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INVESTIGATOR: Ezekwe, M. O.; Liao, S. F.; Walker, C. R.

PERFORMING INSTITUTION:
ALCORN STATE UNIVERSITY
ALCORN STATE, MISSISSIPPI 39096

DEVELOPMENT AND EVALUATION OF PURSLANE AS FUNCTIONAL FEED FOR PRODUCTION OF OMEGA-3 HEALTHY PORK

NON-TECHNICAL SUMMARY: The prevalence of obesity worldwide is a big contributing factor to the high mortality of patients, especially, of cardiovascular diseases. Consumers today are more intelligently seeking alternative nutrient supplies to balance diets for their health and fitness. Although commonly known as a weed in the US, purslane is eaten as a vegetable in many other countries. Preliminary animal and human studies have shown that purslane beneficially improved blood lipid profile and body fat compositions, which can be attributed to its highest omega-3 fatty acid content amongst leafy vegetables. Increasing omega-3 fatty acid, while reducing cholesterol, levels in pork will provide nutritional benefits to humans. This project will be conducted to reach four objectives: (1) A dose-response study with experimental diets containing 0, 4, 8, and 16% purslane meal will be conducted with 144 finishing pigs to determine the optimal level of purslane inclusion to reduce pig blood cholesterol and triglyceride levels while enriching omega-3 fatty acid content in pork. (2) These 144 finished pigs will be slaughtered at 100 kg body weight to examine carcass quality, meat/sensory quality, and fatty acid profiles of fat and lean tissues. (3) Study the effect of dietary purslane on the expression of five key genes/mRNAs involved in lipid metabolism. (4) Two graduate students will be recruited and trained for conducting this joint research project. The significant impact of this project is on its synergistic benefits to pork quality and human health while providing new opportunities for the socioeconomic wellbeing of agricultural communities.

OBJECTIVES: The overall objective of this project is to evaluate the use of an under-utilized crop, purslane, as a functional feed for production of value-added pork enriched with omega-3 fatty acids (omega-3 pork). At the same time, this project will also be conducted to enhance/build the research and education capacities in the field of Animal Nutrition and Production at ASU. Specific objectives include the following: (1) Determine the optimal level of purslane inclusion in swine diet required to reduce blood cholesterol and triglyceride levels in swine and to enrich omega-3 fatty acid content in pork. It is hypothesized that higher levels of purslane present in swine diets will elicit greater omega-3 fatty acid storage to a certain level, and this objective is to evaluate and determine the optimal level of purslane supplementation needed to maximize pork/meat omega-3 fatty acid content. (2) Determine the carcass and meat qualities of the pork from the market hogs fed purslane supplemented diets. We hypothesized that purslane fed to pigs will reduce the ratio of omega-6 to omega-3 fatty acids in pork, and also increase the leanness of carcass and the meat-eating quality as well. Production of omega-3 pork with the same or better sensory quality will definitely attract more health-conscious consumers. (3) Explore the molecular mechanisms by which purslane alters lipid metabolism in animal tissues. Substantial amount of omega-3 fatty acids and antioxidant vitamins justifies the studies to determine the effect of this vegetable on lipid metabolism in animal tissues. This specific objective is to study the effect of dietary purslane on the expression of key genes involved in lipid metabolism in liver, muscle, and adipose tissues. At least four genes (five mRNA species), peroxisome proliferator-activated receptor (PPAR) alpha (PPARalpha), PPARgamma, lipoprotein lipase (LPL), sterol-regulatory-element binding protein 2 (SREBP-2), and microsomal triglyceride transfer protein (MTP) will be studied. (4) Train graduate students to conduct comprehensive scientific project. At least two students with Animal or Meat Science undergraduate background will be recruited to join this project to pursue their MS degrees in Animal Science, Meat Science, or Molecular Animal Nutrition.

APPROACH: A typical basal diet will be formulated for finishing pigs (NRC, 1998), and the purslane meal will replace 0, 4, 8, 16% of the diet. 72 gilts and 72 barrows at 50 kg will be used in 2 gender blocks. Within each block pigs will be stratified by BW and randomly assigned to 4 treatments with 18 in each. Four experimental diets will be fed respectively to the 4 treatment groups housed in 12 pens with 6 pigs in each (3 pens/treatment). The feeding trial will last 6 weeks, during which BW and feed consumption will be measured. Blood samples will be taken weekly, and the serum and plasma will be obtained for lipid and metabolite analyses. All pigs will be probed at 90 kg to determine back fat thickness. Pigs will be slaughtered, and the tissues of back fat, loin and semitendinosus muscle, and three internal organs (liver, heart, and kidney) will be collected for cholesterol, total lipid, and omega-3 fatty acid analyses. Liver total, free, and esterified cholesterol will be measured. Carcass will be evaluated for weight, length, back fat thickness, dressing percentage, and loin eye area. Carcasses will be graded at the last rib 7 cm off the midline. Carcass lean yield will be estimated. Measures of meat quality will include loin muscle color, pH, lipid content, marbling score, firmness score, water holding capacity, and drip loss. Measures of loin muscle eating quality will include cooking loss and moisture content of cooked chops. The fatty acid compositions of the fat and lean tissues will be analyzed. Dissections of carcasses will be conducted 24 hr postmortem. Fat percentage will be calculated. Two 1.5-cm thick loin chops will be cut at the last rib, and the pH, color, and drip loss will be determined. Initial pH will be measured immediately after muscle is removed, and ultimate pH will be measured after 24 hr drip loss determination. Following a 10-min bloom period, L*, a*, and b* values will be measured. Sensory panelists will be recruited to evaluate meat eating quality. Meat samples will be cooked and cut to uniform cubes. Two 0.5-inch cubes will be given to each panelist for evaluation involving 3-point scales for juiciness, tenderness, flavor intensity, pork flavor, and overall mouth feel. The scores will range from 3 (extremely juicy, tender, intense, and characteristic mouth feel) to 1 (extremely dry, tough, bland, unsavory, and uncharacteristic mouth feel). For gene expression study, total RNA will be extracted from 200 mg frozen muscle, adipose and liver tissues. The RNA should be of high purity with 260/280 ratios > 1.80 and 260/230 ratios > 1.90. The RNA integrity numbers should be > 7.5 and 28S/18S rRNA ratios > 1.80. The RNA samples will be treated with DNase-I and reverse transcribed to cDNA. The sequences of 5 mRNA species (PPAR-alpha, PPAR-gamma, LPL, SREBP-2, and MTP) and 2 housekeeping genes (18S rRNA and beta-actin) have been retrieved from GenBank. Primer and probe sets will be designed and manufactured (ABI Assays-by-Design Service). All the sets will be designed to produce amplicons that will bridge exon-exon junctions. PCR operation will follow standard protocols (Liao et al., 2009, 2010).

PROGRESS: 2012/09 TO 2017/08
Target Audience:Agriculture faculty, researchers, scientists, graduate students, undergratuate students swine producers and feed manufacturers Changes/Problems:During the implementationof the project, it was realized that freeze-drying the vegetable biomass using laboratory freeze-dryer could not provide rapid drying for the vegetable purslane. We changed to an air-drying method which involved indoordrying with the supportof humidifiers. Chemical analysis of the composition of purslane dried by both methods was identical. Other problems encountered include environmental and pest problems the delayedexpected biomass production for the experiment. The spring and summer months in Mississippi are too wet or too dry, thus hampering the production of purslane. Even when the plantswere growingwell, the plots were infested with armyworms(Spodoptera frugiperda) which destroy the vegetable instantly. The delay in generating adequate purslane biomass contributed tochanges in the number and level of purslane utilized for the trials. This may have been the primary limitationsof the project findings. What opportunities for training and professional development has the project provided?The projectprovided opportunities for training of a graduate student and other undergraduate students in new biology techniques and understanding of biotechnologyapplications in nutrition research.It also afforded students opportunities to attend and present scientific findingsat professional meetings, training in data collection and analyses., and interpretation of research results. How have the results been disseminated to communities of interest?The results have been presented at various conferences both at domestic meetings and international conference. Results were also presentedat agriculturalfield days.students seminars among scientists,faculty, and students.A manuscript is under preparation. What do you plan to do during the next reporting period to accomplish the goals?The lipid metabolic gene expression studies will be completed andsummarized.

IMPACT: 2012/09 TO 2017/08
What was accomplished under these goals? Feeding trials utilizing growing pigs fed air-dried purslane vegetable diet was completed during spring /summer of 2017. The three levels of purslane supplementation,0, 3,6%, demonstratedthat highest level of purslane (6%) electedhigher blood concentration of HDL- cholesterol while reducing LDL-cholesterol. in pigs. There were no significant differences between control animals and 3% group. No differences wereobservedin omega-3 fatty acidsstorage in pork. Carcass and meat quality traits in pork were notsignificantly affected bytreatments, however, belly weight ofhigher purslane group was significantlyhigher (P<0.05). The effect of dietarypurslane on lipid metabolic gene expression is being analyzed. A graduatestudent working on this molecularaspect of the project and will generate masters thesis. Other funds from our programare being used to accomplishthis objective.

PUBLICATIONS (not previously reported): 2012/09 TO 2017/08
Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: Effect of a sustainable(pasture pork)pork production system on the expression of genes involved in lipid metabolism. MS Thesis , Alcorn State university

PROGRESS: 2015/09/01 TO 2016/08/31
Target Audience:Agriculture faculty, graduate students and undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provieded opportunities for graduate and undergraduate student trainings. Undergraduate students employed on the project have recived hands-on traing in planting, harvesting and processing ofpurslane vegetable as feedingredients for porcine animals. Presentations at Agriculture field days have provided also training opportunities for swine and crop farmers in the Mississippi counties. How have the results been disseminated to communities of interest?The findinds from studies so far completed have been presented at scientifc meetings for research scientists, agriculture faculty and students. Demontrations done at field days and workshops have provided producers/farm families with methods for adoption purslane cultivation. What do you plan to do during the next reporting period to accomplish the goals?The objectives to be completed during the next reporting period: 1) Determine the optimal level of purslane inclusion in swine dietrequired to reduce blood cholesterol and triglyceride levels to enrich omega-3 fatty acid content of pork. 2) Determine the carcass and meat qualities of pork from market hogs fed purslane supplemental diet. 3) Explore the molicular mechanisms by which purslane alters lipid metabolism in animal tissues.

IMPACT: 2015/09/01 TO 2016/08/31
What was accomplished under these goals? The accumulation of biomass suffient to provde supplemental feed ingredient tp experimental animals is continuig. We expect to conduct feeding trials this coming fall.Preliminary work conducted to develop and molicular biology techniques to determine molicular mechanisims influencing lipid metabolism has been achieved.One graduate masters students has completed his thesis successfully. We are looking forward to carrying out the remaing objectives of the project this fall.

PUBLICATIONS: 2015/09/01 TO 2016/08/31
1. Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: Effect of a sustainable ( pasture-based )pork production system on the expression of gene involved in lipid metabolism.Alcorn State university
2. Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: Effects of sustainable(pasture-based) pork production system on the expression of genes involved in lipid metabolism. Masters Thesis, Alcorn State University

PROGRESS: 2014/09/01 TO 2015/08/31
Target Audience:Farmers, swine producers, students , and scientists/faculty Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided opportunity for graduate and undergraduate training and faculty development. How have the results been disseminated to communities of interest?Results have been presented at scientific meetings and at Alcorn Faculty Seminar Series. What do you plan to do during the next reporting period to accomplish the goals?Determine the optimal level of purslane inclusion in swine diet required to reduce blood cholesterol and triglyerides in swine and enrich omega-3 fatty acid content of pork. Determine the carcass and meat qualities of pork from market hogs fed purslane.

IMPACT: 2014/09/01 TO 2015/08/31
What was accomplished under these goals? Preliminary effort to train a graduate student to standardize and establish molicular biology methodology for evaluating experimental animals have been accomplished. A masters thesis generated by the graduate student is under committee review.

PUBLICATIONS: 2014/09/01 TO 2015/08/31
Type: Theses/Dissertations Status: Under Review Year Published: 2015 Citation: Effect of a sustainable (Pasture Produced) pork production on the expression of genes involved in lipid metabolism.

PROGRESS: 2013/09/01 TO 2014/08/31
Target Audience: Target audiences reached during this reporting period are undergraduate students, graduate students and swine producers who visit our research facility at the annual field days. Graduate and undergraduate students participated in growing, harvesting and processing purslane plats for feed formulation. Reserch scientist s in plant soil sciences also were consulted and assisted in purslane planting and production.Producers had opportunity to view purslane vegetable. Changes/Problems: We have experienced some botltlenect in processing harvested vegetable. It became obvious that freezing drying with any laboratory freeze dryer was not fast enough to handle the quantity being produced. We have tryed air drying and it has prvided us with faster products than freeze drying Furthermore we compered the quality of the products by analyizing the composition of alpha linolenic acid content. The result showed that omega-3 fatty acid content of the product was not compromized by our methods. We plan to utilize air-drying in indoors in homidified rooms. What opportunities for training and professional development has the project provided? One graduate student presentation at a scientific conference has bee acommplished, and other meetings are being planned. How have the results been disseminated to communities of interest? The results of the preliminary morlicular biology studies on muscle and adopse tissuies in porcine animals have bee presented at a seminar on campus to students and scientists audience. What do you plan to do during the next reporting period to accomplish the goals? Grow purslane vegetable at lrger sites than previusly done in order to produce enough biomass.Formulation of diets for experimental trials. Conducting the experiments to accomplish the objectives of the study.

IMPACT: 2013/09/01 TO 2014/08/31
What was accomplished under these goals? The production of purslane vegetable A is continuing. We have been able to grow, proceee purslane biomass seasonally for two consective seasons.However we are still short of the dry biomass needed toformulate the ration to conduct the experiment.Over 3500 kg of dried biomass si required for diet formulation. A graduate student training in molicular biology analyses methods has been successful . We have identified (PPAR)alpha, (PPAR)gamma, SRBP-2, and LPL in Muscle and adepose tissue of pigs subjected to differential raising experiences. TheqPCR analysis of muscle and adepose tissues showed that these genes were tissue specific in the response to exercise imposed by graizing. These preliminary results indicate the morlicular techniquics developed are precise and working. A masters thesis is being completed by a graduate student.

PUBLICATIONS: 2013/09/01 TO 2014/08/31
Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Effect of purslne on the expression of gene involved in lipid metabolism in pork. Okoli, Nnamdi B; Y, Meng and M.Ezekwe. Professional Agricultural Conference, Tuskegee University, Alabama, December 8-10, 2013

PROGRESS: 2012/09/01 TO 2013/08/31
Target Audience: Target audience reached at this period include undergraduate students, graduate students limited resource audiences that visted at our farm during annual field days.Graduate and undergraduate students were engaed in assisting in purslane production and processing for feed formulation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student is being trained in biotecnology to produce MS thesis in molicular and gege responses to purslane supplemented feed. Animal biotecnology training has been introduced at the center in ASU research laboratories. Students are being trained to present research work at conferences and field days How have the results been disseminated to communities of interest? Paper has been presented by a garduate student at a scientific conference. Producers were introduced to potential of purslane crop for animal/ human nutrition during Swine Field days. What do you plan to do during the next reporting period to accomplish the goals? Produce purslne biomass for experiment trials. Conduct animal trial. collect tissue sample for analysis. Conduct carcass Analysis and meat quality traits at Southern University collaborators

IMPACT: 2012/09/01 TO 2013/08/31
What was accomplished under these goals? The major activity during this reporting period is the production of purslane biomas for manufacture of feed for experimental animals. Planting and harvesting of purslne vegetable is contuing this planting season.However preliminary work on molicular responses to swine species sujected varying feed composition is in progerss to standardise and implement the molicular methodologies specified in the progect. The objective was to develop laboratory techniques for use in determing molicular mechanisms that affect lipid metabolism in pork tissue. Total RNA was extrcted from the frozen tissues and the consentrationand purity was annalyzed using a spectrophotometer and the intergrity of the samples were examined by gel electrophoresis. All the molicular analysis methods for swine tissue appear to have been standardized and working. Plans are in progress to conduct the project experiments when the sufficient feed materials are assembled.Graduate training in molicular biology techniques is in progress.

PUBLICATIONS: 2012/09/01 TO 2013/08/31
Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Effect of purslane on the expression of genes involved in lipid metabolism in pork. Okoli, Nnamdi B; Y. Meng and M. Ezekwe. Professional Agricultural Conference, Tuskegee University,Alabama, December 8-10, 2013