Food Sci Nutr. 2018;1–20.  | 1www.foodscience-nutrition.com

Received:31October2017  |  Revised:10January2018  |  Accepted:17January2018DOI: 10.1002/fsn3.610

O R I G I N A L R E S E A R C H

Enhancing the fatty acid profile of milk through forage- based rations, with nutrition modeling of diet outcomes

Charles M. Benbrook1,2 | Donald R. Davis3*  | Bradley J. Heins4 | Maged A. Latif5 |  Carlo Leifert6 | Logan Peterman5 | Gillian Butler7 | Ole Faergeman8 |  Silvia Abel-Caines5 | Marcin Baranski6

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2018TheAuthors.Food Science & NutritionpublishedbyWileyPeriodicals,Inc.

*Retired.

1BloombergSchoolofPublicHealth,JohnsHopkinsUniversity,Baltimore,MD,USA2BenbrookConsultingServices,Troy,OR,USA3BiochemicalInstitute,UniversityofTexasatAustin,Austin,TX,USA4WestCentralResearchandOutreachCenter,UniversityofMinnesota,Morris,MN,USA5OrganicValley/CROPPCooperative,Lafarge,WI,USA6CentreforOrganicsResearch,SouthernCrossUniversity,Lismore,NSW,Australia7SchoolofNaturalandEnvironmentalScience,NewcastleUniversity,NewcastleuponTyne,UK8DepartmentofCardiology,AarhusUniversityHospital,Aarhus,Denmark

CorrespondenceCharlesM.Benbrook,90063TroyRoad,Enterprise,OR97828.Email:charlesbenbrook@gmail.com

Funding informationSheepdroveTrust,UK;CROPPCooperative,Lafarge,Wisconsin;LowInputBreeds,SixthFrameworkProgrammeforResearch,TechnologicalDevelopmentandDemonstrationActivities,EuropeanCommunity,Grant/AwardNumber:Grantno. 222623

AbstractConsumerdemandformilkandmeatfromgrass-fedcattleisgrowing,drivenmostlybyperceivedhealthbenefitsandconcernsaboutanimalwelfare.InaU.S.-widestudyof1,163milksamplescollectedover3years,wequantifiedthefattyacidprofileinmilkfromcowsfedanearly100%forage-baseddiet(grassmilk)andcomparedittoprofilesfromasimilarnationwidestudyofmilkfromcowsunderconventionalandorganicmanagement.WealsoexploredhowmuchtheobserveddifferencesmighthelpreversethelargechangesinfattyacidintakesthathaveoccurredintheUnitedStatesoverthelastcentury.Keyfeaturesofthefattyacidprofileofmilkfatincludeitsomega-6/omega-3ratio(lowerisdesirable),andamountsoftotalomega-3,conju-gatedlinoleicacid,andlong-chainomega-3polyunsaturatedfattyacids.Foreach,wefindthatgrassmilkismarkedlydifferentthanbothorganicandconventionalmilk.Theomega-6/omega-3 ratioswere, respectively, 0.95, 2.28, and 5.77 in grassmilk, or-ganic, and conventional milk; total omega-3 levels were 0.049, 0.032, and0.020g/100g milk; total conjugated linoleic acid levels were 0.043, 0.023, and0.019g/100g milk; and eicosapentaenoic acid levels were 0.0036, 0.0033, and0.0025g/100gmilk.Becauseofoftenhighper-capitadairyconsumptionrelativetomostothersourcesofomega-3fattyacidsandconjugatedlinoleicacid,thesediffer-encesingrassmilkcanhelprestoreahistoricalbalanceoffattyacidsandpotentiallyreduce the riskof cardiovascular andothermetabolicdiseases.Althoughoily fishhavesuperiorconcentrationsoflong-chainomega-3fattyacids,mostfishhavelowlevelsofα-linolenicacid(themajoromega-3),andanomega-6/omega-3rationear7.Moreover,fishisnotconsumedregularly,oratall,by~70%oftheU.S.population.

K E Y W O R D S

dairyfarming,dairyfattyacids,grassmilk,GrassmilkTM,omega-6/omega-3ratio,organicmilk

2  |     BENBROOK Et al.

1  | INTRODUC TION

NearlyhalfofAmericanssufferfromoneormorediet-drivenchronicconditions includingcardiovasculardisease (CVD),overweightandobesity, and diabetes (DHHS, 2015; Massiera etal., 2010; FDA,2015;ARS,2010).Sevenof the10 leadingcausesofdeath in theUnitedStateswerediet-relatedin2013(IHME,2016a),andnonearecurableviamedicalinterventionalone,despitehealthcarespendingintheUnitedStatesthatisthehighestpercapitaintheworld(IHME,2016b).Thesesoberfactsareamongthereasonswhythereisgrow-inginterestintheUnitedStatesamongscientistsandconsumersinalteringdietstopreventorslowtheprogressionofmetabolic,car-diovascular,andotherchronicdiseases.

Potentialdietalterationsincludereducingintakesofomega-6(ω- 6)fattyacids(FAs)andincreasingintakesofomega-3(ω-3)FAs,thusdecreasingdietaryω- 6/ω-3ratios.Theseratioshavebecomehistori-callyhighinWesterndietsduringthelastcentury,reachingabout15,comparedtoestimatedevolutionaryratiosnear1(Hibbeln,Nieminen,Blasbalg, Riggs, & Lands, 2006; Simopoulos, 2006). These largechangesareduetobothincreasedintakesofω-6FAsanddecreasedintakesofω-3FAs.Moderngrainfeedingoffarmanimalshascontrib-uted to these ω-6increasesandω-3decreasesinWesterndiets.

ThemorenaturalFAprofileoforganicandgrass-fedmeatandmilkhas receivedmuchattention in recentyears (Średnicka-Toberetal.,2016a,b).TheFAprofileinmodernmeatandmilkcanbesub-stantially changed by shifting animals from grain- or concentrate-rich rations to diets largely based on grass and legume forages(Butleretal.,2011;Daley,Abbott,Doyle,Nader,&Larson,2010;O’Callaghanetal.,2016;Schwendeletal.,2015;Stergiadisetal.,2012).Thisshiftincreasesω-3FAsandconjugatedlinoleicacid(CLA)anddecreasesω-6FAsinmeatandmilk,changesthatmayhelppreventCVD and other chronic conditions (Leikin-Frenkel, 2016; Hibbelnetal.,2006;Simopoulos,2006).Themagnitudeofthesechangesismarkedlygreaterthanmostofthenutritionaldifferencesbetweenorganicallyandconventionallygrownplant-basedfoods(Benbrook,Butler,Latif,Leifert,&Davis,2013;Baranskietal.,2014;Średnicka-Toberetal.,2016a,b).

Thereisrisingdemandforbeefanddairyproductsfromgrass-fedcattle.In2016,natural-foodretailleaderWholeFoodsMarketiden-tifiedgrass-fedmeatanddairyasa top trend,basedonconsumerinterestandrapidsalesgrowth(PRNewswire,2016;WholeFoodsMarket Blog, 2015). Three-quarters of self-identified natural-foodandorganicconsumerspurchasegrass-fedbeefanddairy (MarketLohas,2016).Similar interests inhumanhealthandanimalwelfareled in 2015 to themarketing in Italy, and laterMexico, of “LatteNobile”(NobleMilk),producedbycowsfedprimarilygrassandhay(Rennaetal.,2015;Lombardietal.,2014;AssociazioneLatteNobileItaliano(http://www.lattenobile.it/).

1.1 | Launch of grassmilk brand

TheWisconsin-basedcooperativeCROPPistheleadingU.S.supplieroforganicmilk.In2011,CROPPlaunchedanew,wholemilk,organic

productcalledGrassmilk.TMThismilkcomesfromcowsfedanearly100%forage-baseddiet.Theonlyexceptioniscertainmineralanden-ergysupplements,suchasmolasses.Inthispaper,theterm“grassmilk”referstoCROPP’sproduct,and“grassmilk”referstootherbrandsofmilkfromcowsfedanearly100%forage-basedration.

Besidesprohibitinggrain incowrations,CROPPsetsgrassmilkstandardsforpastureaccess,supplemental feeds,andanimalcare(see Appendix for details). Farmers in the grassmilk program re-ceiveapricepremiumof~15%comparedtotheorganicmilkprice.CROPPcloselymonitorstheFAcontentofrawgrassmilk,toassurecompliancewithitsminimumrequirementsof(1)39to41mgtotalω-3FA/100gofmilk,dependingongeographicalregion,(2)26.6to32.8mgtotalCLA/100gofmilk,and(3)anω- 6/ω-3ratio≤1.2.

The number of farms shipping grassmilk to CROPP proces-sors has grown from five California producers in 2011 to 140farmsthroughouttheUnitedStatesattheendof2016.These140farms represented about 9% of CROPP’s 1,618 dairy-farm mem-bers(CROPP,2016).Milk,yogurt,andcheesemadefromgrassmilkaremarketedunderCROPP’sOrganicValleybrand. In addition tomeetingUSDA’sorganicgrazingstandard(Rinehart&Baier,2011),CROPP’sgrassmilksuppliersmaynotfeedgrainorsilagefromgraincropsharvestedfromfields thathavereachedthe“boot”stageofdevelopment(whenseedheadsformandstarttofillout).Nongrainsupplementsincludingmolasses,alfalfapellets,sugarbeets(chippedorwhole),mineral supplements, andkelpareallowed tomeet theenergyneedsoflactatingcowsandsupportanimalhealth.

Somesupplementalfeedisoftenneededtosustaincowhealthduring months of peak production, or when high-quality, foragefeeds are not available in sufficient quantity. Despite reliance onsomesupplementalfeeds,forage-basedfeedsmakeupthevastma-jorityofannualDryMatterIntake(DMI)ongrassmilkfarms.

Farmersinthegrassmilkprogramarealsorequiredtodocumentthatlactatingcowsconsumeover60%ofDMIfrompastureduringthe grazing season (compared to 30% under the USDA organicstandard),withagrazingseasonofat least150days(comparedto120daysunderfederalorganicrules).Thelengthofthegrazingsea-soncanbereduced incasesofextremedroughtorotherweatherevents or natural disasters, or by the tolerance of soils to animaltraffic.

Thenongrazingportionofrationsongrassmilkfarmsmustcomefrom conserved, organic, forage-based feeds, including dried orfermentedforages(alfalfa,clover,grasshay,etc.).Cerealcropshar-vestedpriortotheirbootstage,suchasbarley,oats,andBMRcorn(“brown mid-rib” phenotypes developed for early silage harvest),canalsobe fed, as theFAprofileof such immaturegrain crops issimilartowidelygrowngrassspeciesincowpastures(see“ResultsandDiscussion”formoredetail).Harvestedfeedstuffsaretypicallypreservedbyfermentationon-farmtoproducebaleageorsilage,orstoredasdryhay.

ThementionedNobleMilkprotocol requiresat least150daysperyearofgrazingand70%DMIfrompastureandhaythroughouttheyear,withupto30%DMIfromgrainsandconcentratesallowed.Silages,supplements,andgeneticallymodifiedfeedsareprohibited.

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Themilkfatmustcontainat least0.25%oftotalCLAand0.5%oftotalω-3FAs,andtheLA/ALAratiomustbelowerthan4yearround(Renna etal., 2015). Pastures must be diverse, with at least fourmajorplantspecies,andthequalityofhayismonitoredwithasen-soryanalysis(Rubino,2014).

Benbrooketal.(2013)reportedsubstantialimprovementsintheFAcompositionofOrganicValleywhole,organicmilk,comparedtowholemilkfromconventionallymanagedcows.Basedon12-monthaverages,theyfoundhigherlevelsofω-3FAsα-linolenicacid(ALA)andeicosapentaenoicacid (EPA)of, respectively,+60%and+33%.Therewasalso18%moretotalCLA,andlessoftheω-6FAslinoleicacid(LA)andarachidonicacid(AA)(25%and17%,respectively),re-sultingina60%lowerω- 6/ω-3ratio(5.77downto2.28).

As estimated below, most organic milk analyzed in Benbrooketal. (2013) came from cows receiving ~20% of their yearlyDMIfromgrain-basedfeeds.Onanannualbasis,theUSDAorganicstan-dard technically allows up to ~90%DMI from sources other thangrazing(asonly30%ofDMImustcomefromgrazingduringamini-mumof120daysperyear),althoughonmostorganicdairiesintheUnitedStates,forage-basedfeedsplayamuchgreaterrolethanisminimallyrequired(CROPP,2016;Rinehart&Baier,2011).

Here,wereporttheaddedimpactsonnutritionallyimportantFAlevelswhenlactatingcowsarefedanearly100%forage-baseddietyear round,andwemodel the impactof thesechangeson typicalU.S.diets.WealsocomparetheimpactsofgrassmilkdairyproductsandfishonFAintakes.

2  | METHODS

MilkFAanalysesreportedinthisstudycomefromCROPP’squality-controltestingofitsgrassmilk.Bulk-tank,rawmilksamplesfromeachparticipatingfarm(140in2016)werecollectedatleastbimonthlyinsterileplasticbottles,packedwithice-packs,andshippedovernightto Silliker, Inc., an ISO/IEC 17025 accredited laboratory in Crete,Illinois. It used AOACmethod 996.06, as revised in 2001 (AOACInternational, 2012), with modified internal standard (C13:0) andtemperatureprogram[initialT=100°(nohold),ramp2°/minto214°(hold 10min), ramp 3°/min to 240° (hold 16min)]. The laboratoryused capillary columnSupelcoSP-2560,100m×0.25mm,0.2μm film.Inunitsofg/100gofmilk,thelaboratorydidnotquantifyindi-vidualFAamounts<0.001,butitdidquantifythosesmallamounts(ifdetected) inunitsof%of totalFA, togivethebestmeasureoftotalFA.This laboratoryanditsmethodsandreportsareidenticaltothoseusedinBenbrooketal.(2013).However,inthispaper,wereportamountsofadditional,minorFAthatwerenotreportedinthe2013paper,duetotherelativelysmallnumberofsamplesin2013.

ThedetectedandsummedisomersofreportedtotalCLAincludecis-9, trans-11 (commonly 75–90% of the total); trans-9, trans- 11; cis-9,cis- 11; trans-10,cis-12;andcis-11,trans-1318:2.Thereportedtrans-18:1includesmainlytrans-1118:1,vaccenicacid.

ThisstudyincludesFAanalysesof1,163rawmilksamplescol-lectedmonthlyorbimonthlyduringthreefullyears,2014–2016.They

comeprimarily fromthree regionsof theUnitedStates—Midwest,Northeast,andCalifornia.AsmallgroupofsamplescamefromtheMiddle-EasternUnitedStatesbeginninginJune2016.Forcompari-sonwithrawgrassmilk,wealsoreportFAresultsfrom69samplesofprocessed,wholegrassmilk,takenfrompasteurized,retailcontain-ers (not homogenized). These sampleswere taken in a systematicmanner similar in locationand season to the rawmilk samples. In2014, therewere 22 samples—12 from theMidwest and 10 fromCalifornia;in2015,therewere23samples—fivefromtheMidwest,sixfromCalifornia,and12fromtheNortheast;andin2016,therewere24samples—sixfromtheMidwest,sixfromCalifornia,and12fromtheNortheast.

Asinthe2013study(Benbrooketal.,2013),wereportaveragesof three ω- 6/ω-3ratios:LA/ALA,ω- 6/ω-3,andω- 3/ω-6,whereω- 6 includessevenFAs(18:2LA+18:3γ-linolenic(GLA)+20:2eicosa-dienoic+20:38,11,14-eicosatrienoic+20:4arachidonic(AA)+22:2docosadienoic+22:4docosatetraenoic),andω-3includes7FAs(18:3ALA + 18:4 stearidonic/moroctic + 20:3 11,14,17-eicosatrienoic +20:5EPA+22:3docosatrienoic+22:5DPA+22:6DHA).Weincludetheaverage ratio,ω- 3/ω-6, for comparisonwithotherpapers thatreportthisinvertedratio.

2.1 | Statistical analysis

DigitallaboratoryresultsweretransferredtoanExcelspreadsheetandspot-verifiedagainstprintedlaboratoryreports.(Therawdataare available fromMALorDRD.) The reportedFA concentrations(g/100gofmilk)in1,163rawmilksampleswereinspectedforoutli-ersbynormalprobabilityandboxplots,andfivesevere,highoutlierswereexcluded,mostlybyconsensusamongDRD,MB,BH,andCMB(18:3γ-linolenic=0.028,trans-18:3=0.075,20:1=0.070,11,14,17–20:3=0.040,and22:6DHA=0.025,alling/100gofmilk).WealsoremovedthecorrespondingvaluesexpressedasapercentoftotalFAs.Anadditionalfouroutlierswerefoundandremovedonlyinthevalues expressed as a percent of total FA: sumof FA=102.998%(high), sum of saturated FA=87.690% (high), 18:1=0.330% (low),andsumofcis-monounsaturated=4.89%(low).Theremovedvaluesrepresentineachcaseonly1in1,163samples(<0.1%).Nooutlierswerefoundinthe69samplesofretailgrassmilk.

Means, counts, standard deviations (SDs), coefficients of vari-ation (CVs),andstandarderrors (SEs)werecalculated inMicrosoftExcel.We reportSDs, CVs, andSEswith 1 or 2 significant digits.BecausethestatisticaluncertaintyofameanismeasuredbyitsSE,wereportmeanstothesamenumberofdecimalplacesastheSEs. Withsamplecountsashighas1,163,SEsandthestatisticaluncer-taintyofmeanscanbemuchsmallerthanthelaboratoryprecisionforindividualmeasurements.

Analysesofannual,monthly,andregionalvariationinFAconcen-trationsusedPROCMIXEDofSAS(SASInstitute,2014).Thefixedeffectswereyearofstudy,regionoftheUnitedStates (California,Midwest,Mideast,andNortheast),andmonthofsampling,withfarmasarandomeffectforrepeatedmeasures.Thecompoundsymme-trycovariancestructurewasused,becauseitresultedinthelowest

4  |     BENBROOK Et al.

Akaike informationcriterion for repeatedmeasures (Littell,Henry,&Ammerman,1998).WereliedontheSatterthwaitecorrectiontoadjustthedegreesoffreedomforunequalvariances.AlltreatmentresultsarereportedasleastsquaresmeansseparatedbytheTukeyprocedurewithsignificancedeclaredatp < .05.

2.2 | Diet scenarios and LA/ALA ratios

WemodeledhypotheticaldietscenariosbasedonthoseinBenbrooketal.(2013)totestthepotentialeffectsofswitchingwhole-fatdairyproductsmadefromconventionalmilk,toorganicmilk,and,finallyinthisstudy,tograssmilk.Forthesediets,wecalculatedoveralldietaryintakesofLAandALA, and theLA/ALA ratio, themajordetermi-nantoftheω- 6/ω-3ratio.Wecouldnotcalculatetheω- 6/ω-3ratioitself, because there is insufficient data on totalω-6 andω-3 FAsinmostof the foods inour scenarios.However, theω- 6/ω-3 ratiocloselytrackstheLA/ALAratio,andbothratiosarehistoricallyhighinmostWesterndiets,duetoincreasedω-6intakesanddecreasedω-3intakes(Hibbelnetal.,2006;Simopoulos,2006).

Here,weuse the samemodeldietsandassumptionsas in thepreviousreport(Table1inBenbrooketal.,2013),andaddnewdietscenariosusingdairyproductswiththeFAprofileofgrassmilk.AsinBenbrooketal.(2013),weusefull-fatdairyproducts(exceptforyogurt),toquantifythemaximum,realisticallyattainableshiftinFAintakes from a switch to grass-milk-based dairy products. For yo-gurt,weusethehighest-fatformgenerallyconsumedintheUnitedStates,sweetened“low-fat”yogurtwithfruit,containing1.41gfatper100g.Sweetenedwhole-fatyogurtisnotusuallyavailable.

Wemodeleddiets foramoderatelyactivewoman,age19–30,consuming 2,100kcal/day. In threemain scenarios, 20%, 33%, or45%ofthatenergycamefromfat.Withinthosescenarios,wecon-structeddietsthatcontaineithermoderateamountsofdairyprod-ucts(threedailyservings,asrecommendedintheDietary Guidelines for Americans(DHHS,2015),or50%higheramounts(4.5servings/day).Whole milk, Cheddar cheese, low-fat yogurt, and ice creamas a “dairy dessert” were the dairy products included (Table 1 inBenbrooketal.,2013).

For the LA andALA contents of dairy fat, the previous studyuseditsmeasured12-monthaverageconcentrationsinconventionaland organicmilks (Benbrook etal., 2013). For the LA andALA innondairy foods, theauthorsusedUSDA’s standard referencedatafor 8 common foods to represent “typical-LA nondairy sources”(USDA,2015).Thosefoodsaveraged23.23gLAand1.841gALAper 100kcal of fat, for an LA/ALA ratio of 12.6. To illustrate theeffects of reducing LA intake, they substituted three of the eightfoodswithsimilar,low-LAfoodsandingredients(e.g.,canolaoilin-steadof soyoil, themajor oil used inmany foods). These revisedeight “low-LAnondairy sources”averaged13.84gLAand2.731gALAper100kcaloffat,withanLA/ALAratioof5.07.

With these assumptions, the2013 authors calculated the LA/ALAratiosfor12dietswithtypical-LAnondairysources(3fatlevels×2 levelsofdairyconsumption×2 typesofdairy fat) and foranadditional12dietswithlow-LAnondairysources.Here,weaddto

thesecalculationsa3rdtypeofdairyfatwiththeaverageFAprofilefoundherein1,163grassmilksamples.

3  | RESULTS AND DISCUSSION

Wesetouttoanswertwokeyquestions.First,towhatextentdoesshifting lactating dairy cattle to nearly 100% forage-based feedsalter the FA profile of their milk compared to currently availableconventionalandorganicmilksintheUnitedStates?Thefollowingsubsectionpresentsresultsfrom3yearsofnationwidesampling,in-cludingseasonalandregionalvariations.

OursecondcorequestionishowmuchcanimprovementsintheFAprofileofgrassmilkhelpreversehistoricallyhighdietaryω- 6/ω- 3 ratios?Weaddressthisquestionwithnutritionmodelingresultsinthethirdsubsectionbelow.

3.1 | Altering the fatty acid profile of milk

Table1showsconcentrationsof37mainFAs (quantifiedamounts>0.001g/100gmilk)inraw,wholegrassmilk,averagedover3years(2014–2016), reported as g/100g ofmilk and as a percentage oftotalFAs.ForeachFA,thereare1,163values, lessthenonquanti-fied samples and any outliers removed (as explained inMethods).SeeTable1footnotesaandbfordetails.Thecoefficientsofvariation(CV=SD/mean)areameasureofvariabilityamongsamples.

TableS1showsthesameinformationfor14minorFAsingrass-milk. Table2 shows the same information as Table1 for 69 retailsamples of grassmilk taken during 2014–2016. The FA profiles ofthese samples of processed, whole-fat grassmilk were measuredto determine whether there were any significant changes in theFAprofileofgrassmilkasaresultofprocessingandpasteurization.ForFAconcentrationsexpressedasapercentageoftotalFAs,theamountsinTables1and2areverysimilar,asexpected:ThemeansinTable2average101±SD5%ofthemeansinTable1(for33FAswith n>50%oftheanalyzedsamples).However,forFAconcentra-tionsexpresseding/100gmilk,themeansinTable2averageonly75±SD4%ofthemeansinTable1(for38FAswithn>50%oftheanalyzedsamples).Thesevaluesare<100%,mainlybecause fat isremovedfromrawmilktoproduceretailwholemilkwithastandard-ized3.25%fatcontent.

Table3comparesselectedFAlevelsandratiosinorganicgrass-milk to those in retail conventional milk and organic milk fromBenbrooketal.,2013.WeincorporatedinTable3resultsfromthe1,163samplesofrawgrassmilk(Table1)ratherthantheresultsfrom69samplesofprocessedgrassmilk(Table2).Wedidsobecausethe1,163samplesofgrassmilkprovideamoreaccurate,year-roundFAprofile of grassmilk than the69 retail samples. The retail conven-tionalandorganicmilk samplesaverage~3.1%totalFAs,whereastheraw,grassmilksamplesaverage~3.6%FAs.Duringtheprocess-ingofrawgrassmilk,~0.5%offatisremovedtomeetthestandardofidentityforfatinwholemilk.Accordingly,inTable3,weadjustedtherawgrassmilkFAamountstoequaltheaveragetotalFAcontent

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TABLE  1 Fattyacidsinraw,wholegrassmilk,36-monthaverage,2014–2016(1,163samples)

g/100 g milka Percent of total fatty acidsb

Mean n SD CV (%) SE Mean n SD CV (%) SE

Fatmeasuredatfarm 4.237 1162 0.49 12 0.014

Totaltriglyceride(calculated)

3.881 1161 0.53 14 0.016

Totalfattyacids 3.585 1161 0.48 13 0.014 99.996 1160 0.15 0.1 0.004

Saturatedfattyacids

4:0butyric 0.09202 1163 0.015 17 0.00045 2.572 1163 0.31 12 0.009

6:0caproic 0.06967 1158 0.013 18 0.00037 1.940 1158 0.21 11 0.006

8:0caprylic 0.04088 1160 0.009 21 0.00025 1.136 1160 0.16 14 0.005

10:0capric 0.0963 1163 0.022 23 0.0007 2.671 1163 0.42 16 0.012

12:0lauric 0.1106 1162 0.027 24 0.0008 3.066 1162 0.50 16 0.015

14:0myristic 0.3997 1160 0.07 18 0.0021 11.114 1160 1.1 10 0.032

15:0pentadecanoic 0.05619 1163 0.012 21 0.00034 1.567 1163 0.24 16 0.007

16:0palmitic 1.116 1162 0.24 21 0.007 31.00 1162 4.3 14 0.13

17:0margaric 0.0315 1163 0.007 21 0.00019 0.878 1163 0.13 15 0.004

18:0stearic 0.3738 1163 0.08 22 0.0024 10.455 1163 2.0 19 0.057

20:0arachidic 0.006756 1149 0.0016 24 0.000047 0.1878 1149 0.035 19 0.0010

22:0 behenic 0.004552 1155 0.0016 34 0.000046 0.1269 1155 0.040 32 0.0012

24:0lignoceric 0.002443 1153 0.0007 28 0.000020 0.0682 1153 0.015 22 0.0004

Totalsaturatedc 2.399 1163 0.39 16 0.011 66.71 1162 4.2 6 0.12

Monounsaturatedfattyacids

14:1myristoleic 0.03404 1160 0.0095 28 0.00028 0.944 1160 0.20 22 0.006

16:1palmitoleic 0.05643 1159 0.014 26 0.00043 1.571 1159 0.33 21 0.010

17:1margaroleic 0.01052 1157 0.0029 28 0.00009 0.295 1157 0.08 26 0.002

18:1includingoleic 0.7258 1163 0.12 16 0.0034 20.36 1162 2.8 14 0.08

20:1includinggadoleic 0.00724 1161 0.0020 28 0.00006 0.2024 1161 0.05 25 0.0015

Totalcis-monounsaturatedc

0.8352 1163 0.12 15 0.0036 23.41 1162 2.6 11 0.08

ω-3fattyacids

18:3 α-linolenic,ALA 0.04409 1163 0.011 25 0.00032 1.229 1163 0.26 21 0.008

18:4stearidonic/moroctic 0.002636 841 0.0010 37 0.000034 0.0729 844 0.025 35 0.0009

20:3 11,14,17-eicosatrienoic

0.001139 736 0.00036 32 0.000013 0.0306 747 0.009 31 0.0003

20:5eicosapentaenoic,EPA

0.004132 1157 0.0010 23 0.000029 0.1148 1157 0.021 18 0.0006

22:3docosatrienoic 0.00114 14 0.00036 32 0.00010 0.028 16 0.015 53 0.004

22:5docosapentaenoic,DPA

0.005432 1158 0.0012 23 0.000036 0.1519 1158 0.030 20 0.0009

22:6docosahexaenoic,DHA

0.001064 249 0.0005 50 0.000034 0.0266 258 0.018 67 0.0011

Totalω- 3d 0.05645 1161 0.013 23 0.00038 1.573 1161 0.30 19 0.009

ω-6fattyacids

18:2linoleic,LA 0.04469 1156 0.010 22 0.00029 1.254 1156 0.27 22 0.008

18:3 γ-linolenic,GLA 0.001100 649 0.00031 28 0.000012 0.0299 670 0.009 31 0.0004

20:2eicosadienoic 0.001031 717 0.00017 17 0.000006 0.0258 743 0.007 28 0.0003

(Continues)

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in the retail conventional andorganicmilks. The last twopairs ofcolumnsshowtheoften-largepercentagedifferencesbetweencon-ventionalandadjustedgrassmilk,andbetweenorganicandadjustedgrassmilk.

The p-valuesinTable3arefromtwo-tailedttests.Theseareac-curate for individualpairwisecomparisonswithineachof the twopairs of columns considered alone, but they somewhat overstatethestatisticalsignificanceofdifferencesbetweenpairsofcolumns,andtheydonotaccountformultiplecomparisonswithincolumns.However,thesecaveatsareminorinviewoftheusuallyextremelysmallp-valuesbyttest.anovamethodsarequestionableduetotheunbalanced data and several years betweenmeasurements. TotalsaturatedandmonounsaturatedFAlevelsintheadjustedgrassmilkshowonlysmallpercentagedifferenceswith those in theconven-tionalandorganicmilks.But largepercentagedifferencesoccur in

theamountsoftotalω-3andω-6FAs,andtotalCLA.Themeanlevelof totalω-3 in the adjusted grassmilk samples ismore than twicethatintheconventionalsamples(up147%).Theshiftfromorganicmanagementtonearly100%forage-baseddietsongrassmilkfarmsincreasestheleveloftotalω-3FAsby52%.Inthecaseoftotalω- 6 FAs, the level drops52% in adjustedgrassmilk samples comparedto conventional samples and drops 36% from organic to adjustedgrassmilk.

Theincreaseinω-3FAsfromconventionaltoadjustedgrassmilk,coupledwith thedecreases inω-6FAs, reduces theω- 6/ω-3 ratiofrom5.8inconventionalmilkto2.3inorganicmilkand0.95inad-justedgrassmilk.ComparablechangesoccurintheLA/ALAratio.

Significantly, lactating cows fed a nearly 100% grass- andlegume-based diet producemilkwith substantially elevated lev-els of two long-chain ω-3 FAs. Compared to conventional milk,

g/100 g milka Percent of total fatty acidsb

Mean n SD CV (%) SE Mean n SD CV (%) SE

20:3 8,11,14-eicosatrienoic(γ)

0.001964 1113 0.0006 32 0.000019 0.0548 1114 0.014 26 0.0004

20:4arachidonic,AA 0.003453 1131 0.0015 43 0.000045 0.0920 1132 0.043 46 0.0013

22:2docosadienoic 0.001803 969 0.0007 39 0.000023 0.0502 975 0.017 33 0.0005

22:4docosatetraenoic 0.00118 91 0.0005 47 0.00006 0.024 108 0.016 70 0.002

Totalω- 6d 0.05250 1163 0.012 22 0.00034 1.467 1163 0.31 21 0.009

Totalcis-polyunsaturatedc 0.10885 1161 0.022 21 0.00066 3.045 1161 0.5 17 0.015

transfattyacids

trans- 14:1 0.01335 1162 0.0029 22 0.00008 0.372 1162 0.07 17 0.002

trans-16:1,trans-palmitoleic

0.01964 1159 0.0042 21 0.00012 0.551 1159 0.11 20 0.003

trans-18:1includingelaidic 0.1381 1160 0.05 39 0.0016 3.885 1160 1.5 39 0.045

trans- 18:2 octadecadienoic

0.02203 1159 0.008 35 0.00022 0.618 1159 0.21 34 0.006

Totaltransfattyacidsc 0.1934 1163 0.06 31 0.0018 5.430 1163 1.7 31 0.049

Conjugatedlinoleicacid,CLA

18:2conjugated,total 0.0498 1163 0.019 38 0.0006 1.403 1163 0.5 38 0.016

Sum

ALA+CLA 0.0939 1163 0.022 24 0.0006 2.633 1163 0.6 22 0.017

Ratios

LA/ALA 1.042 1156 0.21 21 0.006 1.042 1156 0.21 21 0.006

ω- 6/ω- 3 0.954 1154 0.18 19 0.005 0.947 1161 0.19 20 0.006

ω- 3/ω- 6 1.083 1154 0.20 18 0.006 1.131 1161 0.6 54 0.018

aForFAsreportedinunitsofg/100gmilk,meansandtheotherstatisticsarebasedonquantifiedamounts≥0.001g/100g(samples<0.001g/100gnot included).Hence,forminorFAswithnsubstantially<1,163,meansareelevated,andotherstatisticsarebasedonthedistributionofsamples≥0.001g/100g.bForunitsof%oftotalFAs,meansandotherstatisticshavethesamepropertiesasnotedaboveforunitsofg/100gmilk.ForafewminorFAs,thelaboratoryquantifiedupto26moresamplesinunitsof%oftotalFAsthanitdidinunitsofg/100g,increasingthen-valuesshownhere.Inrarecases,the n-valuesdifferalsoby±1duetodifferencesinthenumberofoutliersremoved.cAnaverageofsumsreportedbythelaboratoryforeachsample.ThelaboratorysumsincludeminorFAsreportedinTableS1butnottabulatedhere,sotheyusuallyslightlyexceedthesumofmeansfortheindividualFAslistedhere.dAnaverageofsumsofall7FAsforeachsample.ThisaverageisslightlysmallerthanthesumofmeansshownforeachFA,becausesomeofthelattermeansaresubstantiallyelevatedbyexclusionofvalues<0.001mg/100gmilk(footnotea).

TABLE  1  (Continued)

     |  7BENBROOK Et al.

TABLE  2  fattyacidsinretail,wholegrassmilk,36-monthaverage,2014–2016(69samples)

g/100 g milka Percent of total fatty acidsb

Mean n SD CV (%) SE Mean n SD CV (%) SE

Fatreportedbyprocessor 3.384 69 0.14 4 0.016

Totaltriglyceride(calculated)

2.758 69 0.44 16 0.053

Totalfattyacids 2.616 69 0.42 16 0.050 100.000 69 0.006 0.01 0.0008

Saturatedfattyacids

4:0butyric 0.0696 68 0.012 17 0.0014 2.656 68 0.25 9 0.030

6:0caproic 0.0516 69 0.010 19 0.0012 1.965 69 0.18 9 0.022

8:0caprylic 0.0306 69 0.006 21 0.0008 1.162 69 0.14 12 0.017

10:0capric 0.0728 69 0.014 19 0.0017 2.775 69 0.28 10 0.034

12:0lauric 0.0830 68 0.016 19 0.0019 3.170 68 0.29 9 0.036

14:0myristic 0.298 68 0.049 17 0.006 11.33 68 0.7 6 0.09

15:0pentadecanoic 0.0402 68 0.008 20 0.0010 1.538 68 0.18 12 0.022

16:0palmitic 0.812 69 0.15 18 0.018 31.09 69 3.2 10 0.39

17:0margaric 0.0225 69 0.0045 20 0.0005 0.858 69 0.09 10 0.011

18:0stearic 0.276 69 0.06 22 0.007 10.52 69 1.5 14 0.18

20:0arachidic 0.00496 67 0.0007 15 0.00009 0.1879 67 0.021 11 0.0025

22:0 behenic 0.00338 66 0.0013 37 0.00016 0.126 66 0.042 34 0.005

24:0lignoceric 0.00191 65 0.0005 24 0.00006 0.0715 65 0.014 20 0.0017

Totalsaturatedc 1.758 69 0.29 17 0.035 67.18 69 2.7 4 0.33

Monounsaturatedfattyacids

14:1myristoleic 0.0244 68 0.005 21 0.0006 0.935 68 0.13 14 0.016

16:1palmitoleic 0.0409 69 0.010 24 0.0012 1.560 69 0.26 17 0.031

17:1margaroleic 0.00765 68 0.0017 23 0.00021 0.289 68 0.045 15 0.005

18:1includingoleic 0.506 69 0.12 25 0.015 19.40 69 3.6 19 0.43

20:1includinggadoleic 0.00530 67 0.0013 25 0.00016 0.201 67 0.046 23 0.006

Totalcis-monounsaturatedc

0.585 69 0.13 23 0.016 22.41 69 3.6 16 0.43

ω-3fattyacids

18:3 α-linolenic,ALA 0.0312 69 0.005 17 0.0006 1.196 69 0.14 12 0.017

18:4stearidonic/moroctic 0.00206 35 0.0009 46 0.00016 0.076 35 0.030 39 0.005

20:3 11,14,17-eicosatrienoic

0.001000 35 0.00000 0 0.000000 0.0297 39 0.009 32 0.0015

20:5eicosapentaenoic,EPA

0.00316 63 0.0006 19 0.00008 0.1208 63 0.020 16 0.0025

22:3docosatrienoic 0.00100 2 0.040 2

22:5docosapentaenoic,DPA

0.00408 65 0.0009 21 0.00011 0.1551 65 0.024 16 0.0030

22:6docosahexaenoic,DHA

0.00108 12 0.0003 27 0.00008 0.0325 13 0.015 48 0.0043

Totalω- 3d 0.0397 69 0.007 19 0.0009 1.515 69 0.20 13 0.024

ω-6fattyacids

18:2linoleic,LA 0.0332 69 0.007 20 0.0008 1.272 69 0.18 14 0.022

18:3 γ-linolenic,GLA 0.001031 32 0.00018 17 0.000031 0.0302 37 0.012 39 0.0020

20:2eicosadienoic 0.00113 31 0.00034 30 0.00006 0.0332 34 0.018 54 0.0031

(Continues)

8  |     BENBROOK Et al.

adjusted grassmilk averages43%more20:5EPA and27%more22:5DPA, twoof threecritical long-chainω-3FAs.Thepercentincrease in 22:6 DHA cannot be calculated, because there wastoo little found in the conventional and organic samples testedin 2011–2012. We estimate that the absolute average increaseinDHAisabout0.0006g/100gofmilk (Table3footnote f). It iswidelyagreedthattypicalWesterndietsprovideinsufficientsup-pliesoflong-chainω-3FAs, leadingtheEuropeanFoodStandardAgencytorecommendatleastadoublingofaveragedailyintakesof long-chain ω-3 FAs (EPA, DPA, and DHA), especially duringpregnancy(EFSA,2010).

LimiteddatafromfoursmallfarmsforNobleMilkinItalyshowshifts in FA profile qualitatively similar to those in grassmilk, butsmaller,asexpectedgiventheupto30%grainandconcentratesal-lowedincowrations(Lombardietal.,2014).InNobleMilk,theratios

LA/ALAandω- 6/ω-3areabout30%to50%higherthaningrassmilk.Inthesummer,NobleMilkreachestheannual averagelevelingrass-milkforω-3FAsandalmosttheannualaveragefortotalCLA,butbotharesubstantially lower inother seasons.Totalω-6 isnotablylowbothinNobleMilk(aboutaslowasingrassmilk)andinItalianconventionalmilk (about30% less thanU.S. conventionalmilk re-portedbyBenbrooketal.,2013).

3.2 | Trans fatty acid concentrations

Total transFAconcentrations (excludingCLA)wereone-thirdhigherin grassmilk compared to the similar levels in the organic and con-ventionalmilksshowninTable3.Otherstudieshavealsofoundthatpastureandforage-basedfeedsincreasethelevelsoftransFAinmilk,mainly trans-18:1, simultaneouslywith increases inCLA, a groupof

g/100 g milka Percent of total fatty acidsb

Mean n SD CV (%) SE Mean n SD CV (%) SE

20:3 8,11,14-eicosatrienoic(γ)

0.00159 63 0.0005 33 0.00007 0.0602 63 0.017 29 0.0022

20:4arachidonic,AA 0.00245 65 0.0009 36 0.00011 0.0910 65 0.034 38 0.0043

22:2docosadienoic 0.00141 54 0.0005 35 0.00007 0.0551 54 0.015 28 0.0021

22:4docosatetraenoic 0.00150 2 0.031 4 0.030 95 0.015

Totalω- 6d 0.0390 69 0.008 20 0.0009 1.490 69 0.22 14 0.026

Totalcis-polyunsaturatedc 0.0783 69 0.014 18 0.0017 3.012 69 0.37 12 0.044

transfattyacids

trans- 14:1 0.00975 69 0.0018 19 0.00022 0.3709 69 0.035 9 0.0042

trans-16:1,trans-palmitoleic

0.01433 69 0.0033 23 0.00039 0.549 69 0.08 14 0.009

trans-18:1includingelaidic 0.120 69 0.11 95 0.014 4.53 69 4.0 89 0.48

trans- 18:2 octadecadienoic

0.0156 68 0.0047 30 0.0006 0.596 68 0.16 26 0.019

Totaltransfattyacidsc 0.159 69 0.12 74 0.014 6.05 69 4.1 67 0.49

Conjugatedlinoleicacid,CLA

18:2conjugated,total 0.0353 69 0.010 30 0.0013 1.352 69 0.33 24 0.040

Sum

ALA+CLA 0.0665 69 0.014 20 0.0016 2.548 69 0.35 14 0.042

Ratios

LA/ALA 1.069 69 0.15 14 0.018 1.070 69 0.15 14 0.018

ω- 6/ω- 3 0.992 69 0.13 14 0.016 0.991 69 0.13 13 0.016

ω- 3/ω- 6 1.026 69 0.14 14 0.017 1.028 69 0.14 14 0.017

aForFAsreportedinunitsofg/100gmilk,meansandtheotherstatisticsarebasedonquantifiedamounts≥0.001g/100g(samples<0.001g/100gnot included). Hence, forminor FAswith n substantially <69,means are elevated, and other statistics are based on the distribution of samples≥0.001g/100gmilk.bForunitsof%oftotalFAs,meansandotherstatisticshavethesamepropertiesasnotedaboveforunitsofg/100gmilk.ForafewminorFAs,thelaboratoryquantifiedupto5moresamplesinunitsof%oftotalFAsthanitdidinunitsofg/100gmilk,increasingthen-valuesshownhere.Inrarecases,then-valuesdifferalsoby±1duetodifferencesinthenumberofoutliersremoved.cAnaverageofsumsreportedbythelaboratoryforeachsample.ThelaboratorysumsincludeminorFAsreportedinTableS1butnottabulatedhere,sotheyusuallyslightlyexceedthesumofmeansfortheindividualFAslistedhere.dAnaverageofsumsofall7FAsforeachsample.ThisaverageisslightlysmallerthanthesumofmeansshownforeachFA,becausesomeofthelattermeansaresubstantiallyelevatedbyexclusionofvalues<0.001mg/100gmilk(footnotea).

TABLE  2  (Continued)

     |  9BENBROOK Et al.

TABLE 3 Keyfattyacidsandratiosinwholemilk—conventional,organic,andgrassmilk(g/100gmilk)

Conv

entio

nala

Org

anic

aG

rass

milk

bG

rass

milk

(adj

uste

d to

tal

FA)c

% d

iffer

ence

Conv

entio

nal t

o ad

just

ed

gras

smilk

(%)

pdO

rgan

ic to

adj

uste

d gr

assm

ilk (%

)pd

Totalfattyacids

3.098

3.10

83.

585

3.10

30.

2ns

−0.2

ns

Saturatedfattyacids

2.04

32.

116

2.399

2.07

61.

6ns

−1.9

ns

14:0myristic

0.32

740.3490

0.3997

0.34

605.

7.0

02−0.9

ns

16:0palmitic

0.8995

0.9344

1.11

60.966

7.4

.000

73.

4ns

18:0stearic

0.3559

0.34

340.

3738

0.32

35−9.1

.000

00−5.8

0.00

1

Monounsaturated

fattyacids

0.7944

0.74

100.

8352

0.7229

−9.0

.000

00−2.4

ns

18:1incl.oleic

0.70

740.

6505

0.72

580.

6282

−11.2

.000

00−3.4

.010

Totalω-3FattyAcids

0.0198

0.03

210.

0565

0.0489

147

.000

0052

.000

00

α-linolenicacid,ALA

0.0159

0.02

550.

0441

0.03

8214

0.0

0000

50.0

0000

20:5eicosapentae-

noic,EPA

0.00

250.

0033

0.00

413

0.00

357

43.0

0000

8.3

.000

8

22:5docosapentae-

noic,DPA

0.00

370.

0044

0.00

543

0.00

470

27.0

0000

6.8

.004

22:6docosahexae

-noic,DHA

ee

0.00

106f

0.00092f

Totalω-6fattyacids

0.0948

0.07

110.

0525

0.04

54−52

.000

00−36

.000

00

18:2linoleic,LA

0.08

560.0639

0.04

470.

0387

−55

.000

00−39

.000

00

Totalpolyunsaturated

fattyacids

0.11

470.

1037

0.10

880.0942

−18

.000

00−9.2

.000

00

Totalt

ransfattyacids

0.12

810.

1254

0.1934

0.16

7431

.000

0033

.000

00

TotalCLA

0.0192

0.02

270.0498

0.04

3112

5.0

0000

90.0

0000

LA/ALA

6.27

22.

568

1.04

21.

042

−83

.000

00−59

.000

00

ω- 6/

ω- 3

5.77

42.

276

0.954

0.954

−83

.000

00−58

.000

00

ω- 3/

ω- 6

0.219

0.45

61.

083

1.08

3395

.000

0013

8.0

0000

a FromBenbrooketal.(2013).

b FromTable1.

c Adjustedtothemeanofconventionalandorganic.

d Pairwise

p-valuesby2-tailedttest;ns,notsignificant(

p>.05).

e Notreportedbecausequantifiableamounts≥0.001g/100gmilkwerefoundinonly2of160conventionaland4of218organicsamples.

f Meanof249samples(21%of1162)withquantifiedamounts≥0.001g/100gmilk.WeestimatetheaverageDHAcontentofall1162samplesisabout0.0006g/100gmilk,roughlyhalftheunadjusted

amountshownhere.Forthisestimate,weassumedthatthe913unquantifiedsamplescontainedanaverage0.0005g/100gmilk,halfoftheminimumquantifiedamountof0.001g/100gmilk.

10  |     BENBROOK Et al.

18:2isomers,nearlyallcontainingconjugatedtransbonds(Daleyetal.,2010; Mansson, 2008; Vargas-Bello-Pérezz & Garnsworthy, 2013).IncreasesintotalCLAareassociatedwiththehighALAcontentoffor-agegrasses(Elgersma,2015),soforagefeedingincreasesnotonlyCLAandω-3FAlevelsinmilk(Slotsetal.,2009;Molkentin,2009,butalsotransFAs.IncreasedlevelsofCLAandω-3FAsinmilkhavebeenas-sociatedwithhealthbenefits(Larsson,Bergkvist,&Wolk,2005;Smit,Baylin, & Campos, 2010; Stender & Dyerberg, 2003; Vargas-Bello-Pérezz&Garnsworthy,2013).

TransFAhasabad reputationbecauseofevidence that sourcesfrom partially hydrogenated vegetable oils strongly increase LDLcholesterol,decreaseHDLcholesterol,andhavemultipleothermet-aboliceffectsassociatedwithCVD.Theseadversefindingsareoftenassumed toapplyequally tonatural sourcesof trans FAs.However,such assumptions are unwarranted, because there are large differ-ences between the two sources of transFAs.First,industrialsourcescontainupto60%transFAs,comparedtoamaximumof5to8%ofFAs inmilk (5.4%and6.0% inTables1and2) (Stender&Dyerberg,2003).Second,thedistributionofisomersdiffersgreatlyinthemaintransFAinmilk,trans-18:1(about72%and75%oftotaltransFAsinTables1and2).Inindustrialsources,thepositionofthetransbondhasabroad,near-Gaussiandistributionfromthe6thtothe16thcarbonatom,whereasmilkandotherruminantsourcespeakstronglyatthe11thcarbonatom,withonlysmallamountsatotherpositions(Stender&Dyerberg,2003).

Trans- 18:1 with the transbondatthe11thcarbonatomisvacce-nicacid(VA),themajorprecursortoCLA(rumenicacid)inmilk.Atthehighrangeofhumanintakes,VAhaslittleornoadverseeffectonriskfactorsforCVD(Lacroixetal.,2011).VAinthecow’sudderispartiallyconvertedtorumenicacid,themajorCLAinmilk(75%to90%)(Lock&Bauman,2004;Tyburczyetal.,2008).HumansarealsoabletoconvertsomeVAinmilktothisformofCLA(Lock&Bauman,2004;Turpeinenetal.,2002;Tyburczyetal.,2008).Despitehavingatransdoublebond,rumenicacidhasprovenbenefitsinanimals,es-peciallyanticarcinogenicactivityagainstdiversecancertypes(Lock&Bauman,2004).Inhumans,thereissuggestivesupportforactivityagainstcoloncancerfromalarge,epidemiologicalstudyinSweden(Larsson etal., 2005) and possibly against breast cancer (Dilzer&Park,2012).

Forthesereasons,theFDAexemptsCLAsfromitsdefinitionoftransFAforpurposesoffoodlabeling(FDA,2003).Severalcountries

andNewYorkCityexemptnotonlyCLAs,butalsoruminanttrans FAssuchasVA(Larssonetal.,2005;Table2).

Motard-Bélanger etal. (2008) conducted a double-blind, ran-domizedcrossoverstudyof“high”and“moderate”dietary intakesof trans FAs from specially produced milk. They concluded thathigh intakes of these trans FAs “may adversely affect cholesterolhomeostasis,”but thatmoderate intakes “that arewell above theupperlimitofcurrenthumanconsumptionhaveneutraleffectsonplasmalipidsandothercardiovascularriskfactors.”Their“moder-ate”intakewas4.2g/2,500kcal,where4.2gistheamountoftotaltrans FAs in2.64kgof retail grassmilk (Table2), or10.8 servingsofonecup(244g).The“high”amountwas10.2g/2,500kcal,2.43timeshigherthanthe“moderate”amountandfarbeyondevenex-ceptionallyhighlevelsofdairyproductconsumptionintheUnitedStates.

Moreover,thereissomeevidenceofbenefitsfromVAassociatedwith its conversion toCLA (Kuhnt,Degen,& Jahreis, 2016).A re-centmeta-analysisincluded13randomized,controlledinterventiontrials that used dairy products as the primary source of trans FA,inamountsashighas4.2%ofenergy (10.9g trans FA/2,500kcal)(Gayet-Boyer,Tenenhaus-Aziza,Prunet,&Chardigny,2014).Theau-thorsfoundthattheselevelsoftransFAhavenoharmfuleffectsonHDLcholesterol,LDLcholesterol,ortheirratio.

Hence,thereisaclearneedtodistinguishbetweennaturalandindustrialsourcesoftransFA,butthiswilltaketimeandcarefulre-porting,becauseoflong-standingassumptionstothecontrary.

3.3 | Regional and seasonal differences

Our large, nationwide, 3-year dataset allows assessment of theregional and seasonal consistency in the impact of nearly 100%forage-based feedon theFAprofileof grassmilk.Table4 showsmodest, but sometimes statistically significant, regional differ-encesingrassmilkcompositionfortotalω-6andω-3FA.Thehigh-estaveragelevelsofω-3FAsingrassmilkcamefromtheMidwestand Northeast (1.60% and 1.58% of total FA), while Californiahad the lowest (1.40%), about a 14% difference. Likewise, theMidwestandNortheasthadthetwohighestaverageconcentra-tions of total ω-6 FAs. For total CLA, there are no statisticallysignificantregionaldifferences.AverageratiosofLA/ALAandω- 6/ω-3 varied by 7% across the 4 regions, but these differences

California Mideast Midwest Northeast SEM p- value

Observations 85 54 582 442

Totalω- 3 1.40c 1.434bc 1.601a 1.575ab 0.04 .002

Totalω- 6 1.364ab 1.309b 1.477a 1.495a 0.04 .002

TotalCLA 1.282 1.165 1.300 1.379 0.07 .09

LA/ALA 1.091 1.022 1.035 1.047 0.03 .62

ω- 6/ω- 3 1.189 1.232 1.206 1.151 0.07 .75

*Least squaremeans.Meanswithin a rowwithout common superscripts aredifferent atp < .05. MeanswereevaluatedusingTukey’smultiplecomparisonstest.

TABLE  4 Regionalvariationinselectedgrassmilkfattyacids,2014–2016(%oftotalFA)*

     |  11BENBROOK Et al.

are not statistically significant (p > .05). There were no regionaldifferencesforthemajorFAsinmilk—totalsaturatedandtotalcis-monounsaturatedFAs(notshown).

SomeregionalandseasonalvariationintheFAprofileofgrass-milkisexpected,drivenbydifferencesinthequalityandinbotanicalcompositionof freshandstored forage (RavettoEnrietal.,2017).Suchvariationsareoften triggeredby climatic conditions that aremost extreme during extended drought or heavy rains leading towater-logged soils or flooding. The length of the outdoor grazingperiodalso impacts foragequality, asdoesmanagementattentiontosustainingapropermixofgrassandlegumespeciesinpastures(sothathigh-quality,immatureforagesarepresentinpasturesfromspring through the fall). The timing of forage harvests and how,and howwell, forage-based feeds are conserved also impact for-age composition. Despite all these factors, our results show thatCROPPfarmersswitchingtograssmilkstandardshaveconsistently

improvedmilkFAcompositionover thebroadrangeofagronomicandpedo-climaticconditionsfoundintheUnitedStates.

Table5reportssmallbutsometimesstatisticallysignificantdif-ferencesbetweenyears.Theaverageω- 6/ω-3 ratiodeclinedeachyearfrom2014to2016,foranoveralldeclineof6%(p < .05).Thisdeclineresultsfroma3.2%declineintotalω-6FAsanda1.5%in-creaseintotalω-3FAs(p > .05forboth).Manyfactorsmightcontrib-utetothesechangesover3years,includingimprovingmanagement,changingclimateorpastureconditions,ortheincreasingnumbersofparticipatingfarms.

SeasonalhighsandlowsingrassmilkFAsareshowninTable6,averaged over all 1,163 samples (2014–2016). The ω- 6/ω-3 ratiopeakedinJulyandbottomedinDecember,withavariationof30%fromlowtohigh.Forω-6andω-3levels,themaximumvariationwassomewhatless,21%to22%.ThelargestseasonalvariationoccurredintotalCLAconcentration,whichmorethandoubledinSeptembercomparedtoApril.SaturatedandmonounsaturatedFAlevelsdidnotvarysignificantlybymonth(notshown).

Figure1 shows themonthly variation in averageω- 6/ω-3 ratioinallgeographicalregionsduring2014–2016.FigureS1showssim-ilarplotsforthethreeseparategeographicalregionswiththemostsamples (sample numbers are 85 for California, 582 forMidwest,and442 forNortheast).TheCalifornia regionshowsnotably littlemonthlyvariationinω- 6/ω- 3.

For Noble Milk, reported seasonal variations from four smallfarmsinItalyareconsiderablylargerthaningrassmilkfortotalω- 3 FAs(twofoldlarger)andtotalCLA(nearlythreefoldlarger).However,seasonal variations in LA/ALA andω- 6/ω-3 aremodest (Lombardietal.,2014).

TABLE  5 Yearlyvariationinselectedgrassmilkfattyacids(g/100gmilk)*

2014 2015 2016

Observations 364 370 429

Totalω- 3 0.0519b 0.0558a 0.0527b

Totalω- 6 0.0493b 0.0538a 0.0477b

TotalCLA 0.0454 0.0444 0.0453

LA/ALA 1.0583 1.0566 1.0284

ω- 6/ω- 3 0.9888a 0.9720a 0.9276b

*Leastsquaremeans.Meanswithinarowwithoutcommonsuperscriptsaredifferentatp < .05.

High Month Low Month High/Low

ω- 3 0.0594 December 0.0486 August 1.22

ω- 6 0.0548 October 0.045 February 1.21

CLA 0.0635 September 0.0310 April 2.05

ω- 6/ω- 3 1.093 July 0.838 December 1.30

aLeastsquaremeansinregion-and-yearmixedmodel.

TABLE  6 Seasonalvariationsofkeyfattyacids,2014–2016means(g/100ggrassmilk)a

F IGURE  1 Monthlyvariationinmeanω- 6/ω-3ratiooverallgeographicalregions,2014–2016(429samples).TheverticalbarsshowSEsfromtheleastsquaresanalysis

12  |     BENBROOK Et al.

3.4 | Relationship between dairy rations and milk FA profile

Allcommercialdairybreedsdescendedfromgrazingherbivores.Themore theirdiet strays from leafyvegetation, thegreater thechal-lengetomaintainguthealth.Thewell-knowneffectsofgrainfeed-ingonmilkFAsarecausedbyalterationsinthenormalfunctionsofthecowrumenanditsmicrobes(McDonaldetal.,2006).

CombiningtheresultsofthisstudyofCROPPgrassmilkwithourpriorstudyofconventionalmilkandCROPPorganicmilk(Benbrooketal., 2013), we can estimate quantitative relationships betweenvarious levelsofgrain feedingofdairycowsand theFAcomposi-tionoftheirmilk.Table7showstheserelationshipsfortheω- 6/ω- 3 ratioandthecontentoftotalCLA.InadditiontoGrassmilk,Organicmilk,andConventionalmilk,weincludeanestimatefor“MinimumForages”milkfromcowswithnograzingandmaximumamountsofgrain.MostdairycowsintheUnitedStatesnowreceiveaverysmallshare,ornoneoftheirannualDMIfromgrazing(0to3%).Weesti-matethatcowsunder“MinimumForages”managementgetapproxi-mately60%ofDMIfromcornsilageandconcentratefeeds,and40%fromdryor fermentedalfalfahay ina“totalmixedration” (mostlychopped,dryalfalfahay).MorecommonlyintheUnitedStates,cowsunderConventionalmanagement receivesomewhat lesscornplusconcentratefeeds (47%)andsomewhatmorestoredforage (50%),withabout3%ofannualDMIfromgrazing(totalforages,53%).

AccordingtoCROPPrecords,cowsunderOrganicmanagementon its farms receive about 56%of dailyDMI frompasture duringan average 183-day season and hence about 28%of their annualDMI fromgrazing.OnCROPPfarmsproducingGrassmilk,pastureaccountsforanaverage80%ofDMIovera190-daygrazingseason,or42%ofannualDMI.Stored,forage-basedfeedsaddnearly52%ofdailyDMIonOrganicfarms,and58%onGrassmilkfarms,bringingtheirtotalsfromforage-basedfeedsto,respectively,about80%andnearly100%ofDMI.

In the milk from these four management systems, the ratiosof ω- 6/ω-3decline fromanestimated8withMinimumForages tomeasured values of 5.8 inConventionalmilk, 2.3 inOrganicmilk,and0.95inGrassmilk(Table7).Simultaneously,theannualaverage

amounts of total CLA in conventional to retail grassmilk increaseabout fourfold from about 0.010 to 0.043g/100gmilk. For totalCLA,theimpactofpastureandforagefeedingappearstoincreaseastheirproportionofannualDMIincreasesbeyond80%,anobser-vationthatdeservesfurtherexploration.

Ingraincrops, stageofgrowth impacts theFAcompositionoffeedstuffs in cow rations, aswell as theFAprofileofmilk (Darbyetal.,2012;Darbyetal.,2013;Duvicketal.,2006).SupplementalTextS1andTableS2comparetheFAsincommonforagegrassandlegumecropswiththoseofseveralcerealcropsatvariousstagesofmaturity.

3.5 | Nutrition modeling of grass milk effects on dietary LA/ALA ratios

Tables1–3 show that increasing forage-based feeds in rations forlactatingcowscansignificantlyaltertheFAprofileofmilk;however,a key question remains.Will consumption of dairy products fromcowsfedall,ormostly,forage-basedfeedshaveameaningfulimpactonhumanintakesofFAs,andpotentiallyonpublichealth?

Toaddressthisquestion,wemodeledtotalLAandALAintakesin thedailydietofamoderatelyactive19- to30-year-oldwomenacross36dietscenarios—18dietswithtypical,high-LAfoodssuchasregularmargarineandotherfoodscontainingsoyoil,and18mostlyidenticaldietsinwhichthreefoodslowerinLAcontentweresubsti-tuted(e.g.,pitachipsinsteadofcornchips,andmargarinemadewithcanolaoilinsteadofsoyoil).

The18scenariosineachofthesetwocases(high-andlow-LAintakes)entailed three levelsof fat intake (20%,33%,and45%oftotal energy), two levels of dairy product consumption (3 and4.5servings/day),andthreevariationsofdairyfat(fromcowsmanagedunder the conventional, organic, and grassmilk systems discussedhere,withtheirvaryingrelianceongrazingandforagerationsshowninTable7).

Ourmodeling focuseson total intakesofLAandALAand theLA/ALAratio(ratherthanω-6,ω-3,andω- 6/ω-3),becausetheUSDAdoesnotpublishsufficientandreliabledataonthetotalω-6andω- 3 contentsofmanycommonfoods.Butformanyfoods,itdoesreport

TABLE  7 Estimatedaveragedailydrymatterintakefromgrazing,forage,andgrainunderfourmanagementsystems:impactsonω- 6/ω- 3 andtotalCLAinretailwholemilk

Management system

Average daily dry matter intake (DMI) Milk fatty acids

In season Annual basis

ω- 6/ω- 3 CLA (g/100 g)Grazing (%) Grazing (%)Stored forages (%)

Grazing plus forages (%)

Grains and concentrates (%)

Minimumforages 0 0 40 40 60 8.0 0.010

Conventional 6 3 50 53 47 5.8a 0.019a

Organicb 56 28 52 80 20 2.3a 0.023a

Grassmilkb 80 42 58 100 0 0.95 0.043

aBenbrooketal.(2013).bEstimatedfromannualpastureandlactatingcattlefeedsurveysbyCROPPcooperative.

     |  13BENBROOK Et al.

reliable (fullydifferentiated)contentsofLAandALA.LAandALAare,respectively,byfarthemajordietaryω-6andω-3FAsinnearlyallfoods,sodietaryratiosofLA/ALAareareliableproxyfordietaryω- 6/ω-3ratios.

Weassessthedegreetowhicheachofthe36dietaryscenariosreducestheLA/ALAfromthebaselinelevelof11.3for3servings/day of conventional milk, typical-LA sources of nondairy fat, and33%ofenergyfromfat.Thelowertheratio,thegreaterthebody’sabilitytoconvertdietaryALAtotheessential,longer-chainω-3FAs.Thisconversionismostimportantforpregnantandlactatingwomenand for those who consume little or no oily fish (Brenna, 2002;Burdge&Calder,2005).Oilyfishare,perserving,superiorsourcesfor EPA andDHA, but even oily fish do not contain enoughALAtosignificantlyalterdietaryratiosofLA/ALAorω- 6/ω-3(Benbrooketal.,2013;USDA,2015).

Table8givessample results fromournutritionmodelingcal-culationsforfourdietswithtypicalintakesoftotalfat(33%ofen-ergy)anddairyfatwiththeFAprofileofadjustedgrassmilk.Thesefour diets include those with moderate (recommended) (DHHS,2015)dairyintake(3servings/day)andhighdairyintake(4.5serv-ings/day), with either typical-LA or low-LA sources of nondairyfat.Forthesefourdiets,thetableshowsthedairyandnondairycontributionstodietaryintakesofLAandALA,theLA/ALAratioandchangesinthisratiorelativetothebaselineratioof11.33forrecommended intakesofconventionaldairyproducts (Benbrooketal.,2013).Thus,itshowstheimpactondietaryLA/ALAratiosofswitchingfromconventionaltograssmilkdairyproductsforthesefour diets.We performed similar calculations for correspondingdietswithlowandhighamountsoftotaldietaryfat(20%and45%ofenergy).

Foradietwithtypicaltotaldietaryfat(33%ofenergy),moder-atedairy servings,and typical-LAsourcesofnondairy fat,Table8

showsthataswitchfromconventional toadjustedgrassmilkdairyproductswoulddecreasetheoveralldietaryLA/ALAratioby2.68to8.64 fromthebaseline ratioof11.33.Adding1.5servings/day,foratotalof4.5servings/dayofdairyproducts,wouldfurtherlowertheLA/ALAratioto5.95—atotaldropof5.37.Thesearesubstantialdecreases.Forcorrespondingdietswithlow-LAsourcesofnondairyfat,thereductionsinLA/ALAratioareevenlarger,by7.31and8.19,respectively,formoderateandhighconsumptionofdairyproducts.

Aswediscussbelow,reductionsindietaryLA/ALAratiosofthismagnitudeseemofpotentialpublichealthsignificance.Muchofthereductions can be achieved with grassmilk dairy products alone,withoutreducingintakesofnondairyLA.TheopportunitytoreducetotaldietaryLA/ALAratiosfrom11.33toaslowas3.14,andwithoutmajorchangesindietarypatterns,seemsnotabletous.Inourmodeldiets, there are no changes inmost foods, including French fries,chocolatechipcookies,chicken,pork,andbeef.ThemodeledfoodchoicesrepresentanattainableoptiontoimproveFAintakesinwaysthatwilllikelyreducetheriskforcardiovascularandothermetabolicdisorders,atleastforsomeindividuals.Manyotherfactors—genet-ics, age, health status, and environmental exposures—will interactindeterminingthemagnitudeofsuchimpacts(Simopoulos,2006).

Figure2showsthefullresultsofournutritionmodeling,includ-ingdietswithlowandhighintakesoftotaldietaryfat(20%and45%ofenergy).Fordietswithtypical-LAnondairyfatsources(leftsideofFigure2),thedecreasesindietaryLA/ALAratiosareenhancedinthedietswithonly20%ofenergyfromfatandattenuatedinhigh-fatdiets.Fordietswith low-LAnondairy fatsources (rightsideofFigure2), there is littledependenceon theoverall levelofdietaryfat, but the reductions in dietary LA/ALA ratio are much larger,including evenwith conventional dairy fat.Organic and grassmilkdairyfathavethemostimpactondietswithtypical-LAnondairyfat,comparedtodietswithlow-LAnondairyfat.

TABLE  8 LAandALAcontributionstoaverage-fatdietswithgrassmilkdairyfatandtypical-LAandLow-LAnondairyfatsourcesa

LA from dairy fat (g)b

ALA from dairy fat (g)b

LA from other fat (g)c

ALA from other fat (g)c Total LA (g) Total ALA (g)

Total LA/Total ALA ratio

Typical-LAnondairyfatsources

Moderatedairyintake

0.41 0.41 9.91 0.79 10.32 1.19 8.64

Highdairyintake

0.62 0.62 5.77 0.46 6.39 1.07 5.95

Low-LAnondairyfatsources

Moderatedairyintake

0.41 0.41 5.90 1.17 6.32 1.57 4.01

Highdairyintake

0.62 0.62 3.44 0.68 4.06 1.29 3.14

aThistableextendsTable3inBenbrooketal.,2013toincludegrassmilk.ThemodeleddairyservingsareinTable1ofthatpaper.Init,thebaselineLA/ALAratiois11.33,formoderateconsumptionofconventionaldairyfat.bBasedonLA,ALA,andtotalFAfromTable1,8.79kcal/gdairyfat,and0.933gmilkFA/gdairyfat.Forexample,LA0.41=313kcal(2013Table1)/8.79×0.0447/3.585×0.933.cBased on 23.23g LA and 1.841g ALA per 100kcal nondairy fat and 8.90kcal/g nondairy fat For example, LA 9.91=380kcal (2013Table1)/8.90×23.23/100.Correspondingcalculationsforlow-LAnondairyfatuse13.84gLAand2.731gALAper100kcalnondairyfat.

14  |     BENBROOK Et al.

Fortypicaldietswith33%ofenergyfromfatandtypical-LAnon-dairyfat(leftsideofFigure2),switchingfrommoderateamountsofconventional tohighamountsofgrassmilkdairyproducts reducestheLA/ALA ratio from11.33 to5.95, a47% reduction.The sameswitch for diets with low-LA nondairy fat decreases the LA/ALAratiofrom5.11to3.14.

LAandALAareessentialhumannutrients,buttheybothcomple-mentandcompetewitheachother,andtheirdietaryratiomatters.Theyareelongatedbyparallelandcompetingpathwaysto,respec-tively,AA(fromLA)andEPA(fromALA),whichinturnareconvertedinto eicosanoids that regulate many body functions. EicosanoidsderivedfromAAareproinflammatoryandthrombogenic,andsev-eralhavebeenlinkedtocarcinogenesis,whereasthosederivedfromALAtendtosuppressinflammation,thrombosis,andcarcinogenesis,especiallywhen theω- 6/ω-3 ratio approaches 1 (Larsson,Kumlin,Ingelman-Sundberg,&Wolk,2004).

Thus,alargeexcessofdietaryLAcomparedtoALAcanincreasetheriskofCVD,cancer,andotherdiseases(Burdge&Calder,2005;Ramsden, Hibbeln, Majchrzak, & Davis, 2010; Siri-Tarino, Chiu,Bergeron,&Krauss,2015).Forsome,andperhapsmostpeople intheUnitedStates,high-LAintakesreducethequantityofALAcon-vertedtoEPAanditsrelatedeicosanoidsandalsoreducethecon-versionofALAtoDHA.

DHAisindependentlyimportant,becauseitisrequiredinthede-velopmentoftheinfantbrainandocularsystem(Ailhaud,Massiera,Alessandri, & Guesnet, 2007; Donahue etal., 2011), as discussedfurtherbelow.

ImpairedconversionofALAtoEPAandDHAisofconsiderableconcernintheUnitedStates,becausemostAmericansdonotcon-sume adequate fish tomeet the recommended average intake of250mg/dayofEPA+DHA(DHHS,2015;EPA,2002).Hence,theymustpartlyrelyondietaryintakeofEPAandDHAfrommeatanddairyproductsorsupplements.Indeed,inthelate1990s,over70%ofAmericansage18orolderconsumednofishandshellfish(EPA,2002).

3.6 | Contribution of grass milk dairy products and fish to fatty acid intakes

Oilyfisharetheultimate,directsourceofthelong-chainω-3PUFAs,EPA,DPA,andDHA.DHAispresentatverylowconcentrationsinotherfoods,includinggrassmilk,butitplaysavitalroleinthede-velopment of an infant’s and child’s brain, eyes, and nervous sys-tem(Bondietal.,2013;Moonetal.,2013;Ryanetal.,2010).Forthe70%ofAmericanswhoconsumeessentiallynofish,theefficiencyofconversionofALAto long-chainω-3FAs iscritically important,especially for thosewith elevated need, such as growing childrenandwomenwhoarepregnantorbreastfeeding.Forthisconversion,ALAfromdairyproductsandotherfoodsplaysdualroles.FirstasaprecursortoEPA,DPA,andDHA,andsecondbydecreasingtheLA/ALAratio,andhencethetendencyofLAtocaptureandutilizetheenzymesneededtoconvertALAtolong-chainω-3FAs.

Although high in long-chain ω-3 FAs, oily fish do not containsignificant amounts of either LAorALA, and for this reason, fish

F IGURE  2 DecreasesindietaryLA/ALAratiosforanadultwomanconsumingtwolevelsofconventional,organic,andgrassmilkdairyproductsandtwotypesofnondairyfat.Thedietscontainmoderate“Mod.”(3servings/day)or“High”(4.5servings/day)amountsofdairyproductsmadefromconventional(“Conv.”),“Organic,”or“Grassmilk,”inthecontextsoftotalfatcontributing20%,33%,or45%ofenergy,andnondairyfatcontainingtypicalamountsofLA(leftside)orlowamountsofLA(rightside)

     |  15BENBROOK Et al.

TABLE 9 Dailyfattyacidcontentofrecommendedandper-capitaintakesofdairyproductsandfish

Dai

ry o

r fis

h so

urce

s of f

atty

aci

dsD

aily

fatt

y ac

id c

onte

nt (m

g)

Port

ion

size

aD

airy

se

rvin

gs/p

ortio

n

Dai

ly a

mou

nts t

hat s

uppl

y re

com

-m

ende

d 3

dairy

ser

ving

s

ALA

LALA

/ALA

ratio

Long

- cha

in ω

- 3 fa

tty

acid

s

Port

ions

aD

airy

Ser

v.W

eigh

t (g)

EPA

DPA

DH

Ab

EPA

+D

PAEP

A +

DPA

+

DH

A

Dairyfatfromgrassmilka

Wholemilk

1cup

1.0

1.5

1.5

366.

013

6.5

138.

40.954

12.8

16.8

1.9

29.6

31.5

Cheddarcheese

1.5oz.

1.0

1.0

1.0

42.5

316

1.7

163.9

0.954

1.5

2.0

0.2

3.4

3.7

Yogurt,low-fat

with

frui

t6oz.

0.5

1.0

0.5

170.

127

.527.9

0.954

5.9

7.8

0.9

13.8

14.6

Icecream

0.5cup

––

–66

.084

.185

.20.954

2.3

3.0

0.3

5.3

5.7

Totals

3.0

644.

6409.9

415.

40.954

22.5

29.6

3.3

52.1

55.4

ScaledtoU.S.

per-capitadaily

intakeofmilk,

cheeseandother

c

270

171.

717

4.0

0.954

9.4

12.4

1.4

21.8

23.2

Dai

ly P

ortio

nD

aily

am

ount

that

sup

plie

s rec

om-

men

ded

8 O

z. F

ish/

wee

k

Port

ions

Wei

ght (

g)

Fatfromfish

Meanof7common

speciesd

1.143oz

1.0

32.4

19.6

138

6.5

89.4

37.2

155

127

282

ScaledtoU.S.

per-capitadaily

consumptionof

finfis

he

–9.12

5.5

38.8

6.5

25.2

10.5

43.6

35.6

79.3

a Samefoodsandportionsasinthemodeleddietsin(Benbrooketal.,2013;Table1)andTable8.Calculationsusedailyservingweight,Table1amountsper100g,0.933gFApergdairyfat,andthefollow

-ingUSDAdata:wholemilk244g/cupand3.25%fat,cheese33.14%fat,low-yogurt1.41%fat,vanillaicecream11.1%fat.

b UsinganestimatedaverageDHAcontentof0.0006g/100gmilk,roughlyhalfofthe0.00106inTable1forthe249highestsamples.Forthisestimate,weassumedthatthe913unquantifiedsamples

containedanaverage0.0005g/100gmilk,halfoftheminimumquantifiedamountof0.001g/100gmilk.

c Table5in(Linetal.,2003).

d Table4in(Benbrooketal.,2013)(averageofcannedtuna,tilapia,halibut,sockeyesalmon,catfish,trout,&Atlanticsalmon).

e Table1in(EPA,2002).

16  |     BENBROOK Et al.

consumptiondoesnotsignificantly impactoveralldietaryratiosofLA/ALAorω- 6/ω-3.Benbrooketal.(2013)usedUSDAdataontheFAcontentsofsevencommonlyconsumedfishspecies(cannedtuna,tilapia,halibut,sockeyesalmon,catfish,trout,andAtlanticsalmon)tocalculate theamountsofLA,ALA,EPA,DPA,andDHAfrom8ouncesof fishperweek, theamount recommended in theDietary Guidelines for Americans (DHHS,2015).Thisweeklyamountofthe7fishspeciessuppliesbetween1(cannedlighttuna)to58mg/day(Atlanticsalmon)ofALA,withanaverageof20mg/day(Table4inBenbrooketal.,2013).ThisdailyamountofALAissmallcomparedtothe137mgin1.5cupsofgrassmilk,orthe162mgina1.5-ounceservingofcheddarcheesemadefromgrassmilk(seebelow).

Table9showstheamountsofkeyFAfromgrassmilkdairyprod-uctsinourdietarymodeling.Theseamountscomplementthedatapre-sentedinTable3ofBenbrooketal.(2013)forconventionalandorganicdairyproducts.Table9alsoshowstheFAcontentofthe7commonlyconsumedfishmentionedabove.Inadditiontotheserecommendedamountsofdairyand fish (DHHS,2015),Table9alsoshowstheFAcontentof the lower, actual per-capita consumptionsofdairyprod-ucts(270g/day)andfish(9.1g/day).Actual,averageper-capitaintakesare28%ofrecommendedforfishand42%ofrecommendedfordairyproducts(Lin,Variyam,Allshouse,&Cromartie,2003).

Basedonaverageper-capitaconsumptionofdairyproductsandfish,grassmilkdairyproductswouldsupply31timesmoreALAthanfish,4.5timesmoreLA,37%asmuchEPA,1.2timesmoreDPA,butonlyabout3%oftheDHA.Grassmilkdairyproductssupply29%asmuchtotal long-chainFA(EPA+DPA+DHA)asfish,withamuchloweroverallLA/ALAratio(0.95versus6.5).

4  | CONCLUSIONS

Wefindthatnearly100%grass-andlegume-basedfeedingoflac-tatingdairycowstypicallyyieldsmilkfatwithratiosofLA/ALAandω- 6/ω-3closeto1,comparedto5.8formilkfromcowsonconven-tionallymanaged farms, and 2.3 for typical (but not nearly 100%grass-fed)organicdairyfarms.Ourdietarymodelingscenariosshowthat replacing recommended daily servings of conventional dairyproductswithgrassmilkproductsandavoidingsomefoodshighinLA could substantially decrease historically high dietary ratios ofLA/ALA(andthusω- 6/ω-3ratios)fromcurrentvaluesof>10toaslowas3.1. Suchdecreaseshave several potential healthbenefits,including an enhanced ability to convert dietaryALA to the long-chainω-3FAsEPA,DPA,andDHA.Thesenutrientsaretypicallynotconsumedatrecommendedlevels(DHHS,2015),andareespeciallyneededduringpregnancyandlactation,bychildren,andbythema-jorityofAmericanswhoeatlittleornofish.

BecauseofthewidelyvaryingFAprofileofdairyproductsde-pending on production systems, coupled with large variations intheir fatcontent (whole, reducedfat,andfat free), thewidelydis-seminatedpromotionalclaim“milkismilk”(DairyReporter,2003)ishardtosquarewiththenatureofdairyproductscurrentlysoldandconsumedintheUnitedStatesandelsewhere.

Shifting lactating dairy cows to rations containing substantialportionsofforage-basedfeedsandlessgraindramaticallydecreasestheamountsofLAinmilk,whilealsoelevatinglevelsofALA,long-chainω-3FAs,andtotalCLA.TheseattainableshiftsintheFApro-fileofmilkanddairyproductsareoneofseveralpracticalwaystopotentiallyimprovethequalityofAmericandiets.Theshiftscanbeaccomplishedwith existing dairy industry infrastructure andwithlikelymodestimpactonfoodexpendituresafteratransitionperiod.

Improvedmessagesfromgovernmentdietaryrecommendations(Nissen,2016)andfoodlabelingreformsshould,overtime,increaseconsumerdemandforgrass-fedbeef,milk,andotherlivestockprod-ucts.DifferentiatingmoreclearlybetweentheFAsimplicatedornotimplicatedintherisksforobesity,CVD,andmetabolicsyndromewillbeanadditionalimportantstepforward.

Furtherresearchisneededtodeterminerealisticallyattainableshifts inFA consumption in thewidediversityof diets across theU.S.populationandtoassessthecostofalternativepathstowardhealthierfatintakes.Likewise,furtherresearchisneededtoidentifyprofitable and scalable changes in livestock feed rationsand foodmanufacturingthatwilllowerdietaryω- 6/ω-3ratiosandincreasein-takesoflong-chainω-3FAsandCLA.Improvedunderstandingoftherelationshipbetweenfatqualityandhealthoutcomeswillhelpguidelivestockanddairyfarmers,thefoodindustry,governmentagencies,scientists,andphysicianssearchingforpromisingwaystopromotepublichealth.

ACKNOWLEDG EMENTS

We appreciate the “jobwell done” by the CROPPCooperative re-gionalpoolstaffincollectingthebulk-tanksamplesandgettingthemtothelaboratory.WethankthetechnicalstaffatSilliker,Inc.,fortheirattentiontodetailandhelpworkingthroughtechnicaldetails.Wealsoacknowledge andhonor the commitment and skill of the grassmilkfarmersinCROPP,allofwhomfacedaraftofchallengesinconvertingtheiroperationstonearly100%forage-basedfeed.Theauthorsaregrateful for funding fromCROPPCooperative, Lafarge,Wisconsin;LowInputBreeds, Sixth Framework Programme for Research,TechnologicalDevelopmentandDemonstrationActivities,EuropeanCommunity(grantno.222623);andtheSheepdroveTrust,UK.

E THIC AL S TATEMENTS

Thisstudydoesnot involveanyhumanoranimal testing.Regardingconflicts of interest, CROPP Cooperative sells grassmilk via itsOrganicValley brand.MAL is the ExecutiveDirector of Research&Development and Quality Assurance at CROPP Cooperative. LPandSA-CareontheresearchandtechnicalservicesstaffofCROPPCooperative.BJHisfacultysupervisoroftheUniversityofMinnesotaWest Central Research andOutreach Center’s organic dairy,whichmarkets its milk through CROPP Cooperative and Organic Valley.CMBwasChiefScientistofTheOrganicCenter,2005–2012,fundedinpartbyCROPPCooperative;DRDwasaconsultanttosamecenter,2011–2012. CMBwas program leader for theMeasure toManage

     |  17BENBROOK Et al.

programatCenterforSustainingAgricultureandNaturalResources,Washington State University, 2012–2015, forwhich CROPPwas afunder;DRDwasaconsultanttothesameprogram,2012–2015.

ORCID

Donald R. Davis http://orcid.org/0000-0001-8343-1268

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APPENDIX Grass milk standards and oversightIn2016,acoalitionofdairy industryandcertificationorganiza-

tionsdefinedabroadnationalstandardfor“100%Grass-feddairy”(AGA, 2016). These organizations included Pennsylvania CertifiedOrganic, American Grassfed Association, Northeast OrganicFarmingAssociation[NewYork(NOFA-NY,2016)&Vermontchap-ters],MapleHillCreamery,andCROPPCooperative.Thatcoalitionadopted CROPP’s Grassmilk standards as part of its consensusstandard.CROPP’sinternalstandardscurrentlycomplywiththena-tional consensus, and the cooperative continues to takeanactiveroleinsolidifyingthelanguageandcertificationrequirementsasso-ciatedwithnearly100%grass-feddairyclaims.

Feeding requirementsInaddition to thedryhayand fermentedhay feeds thatareal-

lowedongrassmilkfarms,CROPP’sgrassmilkstandardallowsthefeedingofprebootcerealcrops.Theseplantsdonotcontainmatureseeds,onlyplantfoliageandstemmaterial.Thedistinctionbetweengrainandfoliagefromprebootcerealgraincropsisbasedonstarchcontent.Starchisassociatedwithanincreaseinω-6FAsinthefeedandintheresultingmilk.Somefarmerstransitioningcowstonearly100%-foragediets,or

managinggrassmilkherds,needtheoptiontoincludeprebootcereal

cropsintheirconservedfeeds.Suchfeedsincreasetheenergycon-tentof therationandhelpsustaincowbodycondition.Theyhelpminimizeω-6FAsintherationandprotecttheintegrityof“nograin”claimsingrassmilkmarketing.

Animal-care requirementsBeyondfatquality,consumersarealsoincreasinglyinterestedin

animalwelfare.Avarietyof indicatorsassociatedwithanimalwel-fareandherdhealtharefindingtheirwayintocertificationrequire-ments.Thesemetricsincludethebodyconditionoflactatingcows,outdoor access, use of antibiotics and hormones, physical altera-tions,lameness,livingconditions,andsourcesofanimalstress.CROPPgrassmilkfarmersaresubjecttothesameanimal-carere-

portingandherd-healthverificationsthatareusedthroughoutthecooperative. These include the National Milk Farmers AssuringResponsibleManagement(FARM)requirementsforbodycondition,lameness,access tooutdoorsandwater,ventilation,andhandling.Some CROPP animal-welfare requirements go beyond those im-posedbyFARM,suchasprohibitionofoxytocin,adrugusedtoas-sistincalving.On-siteauditsareperformedbystaffanimal-carespecialistsand

qualifiedfieldstaffatleasteverythirdyearthroughoutafarm’stran-sitionandparticipation in theprogram.Audit follow-upaddressesanyconcernsandsetsforthrequiredimprovements.Routinevisits

Slots,T.,Butler,G.,Leifert,C.,Kristensen,T.,Skibsted,L.H.,&Nielsen,J.H.(2009).Potentialstodifferentiatemilkcompositionbydifferentfeedingstrategies.Journal of Dairy Science,92,2057–2066.https://doi.org/10.3168/jds.2008-1392

Smit, L. A., Baylin, A., & Campos, H. (2010). Conjugated linoleic acidin adipose tissue and risk of myocardial infarction. American Journal of Clinical Nutrition, 92, 34–40. https://doi.org/10.3945/ajcn.2010.29524

Średnicka-Tober,D.,Barański,M.,Seal,C.J.,Sanderson,R.,Benbrook,C.,Steinshamn,H.,…Leifert,C.(2016a).HigherPUFAandn-3PUFA,conjugatedlinoleicacid,α-tocopherolandiron,butloweriodineandseleniumconcentrationsinorganicmilk:Asystematicliteraturere-viewandmeta-andredundancyanalyses.British Journal of Nutrition,115,1043–1060.https://doi.org/10.1017/s0007114516000349

Średnicka-Tober,D.,Barański,M.,Seal,C.J.,Sanderson,R.,Benbrook,C.,Steinshamn,H.,…Leifert,C. (2016b).Compositiondifferencesbe-tweenorganicandconventionalmeat;asystematicliteraturereviewandmeta-analysis.British Journal of Nutrition,115,994–1011.https://doi.org/10.1017/S0007114515005073

Stender,S.,&Dyerberg,J.(2003).Theinfluenceoftransfattyacidsonhealth,4thed.,DanishNutritionCouncil,Arrild,Denmark,Figure3.Retrieved fromhttps://sst.dk/publ/MER/2003/THE_INFLUENCE_OF_TRANS_FATTY_ACIDS_ON_HEALTH-FOURTH_EDITION2003.PDF

Stergiadis,S.,Leifert,C.,Seal,C.J.,Eyre,M.D.,Nielsen,J.H.,Larsen,M.K.,…Butler,B.(2012).EffectoffeedingintensityandmilkingsystemonnutritionallyrelevantmilkcomponentsindairyfarmingsystemsinNorthEastofEngland.Journal of Agricultural and Food Chemistry,60,7270–7281.https://doi.org/10.1021/jf301053b

Turpeinen,A.M.,Mutanen,M.,Aro,A.,Salminen,I.,Basu,S.,Palmquist,D. L., & Griinari, J. M. (2002). Bioconversion of vaccenic acid to

conjugated linoleic acid in humans. American Journal of Clinical Nutrition,76,504–510.https://doi.org/10.1093/ajcn/76.3.504

Tyburczy,C., Lock,A. L.,Dwyer,D.A.,Destaillats, F.,Mouloungui, Z.,Candy, L.,&Bauman,D.E. (2008).Uptake andutilizationofTrans octadecenoicacidsinlactatingdairycows.Journal of Dairy Science,91,3850–3861.https://doi.org/10.3168/jds.2007-0893

USDA. (2015). National nutrient database for standard reference, re-lease 28. U.S. Department of Agriculture (USDA). Retrieved fromhttp://ndb.nal.usda.gov

Vargas-Bello-Pérezz, E.,&Garnsworthy,P.C. (2013).Trans fatty acidsand their role inmilkofdairy cows.Ciencia e Investigación Agraria,40,449–473.https://doi.org/10.4067/S0718-16202013000300001

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SUPPORTING INFORMATION

AdditionalSupportingInformationmaybefoundonlineinthesup-portinginformationtabforthisarticle.

How to cite this article:BenbrookCM,DavisDR,HeinsBJ,etal.Enhancingthefattyacidprofileofmilkthroughforage-basedrations,withnutritionmodelingofdietoutcomes. Food Sci Nutr. 2018;00:1–20. https://doi.org/10.1002/fsn3.610

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by CROPP staff also help track and troubleshoot any problemsuniquetoagivenfarmorregion.Expertadvice isavailabletograssmilkfarmerstohelpthemim-

prove their forage quality and production levels, cow health, andprofitability. Regular testing of bulk-tankmilk FA levels is used tomonitor compliance with the nearly 100% forage-based feedrequirement.

Challenges and benefits of increasing reliance on forage-based feedsIncreasing forage-based feeds usually reducesmilk production.

Acrossfarmsofallsizesin2014,rolling-herd-average(RHA)305-daymilkproductionwas14,513lb/cowongrazing farms,14,758lbonorganicfarms,and21,862lbonconventionalfarms(APHIS,2014).WithinCROPP,organicdairiesfeeding~20%ofDMIfromgrains

have RHAs in the range 14,000 to 18,000lb/cow, while mostgrassmilkoperationsachieveRHAsintherange6,000to16,000lb/cow.During the spring and early summer, cows in early lactationtendtoproducemoremilk,makingitdifficulttomeetenergyneeds,especiallywithoutasupplementalenergysource(e.g.,molasses).Increased reliance on grazing and forage-based diets requires

careful management of soil fertility, pasture composition, forageproduction, and animal health (especially locomotion).Annual and

perennialforagecropsaremanagedcollectivelythroughouttheyeartoprovideforbothgrazingandconservedwinterfeed.Theextremerelianceonpastureandconserved foragesmaymake these farmslessresilientinthefaceofprolongeddroughtconditions.Itremainsto be seen whether availability and cost of high-quality, off-farmconservedforageputthesefarmsatincreasedriskduringperiodsofprolonged drought, leading to near-complete forage crop/pasturelosses.In addition to challenges, grass milk farmers receive benefits.

Theseincludeapricepremium(about15%onCROPPfarms),gen-erally reducedfeedcosts,andprotectionfrompricespikes inor-ganic grains and grain-based supplements. Agronomically, thesefarms generally havemore tolerance for wet springs, which candelayrow-cropfarmingpracticesandincreaseweedmanagementchallenges.Environmentalbenefitsalsoaccompanytheshifttogreaterreli-

anceonforage-basedfeedsandtheincreasedacreageinperennialgrasscommunities.Soilhealthtendstoimprove,becauseofreducedtillage and year-round perennial grass cover. This improvement insoilhealthgenerallyresultsinreducedsoilerosionandlesssedimentandnutrientrun-offintolocalwatershedscomparedtoconventionaltillage,orconservationtillage-basedcroprotations.

Graphical AbstractThecontentsofthispagewillbeusedaspartofthegraphicalabstractofhtmlonly.

Itwillnotbepublishedaspartofmainarticle.

Froma3-year,U.S.-widestudy,wereportthefattyacidprofilefrom1,163samplesofmilkfromnearly100%forage-fedcows(grassmilk)andcompareittoprofilesfromasimilarstudyofconventionalandorganicmilk.Wefindincreasesinomega-3fattyacidsandconjugatedlinoleicacidandadecreaseinomega-6fattyacids.Inmodelhumandiets,thesechangesaresufficienttosubstantiallyreducehistoricallyhighome-ga-6/omega-3ratiosinmoderndiets.