cardiac arrhythmias and sudden cardiac death (3). Despite this, consumers are unaware as to how much n-3 PUFA should be consumed, either in terms of absolute amounts, the forms of dietary n-3 PUFA, or the importance of the dietary n-3/n-6 ratio. Furthermore, consumers are unaware that different forms of dietary PUFA have differing health benefits based on their ability to impact the functional tissue long chain n-3 PUFAs, EPA and DHA. In addition, the approach of the National Academy of Sciences in setting an AI for n-3 fatty acids based on current US consumption of ALA and the absence of deficiency symptoms does little to emphasize the importance of dietary long-chain n-3 fatty acid such as EPA and DHA in cardiovascular health, cancer, gestational health, obesity induced inflammatory-mediated CVD, arthritis, bone health, and fertility. Members of the NC-1167 Committee are strongly convinced that the approach of the NAS in setting n-3 recommendations based on current ALA intakes contributes to confusion at the level of the consumer as to what forms and amounts of n-3 fatty acids confer health protection and disease prevention benefits, and also promotes the impression that all forms of n-3 fatty acids are metabolically equal.
Research Objective 3: Design effective nutrition education programs and delivery methods that modify human behavior such that individuals including those most at risk choose healthier diets.
Members of the NC-1167 Committee (CO, KS, NJ, NB) have developed a questionnaire that shows over 80% of dieticians in these four states indicated a need for more education and knowledge about n-3 fatty acid and the different effects of both n-3 forms and amounts. These results show that practicing dieticians need the scientific information on the effectiveness of n-3 PUFA forms and intakes in order to devise effective, theory-driven, education programs and intervention strategies such that targeted nutrition education will result in improved n-3 intakes and consumer health.

Importance of the work: As part of the Dietary Reference Intakes (DRI) for macronutrients, recommendations for n-3 intakes were made by the National Academy of Sciences (NAS) in 2002 (1,2). These recommendations were based on current US n-3 fatty acid intakes and the absence of deficiency symptoms. An Adequate Intake (AI) was set for ALA at 1.6 g/day for men and 1.1 g/day for women in the 19-50 year age group. In making these recommendations the NAS recognized that up to ten percent of the AI for ALA can be met from EPA and DHA (1,2). The NAS also recognized that a dietary requirement for n-3 fatty acids could not be set due to insufficient data for establishing an Estimated Average Requirement (EAR) which is the basis for determining a Recommended Dietary Allowance (RDA). While the DRI report noted that intakes of n-3 fatty acids above the AI confer additional health benefits, recommendations emphasizing greater intakes of both specific forms and amounts of n-3 fatty acids were not made because of a paucity of robust data at the time of the report. NC-1167 proposes to address this lack of data such that a clear picture of specific n-3 fatty acid forms and amounts consistent with health promotion and disease prevention can form the basis for modification of the NAS n-3 fatty acid recommendations. Members of NC-1167 will focus their efforts on identifying both the dietary n-3 forms and amounts that confer health promotion/disease prevention benefits rather than the NAS approach of focusing on the prevention of deficiency symptoms (1,2).

NC-1167 maintains its focus in delineating the impact of dietary PUFA in the maintenance of health and the prevention of disease in the US population, with an emphasis on identifying the health benefits of various dietary forms and amounts of n-3 PUFA. NC-1167 members have been amongst the first to show differing effects of n-3 fatty acid forms and amounts in a variety of health problems. NC-1167 scientists have shown that high n-6 PUFA (linoleic acid) inhibits bone formation and mineralization (CO, IN) and that long chain n-3 such as EPA and DHA blunts bone resorption and favors bone mineralization. The NC-1167 group was among the first group of scientists to demonstrate that high n-6 PUFA diets promote tumor growth (TN) and that n-3 PUFA such as EPA and DHA inhibited tumor growth (TN). Members of the NC-1167 group were amongst the first to show that modest supplementation of pregnant women with DHA increased gestational duration and infant growth rates (CO) and that high maternal tissue n-6 PUFA was associated with premature delivery (CO). Other members of NC-1167 have shown that the long-chain n-3 PUFA EPA and stearidonic acid (SDA, a vegetable oil based n-3 PUFA that enhances EPA levels) inhibit adipose tissue release of inflammatory mediators that contribute to obesity-associated cardiovascular disease (MI). Infertility is also associated with nigh n-6 PUFA diets (WY) and n-3 PUFA consumption increases egg release to the oviduct.

Human diets in the US provide 6% to 7% of calories as LA and only 0.7 % of calories from n-3 PUFA, with 90% of this provided by ALA (4). Several studies indicate that humans are not efficient in converting ALA to the functional long-chain n-3 PUFA such as DHA and EPA, regardless of the n-6/n-3 dietary ratio (5). The reasons for this inefficient conversion may be competition between the n-6 (LA) and n-3 (ALA) fatty acids for the ?-5 and ?-6 desaturase enzyme conversions (5), or the suppression of ?-5 and ?-6 expression by high PUFA diets (6). Work by NC-1167 scientists has shown that there are threshold levels of n-6 PUFA with increased risks for coronary thrombosis, cancer proliferation, immunosuppression, bone loss, infertility, premature delivery and perinatal health, and modest supplements of long-chain n-3 PUFA can reverse many of the detrimental effects of elevated n-6 PUFA consumption, even without reductions in the dietary n-6 PUFA content. Work by members of the NC-1167 committee has shown that increasing ALA consumption dramatically does not increase tissue phospholipid DHA, the functional form of n-3 PUFA (TN, CO), and only modestly increases the low levels of phospholipid EPA (TN). Modest supplements of DHA, provided at the current US intake of ALA and without reduction in the LA content of the diet, significantly increase tissue phospholipid DHA levels (CO). These results indicate that ALA consumption is not an effective way to optimize tissue phospholipid EPA and, especially, DHA concentrations  this can only be achieved by modest amounts of dietary EPA or DHA (TN, CO). These findings indicate that ALA cannot support tissue EPA and DHA levels at which health outcomes are improved. The Food and Drug Administration has recently cautioned consumers to limit fish consumption because of concern with environmental heavy metal and pesticide contamination. Thus, the investigation of agricultural-based sources of dietary n-3 PUFA in addition to marine based n-3 PUFA for achieving health promotion and disease prevention benefits in the US population is crucial. While no single station has the expertise to investigate all the n-3 PUFA linked wellness outcomes, the expertise of the NC-1167 Committee ensures that our search for an appropriate dietary n-3 PUFA intake, both form, amount, and useful consumer information will focus on major health outcomes such as CVD, cancer, bone diseases, prematurity and perinatal health, emphasized in the Health People 2010 Report (7).

Consequences if not done: Without this research, the scientific rationale supporting the levels and types of dietary n-3 PUFA needed to reduce risk of chronic diseases, such as cancer inflammatory and cardiovascular diseases, will remain a matter of interpretation. This is important because of the prevalence of these diseases in the American population and their serious impact on the healthcare economy. It is imperative that we not lump all the n-3 fatty acids together with the assumption that they have similar biological properties.
Equally, it is a mistake to assume that it is sufficient to recommend more fish in the diet with little hope that these recommendations are achievable. The most effective methods to implement dietary behavior change are being rigorously studied, particularly in relation to reducing total fat intake. However, these educational theories have not been applied to the area of n-3 fatty acid intake and without this data success is questionable. In addition, education to increase intake of n-3 fatty acids is further complicated because it contradicts previous nutrition communications recommending dietary fat reduction.

Therefore, without these studies, n-3 PUFA as a tool for health promotion and disease prevention will remain an interesting curiosity to the biomedical community. There is a need to determine how much and what type will result in a predictable outcomes and how to educate and motivate the public to achieve these desired outcomes.

Technical feasibility of the research: The scientists that make up the technical committee on NC-1167 are the leading authorities in their respective disciplines as it relates to the biological impact of dietary n-3 fatty acids. A review of annual reports and their publication record provides the basis for this statement. The methods outlined in this proposal are standard procedures developed and perfected by each of the investigators. As a result, each member provides a unique perspective to the problem, when seamlessly blended, will result in definitive answers. This applies to the natural as well as the social science portions of the proposal. For example, regarding the nutrition education component, this project is feasible because information will be sought from nutrition professionals who are highly organized into state and local districts. They meet on a regular basis and seek continuing education opportunities to maintain credentials. Specialized practice among nutrition professionals is common, thereby enabling us to identify practices specific to the diseases of interest. Development of an educational intervention is possible because members of the NC-1167 regional project possess n-3 fatty acid expertise and nutrition education experience, assuring scientific validity for both content and delivery components.

Advantages for doing the work as a multistate effort: The NC-1167 Research Project addresses the role of dietary n-3 PUFA in the promotion of health. No single station has the expertise or resources to investigate all the components needed to establish health driven guidelines for n-3 intakes. We will leverage our collective results into an integrative recommendation outlining the types and levels of n-3 fatty acids and their potential health outcomes. We will use this information in conjunction with the nutrition education component as a means to increase n-3 PUFA consumption by Americans and to insure that the amount and types of n-3 PUFA are biologically meaningful. To accomplish this, data have to be generated using different levels and types of n-3 PUFA under a variety of experimental conditions with multiple clinical and biochemical endpoints involving conditions such as cancer (TN, ), inflammation (MI, MO), diabetes (MI), reproductive issues and gestational/perinatal health (CO, WY), and Alzheimers disease (MO). Similarly, a multistate effort (directed by KS) makes it more technically feasible to have a large enough sample size of nutrition professionals for the nutrition education component. Intake of foods containing n-3 PUFA may vary by geographic locations, cultural background and demographic characteristics. A multistate effort provides the opportunity to address issues related to demographics, geography and ethnic diversity. Another advantage is the availability of a broad scope of technical and educational expertise for use in designing the intervention. A multistate effort will result in a larger sample size for the intervention. This will mean greater feedback on the nutrition education intervention practices and the ability to make statistically relevant group comparisons. This vigorous feedback increases the likelihood that nutrition education programs designed to increase n-3 PUFA intake will be efficacious.

Likely impacts from successful completion of the work: Health outcomes and biomarkers will be identified thereby enabling successful educational interventions to be documented. Effective educational programs that increase nutrition practitioner n-3 PUFA counseling will increase consumption of food-based sources of n-3 fatty acids to improve the quality of life and help reduce diseases, such as heart disease and cancer. The resulting economic impacts include decreasing healthcare costs and those related to an increased need for production of agriculture-based n-3 fatty acid sources, such as flaxseed, stearidonic (SDA)-containing vegetable oils and n-3 enriched animal products. The potential for negative health and economic consequences of excessive and unsafe n-3 supplementation practices will be reduced.

Related, Current and Previous Work

Related, Current and Previous Work:

Members of NC 1167 searched the literature in formulating this section of the proposal. In addition, Susan Welsh (USDA, CSREES) searched the CRIS database for active projects using the search terms omega-3 fatty acids and n-3 polyunsaturated fatty acids. Twenty nine projects were identified, and of these 11 were conducted by NC 1167 investigators. Several of these NC 1167 investigators were also supported by competitive USDA funding in the n-3 PUFA area. A similar search of the CRIS database, coded for Nutrition Education and Behavior, identified 6 active projects, 3 of which were conducted by NC 1167 investigators.

The overall goal of the previous NC 1167 project (2002-2007) was to identify and evaluate agricultural and marine sources of n-3 PUFA to meet the new dietary guidelines for optimal health and the reduction in the risk of disease throughout the life cycle. At the time of preparation of this proposal, members of NC 1167 fully expected that the NAS and the Institute of Medicine (IOM) would formulate recommendations for n-3 fatty acids that recognized the unique benefits of long-chain n-3 PUFA. In 2002 the Dietary Reference Intakes (DRI) for n-3 intakes were published and an Adequate Intake (AI) for n-3 fatty acids was set for ALA (1.1 to 1.6 g/day) based on current ALA intakes and the absence of deficiency symptoms. While this DRI report did recognize that intakes of specific forms and amounts of n-3 fatty acids above the AI conferred additional health benefits, recommendations for long-chain n-3 PUFA intakes were not made because of a lack of data at the time of publication of the report (1,2).

The following objectives were proposed for NC 1167 for the 2002-2007 period to address our overall goal:

Objective #1: Evaluate the effect of different n-3 fatty acids, both form (source) and amount, on tissue functions and correlate these effects with changes in putative biomarkers relevant to health promotion and disease prevention.
Objective #2: Experimental diets used in animal studies will examine dietary levels of n-3 PUFA that are achievable in human diets, based on human equivalent amounts (allometric scaling) in rodent models.
Objective #3: Develop effective means for translating research knowledge about n-3 PUFA into consumer food choices.

We have made significant progress in meeting these objectives and the following sections highlight our progress and review the significance of these findings to the area n-3 fatty acid form and amount in human health and disease.

Objective #2: Rodent models of dietary intakes of n-3 PUFA are of limited use unless their applicability to human intakes can be clearly established. Numerous rodent dietary models in the n-3 PUFA field have fed dietary levels of n-3 PUFA that are clearly unachievable in humans (i.e., equivalent to 8-90 g/d). The inability to translate data from experimental models to human clinical outcomes was a major criticism of the HHS Agency for Healthcare Research and Quality (AHRQ) Technical report # 113, Effects of Omega-3 Fatty Acids on Cancer (8), and its recently published adjudicated manuscript (9). Work at the TN station has established a mathematical model for the allometric scaling of dietary lipids between human and rodent diets. Extrapolations based on caloric density (% of calories or energy %) was far superior to that of body weight and body weight to the 3/4 power (10) which are typical metabolic size models (11) which have not been validated for nutrients.
Furthermore, the studies at TN established the fact that when control rodent diets are devoid of n-3 PUFA (i.e., containing only corn oil), and when corn oil in control diets is replaced by n-3 PUFA-containing oils, these additions of n-3 PUFA to a n-3 deficient diet results in an exaggerated biological response. These data suggest that providing inadequate levels of n-3 PUFA in the background diets dramatically increases the risk of generating false positive results that have little preclinical value for humans. Importantly, this model provides a recommended upper limit for rodent diets that correspond to the upper limits recommended by the FDA with regards to daily n-3 PUFA supplementation for the public (i.e., 3 g/d or 1.35% of energy). Therefore, inadequate dietary designs are a likely explanation, in part, for the discordant results between rodent and humans with regards to cancer risk (9), a concept that can be applicable elsewhere (12,13). This is the first study to directly evaluate pharmacodynamic data of n-3 PUFA between humans and rodents and provides a guideline for allometric dosing of n-3 PUFA in rodent diets (10).

When applying the validated allometric scaling model in pregnant rats CO Station has shown significant improvements in prostaglandin (PG) and matrix metalloproteinase (MMP) biomarkers of extended gestation duration, simply by substituting DHA for ALA at the current US dietary intake of 0.7 en% (energy %) (6). Thus, simply changing the n-3 PUFA form, DHA instead of ALA, without altering either the amount of dietary n-3 PUFA or the n-6 PUFA, demonstrates the applicability of the validated rodent allometric scaling model. Tripling ALA from 0.7 energy % to 2.0 energy % was not effective (6). These findings are supported by recent human results (14) showing that increases in tissue phospholipid DHA are only achieved with diet DHA and saturate at approximately 2.0 g/day (0.9 en%). Important unanswered questions remain  what is the threshold level of dietary DHA or other long-chain n-3 PUFA forms, and can long-chain n-3 PUFA forms be simply added to diets as opposed to replacing ALA? We now feel confident that the allometric scaling model provides a validated approach to addressing these issues.

Objective # 1:
Role of n-3/n-6 PUFA in Cardiovascular Disease: Cardiovascular disease (CVD) remains the leading cause of death in the US (3,15) and obesity-associated type 2 diabetes is a significant contributor to CVD (16). Increasing obesity rates in the US population (16) is a significant health concern and obesity is an independent risk factor in the developments of CVD (17). CVD is an inflammatory disease (15) and obesity contributes to inflammation by increasing the production of pro-inflammatory cytokines and adipokines (15). In in vitro experiments, MI Station has shown that primary adipose tissue inflammatory cells (stem cells and preadipocytes) from obese mice, the pro-EPA fatty acid stearidonic acid (SDA, 18:4 n-3) and EPA have significantly higher anti-inflammatory properties compared to n-6 PUFA (LA, 18:2, n-6 and AA, 20:4, n-6). Preadipocytes incubated with either SDA or EPA secreted significantly lower interleukin (IL)-6 when stimulated with lipopolysaccharide (18). N-3 PUFA were also shown to inhibit adipose cell inflammatory markers via down regulating toll-like receptor-2 (TLR-2) and TLR-4 expression. These findings suggest that n-3 PUFA rather than n-6 PUFA play a beneficial role in the reduction in obesity-associated increases in cardiovascular disease risk by lowering adipose tissue production of inflammatory factors. Follow up studies will address whether human primary adipose tissue cell productions of inflammatory markers are regulated in the similar manner as in the allometric scaling animal model studies.
NC Station examined the influence of both unoxidized and oxidized AA, EPA and DHA on the inflammatory markers of CVD, C-reactive protein (CRP) and IL production in lipopolysaccharide stimulated cultured macrophages. Incubation with 50 ¼M unoxidized EPA and DHA, but not AA, significantly reduced CRP production but did not alter IL-2 and IL-6 production. All three oxidized fatty acids reduced CRP and IL secretion, but oxidized EPA was far more potent than oxidized DHA and AA. These results suggest an anti-inflammatory benefit in CVD for n-3 fatty acids while n-6 fatty acids are pro-inflammatory. Translation of these in vitro findings to human equivalent dietary intakes (allometic scaling) is an important area for further investigation.

N-3 PUFA, Inflammation and Immunity:
MO Station investigated the impact of dietary n-3 PUFA on the immune response to infection (19). EPA and DHA were equally effective at reducing in vivo production of two key pro-inflammatory cytokines, interleukin (IL)-12 and interferon (IFN)g, during the early host response to infection from the food borne pathogen, Listeria monocytogenes, and was associated with impaired bacterial clearance and reduced survival in fish oil treatments (20). Mechanistic studies of n-3 PUFA effects on host response to this pathogen suggest that expression/function of (IFN)g receptors are diminished by these fatty acids (21). In an effort to enhance the pre-clinical value of this research collaboration between MO and TN Stations examined the impact of a more modest increase in the intake of n-3 PUFA in this infectious disease model. The background diet used in this study mimicked current total fat and fatty acid profiles of adults in the U.S, including LA, ALA, AA, EPA and DHA. Healthy mice were fed diets with an additional 1.4 energy% EPA (a HED of 3 g/d in humans). At three days post-challenge with L. monocytogenes, +EPA mice had 25 fold (p<0.05) more bacteria in their liver compared to mice fed the basal diets. However, supplemental EPA did not affect the in vivo production of the cytokines TNF-a, IL-6, IL-12p70, IFNg, or the chemokine, MCP-1. These data suggest that a diet enriched with a modest amount of EPA (i.e., equivalent to ~3 g/d, or 1.4 en% in an adult human) can diminished host resistance (innate immunity) to a common food borne pathogen, and that in vivo pro-inflammatory cytokine production may not be a sensitive biomarker for n-3 PUFA modulation of host response to infection. Thus, not all aspects of n-3 PUFA nutrition are beneficial and these findings have important implications for innate immunity in the elderly and other immuno-compromized populations.

TX Station has examined aspects of cell-mediated immunity and how these are influenced by DHA incorporation into cellular plasma membranes. The maintenance of a healthy state in normal individuals requires optimal activation of a balanced immune system, so that protective host responses (e.g., to infectious agents) can be maintained, while potentially detrimental host responses (e.g., chronic inflammation, hypersensitivity, etc) can be controlled appropriately (22,23). Establishing and maintaining the appropriate balance of T-cell subsets is likely a critical component of health maintenance, as evidenced by disease conditions in which the correct balance has been perturbed (24). The anti-inflammatory properties of dietary n-3 PUFA are the result of a direct suppressive effect on T-cell receptor (TCR) activation. In addition, we have confirmed that n-3 PUFA feeding alters the balance between CD4+ T-helper (Th1 and Th2) subsets by directly suppressing Th1 cell development (25-27). However, the precise mechanisms by which dietary PUFA influence the maintenance of appropriate T-cell subset balance for a healthy immune system have not been elucidated. To elucidate the molecular mechanism(s) through which DHA inhibits T-cell activation, we have recently demonstrated that dietary n-3 PUFA alter the phospholipid and signaling protein composition of lipid rafts. This is noteworthy because these specialized microdomains within the plasma membrane play an important role in the maintenance and amplification of TCR signaling pathways (28-30).

N-3 PUFA and Reproductive health: Birth weight and gestational age at birth are critical determinants of infant mortality and morbidity, and in the United States preterm birth resulting in low birth weight comprises 6% to 10% of all births  approximately 300,000 per year (1,3, 31). Increasing rates of late preterm birth over the 1992-2002 period adds to this health risk (32). CO Station, employing the HED (allometric scaling model) in pregnant rats, has shown DHA, fed at 0.7 en% - the current US intake of n-3 fatty acids  significantly reduced maternal reproductive tissue prostaglandin (PG) and matrix metalloproteinase (MMP) indices of gestational duration (6). Uterus and placenta DHA were significantly increased by 160% to 180% by the 0.7 en% DHA diet, and arachidonic acid content was significantly reduced. Placenta and uterus PGE2, placenta PGF2± , placenta active MMP-2 and MMP-9 , and placental collagenase production were significantly reduced by 30% to 60% by 0.7 en% DHA in comparison to 0.7 en% LnA (6). Increasing ALA to 2.0 en% was without effect. Providing DHA at an enhanced 2.0 en% did not significantly enhance the suppression of PG and MMP production. These results show that substituting DHA for ALA at the current US n-3 fatty acid intake of 0.7 en% is effective in suppressing PG and MMP indices of premature delivery and shortened gestation, and indicates that form of dietary n-3 fatty acid, DHA versus LnA, appears to be more important than the amount (6). In contrast to DHA, SDA was without effect on MMP expression at either 0.7 en% or 2.0 en% (33). The tissue fatty acid profiles indicated that SDA was converted to EPA, but not to DHA (33). SDA, a plant based n-3 PUFA, is without effect on indices of shortened gestation at achievable human intakes and these data suggest that DHA, the major long-chain n-3 PUFA of human phospholipids, is the important n-3 PUFA. The influence of EPA still needs to be addressed using this model.

In a human clinical trial funded by the USDA-IFAFS Functional Foods Initiative, CO Station has demonstrated that 300 mg and 600 mg supplementary DHA  equivalent to an additional 0.14 en% and 0.27 en% respectively  provided in the last trimester of pregnancy significantly increased red blood cell phospholipid DHA and increased gestation duration by 4 days. Thus, translation of the rodent allometric scaling model to a human application has been successful. Furthermore, the amount of additional dietary DHA necessary to achieve this improvement appears to be quite modest (34) and is corroborated by recent human studies showing enhancement of tissue DHA is achieved only by dietary DHA (not ALA or EPA) and is saturated at a modest 2 g/day (i.e. 0.9 en%) (14).

WY Station employed the allometric scaling approach were dietary n-3 fatty acids are included in rodent diets at an amount, percent of calories (energy % or en %), equivalent to human dietary n-3 intakes. Using this approach, and feeding EPA and SDA at 0.7 en%, it appears that there is a differential effect of the type of long-chain n-3 PUFA (EPA vs. SDA) on PGE and PGF production, and on COX-1 and COX-2 expression in ova. Thus, depending on the form of the form of the n-3 polyunsaturated fatty acid (PUFA) ingested, there may be an influence on ovulation and a concomitant alteration on reproductive capability. These results also indicate that the dietary form of n-3 (EPA vs. SDA) has an important influence on reproduction.

Objective #3: KS, CO, NJ and NB Stations worked as a team to develop a questionnaire for assessing the n-3 fatty acid knowledge and practices of dietitians. For the formative phase of development, 10 dietitians from each state were called and ask to respond to open-ended questions. Responses were grouped and a detailed questionnaire was developed and adapted for completion using both telephone and web survey methodologies. Results showed that > 80% of the dietitians surveyed in the four states indicated a need for more education and knowledge about n-3 fatty acids, thus demonstrating the need for translational research on n-3 fatty acids aimed at nutrition practitioners in the next stage of the project (35). They were unaware of the various types of n-3 PUFA. The majority were aware that n-3 PUFA were related to heart disease and lipid levels in some way, but they were unaware of the effects of n-3 PUFA on pregnancy outcomes, inflammation, diabetes, and cancer. Those who regularly worked with heart disease patients encouraged increased n-3 PUFA consumption, and were more aware than others of which foods to recommend. However, other than fish and fish oils supplements, most were unaware of other good sources, and no one knew how to counsel clients to select the best supplements. Nearly all were interested in receiving more information about n-3 PUFA; and, most indicated that they most wanted to receive information in the form of fact sheets or handouts that they could share with those who they educated. They indicated they would be interested in receiving these handouts via multiple venues, i.e., the web, listserves, conferences, etc. Although the last year of the project is still underway, four handouts are being created based on needs identified through this research coupled with input from the bench scientists working on this project regarding the most important information that should be taught.

NB Station developed and validated a method for assessing total n-3 fatty acid intakes using a food frequency and developed a nutrition education intervention for adults with cardiovascular disease using the food frequency to assess the impact of the educational intervention on n-3 fatty acid intakes (36,37). Nutrition education intervention doubled consumption of n-3 fatty acids, and the food frequency was effective in measuring changes in intake. NB Station also demonstrated that both total and long-chain n-3 fatty acid consumption was significantly greater in coastal areas than in internal regions of Saudi Arabia (38,39), indicating the need to consider regional food culture when designing nutrition education interventions to increase n-3 fatty acid intake.

CO Station explored nutrition education, designed to provide an additional 300 mg DHA/day, on pregnancy duration as part of a USDA-IFAFS funded Functional Foods initiative (34). Nutrition education included formative assessment, validation of a pictorial food frequency questionnaire and development and implementation of a nutrition education intervention using designed bi-lingual materials in a inner city Denver WIC program with a predominately Hispanic population of limited resources. Nutrition education/intervention increased gestation duration by 4 days, and was as effective as 600 mg/day supplementary DHA used in the clinical phase of the trial. The cost of providing such nutrition education within existing federal food assistance programs is minimal and should be explored, especially for food assistance and nutrition education programs for pregnant women and those at higher risk for low birth weight and premature deliver.

Objectives

1. To determine the health promotion and disease prevention effects of both the forms and the amounts of n-3 fatty acids by correlation with tissue functions, and with alterations in biomarkers, relevant to optimal health and disease prevention.
   
2. To examine dietary levels of both form and amount of n-3 PUFA that promote health and reduce disease that are relevant and achievable in human diets using the human equivalent dose method (allometric scaling) in rodent models.
   
3. To develop, test and disseminate effective means for translating research on the health promoting and disease preventing effects of n-3 PUFA into consumer food choices.
   

Methods

Objectives for the new proposal: The problem to be addressed is that the DRI for n-3 PUFA is formulated based on an AI for ALA and the absence of deficiency symptoms in the population, despite evidence indicating that long-chain n-3 PUFA confer distinct health promotion benefits. The overall goal of this project is to provide a data base to establish the n-3 PUFA forms and amounts that meet dietary guidelines for optimal health and disease prevention. The following objectives are proposed to meet the project goal: Objective #1 To determine the health promotion and disease prevention effects of both the forms and the amounts of n-3 fatty acids by correlation with tissue functions, and with alterations in biomarkers, relevant to optimal health and disease prevention. Objective #2 To examine dietary levels of both form and amount of n-3 PUFA that promote health and reduce disease that are relevant and achievable in human diets using the human equivalent dose method (allometric scaling) in rodent models. Objective #3 To develop, test and disseminate effective means for translating research on the health promoting and disease preventing effects of n-3 PUFA into consumer food choices. Procedures to address objective 1: To investigate and improve the effectiveness of using n-3 PUFA in cell culture (in vitro) as a high-throughput preclinical screening tool and to increase the compatibility of this in vitro approach with the human equivalent dose (HED) in vivo data. Randomized clinical trials (RCT) are expensive approaches to defining the effects of nutrients on disease outcomes and often use supplementary levels that are unachievable in human diets. We have addressed this problem in rodent models by validating the human equivalent dose (HED) allometric scaling approach to rodent experiments. We now propose to extend this to cell culture (in vitro) models, validate this approach, and develop a high-throughput preclinical screening tool that will contribute to defining the forms and amounts of n-3 PUFA in health promotion and disease prevention. Methods and Procedures: All of the proposed experiments utilize approaches, techniques, and data analyses that have been previously subjected to peer review by agencies such as USDA, NIH, NCI, and other non-government funding agencies such as AICR and industry. Station participant publication records in peer-reviewed journals are extensive indicating that the experimental methods to be used are routinely performed in the laboratories of the respective scientists. As such, we will be emphasizing experimental designs in this text. Cell culture experiments provide highly defined conditions with innumerable controls in a design that is impossible using animal models and, most importantly, can be used to elucidate cell signaling mechanisms. Unfortunately, there is little translational value when trying to extrapolate results in vitro to in vivo, viz: experimental animal models and humans. Improving the translational value of in vitro data as it applies to in vivo models and subsequently humans is challenging, and no efforts are being made to address this issue. We have agreed that some of the conflicting data generated by cell culture, experimental animal models and human trials may be explained, in part, by the cell culture environment. Cells are typically grown in standardized, well defined growth media supplemented with fetal bovine serum (FBS) to provide growth factors which has an unrecognized impact on membrane fatty acid composition. We hypothesize in order to truly achieve extrapolatable results the starting membrane composition of the cells in culture should reflect the composition of comparable cells in situ. We propose to address this important issue by revisiting experimental designs that will enhance translational predictability in a manner similar to that we employed in the allometric scaling model to determine human equivalent doses of n-3 PUFA in rodent diets. MO Station has established that membrane phospholipids of cells grown in culture are severely deficient in n-3 PUFA because of the compositions of the culture media. For example, n-3 PUFA and DHA levels in primary rat brain microglia cells make up 5.1% and 4.7%, respectively, of the phospholipids fatty acids, but the comparable levels in standard cultures (DMEM, 5% FBS) of BV-2 microglial cells were 0.32% and 0.32%, respectively. It was determined that these levels could be normalized to the levels found in the primary rat brain cells with the addition of DHA. We propose that experiments should be performed using cells where the PUFA contents are normalized to in vivo levels, depending upon the tissue, and then exposed to additional amounts of n-3 PUFA (i.e. plasma levels). These results can then be compared to un-normalized cells and results observed in vivo to help provide validation. MO station will investigate the impact of n-3 PUFA in microglia cells in vitro using a microglia cell line, BV-2 cells. When exposed to 40 µM arachidonate and 20 µM DHA the fatty acid composition of these cells recapitulate (normalize) the fatty acid composition of primary brain microglia cells from rats fed a standard diet. Long-chain n-3 PUFA are thought to have anti-inflammatory activity in vitro and in vivo. Over-production of pro-inflammatory molecules by microglia exacerbates many CNS diseases such as Alzheimers (40). The hypothesis is that n-3 PUFA will reduce pro-inflammatory mediator production by murine microglia. The in vitro bioactivity of two commonly used n-3 PUFA: DHA and EPA will be evaluated using BV-2 cells (with normalized and un-normalized fatty acid composition) and primary rat brain microglial cells. The production of cytokines (i.e., interleukin-6 (IL-6), IL-1², nitric oxide (NO), and tumor necrosis factor (TNF)-±) will be measured under at three doses of EPA and DHA (10 µM - 50 µM) in both normalized and un-normalized BV-2 cells and will be compared to the primary rat brain microglia cells. These results will be the first to investigate whether normalizing the fatty acid composition of cell membranes better mimics the results of primary cells from a comparable tissue. MI Station will test adipocyte cells lines and compare them to primary adipocytes using their inflammatory model. Obesity contributes to a pro-inflammatory environment by secreting a number of cytokines, including interleukin (IL)-6. Previous data from MI demonstrates that n-3 PUFA inhibits the production of this cytokine. MI station will test the effects of different forms of n-3 PUFA on adipose tissue release of pro-inflammatory mediators that contribute to obesity-associated cardiovascular disease. The membrane fatty acid compositions of primary adipocytes will be recapitulated in immortalized adipocytes and the effects of ALA, LA, SDA, AA and EPA on the production of the pro-inflammatory cytokine interleukin (IL)-6 will be tested and compared. These results will then be compared to data generated in vivo. CO Station proposes to investigate normalized and un-normalized late pregnancy immortalized human myometrial cells (PHM1-41) (41) using the same experimental model. That is, by determining the in situ fatty acid composition of comparable cells, normalizing the fatty acid compositions of equivalent immortalized cells and then determining the effects of ALA, EPA and DHA additions (10 ¼M to 50 ¼M) on oxytocin mediated Ca2+ entry. Modification of this Ca2+ signaling pathway by n-3 fatty acids is important in pregnancy duration and neonatal health. In summary, these first experiments are designed to coordinate and improve the translational value of data generated from cultured cell lines to cells and tissues that are more physiologically relevant. To determine the effects of n-3 PUFA, both forms and amounts, on targeted tissue function, cellular signaling mechanisms and changes in putative biomarkers that are relevant to optimal health and disease prevention. N-3 PUFA effects on cell signaling mechanisms and targeted tissue function are important in setting n-3 PUFA goals for health and disease prevention. Since participating stations propose to develop and validate in vitro (cell culture) methods to assess the health benefits of n-3 PUFA as a preclinical tool (Objective #1), and to test these in vitro findings in the validated in vivo rodent allometric scaling model (Objective #2), these in vitro experiments afford an excellent experimental opportunity to determine the cellular signaling mechanisms that influence tissue functions in a several disease prevention settings. MO Station will investigate the impact of four different n-3 PUFA forms, a-linolenic acid (ALA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), on the inflammatory response of brain microglial cells. Preliminary findings of reduced oxytocin mediated Ca2+ in DHA cultured human myometrial cells suggests that long-chain n-3 PUFA forms can affect the cellular signaling mechanisms of parturition. CO Station proposes to examine how n-3 PUFA can alter plasma membrane oxytocin receptor population, and how these fatty acids alter the inositol trisphosphate (IP-3) Ca2+ signaling system that controls uterine contractions as term approaches. TX Station will determine how DHA can modify lipid raft microdomain composition and how these changes alter the dynamic partitioning of signaling proteins necessary for cell proliferation and apoptosis in T-cells and the modulation of CD4+ Th1-cell activation in response to antigen stimulation. MI station will examine the release of proinflammatory cytokines inteleulin-6 (IL-6) from cultured adipocytes and how n-3 PUFA forms influences this release. Procedures to address Objective #2: With the validation of the HED rodent model, participating stations are now able to extend their contributions to the data base necessary to establish the n-3 PUFA forms and amounts that meet dietary guidelines for optimal health and disease prevention. TN Station plans to extend this research to n-6 PUFA, such as LA and AA. The DRIs suggest that the effectiveness of dietary n-3 PUFA are dependent upon n-6 PUFA levels (LA) and recent evidence using a rodent model suggests that modifying dietary LA levels influences ALA conversion to DHA (42). TN Station will conduct allometric scaling studies in mice supplemented with human equivalent doses of LA (0.5 en% - 3 en%) and AA (0.09 en% - 0.4 en%) and determine changes in tissue (plasma and RBC) fatty acid composition. These data will be compared to archival data in the same fashion as the n-3 PUFA were compared in the previous proposal that validated the allometric scaling model. KS Station will investigate the role of n-3 PUFA in protection from pulmonary and hepatic fibrosis. KS will determine whether the shorter chain length n-3 PUFA, such as ALA, is as effective in inhibiting fibrosis as the long-chain n-3 PUFA using a bleomycin model for inducing fibrosis. Bleomycin chemotherapy is effective in cancer treatment by induction of both single and double strand DNA cleavage but it induces pulmonary fibrosis via some unknown mechanism that may involve eicosanoids. The hypothesis to be tested is: can n-3 PUFA, which alter eicosanoid metabolism, alter the profibrogenic outcomes of bleomycin treatment. Preliminary findings suggest that ALA (flax oil) showed better protection against liver bleomycin than EPA and DHA containing fish oils, a protection not seen in lung. Using the rodent model KS proposes to determine the dose response effects of flaxseed oil on the development of pulmonary fibrosis and define protein markers associated with this lung disease fibrotic injury. TN Station proposes to test the effectiveness of n-3 PUFA on the natural progression of prostate cancer, that is, the length of time it takes hormone-sensitive cells to transform to hormone-insensitive cells (43). In humans prostate removal and androgen ablation often results in metastasis in bone where cells learn to grow without androgens (43). Recent evidence indicates that AA promotes metastatic migration to the bone, and EPA and DHA inhibit this process (44). Epidemiological studies support a role for n-3 PUFA in prostate cancer inhibition (45). The lag phase to androgen independence is typically 3-5 years. We propose to use a xenograft model (using immuno-compromised nude mice) and a prostate cancer cell line (CWR22 cells) that mimics the natural progression of the human disease (46). These cells will grow in the presence of androgens and the resulting tumor will regress following castration (removal of androgens). However, like the human disease, over time these cells will begin to aggressively grow again without androgens (46). We propose to feed animals diets supplemented with EPA to see if the time to progression  the lag phase - will be abated and compare these results to dietary ALA because it has been reported that dietary ALA increase the risk of prostate cancer in contrast to long-chain n-3 PUFA (47). WY Station will examine the influence of both the terrestrial and marine n-3 PUFA on fertility assessed by ova release into the oviducts in rats. Preliminary data has shown that feeding 0.3 en% fish oil to rats significantly decreased ovulation (48). Using the rodent model with HED of ALA, EPA and DHA, WY Station will examine their influence on ova release, ovarian cyclooxygenase (COX)-1 and COX-2 expression, and ovary PGE and PGF production. Impaired fecundity is an important health issue and the influence of n-3 fatty acids on fertility and ovulation is important in setting recommendations for optimal health. CO Station will examine the effect of EPA on PG and MMP markers of shortened gestation using human equivalent doses of EPA. DHA (6), but not SDA (33), is effective in reducing these biomarkers of shortened gestation at human relevant intake of 0.7 en%. Since fish and fish-oil supplements provide both DHA and EPA, determining the independent effects of these long-chain n-3 PUFA on gestation duration biomarkers is an important reproductive health promotion question. Using the HED rat model CO proposes to examine the effect of 0.7 en% - 2.0 en% EPA on uterus and placenta PG production and MMP expression. Since long-chain n-3 PUFA depresses both uterus and placenta AA (6,33), and AA consumption may alter this DHA protection, rats will be fed HED of AA, approximately 0.09 en% equivalent to the 200mg average US daily consumption of AA (49), and uterus and placenta PG and MMP production will be determined at day 20 of pregnancy. MI Station will test effects of different n-3 PUFA forms (ALA, SDA and EPA) on adipose tissue release of inflammatory mediators that contribute to obesity-associated cardiovascular disease. Specifically, effects of ALA on adipose tissue production of proinflammatory cytokine such as inteleulin-6 (IL-6) will be compared to LA, SDA, EPA and AA treated cells. Previously, we have observed that effects of SDA are exerted via its conversion to EPA (50). Thus, whether ALA has independent effects on adipose tissue cell production of proinflammatory cytokine production without being elongated to longer chain n-3 PUFA will also be tested . MO Station will investigate the impact of individual n-3 PUFA on the development and progression of Alzheimers disease (AD) (51) using a mouse model of this human disease. We hypothesize that intake of SDA, EPA and DHA, but not LNA, at modest levels, will diminish the inflammatory responses of brain microglia and subsequently reduce AD progression in mice transgenic for amyloid precursor protein (APP) processing (Tg2576 mouse). This transgenic mouse strain expresses chronically elevated levels of proinflammatory cytokines in brain microglia and serve as an excellent animal model for studying the role of inflammation in human AD. (52) Recent data (53) has shown that 1.2 energy% DHA in the diet of such transgenic mice reduced the development of AD-like lesions in the brains of aged animals, and dietary EPA supplementation (i.e., ~1.2 en%) diminished in vivo activation of rat microglia in the hippocampus region of the brain following intracerebral injection with amyloid-b peptide (54). However, nothing is known about the relative potency of plant-derived n-3 PUFA on microglia activation or AD progression, nor has the beneficial effects of EPA and DHA been directly compared in microglia activation or AD progression. TX Station will examine the anti-inflammatory effects of dietary n-3 PUFA in the immune response, and how n-3 PUFA feeding alters the balance between CD4+ T-helper (Th1, Th2) subsets. The establishment and maintenance of the appropriate balance of T-cell subsets is a critical component of health maintenance, as evidenced by disease conditions in which the correct balance has been perturbed (24). A wealth of published literature supports the contention that DHA containing diets are important in determining the quantity and quality of the hosts immune responses (55-60). Therefore, it is essential to understand precisely how specific n-3 PUFA, e.g., DHA, modulate immune function, so that recommendations regarding the health benefits to be derived from dietary manipulation can be based upon a firm scientific foundation. TX Station will determine the influence of dietary DHA on modulation of CD4+ Th1-cell activation in response to antigen stimulation, and the ability of DHA to alter Th1 cell-mediated inflammatory bowel disease in a murine model. Procedures to address Objective #3: We propose to primarily use an accessible web-based venue, e.g. eXtension, to address this objective (61). The users for the site are intended to be consumers and nutrition professionals, e.g., Extension educators, dietitians, and nutritionists. All materials will be designed with the consumer as the primary target. Basic science researchers working on Objectives 1, 2 and 3 of this proposal will work directly with the nutrition education researchers on this objective to ensure the content on the site constitutes the most up-to-date information possible. Both information on n-3 PUFA and common misconceptions regarding n-3 PUFA will be addressed. The content included on the site will be developed as the science unfolds, and updated as possible. Basic science researchers participating on this project are working on the following areas: heart disease, cancer, inflammation, obesity, gestational health, reproduction, diabetes, and Alzheimers disease. These areas will therefore be the first n-3 PUFA-health/disease topics explored and the nutrition educators will ensure that those scientists with focused research in these areas will assist with the development of corresponding materials. Nutrition educators will ensure that ample resources are available regarding food and supplement selection, and practical consumer concerns with regard to increasing their n-3 PUFA intake, e.g., fish contamination, supplement dosage, form efficacy, and safety. Since n-3 PUFA is a dynamic area of nutritional science, a great deal of time will need to be allocated to material creation, as well as the examination of the validity of currently available materials. It is expected that using web-based venues for information distribution, in addition to those materials created as part of this project, will establish links to other valid information sources. Methods will be executed according to the following timeline: Year 1 (FY 08): " Continue the determination of educational needs in this area and make any needed revisions to previously developed materials. " Apply as a group to be an eXtension community of practice. Year 2 (FY 09): " Developing web-site structure: uploading currently available content. Years 3 and 4 (FYs 10-11): " Further develop site content as a collaborative team, i.e., basic scientists and nutrition educators. " Market site and monitor its use Year 5 (FY 12): " Continue web updates " Assess outcomes, i.e., degree of usage, needs met, increased knowledge regarding n-3 PUFA health benefits, n-3 PUFA forms and amounts for health promotion.

Measurement of Progress and Results

Outputs

• Establishing in virto (cell culture) models of n-3 PUFA effects
• In vitro effects of n-3 PUFA forms on biomarkers of health promotion and disease prevention
• Expansion of in vivo (experimental animals fed HED) data for n-3 forms and amounts for health promotion and disease prevention
• Increased database defining the health benefits of n-3 PUFA forms
• Data on n-3 PUFA intakes in diverse populations
• Output 6 Expansion of food-based n-3 PUFA for optimal health and disease prevention Output 7 Web material development for education and training of nutrition professionals Output 8 Dissemination of data in peer-reviewed journals, scientific and professional meetings

Outcomes or Projected Impacts

• Validation of the in vitro (cell culture) approach to defining n-3 PUFA requirements
• Defining the use of cell culture as a high-throughput preclinical screening tool
• Improved health of the U.S. population
• Establishing more definitive and form-specific DRIs for n-3 PUFA
• Modification of the n-3 PUFA requirements to include forms other than ALA
• Impact 6 Decreased incidence and progression of degenerative and age-related diseases Impact 7 Establishing a Community of Practice so that information from science can be made available through eXtension Impact 8 Education of the clients of nutrition professionals about the health benefits of n-3 PUFA Impact 9 Increased awareness by the medical community of the specific benefits of n-3 PUFA forms and amounts

Milestones

(2008): Submission of a competitive grant application to USDA by one or more of the Station representatives

(2010): Symposium presentation of findings at national/international meetings, eg Experimental Biology meeting, International Society for the Study of Fatty Acids and Lipids (ISSFAL)

(2011): Symposium presentation of findings at national/international meetings, eg Experimental Biology, ISSFAL

(2012): Symposium presentation of findings at national/international meetings, eg Experimental Biology, ISSFAL, American Dietetic Association

(2012): Author a report directed at the governing organizations, such as USDA, IOM, and FDA

(12):Redefinition of the DRI for n-3 PUFA at the conclusion of the project

Projected Participation

View Appendix E: Participation

Outreach Plan

The outreach plan is discussed in Objective #3. Objective #3 is, in its entirety, an outreach plan with a timeline.

Organization/Governance

The committee, comprising the scientists from participating stations, in addition to the Regional Administrative Advisor and the USDA CSREES Representative will be organized and governed as specified in the North Central Regional Association (NCRA) guidelines (62).

At the annual meeting(s) of the Technical Committe a Chair and Secretary is elected for the upcomming year. In addition, members of the Technical Committee will elect members to a committe responsible for organizing national/international presentations (see Milestones) and also to writing committtes for the preparation of reports, position papers and recommendations (see Milestones).

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Attachments

Land Grant Participating States/Institutions

CO, LA, MN, MO, NE, NJ, OH, TX, VA, VI

Non Land Grant Participating States/Institutions

East Carolina University, Energy / Livestock Financial, Laboratory / Livestock Project Integrator, Tennessee State University, Texas Woman's University, University of Wisconsin - Stout, USDA-ARS, USDA-ARS/ND