

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
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Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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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).