Supplementation of Oilseeds to an Herbage Diet High in Condensed Tannins Affects Methane Production with Minimal Impact on Ruminal Fermentation in Continuous Culture

  Ci a ion: Billman, E.D.; Dilla d, S.L.; Roca-Fe nández, A.I.; Sode , K.J. Supplemen a ion o Oilseeds o an He bage Die High in Condensed Tannins A ec s Me hane P oduc ion wi h Minimal Impac on Ruminal Fe men a ion in Con inuous Cul u e. Fe men a ion 2022,8, 109. h ps:// doi.o g/10.3390/ e men a ion8030109 Academic Edi o s: Yeong-Hsiang Cheng, Monika S e aniuk-Szmukie and Qing Zhang Recei ed: 7 Feb ua y 2022 Accep ed: 28 Feb ua y 2022 Published: 3 Ma ch 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). e men a ion A icle Supplemen a ion o Oilseeds o an He bage Die High in Condensed Tannins A ec s Me hane P oduc ion wi h Minimal Impac on Ruminal Fe men a ion in Con inuous Cul u e E ic D. Billman 1,2, S. Leanne Dilla d 1,3 , Ana Isabel Roca-Fe nández 1,4 and Ka hy J. Sode 1,* 1USDA-ARS, Pas u e Sys ems and Wa e shed Managemen Resea ch Uni , Uni e si y Pa k, PA 16802, USA; [email p o ec ed] (E.D.B.); [email p o ec ed] (S.L.D.); [email p o ec ed] (A.I.R.-F.) 2USDA-ARS, Coas al Plains Soil, Wa e , and Plan Conse a ion Resea ch Uni , Flo ence, SC 29501, USA 3Depa men o Animal Sciences and Depa men o C op, Soil, and En i onmen al Sciences, Aubu n Uni e si y, Aubu n, AL 36803, USA 4Depa amen o de P oducción Vege al y P oyec os de Ingenie ía, Escuela Poli écnica Supe io de Ingenie ía, Uni e sidad de San iago de Compos ela, 27002 Lugo, Spain *Co espondence: ka hy[email p o ec ed]; Tel.: +1-(814)-865-3158 Abs ac : Condensed annins (CT) ha e been obse ed o educe en e ic CH 4 p oduc ion when added o uminan die s. Howe e , high concen a ions o CT in o ages such as se icea lespedeza (SL; Lespedeza cunea a (Dum. Cou s.) G. Don) may dep ess nu ien diges ibili y. Oilseed c ops, high in lipid concen a ion, also educe en e ic CH 4 ia oxici y o me hanogenic bac e ia wi h less dep ession o nu ien diges ibili y. Howe e , i is unclea whe he combining hese wo eeds would esul in e en g ea e dec eases in CH 4 wi hou impai ing uminal e men a ion. This s udy used an in i o con inuous cul u e e men o sys em o de e mine i supplemen a ion o g ound oilseeds would u he educe en e ic CH 4 p oduc ion while imp o ing nu ien diges ibili y o high-CT o ages. The expe imen al design was a 4 × 4 La in squa e, wi h ou die s con aining (d y ma e basis) 45% o cha dg ass (OCH; Dac ylis glome a a L.), 45% se icea lespedeza (SL; Lespedeza cunea a (Dum. Cou s.) G. Don), and 10% oilseed supplemen s, using canola (CAN; B assica napus L.), soybean (SOY; Glycine max L.), sun lowe (SUN; Helian hus annuus L.), o a mix o all h ee species (MIX; in equal p opo ions). Fe men o s we e ed 82 g o d y ma e /d in ou equal eedings o e ou 10 d pe iods. Me hane was eco ded e e y 10 min, and e luen samples we e analyzed o pH, ola ile a y acids, d y ma e , o ganic ma e , c ude p o ein, neu al de e gen ibe , and acid de e gen ibe o de e mine appa en and ue nu ien diges ibili ies. The CAN, SUN, and MIX die s had g ea e concen a ions o c ude a (7–8 g/kg) han he SOY die (5.7 g/kg), which con ibu ed o he g ea e educ ion in en e ic CH 4 p oduc ion in hose die s (13–27 mg/d) compa ed o he SOY die (84 mg/d). Appa en and ue nu ien diges ibili ies we e no a ec ed by he addi ion o g ound oilseeds. While N in ake inc eased concomi an wi h c ude p o ein inc eases in he die s, he e we e no addi ional e ec s on N lows. While supplemen ing a high-CT die wi h any o he h ee oilseeds (canola, soybean, sun lowe , o a mix u e o he h ee oilseeds) educed o al CH 4 emission wi hou dep essing nu ien diges ibili y, canola and mixes con aining canola we e mos e ec i e. Fu he esea ch is needed in i o o e alua e whe he hese esul s ansla e o g ea e eed e iciency and animal p oduc ion. Keywo ds: o age; me hane; nu ien diges ibili y; oilseeds; annin 1. In oduc ion G eenhouse gases such as me hane (CH 4 ) a e well-es ablished con ibu o s o deg a- da ion o he ozone laye [ 1 ]. This has led o ising global empe a u es and exace ba ed clima e change o he pas se e al decades [ 2 ]. While he e a e mul iple sou ces o g een- house gas pollu ion, pa icula ly om ossil uels, li es ock CH 4 emissions ha e also d awn Fe men a ion 2022,8, 109. h ps://doi.o g/10.3390/ e men a ion8030109 h ps://www.mdpi.com/jou nal/ e men a ion Fe men a ion 2022,8, 109 2 o 12 subs an ial a en ion om he gene al public [ 3 ]. Da a om he pas decade indica e ha en e ic CH 4 p oduc ion by uminan li es ock comp ises mo e han 25% o all ag icul u al g eenhouse gas sou ces [ 4 ]. Fu he da a om he US and Aus alia indica ed ha bee ca le and dai y cows (Bos au us L.) a e he p ima y CH 4 -p oducing li es ock species [ 5 , 6 ]. The e o e, add essing CH 4 emissions by changing he die a y and nu i ional p o ile o hese uminan li es ock is o c i ical impo ance o educing ag icul u e’s con ibu ion o global clima e change. Nume ous eed addi i es and die a y supplemen s ha e been examined o he po- en ial educ ion in CH 4 emissions in uminan s [ 7 ]. Howe e , his educ ion is o en a he expense o animal pe o mance o eed e iciency. A class o polyphenolic compounds known as condensed annins (CT) exempli y his pa e n o subs an ially educing CH 4 emissions [ 8 ]. O he bene i s o CT include educed isk o bloa and in e nal pa asi e in ec ion [ 9 , 10 ]. Howe e , o ages con aining high le els o CT (>25 g/kg) ha e been shown o ha e de imen al e ec s on nu ien diges ibili y [ 11 ]. Condensed annins a e na - u ally p oduced by se e al leguminous o ages, including se icea lespedeza (SL; Lespedeza cunea a (Dum. Cou s.) G. Don), sain oin (Onob ychis spp. Mill.), and bi ds oo e oil (Lo us co nicula us L.), as de ense om he bi o y. The e is also a wide ange in he concen a ion o CT wi hin many o age legumes, which can al e he e icacy o educing CH 4 emissions o a ec eed diges ibili y [ 12 ]. P e ious wo k ound ha die s con aining 50% o DM as SL ( he highes CT o all o ages e alua ed) had he lowes CH 4 emissions bu also had he lowes DM, OM, NDF, and ADF diges ibili ies compa ed wi h legumes con aining lowe CT concen a ions [ 11 ]. This exempli ies he need o de elop uminan die s wi h balanced CT concen a ions o p o ide he maximum CH 4 educ ion while simul aneously minimizing impac s on diges ibili y [13]. Oilseeds such as soybean (Glycine max L.), canola (B assica napus L.), and sun lowe (Helian hus annuus L.) a e high in lipid con en , wi h o e 80% o hei ene gy ese es being in he o m o a y acids [ 14 ]. The c ude a (CF) o oilseeds ia we chemis y p o ides a measu e o lipid con en , which is he mos likely con ibu o o educing CH 4 emissions and can modi y milk a y acid p o iles in dai y cows [ 15 ]. Fo example, monounsa u a ed a y acids (palmi oleic (16:1) and oleic (18:1)), polyunsa u a ed a y acids (linoleic (18:2), linolenic (18:3)), and medium-chain a y acids a e oxic o he me hanogenic bac e ia in he umen [ 16 , 17 ]. Recen wo k has also shown ha lipid-encapsula ed annins om acacia sh ubs (Acacia pennine is DC) signi ican ly educed en e ic CH 4 p oduc ion a a simila a e o unencapsula ed annins, bu NDF and ADF diges ibili ies we e g ea e han in unencapsula ed annins [18]. Because oilseeds a e p ima ily composed o lipids, hey can be e ec i e a educ- ing en e ic CH 4 emissions in small p opo ions (<10%) o he die . Sode e al. [ 19 ] ound no educ ion in nu ien diges ibili y when laxseed, canola, o sun lowe we e ed in i o a 10% o o al DM o an he bage die , bu CH 4 was no e alua ed in ha s udy. Beauchemin e al. [20] showed a signi ican educ ion in CH4 p oduc ion when sun lowe , laxseed, and canola we e supplemen ed a 3.1 o 4.2% o die DM o lac a ing dai y cows ed TMR. Howe e , diges ible d y ma e (DM) in ake was educed by lax and sun lowe , bu he e was no educ ion in diges ible DM in ake wi h canola. Recen wo k wi h a simila oilseed, hemp (Canabis sa i a L.), has also shown he e icacy o high-lipid seed meal in educing en e ic CH 4 wi h less han 11 mL CH 4 /g/day and o ganic ma e diges ibili ies anging om 30 o 40% [21]. While bo h CT and oilseeds ha e independen ly been e ec i e a educing en e ic CH 4 emissions in ca le, he e has been li le examina ion o hei po en ial addi i e e ec s on CH 4 emissions o uminal e men a ion. I ed in andem wi h CT, he p ope ies o lipids ound in oilseeds may educe he nega i e e ec s o CT on he diges ibili y o consumed o ages while p o iding g ea e educ ions in en e ic CH 4 emission. Howe e , his hypo hesis has ye o be e alua ed. The e o e, he objec i e o his s udy was o e alua e he e icacy o eeding g ound oilseeds o h ee species wi h die s con aining SL o educe en e ic CH 4 emissions and minimize nega i e e ec s on die diges ibili y. We hypo hesized Fe men a ion 2022,8, 109 3 o 12 ha oilseed species con aining he g ea es lipid con en would exhibi simila educ ions in CH4emissions while ha ing he leas ad e se impac on nu ien diges ibili y. 2. Ma e ials and Me hods A umen luid dono cow was housed a he Pennsyl ania S a e Uni e si y Dai y Re- sea ch Fa m (Uni e si y Pa k, PA, USA) and managed unde Pennsyl ania S a e Uni e si y Ins i u ional Animal Ca e and Use Commi ee guidelines (IACUC; p o ocol no. 46212). 2.1. Si e, Expe imen al Design, and Die s This s udy was conduc ed a he USDA-ARS Pas u e Sys ems and Wa e shed Manage- men Resea ch Uni (Uni e si y Pa k, PA, USA) om Sep embe o No embe 2016. The o cha dg ass (OCH) was g own and ha es ed om a 3 y s and loca ed a he Russell E. La son Ag icul u al Resea ch Cen e (Rock Sp ings, PA, USA; 40 ◦ 40 0 00 00 N, 77 ◦ 56 0 24 00 W). Vege a i e biomass was ha es ed in summe 2016, eeze-d ied o p ese e nu i ional alue, and g ound in a Wiley Mill (Thomas Scien i ic, Swedesbo o, NJ, USA) o pass a 2 mm mesh sc een. The biomass o he SL cul i a ‘AU G aze ’ (Sims B o he s, Inc., Union Sp ings, AL, USA) was ha es ed in July 2015 om a 3 y monocul u e, g own a he Uni- e si y o Ken ucky’s Spindle op Resea ch Fa m (Lexing on, KY; 30 ◦ 7 0 40 00 N, 84 ◦ 29 0 39 00 W). The ha es o SL biomass occu ed a he lowe ing s age, and ma e ial was eeze-d ied and g ound in a Wiley Mill o a 2 mm pa icle size. Oilseed c op seeds we e om he ollowing cul i a s: ‘Inspi a ion’ canola (Rubisco Seeds, Philpo , KY, USA), ‘Pe edo ic’ sun lowe (Hancock Seed Company, Dade Ci y, FL, USA), and ‘S onewall’ soybean (Han- cock Seed Company, Dade Ci y, FL, USA). Seeds we e no eeze-d ied bu we e g ound o 2 mm ineness in a cyclone mill (UDY Co p., Fo Collins, CO, USA). The 2 mm ineness was selec ed o p e en oil pa icles om o ming when g inding he oilseeds. Thus, all ma e ial ( o age and oilseeds) we e g ound o his ineness. G ound whole seeds we e used o main ain he a y acid composi ion, compa ed o seed meals which ha e been ex ac ed. A 4 × 4 La in squa e design was used o andomize ou die s wi hin each o ou 10 d pe iods. Each die comp ised iden ical basal o ages, wi h 45% OCH and 45% SL (high-CT legume), wi h he emaining 10% comp ising one o h ee oilseed supplemen s as ollows: 10% canola seed (CAN), 10% soybean seed (SOY), 10% sun lowe seed (SUN), and an e en mix (3.33% each) o canola, soybean, and sun lowe (MIX). 2.2. Con inuous Cul u e Sys em A ou -uni single- low con inuous cul u e in i o e men a ion sys em (Applikon Bio echnology, B.V. Schidam, The Ne he lands) was used o simula e umen diges ion. De ails o his sys em can be ound in Dilla d e al. [ 22 ]. Fe men o s we e ed ou imes daily (20.5 g/ eeding) a 07:30, 10:30, 14:00, and 19:00 h, wi h daily DM ed ixed a 82 g/d. The diges a e en ion ime and bu e dilu ion a e we e adjus ed daily ia egula ion o e luen emo al and bu e low and we e main ained a 24 h and 10%/h, espec i ely. Rumen luid and diges a we e collec ed om a is ula ed, nonlac a ing, nonp egnan , 4-yea -old Hols ein cow (794 kg body weigh ) ed a die o silage, hay, and g ain (3:1 o age- o-concen a e a io, ad libi um). On he mo ning o d 1 o each pe iod, app oxima ely wo hou s a e eeding, 7 L o umen luid was collec ed om he dono cow using a hand pump and placed in o plas ic ai igh con aine s ha we e p e-wa med o 39 ◦ C. Solid umen diges a (o he 3:1 o age- o-concen a e die ) was collec ed by hand om he en al, cen al, and do sal a eas o he umen. Wi hin 30 min o collec ion, umen luid and solid diges a we e anspo ed back o he USDA-ARS lab o p epa e o ans e in o e men o essels. Fluid was i s s ained h ough ou laye s o cheeseclo h and 1.5 L was pou ed in o each o he ou p e-wa med e men o essels. Each e men o was hen inocula ed wi h 32 g o solid diges a. The low o CO 2 was ini ia ed a 20 mL/min o 1.5 h ollowing inocula ion o c ea e anae obic condi ions in each essel and hen lowe ed o 1 mL/min o he du a ion o he expe imen al pe iod. Fe men a ion 2022,8, 109 4 o 12 Each 10 d pe iod consis ed o se en days o die adap a ion ollowed by h ee days o sampling. Fo each e men o , daily e luen was emo ed, pumped in o a 4 L s o age con aine , and cooled o 4 ◦ C o inhibi u he mic obial e men a ion. The con en s o hese 4 L con aine s we e weighed du ing days 1–7 o calib a e e luen emo al o app oxima ely 4 L/d and hen disca ded. A he 10:30 h eeding (second daily eeding) on days 8–10, daily e luen con en s we e weighed, mixed wi h a blende (model 38 LL52 Wa ing; To ing on, CT, USA), and hen subsampled. Fi s , 50 mL o e luen was s ained h ough 8 laye s o cheeseclo h. Then, 2 con aine s con aining 3 mL o 25% m-phospho ic acid we e each illed wi h 15 mL o s ained e luen o de e mina ion o VFA [ 23 ] and NH 3 -N [ 24 ] concen a ions. Finally, 1 L/d o blended e luen was collec ed and composi ed ac oss all h ee sampling days (3 L o al) o assessmen o e luen nu i i e alue pa ame e s. Composi ed e luen samples we e hen eeze-d ied, g ound o pass h ough a 1 mm sie e, and s o ed in sealed plas ic bags o la e analyses. 2.3. Me hane Quan i ica ion Du ing each 10 d pe iod, CH 4 measu emen s we e aken a 10 min in e als on each e men o essel using a pho oacous ic gas moni o (LumaSense Technologies Inc., San a Cla a, CA, USA) connec ed o a mul ipo sample (CAI, Inc., O ange, CA, USA) ha con olled he low o gas om he headspace o each essel. These eadings esul ed in six eadings/ e men o /h, wi h a o al o 2880 eadings pe e men o o e each 10 d pe iod. Each 10 min cycle o he gas moni o equi ed 140 cm 3 o he 1500 mL o headspace gas in he essel. Daily CH4p oduc ion was calcula ed wi h he ollowing equa ion: ∑[CH4 olumea−CH4 olumeb](1) whe e CH 4 olume a was de ined as he headspace olume mul iplied by he CH 4 con- cen a ion, and CH 4 olume b was de ined as he CH 4 olume 10 min p io o measu ing olumea, summed o e each o he h ee 24 h sampling days. 2.4. Nu ien Analyses Fo age and seeds we e analyzed ia we chemis y (Dai y One, I haca, NY, USA) o he ollowing p ocedu es: DM (me hod 930.15; [ 25 ]), CP (me hod 990.03; [ 25 ]), RDP (Co nell S ep omyces g iseus enzyma ic diges ion; [ 26 ]), aNDF (Ankom Technology me hod 6), ADF [ 27 ], lignin [ 27 ], and CF (me hod 2003.05; [ 25 ]). Non- ib ous ca bohyd a e was calcula ed as g/kg NFC =100 −[CP(g/kg)+NDF(g/kg)+CF (g/kg)+ash (g/kg)] (2) To al diges ible nu ien s we e calcula ed om o mulas de i ed om Weiss [ 28 ]. Fo age and seed samples we e sen o he Depa men o Plan s, Soils, and Clima e a U ah S a e Uni e si y (Logan, UT, USA), whe e CT concen a ions we e quan i ied using a bu anol-HCl-i on assay [29]. E luen samples we e analyzed o DM and OM (me hods 930.15 and 942.05; [ 25 ]), CP (mic o-Kjeldahl diges ion using 75 mL calib a ed ubes wi h CuSO 4 ca alys ; me hod 976.06; [ 25 ]), and aNDF [ 27 ] using α -amylase and sodium sul i e (inclusi e o ash). Concen- a ions o o al and indi idual VFA we e de e mined using gas ch oma og aphy (Va ian 330 Gas Ch oma og aph (FID De ec o ), Va ian 4290 In eg a o ; Supelco, 1975, modi ied o use an 80/120 Ca bopack B-DA/4% Ca bowax 20 M column) a he Rumen Fe men a ion P o iling Labo a o y a Wes Vi ginia Uni e si y (Mo gan own, WV, USA). 2.5. S a is ical Analyses Da a we e analyzed as a 4 × 4 La in squa e, using PROC GLIMMIX in SAS 9.4 (SAS Ins i u e, Ca y, NC, USA). Repea ed measu es wi h an au o eg essi e co a iance s uc u e we e used o he esponse a iables CH 4 concen a ion, VFA concen a ions, and e men o pH le els, as hese alues we e eco ded daily o e he h ee sampling days. Fo hese Fe men a ion 2022,8, 109 5 o 12 a iables, pe iod and die we e conside ed ixed e ec s, while e men o and sampling day we e conside ed andom e ec s. The ollowing model was used o hese a iables: Yijkl =µ+Pi+Fj+Dk+PDik +Tl+PTil +ε(ijkl)(3) whe e µ = popula ion mean, P i = mean e ec s o he i h pe iod, F j = mean e ec s o he j h e men o , D k = mean e ec s o he k h sampling day, T l = mean e ec s o he l h die , and ε(ijkl) = expe imen al e o . A second model was used o assess diges ibili y and N me abolism da a, wi hou epea ed measu es, as alues we e only assessed on a pe -pe iod basis. Again, pe iod a die was a ixed e ec , while e men o was conside ed a andom e ec : Yijkl =µ+Pi+Fj+Tk+PTik +ε(ijk)(4) whe e µ = popula ion mean, P i = mean e ec s o he i h pe iod, F j = mean e ec s o he j h e men o , Tk= mean e ec s o he k h die , and ε(ijk)= expe imen al e o . Fo all s a is ical analyses, an alpha le el o α = 0.05 was used o de e mine signi ican di e ences, while ends we e es ablished a an alpha le el o 0.10 > α > 0.05. A e conduc ing analysis o a iance, no pe iod × die in e ac ions we e ound o any a iables es ed; he e o e, only main e ec s a e p esen ed. 3. Resul s 3.1. Die Composi ion and Diges ibili ies The chemical composi ions o he ing edien s and die s a e p esen ed in Table 1. S a is ical compa ison o die s was no conduc ed because he nu ien composi ion o die s was based on pooled samples. The CT concen a ion o he SL o age was 149 g/kg DM , compa ed o 3.7 g/kg DM o OCH, and less han 1 g/kg DM o he g ound canola, soybean, and sun lowe oilseeds. As all die s con ained he same amoun o SL, inal CT concen a ions we e iden ical among die s (68.9 g/kg DM). C ude a concen a ions o canola and sun lowe we e app oxima ely wice he nume ic alue o soybean. This esul ed in he SOY die ha ing only 5.7% CF, compa ed o 7–8% CF in he CAN, SUN, and MIX die s. While he g ound soybean seed used o o mula e he die s was g ea e in CP and RDP and had lowe aNDF, ADF, and lignin han canola o sun lowe , he e we e no dis inc nume ical ends obse ed among he inal die s o any o he p o ein o ibe pa ame e s. This was a ibu ed o hese componen s comp ising only 10% o each die . Table 1. Chemical composi ions o ing edien s and die s ed du ing con inuous cul u e e men a ion. Fo age Die s 1 I em Uni O cha dg ass Se icea Lespedeza Canola Soybean Sun lowe CAN SOY SUN MIX CP g/kg DM 348 178 246 406 139 261 277 251 263 RDP g/kg CP 810 323 651 807 750 574 589 584 581 aNDF g/kg DM 412 460 416 172 283 434 410 421 421 ADF g/kg DM 222 324 290 153 243 275 261 270 268 Lignin g/kg DM 65 100 78 22 94 82 76 84 81 NFC 2g/kg DM 118 271 - 154 139 175 190 189 175 NELMcal/kg DM 1.5 1.3 3.6 3.2 2.6 1.7 1.6 1.5 1.6 C ude Fa g/kg DM 50 29 437 210 404 79 57 76 70 CT 3g/kg DM 3.7 149.2 0.8 0.3 0.3 68.9 68.9 68.9 68.9 1 All die s comp ised 45% o cha dg ass and 45% se icea lespedeza. The emaining 10% was as ollows: CAN = 10% g ound canola seed, SOY = 10% g ound soybean seed, SUN = 10% g ound sun lowe seed, and MIX = 3.33% g ound canola, 3.33% g ound soybean, and 3.33% g ound sun lowe seed. 2 Calcula ed as NFC (%) = 100 − [CP (%) + aNDF (%) + c ude a (%) + ash (%)]. 3CT: condensed annins. No di e ences we e obse ed (p> 0.10) in ei he appa en o ue DM and OM diges ibili ies among oilseed die s (Table 2). Addi ionally, appa en aNDF and ADF di- ges ibili ies we e simila be ween die s (p> 0.10). Fe men a ion 2022,8, 109 6 o 12 Table 2. Nu ien diges ibili ies o ou high-condensed- annin he bage die s con aining g ound canola, soybean, sun lowe seed, o a mix o he h ee oilseeds du ing con inuous cul u e e men a ion. Pa ame e Die 1 SEM p-Value CAN SOY SUN MIX Appa en Diges ibili y OM 0.39 0.41 0.40 0.37 0.044 >0.10 DM 0.39 0.42 0.41 0.39 0.033 >0.10 aNDF 0.52 0.60 0.453 0.54 0.032 >0.10 ADF 0.31 0.52 0.37 0.43 0.067 >0.10 T ue Diges ibili y OM 0.81 0.81 0.78 0.84 0.039 >0.10 DM 0.65 0.67 0.63 0.68 0.032 >0.10 1 All die s comp ised 45% o cha dg ass and 45% se icea lespedeza. The emaining 10% was as ollows: CAN = 10% g ound canola, SOY = 10% g ound soybean, SUN = 10% g ound sun lowe , and MIX = 3.33% g ound canola, 3.33% g ound soybean, and 3.33% g ound sun lowe seed. 3.2. Me hane P oduc ion, VFAs, and pH To al daily CH 4 p oduc ion was he lowes (p< 0.001) in he MIX and CAN die s, in e media e in he SUN die , and he highes in he SOY die (p< 0.001; Table 3). P oduc ion o CH 4 pe g am o OM and aNDF was g ea e (p= 0.01) o he SOY die , compa ed o all h ee o he die s. These e ec s we e magni ied when he amoun o CH 4 p oduced pe g am o diges ible OM and aNDF was examined. Bo h pa ame e s esul ed in he MIX, CAN, and SUN die s ha ing less CH 4 p oduced pe g am o diges ible OM (p= 0.01) o aNDF (p= 0.02) han he SOY die . The SOY die had he g ea es (p< 0.001) o al VFA concen a ion (Table 3). Mola p opo ions o ace a e we e he g ea es (p< 0.001) o SOY and he lowes o CAN and MIX. Mola p opo ions o p opiona e we e he g ea es (p= 0.001) o he CAN and MIX die s and he lowes (p= 0.001) o he SOY die . Bu y a e p opo ions we e he g ea es (p< 0.01) in he MIX die . The CAN and SOY die s had he g ea es (p = 0.01) p opo ions o isobu y a e, while he MIX die had he lowes . Mola p opo ions o ale a e we e he g ea es (p< 0.001) o he CAN and MIX die s and he lowes o he SOY die . The CAN and MIX die s had he g ea es (p< 0.001) p opo ions o ale a e. Iso ale a e was unde ec able o all die s (da a no shown). The CAN and MIX die s had he lowes ( p< 0.001 ) a ios o ace a e/p opiona e (A/P), ace a e and bu y a e/p opiona e (A + B/P), and ace a e and bu y a e/p opiona e and ale a e (A + B/P + V), while he SOY die had he g ea es (p< 0.001) a io in all h ee pa ame e s. The SOY die had he lowes (p< 0.001) mean, maximum, and minimum e men o pH (Table 3). The MIX die had he g ea es (p< 0.001) mean and minimum pH, while he MIX, CAN, and SUN die s had he g ea es maximum pH and minimum pH (Table 3). The SOY die had he g ea es N in ake, ollowed sequen ially by MIX, CAN, and SUN (p< 0.001; Table 3). No o he pa ame e s o N me abolism we e a ec ed (p> 0.10) by he oilseeds added o he high-CT basal die . Fe men a ion 2022,8, 109 7 o 12 Table 3. Me hane (CH 4 ) ou pu , ola ile a y acid (VFA) p oduc ion, and e men o pH o ou high-condensed- annin he bage die s con aining g ound canola, soybean, sun lowe seed, o a mix o he h ee oilseeds du ing con inuous cul u e e men a ion. Mola p opo ions o speci ic VFAs a e gi en as mols pe 100 mols o o al VFAs. Die 1 I em Uni CAN SOY SUN MIX SEM p-Value CH4p oduc ion To al CH4mg/d 17.9 c84.3 a27.4 b13.4 c4.41 0.01 CH4/g OM mg/g 0.2 b1.1 a0.4 b0.2 b0.79 0.01 CH4/g aNDF mg/g 0.5 b2.5 a0.8 b0.4 b1.84 0.01 CH4/g diges ible OM mg/g 0.4 b1.6 a0.6 b0.3 b1.02 0.01 CH4/g diges ible aNDF mg/g 1.2 b4.1 a2.3 ab 0.7 b2.29 0.02 VFA To al mmol/L 38.33 b46.04 a37.52 b38.73 b1.056 <0.001 Ace a e (A) mol/100 mol 66.7 c68.1 a67.3 b66.4 c0.20 <0.001 P opiona e (P) mol/100 mol 22.8 a21.5 c22.3 b22.8 a0.18 <0.001 Bu y a e (B) mol/100 mol 8.7 b8.7 b8.8 b9.1 a0.12 0.01 Isobu y a e mol/100 mol 0.3 a0.4 a0.3 b0.2 c0.038 0.01 Vale a e (V) mol/100 mol 1.5 a1.3 c1.4 b1.5 a0.029 <0.001 A/P mol/100 mol 2.93 c3.18 a3.02 b2.92 c0.032 < 0.001 (A + B)/P mol/100 mol 3.31 c3.59 a3.42 b3.32 c0.036 <0.001 (A + B)/(P + V) mol/100 mol 3.12 c3.38 a3.22 b3.11 c0.031 <0.001 pH Mean pH 6.96 b6.83 c6.95 b7.01 a0.022 <0.001 Max pH 7.50 a7.33 b7.52 a7.53 a0.035 <0.001 Min pH 6.70 b6.58 c6.67 b6.77 a0.015 <0.001 Ni ogen me abolism N in ake g/d 4.21 c4.42 a4.07 d4.24 b0.01 <0.001 NH3-N mg/dL 17.3 18.4 17.4 16.6 1.13 >0.10 N lows To al N g/d 2.6 2.4 2.5 2.3 0.26 >0.10 NH3-N g/d 0.74 0.78 0.74 0.71 0.034 >0.10 Non-NH3-N g/d 1.8 1.6 1.7 1.6 0.28 >0.10 a–d Means wi hin a ow wi h di e en supe sc ip s di e (p< 0.05). 1 All die s comp ised 45% o cha dg ass and 45% se icea lespedeza. The emaining 10% was as ollows: CAN = 10% g ound canola seed, SOY = 10% g ound soybean, SUN = 10% g ound sun lowe seed, and MIX = 3.33% g ound canola, 3.33% g ound soybean, and 3.33% g ound sun lowe seed. 4. Discussion 4.1. Impo ance o Die Composi ion and Diges ibili ies The obse ed p opo ions o CF o indi idual oilseeds (Table 1) ollowed hose es ablished by Liu e al. (2016) [ 30 ]. This esul ed in he SOY die ha ing only 57 g CF/kg DM, compa ed o 70–79 g CF/kg DM in he CAN, SUN, and MIX die s. While he g ound soybean used o o mula e he die s was g ea e in CP and RDP and had lowe aNDF, ADF, and lignin han canola o sun lowe , he e we e no dis inc nume ical ends obse ed among he inal die s o any o he p o ein o ibe pa ame e s. This was a ibu ed o he oilseed supplemen s comp ising only 10% o each die . Fo his s udy, SL was he only app eciable sou ce o CT in he componen s o any die . Thus, he addi ion o he oilseeds, alone, did no a ec he diges ibili ies o a high-CT die (Table 2) as he o al CT concen a ion in he die was nume ically simila ac oss all die s. Se icea lespedeza is known o ha e one o he highes concen a ions o CT among o age legumes [ 31 ], which ou wo k suppo s (149.2 g CT/kg). In ano he con inuous cul u e e men o s udy, Roca-Fe nández e al. [ 11 ] diges ed legumes (al al a (Medicago sa i a L.), bi ds oo e oil (Lo us co nicula us L.), c own e ch (Secu ige a a ia L.), and SL) ha anged om 2.3 o 148 g/kg o DM in CT and ound ha as he CT in he die inc eased, CH 4 dec eased, bu he e was also a co esponding dec ease in nu ien diges ibili y. Wo k Fe men a ion 2022,8, 109 8 o 12 wi h lambs ed acacia (Acacia cyanophylla Lindl.) lea es (app oxima ely 50 g CT/kg) sup- plemen ed wi h soybean meal nea ly doubled a e age daily gains while main aining CP diges ibili ies in excess o 0.70 [ 32 ]. The e o e, oilseeds could po en ially imp o e he diges ibili y o a die wi h a lowe concen a ion o CT han he SL die used in his s udy. Howe e , his migh necessi a e di e en p ocessing o he oilseeds (i.e., using seed meal o concen a ed oil ex ac s ins ead o g ound seeds). 4.2. E ec s on En e ic Me hane P oduc ion, VFAs, Fe men o pH, and N Me abolism The educ ions in CH 4 p oduc ion p o ided by he high-CT o age a e e en g ea e han hose epo ed by Roca-Fe nández e al. [ 11 ] when oilseed supplemen s we e added o he die (Table 3). In he p esen s udy, soybean was no as e ec i e in educing CH 4 p oduc ion as canola, sun lowe , o a mix u e o all h ee species; he SOY die p oduced h ee o ou imes he amoun o CH 4 (~84 mg/d) as he CAN, SUN, and MIX die s. This was likely due o he lowe concen a ion o CF p esen in he g ound soybean used in he SOY die , compa ed o he concen a ions in he g ound canola and sun lowe seed. I is impo an o no e ha soybean is he mos eadily a ailable oilseed a ailable o animal eed supplemen a ion and is commonly used as a p o ein sou ce [ 33 ]. P oduce s in e es ed in lowe ing CH 4 emissions om hei bee ca le o dai y cows should conside ei he canola, sun lowe , o oilseed mixes. This will assis in minimizing he CH 4 impac o a ming sys ems and con ibu e o educed g eenhouse gas emissions. Howe e , hese o he sou ces a e likely mo e expensi e han soybeans, and no as eadily a ailable; he e o e, a me s would need an economic incen i e o inco po a e hese supplemen s in o he d a ions. The c ude a concen a ion in he die has been p e iously shown o educe en e ic CH 4 emissions in uminan s [ 34 ]. The e was a end o CH 4 p oduc ion being nega i ely co ela ed wi h CF (Pea son co ela ion coe icien = − 0.81, p= 0.08, da a no shown). The g ea e CH 4 p oduc ion wi h he SOY die could be a ibu ed o he lowe p opo ion o unsa u a ed a y acids in soybean (~85%; [ 14 ]), compa ed o ha o canola (~92%; [ 35 ]). Unsa u a ed a y acids we e ound o educe he p oduc ion o CH 4 in uminan s as a back as he 1960s, wi h an inc easing concen a ion o hese a y acids causing u he educ ions in CH 4 [ 36 ]. This was a ibu ed o unsa u a ed a y acids compe ing o H + ions in he umen du ing hyd ogena ion, which would o he wise be used o o m CH 4 [ 37 ]. While mo e ecen wo k has ocused on he addi ion o concen a ed soybean oil o canola oil o a ions, ou wo k sugges s ha a educ ion in CH 4 in he CAN, SUN, and MIX die s was s ill impa ed when hese oilseeds we e simply ed as g ound seed. When oilseeds a e combined wi h CT in he die , hese esul s indica e ha CH 4 emissions om uminal e men a ion can be signi ican ly educed, i.e., <30 mg CH 4 /d (CAN, SUN, o MIX die s) s. >200 mg CH 4 /d (50% o cha dg ass, 50% al al a die ) when no CT o g ound oilseeds a e ed [11]. The VFA esul s (Table 3) a e impo an o no e o se e al easons. Fi s , he lowe o al VFA concen a ions ound in he CAN, SUN, and MIX die s could be a ibu ed o he g ea e concen a ion o CF in hose die s (7–8 g/kg), compa ed o he SOY die (5.7 g/kg). This sugges s ha g ea e amoun s o CF may ha e a nega i e ela ion o VFA p oduc ion, bo h on an indi idual and o al VFA basis. Wo k om he 1960s and 1970s ound ei he (a) no e ec o eeding a g ea e a con en on VFA p oduc ion [ 38 , 39 ] o (b) con lic ing esul s o ou indings, i.e., inc eased ace a e, bu dec eased p opiona e, in high- a die s [ 40 ]. Howe e , hese olde ials used ex ac ed and pu e seed oil, molasses, o o he high- a plan p oduc s. Mo e ecen wo k om Paula e al. [ 41 ] ound ha some indi idual VFAs we e dec eased wi h canola meal. Howe e , hese e ec s we e no as consis en as he esul s o he p esen s udy, likely due o he ex ac ion p ocess emo ing much o he a con en . A ecen in i o s udy ound a signi ican dec ease in VFA p oduc ion in esponse o inc easing CT in o age die s con aining legumes di e ing in CT, he highes o which was a 50:50 OCH/SL die [ 11 ]. The esul s o he cu en s udy show ha he addi ion o 10% oilseeds ha con ain high CF concen a ions (CAN, SUN, o MIX s. SOY die s) o 45:45 OCH/SL die s migh also be p o oking signi ican declines in uminal VFA Fe men a ion 2022,8, 109 9 o 12 p oduc ion. Depending on wha VFAs a e a ec ed, his may cause de imen al e ec s on milk p oduc ion o milk quali y componen s o dai y o educe li e-weigh gains in bee p oduc ion. The e men o pH da a (Table 3) we e simila o hose om a p e ious s udy by Kowalczyk e al. [ 42 ] whe e sequen ial a es o allow (high in lipid concen a ion) we e added as supplemen s o uminan die s. Howe e , such small di e ences likely would no be biologically signi ican [ 43 ]. I should be no ed ha he high-CT die s in he p esen s udy had a mean pH ha was sligh ly mo e alkaline (6.9–7.0) han no mal o age die s (~6.5–6.7) [ 44 ]. This accoun s o he inc eased ace a e p oduc ion compa ed o p opiona e ha was obse ed ac oss all ou die s. Howe e , he small biological di e ence in pH likely would no be he cause o he di e ences in he VFA concen a ion among die s, as all pH alues we e well wi hin he no mal ange o op imal umen unc ion [45]. The addi ion o g ound oilseeds o he basal die had no impac on he N lows (Table 3). Howe e , he g ea e CP concen a ions in ce ain oilseed species, e.g., soybean, did a ec he amoun o N p esen in each die . This may be due o soybean p o iding a high-quali y p o ein sou ce o N o enhance he supply o CP and RDP om p e o med AA and pep ides in uminal e men a ion [ 8 ]. The g ound soybean used in his in i o wo k had less CP han no mal soybean c ops, 40% compa ed o 50–55% [ 46 ], likely due o S onewall being an olde , public elease cul i a [ 47 ] ha has no been selec ed o a g ea e CP concen a ion. I a di e en sou ce o g ound soybean was used o his s udy, i is likely ha N lows in ou in i o sys em would ha e di e ed be ween die s, which is suppo ed by he indings o [ 48 ] and esea ch conduc ed by [ 49 ]. Ano he po en ial eason o he minimal e ec o oilseeds was ha hey comp ised only 10% o he die . This limi ed he di e ences in die a y CP ha we e in each die , because OCH and SL we e he p edominan componen s (45% each) o he basal die s. Based on conclusions om Roca-Fe nández e al. [ 11 ], i is likely ha al e ing he sou ce o CT would ha e mo e e ec on N me abolism and lows, a he han which g ound oilseed was added o he die . 5. Conclusions The addi ion o he oilseeds canola, sun lowe , and a mix u e o canola, sun lowe , and soybean o an he bage die high in CT educed CH 4 p oduc ion compa ed o he addi ion o only soybean. This was a ibu ed o he g ea e CF concen a ions ound in canola and sun lowe compa ed o soybean, which equa ed o g ea e uminal a y acid concen a ions ha a e an agonis ic o en e ic me hanogenesis. A he same ime, nu ien diges ibili y was no dep essed, sugges ing ha animal pe o mance may no be impai ed. To al VFA p oduc ion was, howe e , no ably educed in he CAN, SUN, and MIX die s, which was also a ibu ed o he g ea e CF p esen in hose die s and may nega i ely impac milk p oduc ion, milk componen s, and li e-weigh gain. Fu u e wo k o assess di e en le els, as well as di e en combina ions, o oilseeds supplemen ed o high-CT die s on uminal e men a ion, CH4p oduc ion, and animal pe o mance is needed. Au ho Con ibu ions: Concep ualiza ion, A.I.R.-F., S.L.D. and K.J.S.; me hodology, A.I.R.-F., S.L.D. and K.J.S.; alida ion, A.I.R.-F., S.L.D., E.D.B. and K.J.S.; o mal analysis, E.D.B. and A.I.R.-F.; in es i- ga ion, A.I.R.-F., S.L.D.; esou ces, K.J.S.; da a cu a ion, A.I.R.-F., S.L.D., E.D.B.; w i ing—o iginal d a p epa a ion, E.D.B., A.I.R.-F., S.L.D. and K.J.S.; w i ing— e iew and edi ing, E.D.B., A.I.R.-F., S.L.D. and K.J.S.; isualiza ion, A.I.R.-F., S.L.D. and K.J.S.; supe ision, K.J.S.; p ojec adminis a ion, K.J.S.; unding acquisi ion, A.I.R.-F., S.L.D. and K.J.S. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This wo k was pa ially unded by USDA-NIFA-OREI (P ojec Numbe : 8070-21000-008-16) and Xun a de Galicia-Plan 12 C-Modali y A (P ojec Numbe : ED481B-2014/021-0). USDA is an equal oppo uni y p o ide and employe . Ins i u ional Re iew Boa d S a emen : The animal p o ocol used in his s udy was app o ed by he Pennsyl ania S a e Uni e si y Ins i u ional Animal Ca e and Use Commi ee guidelines (IACUC; p o ocol no. 46212).