Omega-3 Purlsane Eggs

Final Report for GS07-062

Project Type: Graduate Student
Funds awarded in 2007: $10,000.00
Projected End Date: 12/31/2008
Grant Recipient: North Carolina State University
Region: Southern
State: North Carolina
Graduate Student:
Major Professor:
Thomas Rufty
NCSU Crop Science Department
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Project Information

Summary:

In this project, we worked to develop key elements of a Purslane – laying hen system for production of high quality omega-3 fatty acids that are important in human nutrition. We defined phenotypic variation in growth, yield and concentrations of α-linolenic acid (ALA). We also documented a strong fertilizer N response with selected Purslane varieties. Purslane is currently being fed to free-range laying hens to determine the extent that DHA content is altered. Additional experiments are examining purslane tolerance to drought conditions and its tolerance to water stress.

Introduction

The purpose of this project is to develop a low-input, sustainable agricultural system that will open the fast-growing omega-3 market to small farmers in the Southeast. The system involves growing the native plant common purslane (Portulaca oleracea L.) that is well adapted to the southeastern environment and was recently found to have omega-3 concentrations that are among the highest of any known plant species. The purslane will serve as a feed source for free-range laying hens that will, in turn, produce eggs with elevated, high quality omega-3s for human consumption. The omega-3 market is already established and rapidly expanding, and thus offers an excellent economic opportunity for small farmers.

Problem

Omega-3 fatty acids are now widely recognized as an essential component of human nutrition. They play an integral role in fetal brain development, mental health, cardiovascular function, retina function, gene expression, inflammation responses, and insulin sensing (6-21). Omega-3 fats are considered essential fatty acids because they cannot be produced by human metabolism. They must be acquired from food.

Currently, the diet of most Americans does not include adequate amounts of DHA (docosohexaenoic acid) (22). Plants produce 18:3 short-chain omega-3s (ALA, α-linolenic acid), which are poorly utilized by the body and contribute only marginally to human nutrition. The essential source for humans is the longer chain 22:6 DHA omega-3 form. Importantly, some animals, including poultry, can efficiently make DHA by lengthening ALA omega-3s originally produced in plants.

Historically, Americans obtained DHA from pasture raised animal products (23). This main source of omega-3s has been lost as the U.S. shifted towards confinement animal systems with mainly grain feed, which has low omega-3 levels.

There are problems with the two major sources of DHA in the Western diet, and neither of the sources are sustainable. Fatty fish are a main source of DHA. However, fish require processing and long distance transport with considerable transportation cost and the associated generation of greenhouse gases. Also, increased fish consumption is leading to rapid depletion of ocean fish populations (24). Perhaps most importantly, fatty fish are increasingly contaminated with heavy metals which are known to cause human health problems, particularly in young children. Heavy metal exposures can result in brain damage during pregnancy and the neonatal stage (14,16,25). The potential damaging effects thus coincide with the period with the most intense requirement for omega-3s, when much brain development occurs (6,9-10,12,14,16,25). The possible alternative of fish farming, where heavy metal accumulation might be minimized and natural populations protected, has also raised serious concerns because of environmental pollution and antibiotic resistance (26).

Flax seed is the second major source of omega-3s in Western diets. Unfortunately, production is confined to the upper mid-west climate (27), so there are substantial economic and environmental costs in transportation to the Southeast and other regions in the U.S. Feeding flax to chickens does increase the DHA concentration in egg yolks (28), but this is done mainly in large industrial farms. In those operations, conditions generally dictate that laying hens have little or no access to outdoors or room to express natural behaviors, and chicks typically are debeaked to prevent pecking other hens to death (29). Also, phosphorus and nitrogen originating from large industrial poultry operations are primary contributors to eutrophication and degradation of water resources (30).

The dietary importance of omega-3s and the problems with existing DHA sources strongly support the need for local sources.

Rationale

Plants are the primary producers of omega-3 fats in terrestrial ecosystems. Omega-3s are mostly concentrated in thylakoid membranes of chloroplasts in plant leaves and in seeds of some species. Almost all omega-3 fatty acids in plants exist as short chain ALA.

Purslane is an excellent source of ALA, with levels that are among the highest found in the plant kingdom up to this time (1,2). Consistent with this contention, recent analyses done in an associated SARE Graduate Student Research Project revealed that ALA accounted for 60-67% of the leaf fatty acids and 26-29% of seed fatty acids in purslane.

Purslane is well adapted to the climate and soils in the Southeast and grows widely as a weed throughout the region. Compelling evidence for adaptation is the pervasiveness of purslane as a problem weed in multiple horticultural crops (31,32). The advantageous physiological mechanisms are somewhat obscure at this time, but several favorable properties are evident. Purslane reproduces indeterminately, so it continually generates seeds and has a substantial amount of reproductive elasticity. Furthermore, drought is one of the biggest problems encountered in southeastern agriculture. Field observations (33) and our preliminary studies indicate that purslane continues to grow and reproduce efficiently under drought conditions that adversely impact vegetable and crop species. And when extreme drought causes defoliation, eventual rainfall results in new shoot development from nodal tissues within days, and new growth and seed production follow.

A unique physiological property of purslane, and one that underscores its uniqueness, is that purslane evidently shifts between C4 and CAM metabolism. The shift depends on nutrient and water availability, making it highly adaptable to a range of environmental conditions (34,35). Under optimal growing conditions with sufficient water, as usually occurs in spring, purslane functions as a rapidly growing C4 plant. When conditions become dryer, as they often do later in the summer, CAM metabolism results in enhanced water use efficiency.

The rapid growth rates, apparent stress tolerance, and reproductive plasticity of purslane make it, potentially, an excellent forage crop. Importantly, preliminary studies indicate that free-range laying hens preferentially eat purslane growing in fields of mixed vegetation and purslane seeds mixed into hen feed. This dietary linkage with poultry is critical for the success of the DHA omega-3 generation system.

Purslane contains ALA omega-3 fatty acids, but the form of omega-3 fatty acids most important to humans is DHA. Humans can obtain DHA in two ways. One is to consume ALA and elongate it metabolically to DHA. However, the enzyme involved in the elongation reaction is functionally inefficient. It also elongates omega-6 fats, which are far more prevalent in the Western diet than ALA omega-3s. Because of enzyme competition and internal catabolism, it is estimated that less than 15% of consumed ALA is converted to DHA in humans (6). As evidence of the problem, nursing women consuming only ALA have drastically lower DHA in their breast milk than women who consume DHA (6).

The second way humans can obtain DHA fatty acids is by consuming DHA directly. Within a nutritional perspective, even though animals such as chickens cannot produce omega-3 fats, they can convert dietary ALA from plants into DHA. It has been repeatedly shown that laying hens fed high ALA diets produced eggs with high DHA yolks (4-5,28). The eggs thus become an inexpensive and concentrated source of DHA for humans.

Raising chickens in open pastures offers benefits over large vertically-integrated commercial systems. Under the integrated contracts, farmers must take out large loans to build expensive buildings. In contrast, free-range rotations have low up-front costs. Production on a relatively small area of land can be maximized by properly rotating plots. Plus, raising chickens on open pasture cycles nutrients and reduces the need for antibiotics and coccidistats because UV light kills nearly all chicken pathogens (36). And importantly, free-range, high omega-3 eggs have a market value that is markedly higher than that of typical, local free-range eggs.

Significance

One of the most challenging aspects of the movement towards sustainability is to integrate plant and animal systems in a profitable, environmentally sensitive manner. The purslane/poultry system will accomplish that. With purslane serving as a key food component for the free-range hens and the hens providing fertility for the purslane, the system cycles nutrients and minimizes the need for external inputs. With appropriate purslane acreage and poultry populations, it also can be optimized for nutrient cycling and nutrient containment to minimize environmental degradation.

The elevation of omega-3 levels will add value to free-range eggs. Based on the premium for high omega-3 eggs currently sold in supermarkets, along with the relatively low cost for establishing purslane, high omega-3 free-range eggs should generate higher profits for small farmers.

Another significant feature of the purslane/poultry omega-3 generation system is the production of a nutrient that is essential to human health but currently deficient in the diet of people in the U.S. While the shift to large-scale, commercial production of meat and dairy products can be justified in economic terms, it does come with costs – the loss of dietary omega-3s being one. Individual and community health benefits from having a local, readily available, lower cost supply of omega-3s could be considerable.

A compelling aspect of the purslane/poultry system is that we know it can work. Field observations over the past decades, recent preliminary experiments, and research literature provide concept proofs: 1) purslane is a rapidly growing species that is well adapted to the Southeast; 2) purslane has high ALA omega-3 fatty acids, 3) free-range chickens preferentially eat purslane vegetation and seeds, 4) a high ALA diet for poultry results in high DHA eggs, and 5) high DHA omega-3 eggs bring a market premium. The goal now is to assemble the sustainable integrated system, make it efficient, and maximize its profitability.

References
1. Simopoulus, A.P. (1992) Common Purslane: A Source of Omega-3 Fatty Acids and Melatonin. J. Pineal Res. 39:331–332.
2. Almazan, A.M. et al. (1998) Fat and Fatty Acid Concentrations in Some Green Vegetables. Journal of Food Composition and Analysis 11:375-380.
3. Simopoulus, A.P. et al. (1989) Letter: n-3 Fatty Acids in Eggs from Range-Fed Greek Chickens. NEJM 321:1412.
4. Zotte, A.D. et al. (2005) The dietary inclusion of Portulaca oleracea to the diet of laying hens increases the n-3 fatty acids content and reduces the cholesterol content in the egg yolk. Italian Journal of Animal Science 4S:157-159.
5. Ayerza, R. and Coates, W. (2000). Dietary Levels of Chia: Influence on Yolk Cholesterol, Lipid Content, and Fatty Acid Composition for Two Strains of Hens. Poultry Science 79:724-739.
6. Lauritzen, L. et al. (2001) Review: The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina. Progress in Lipid Research, 40:1-94.
7. Uauy, Ricardo et al. (2001) Essential fatty acids in visual and brain development. Lipids 36:885-895.
8. Kris-Eterton, P.M. et al. (2002) Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease. Circulation 106:2747-2757.
9. O’Conner, Deborah L. et al. (2001) Growth and Development in Preterm Infants Fed Long-Chain Polyunsaturated Fatty Acids: A Prospective, Randomized Controlled Trial. Pediatrics 108:359-371.
10. Daniels, J.L. et al. (2004) Fish Intake During Pregnancy and Early Cognitive Development of Offspring. Epidemiology 15:394-402.
11. Kitajka, Klara et al. (2004) Effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression. 101:10931-10936.
12. Helland, I.B. (2003) Maternal Supplementation With Very-Long-Chain n-3 Fatty Acids During Pregnancy and Lactation Augments Children’s IQ at 4 Years of Age. Pediatrics, 111:39-44.
13. Hornstra, G. (2000) Essential fatty acids in mothers and their neonates. Am J Clin Nutr. 71(suppl): 1262S-1269S.
14. Oken, Emily. (2005) Maternal Fish Consumption, Hair Mercury, and Infant Cognition in a U.S. Cohort. Environmental Health Perspectives 113:1376-1380.
15. Al Monique D.M. et al. (2000) Long-chain polyunsaturated fatty acids, pregnancy, and pregnancy outcomes. Am J Clin Nutr. 71(suppl): 285S-291S.
16. Oken, Emily et al. (2008) Maternal Fish Intake during Pregnancy, Blood Mercury Levels, and Child Cognition at Age 3 Years in a US Cohort. Am J Epidemiol 167: 1171-1181.
17. Lauretani, F. et al. (2007) Omega-6 and omega-3 fatty acids predict accelerated decline of peripheral nerve function in older persons. European J Neuro 14:801-808.
18. Amminger, G.P. et al. (2007) Omega-3 Fatty Acids Supplementation in Children with Autism: A Double-blind Randomized, Placebo-controlled Pilot Study. Biol Psychiatry 61:551-553.
19. Lee, K.W. et al.(2003) Review: The role of omega-3 fatty acids in the secondary prevention of cardiovascular disease. Q J Med 96:465-480.
20. Tsitouras, P.D. et al. (2008) High Omega-3 Fat Intake Improves Insulin Sensitivity and Reduces CRP and IL6, but does not Affect Other Endocrine Axes in Healthy Older Adults Horm Metab Res 40:199-205.
21. Bazan, N.G. (2007) Omega-3 fatty acids, pro-inflammatory signaling and neuroprotection. Curr Opin Clin Nutr Metab Care 10:136-141.
22. American Heart Association (AHA) (2008) Fish and Omega-3 Fatty Acids. www.americanheart.org
23. Simopoulos A.P. (1999) Evolutionary aspects fatty acids in the food supply of omega-3. Prostalandins, Leukotrienes and Essential Fatty Acids 60:421-429.
24. FAO (2007) The State of World Fisheries and Aquaculture 2006 (SOFIA). FAO Fisheries and Aquaculture Department.
25. Burger, Joanne (2005) Mercury in Commercial Fish: Optimizing Individual Choices to Reduce Risk. Environmental Health Perspectives 113:1-6.
26. Jones, J.G. (2007) Pollution from Fish Farms. Water and Environment Journal. 4:14-18.
27. Berglund, D.R. et al. (2007) Flax Production in North Dakota. North Dakota State University. A-1038.
28. Bean, L.D. et al.(2003) Long-Term Effects of Feeding Flaxseed on Performance and Egg Fatty Acid Composition of Brown and White Hens. Poultry Science 82:388–394.
29. United Egg Producers (2008) United Egg Producers Animal Husbandry Guidelines for U.S. Egg Laying Flocks.
30. EPA. Ag 101: Poultry Production. www.epa.gov
31. Webster, T.M. (2006) Weed Survey-Southern States: Vegetable, Fruit and Nut Crops Subsection. Proceedings, Southern Weed Science Society. 59:260-276.
32. Southern Weed Society. Common Purslane-Portulaca oleracea L.
33. Virginia Tech Weed I.D. Guide. Common Purslane: Portulaca oleracea. www.ppws.vt.edu/scott/weed_id/porol.htm
34. Koch, K. et al. (1980) Characteristics of Crassulacean Acid Metabolism in the Succulent C4 Dicot, Portulaca oleracea L. Plant Physiology 65:193-197.
35. Mazen A.M.A. (2006) Changes in levels of phosphoenolpyruvate carboxylase with induction of Crassulacean acid metabolism (CAM)-like behavior in the C4 plant Portulaca oleracea.

Project Objectives:

1. Determine the variation in ALA omega-3 fatty acids in Purslane acquired from different sources and how best to conserve ALA in processing.

2. Determine alterations in ALA content in Purslane plants subjected to environmental stress.

3. Evaluate the potential for Purslane inclusion into the poultry diet and the effects of Purslane on
DHA omega-3 levels in eggs.

4. Examine the effects of Purslane inclusion in forage rotation on egg production and DHA
accumulation in eggs.

5. Determine the economic feasibility of the proposed production system.

Cooperators

Click linked name(s) to expand
  • Thomas Rufty

Research

Materials and methods:

Genetic Variation

Purslane seeds were collected from governmental organizations, commercial companies, and North Carolina fields. Experiments were conducted in NCSU Method Road Greenhouses. Pots were filled with 1:1 field soil:metro-mix and well fertilized. A complete randomized block design was used to minimize statistical effects of light and temperature gradients. Purslane seeds were planted and thinned to one per pot. Plants were watered twice weekly. Plants were harvested at 30 and 60 days.

At harvest, numbers of mature and immature seedpods were quantified. Plants were shaken in sieves to remove mature seeds. Seeds were weighed, and sub-sampled for analysis. Remaining vegetative material was dried then weighed. Redstem purslane seeds were similarly grown and measured for comparison.

Samples were then processed and analyzed using gas chromatography. Standards of known concentration were run simultaneously. The resulting chromatograph was used to determine the total ALA (18:3 n-3) concentrations in the various tissues.

Drought Stress

Periodic droughts are another major limitation for crop yields in the Southeast. Experiments examined drought tolerance of different purslane lines and the impact of drought on ALA levels.

The study used several, high performing purslane lines. Plants were bagged to prevent evaporative or drainage losses. The amounts of water used was determined by weighing pots. Control plants were weighed daily and the amount of water lost replaced.

Nitrogen

Low nitrogen is the most common limitation for plant growth in the Southeast. Experiments examined the impact of differing N fertility on growth and reproductive output. A similar protocol to phenotypic tests was used, with the exception that granular fertilizer was added at low, medium, and high rates to establish differing growth and developmental rates. Detailed record keeping determined nutrient affects on seed production. Mature seed pods were counted and harvested at regular intervals. Analysis of seeds will determine the extent that N fertility altered ALA content.

Poultry System

The purslane/poultry system is primarily intended for small-diversified farm operations. This includes both established and new farms, and is designed to avoid large start-up costs or tracts of land. It can be scaled up or down as desired and add to farm profitability at any levels of scale. The option also exists of managing the system organically.

The primary source of calories and nutrients for hens should be a balanced corn-soy diet, plus an omega-3 supplement of course! The layer pellet feed was purchased commercially. Fresh potable water was always available.

A model system was developed on the small farm of our collaborator in Johnston County, N.C. The structural apparatus, 60 hens, and the initial purslane plots, were put in place in summer of 2008.
Four movable, four-season convertible coops were designed and built. The design was based on a collaboration between Beth Haarer and Andrew Becker of Wild Onion Farm and Laura Vance. Design ideas were also taken from ATTRA bulletins.

The coops are 5 x 8 feet, made of inexpensive, light-weight, durable materials and are easy to build. The coops can house up to 20 hens. Because chickens produce large amounts of excrement while roosting, the floor was left open to allow waste to fall directly onto the ground, making clean-up unnecessary. One person can easily move the coop around purslane plots, so waste can be systematically distributed around the field. Nest boxes made of pine and metal mesh were placed inside the coops. They have three compartments large enough to fit two birds each and are filled with straw. Hardware cloth bottoms provide some cushion and allowed broken eggs to drop through. Eggs can easily be collected through a slit in the back.

Commercial movable fencing was utilized for ease of rotations among established purslane plots. An electrifiable net fence was constructed to help prevent predation.

In the summer, the hens spent the majority of the day in the shade, so movable 6 x 8 ft shade structures also were assembled, with food and water dispensers located there. Localized waste generation again was an issue, so the lightweight shade structures could be moved within and among purslane plots.

Research results and discussion:

Genetic Variation
This study will examine natural phenotypic variation in growth and ALA content of purslane collected from a wide range of locations, and then will select seeds for further experiments in controlled conditions and the field. Sixteen biotypes were examined.

Figure 1 shows a great amount of diversity in early production of seed pods among seed sources. Numbers in later harvests increased and were more similar. This suggests that selection for early and late seed producing varieties may be an option,

Fourteen commercial Portulaca oleracea and two locally collected Portulaca rubricaulis seed sources or ‘varieties’ were grown, harvested, and analyzed. Fatty acid contents, measured using gas chromatography, showed 26-29% of the fatty acids in Purslane seeds were ALA, compared to 6-8% of the fatty acids in soybean seed checks. This is a much higher ALA percentage than all commercial oils, and it approaches levels in flaxseed. Purslane leaves contained 60-67% ALA as a percentage of total fatty acids, compared to 52% in soybean leaves. As a proportion of dry weight, one ‘variety’ had over twice the amount of ALA in its leaves as soybean and considerable phenotypic variation was seen among seed sources, suggesting good potential for selection of genetically superior lines.

The ratio of omega-3s to omega-6s in seeds was around 0.6 but had some variation. This ratio is rare, and has significant positive health implications.

Drought Tolerance

In figure 4, we see relative transpiration as a function of the remaining fraction of transpirable soil water for two representative purslane phenotypes. Phenotype A was bushy and produces seeds early, while phenotype B is taller with longer branches and produces seeds later. Transpiration, relative to a well watered control, decreased sooner than we see in most crop and weed species. This suggests a possible mechanism for purslane’s drought tolerance: early stomotal closure. Quick response to decreasing water status would allow purslane to conserve more water in the root zone.

Additionally, we withheld water long after FTSW got to zero. Plants dropped all of their leaves but stems remained erect. Then, when we resupplied water, new leaves began to emerge and the plants recovered. We intend to follow up on this in a controlled expriment.

Nitrogen Effects

Nitrogen availability had a significant effect on the number of seedpods produced in two representative phenotypes of purslane. The plants with highest fertilization rates produced between 6 and 7 times more seedpods than the plants at the lowest rate. Clearly proper fertilization would boost yields significantly. There does appear to be a point where additional fertilizer provides no additional increase in yield. The two genotypes had similar relative responses to nitrogen fertilizer, but the magnitude of total production was different.

Poultry Incorporation

In the model system, the hens ate purslane voraciously from established 50 x 50 ft plots during August and September of 2008, and they preferred it to native weeds present in the area. Hens also quickly ate purslane seeds supplied in a feed mix. Clearly, purslane was a preferred feed source. Quantitative data are being collected in spring and summer of 2009 on newly established plots.

The model system is functioning well up to this time. No problems have been observed with structural aspects, although extra anchors were required in August for storms with exceptionally high winds. The hens did not lay eggs during the short adjustment period, so it was not possible to determine if the purslane had an impact on the omega-3 content of their eggs. The main operational questions are ‘how much acreage is needed to support a particular sized hen population?’, and ‘how much can the purslane plots be degraded by hen feeding and still regenerate without replanting?’. Those questions are being addressed at the same site and at others in 2009.

Currently, we are working 52 laying hens divided into four groups at Wild Onion Farms, a small organic farm in Johnston County, NC. Hens were purchased at 4 weeks age from Sumner-Byrd Farm in Holly Springs, NC, where they were then raised to 16 weeks. Hens began laying eggs in December of 2008. When egg production became consistent in February of 2009, egg collection for fatty acid sampling began. Hens are fed Nutrena Laying pellets ad libitum, oyster shell supplements, and small amounts of Nutrena Scratch Grains daily. Between 2/15-3/16, hens were raised outdoors on bare ground to obtain baseline fatty acid numbers without plant tissue. Then, on 3/17, hens were moved to new paddocks planted in rye grass and crimson clover. Grazing was allowed, along with continued provision of previous diet. Eggs produced in each rep were collected and counted daily, and total egg mass and yolk mass measured weekly to show how quickly omega-3 levels increase on a typical cover-crop pasture grazing and how levels are maintained. Then and currently, using a balanced latin-square design, 5 week old purslane plants are being provided in different amounts to three of the four groups. Every four weeks, the supplement level is switched. The latin square design will allow to separate out effects of prior supplementation level, group, and time period. Egg yolks have been sampled and frozen, and GC analyses will begin in early May due to queue for analytical equipment. Supplemental results will be submitted to SARE once statistical analyses can be conducted.

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

We expect that two research publications will be generated from the results of the study. The first will require replication of initial phenotypic and environmental effects on ALA content in Purslane. The second will be based on Purslane incorporation into laying-hen diets.

Project Outcomes

Project outcomes:

Can’t say until we get final results.

Economic Analysis

It is premature for any economic analysis.

Farmer Adoption

Currently, Wild Onion Farm is using this technology. Depending on the results of our egg analyses, we will dissiminate our results to CEFS Seasons of Sustainability, Organic Grower School, Southern SAWG (Sustainable Agriculture Working Group), Carolina Farm Steward Association, and to post project information on the internet.

Recommendations:

Areas needing additional study

Much work is still needed, including expanded experiments testing temperature and daylength effects on Purslane growth and ALA production. Field studies including planting date and planting density are also important. Additionally, controlled studies measuring the relative effects of Purslane seeds and leaves on DHA in eggs would be informative.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.