Ruminal mic obio a is associa ed wi h eed-e ficiency
pheno ype o a ening bulls ed high-concen a e die s
S. Cos a-Rou a
A
, D. Villalba
A,D
, M. Blanco
B,C
, I. Casasús
B,C
,
J. Balcells
A
and A. R. Se adj
A
A
Depa amen de Ciència Animal, Uni e si a de Lleida, A inguda Alcalde Ro i a Rou e 191,
25198, Lleida, Spain.
B
Unidad de P oducción y Sanidad Animal, Cen o de In es igación y Tecnología Ag oalimen a ia de A agón
(CITA), A enida Mon añana 930, 50059, Za agoza, Spain.
C
Ins i u o Ag oalimen a io de A agón –IA2 (CITA-Uni e sidad de Za agoza), Za agoza, Spain.
D
Co esponding au ho . Email: [email p o ec ed]
Abs ac
Con ex . Imp o ing eed e ficiency in li es ock p oduc ion is o g ea impo ance o educe eeding cos s.
Aims. To examine he ela ionship be ween uminal mic obio a and a ia ion in eed e ficiency in bee ca le ed
concen a e-based die s.
Me hods. Residual eed in ake o 389 a ening bulls, supplied wi h co n-based concen a e and o age ad libi um, was
used oes ima eanimals’ eede ficiency.Faecesand uminalfluidsamples, om48bullschosena andom,we ecollec ed
o es ima e hei o age in ake and o de e mine hei appa en diges ibili y, uminal e men a ion and mic obio a. Those
animalswi hex eme alueso eede ficiency(high-e ficiency(HE,n=12)andlow-e ficiency(LE,n=13))we esubjec ed
o u he compa isons. Alphabiodi e si y was calcula ed on he basis o heno malised sequence da a. Be a di e si y was
app oached h ough pe o ming a canonical co espondence analysis based on log- ans o med sequence da a. Gene a
di e en ial abundance was es ed wi h an ANOVA-like di e en ial exp ession analysis and gene a in e ac ions we e
de e mined applying he spa se co ela ions o composi ional da a echnique.
Key esul s.No di e ences in d y ma e in ake we e ound be ween he wo ca ego ies o eed e ficiency (P=0.699);
howe e , HE animals had highe appa en diges ibili y o d y ma e (P= 0.002), o ganic ma e (P= 0.003) and c ude
p o ein (P= 0.043). The concen a ion o ola ile a y acids was una ec ed by eed e ficiency (P= 0.676) bu bu y a e
p opo ion inc eased wi h ime in LE animals (P= 0.047). Ruminal mic obio a was di e en be ween HE and LE
animals (P= 0.022); bo h abiodi e si y and gene a ne wo k connec ance inc eased wi h ime in LE bulls (P= 0.005 o
Shannon index and P= 0.020 o Simpson index), which sugges s ha LE animals hos ed a mo e obus uminal
mic obio a. Ce ain gene a usually ela ed o high ene gy loss h ough me hane p oduc ion we e ound o es ablish mo e
connec ions wi h o he gene a in LE animals’ umen han in HE ones. Mic obio a unc ion capabili y sugges ed ha
me hane me abolism was dec eased in HE finishing bulls.
Conclusions.Rumenmic obio awasassocia edwi h eede ficiencypheno ypesin a eningbulls edconcen a e-based
die s.
Implica ions. The possible ade-o be ween eed e ficiency and obus ness o uminal mic obio a should be aken
in o accoun o he op imisa ion o ca le p oduc ion, especially in sys ems wi h in insic cha ac e is ics ha may
cons i u e a dis u bance o umen mic obial communi y.
Keywo ds: appa en diges ibili y, bee ca le, eed e ficiency, umen mic obial communi y.
Recei ed 5 June 2020, accep ed 27 No embe 2020, published online 28 Janua y 2021
In oduc ion
Imp o ing eed e ficiency (FE) in bee ca le p oduc ion
sys ems p o ides an oppo uni y o cu down on he cos o
eeding li es ock. In ha sense, esidual eed in ake (RFI) can
be used as an index o FE ha is independen o a ia ion in
bodyweigh (BW) and a e age daily gain (ADG; A hu e al.
2001; Schenkel e al.2004; A hu and He d 2008), being he
gold s anda d index o examine biological mechanisms
associa ed wi h in e -animal di e ences in FE. Mo eo e ,
some s udies ha e demons a ed he possibili y o selec ion
o low RFI as a s a egy o g eenhouse gas mi iga ion, as i
has been co ela ed wi h lowe me hane emission and g ea e
die diges ibili y (He d and A hu 2009). Limi a ions in
conduc ing RFI ials ( eco ding BW and eed in ake o a
CSIRO PUBLISHING
Animal P oduc ion Science
h ps://doi.o g/10.1071/AN20344
Jou nal compila ion CSIRO 2021 Open Access CC BY www.publish.csi o.au/jou nals/an
SPECIAL ISSUE
long ime) and sea ching o umen mic obial ma ke s o
iden i y e ficien animals wi h low RFI ha e become a
con empo a y challenge.
Resea ch in ca le has ocused mos ly on he mic obial
esponse o die a y changes and managemen p ac ices,
whe eas ials o unde s anding he ela ionship be ween
hos FE pheno ype and umen mic obio a a e sca ce and ye
o be unde aken (Mye e al.2015). P e ious s udies ha e
shown ha umen mic obes a e esponsible o ene gy supply
h ough p oducing o ganic acids (Hun ing on 1990), and mos
axa associa ed wi h a ia ion in FE ha e been ela ed o
celluloly ic, e men a i e and me abolic ac i i ies (Mye
e al.2015). The e o e, di e ences in he p oduc ion a e o
o ganic acids lead o a ia ion in nu ien diges ibili y and
e men a ion ha ul ima ely change animals’pheno ypic
e ficiency (He d and A hu 2009).
This expe imen aimed o unde s and he ela ionship
be ween uminal mic obio a and a ia ion in FE o bee ca le
ed concen a e-based die s.
Ma e ials and me hods
Animals, die s and housing
Residual eed-in ake da a om wo eeding expe imen s
comp ising 389 a ening bulls we e used o explo e
ela ionships be ween uminal mic obio a and FE. This
da ase included 317 animals aised a he esea ch acili ies
o Coope a i a d’I a s d’U gell,SCCP (I a s d’U gell, Spain,
414105000N, 05805300E) and 72 animals om he CITA-La
Ga cipolle a Resea ch S a ion (Jaca, Spain, 423703400N,
03001000W). All p ocedu es we e pe o med unde P ojec
Licence CEEA 01-07/16 and app o ed by he in-house
E hics Commi ee o Animal Expe imen s a he Uni e si y
o Lleida. Ca e and use o animals we e in acco dance wi h he
Spanish Policy o Animal P o ec ion RD 53/2013, which
mee s he Eu opean Union Di ec i e 2010/63 on he
p o ec ion o animals used o expe imen al and o he
scien ificpu poses.
Animals aised a he esea ch acili ies o Coope a i a
d’I a s d’U gell,SCCP we e dis ibu ed in he ollowing ou
ba ches: ba ches Numbe 1 o Numbe 3 included 231 Hols ein
bulls (63–83 animals pe ba ch) and ba ch Numbe 4 included
86 Mon belia d bulls. BW and eed in ake da a we e collec ed
on a daily basis. Animals aised a CITA-La Ga cipolle a
Resea ch S a ion we e dis ibu ed in he ollowing h ee
ba ches: ba ches Numbe 5 and Numbe 6 included 28 and
32 Pa da de Mon aña bulls espec i ely, and ba ch Numbe 7
included 12 Pi enaica bulls. Fo hese animals, BW was
measu ed weekly and eed in ake da a we e collec ed on a
daily basis.
Bodyweigh and eed in ake da a we e eco ded h oughou
he en i e a ening phase; he fi s 150 days we e conside ed as
he g owing phase (121 days old, s.d. 37 days; and 162 kg BW,
s.d. 49 kg), ollowed by a finishing phase, which las ed un il
animals eached slaugh e weigh (336 days old, s.d.: 31; and
501 kg BW, s.d. 56 kg).
Animals we e ed concen a e and o age ad libi um,which
we e p o ided sepa a ely in wo di e en bunke s, and hey
had ee access o d inking wa e , ollowing he con en ional
bee ca le eeding sys em in Spain. The concen a es used
we e e y simila in composi ion and hei main ing edien s
we e aw co n, co n glu en eed, aw ba ley, co n d ied
dis ille s g ains wi h solubles and aw chickpea; whe eas
o age used was mainly ba ley s aw (349 animals), oa s
haylage (20 animals) and e ch haylage (20 animals). Feed
chemical and nu i ional composi ion is shown in Table 1.
Measu emen s and sampling
In ake o concen a es was eco ded au oma ically a bo h
esea ch acili ies, by using au oma ic eed s a ions ha we e
equipped wi h a eedbunk (p o ided wi h a scale) and an
indi idual eede . When a cal en e ed he eede , i was
iden ified and i s concen a e in ake was ob ained by
di e ence be ween ini ial and final eedbunk weigh . Feed
s a ions a ailable a he esea ch acili ies o Coope a i a
d’I a s d’U gell,SCCP we e addi ionally equipped wi h a
scale unde he indi idual eede by which he animals we e
au oma ically weighed a each isi , whe eas a CITA-La
Ga cipolle a Resea ch S a ion, BW da a we e eco ded
manually once a week.
Faeces and uminal fluid samples om 48 bulls (selec ed a
andom wi hin ba ches) we e collec ed wice, a mid-g owing
pe iod (GRO, 159 days old and 225 kg BW) and mid-finishing
pe iod (FIN, 266 days old and 434 kg BW), o o age in ake
es ima ion and diges ibili y, uminal e men a ion and
mic obial communi y cha ac e isa ion.
Faecal exc e ion and o age in ake we e calcula ed on he
basis o concen a e in ake and adap ing he wo indiges ible-
ma ke sys em (Owens and Hanson 1992), by using ch omium
oxide as an ex e nal ma ke and acid insoluble ash as in e nal
ma ke . Then, appa en diges ibili y o d y ma e (DM),
o ganic ma e (OM) and c ude p o ein (CP) we e es ima ed.
De ailed in o ma ion abou ma ke adminis a ion, eed and
aeces analy ical de e mina ions and appa en diges ibili y
calcula ions ha e been desc ibed in Cos a-Rou a e al.(2020).
Ruminal fluid was sampled in he mo ning by using an o al
s omach ube connec ed o a acuum pump. Each sample was
Table 1. Feed chemical and nu i ional composi ion
Valuesa emeans, wi h minimumandmaximum gi en inpa en heses. ADF,
acid de e gen fib e; CP, c ude p o ein; DM, d y ma e ; EE, e he ex ac ;
NDF, neu al de e gen fib e; OM, o ganic ma e ; PDIN and PDIE, p o ein
diges ible in he small in es ine allowed by p o ein and ene gy; UFV, o age
uni o mea p oduc ion
Pa ame e Concen a e Fo age
Chemical composi ion (%DM)
DM (% esh weigh ) 87.1 (85.6–87.9) 65.8 (48.9–85.7)
OM 94.6 (94.0–94.8) 88.9 (84.3–92.6)
CP 13.0 (11.2–14.0) 11.2 (7.2–16.4)
EE 4.2 (2.5–7.2) 2.0 (1.4–2.7)
NDF 16.6 (13.5–20.7) 57.1 (44.2–75.5)
ADF 5.9 (4.7–7.9) 33.5 (28.2–43.8)
Nu i ional composi ion
UFV (UFV/kg DM) 1.02 (0.97–1.03) 0.55 (0.36–0.73)
PDIN (g/kg DM) 91.8 (79.8–95.9) 65.4 (40.8–93.7)
PDIE (g/kg DM) 87.4 (80.3–94.9) 56.0 (52.6–58.0)
BAnimal P oduc ion Science S. Cos a-Rou a e al.
ob ained h ough wo sequen ial collec ions. Fi s , uminal
fluid (~200 mL) was collec ed and disca ded o a oid
sample con amina ion wi h sali a ha could ge in o he
ube du ing i s in oduc ion h ough he animal’s mou h and
oesophagus. A e ha , uminal fluid (~200 mL) was e-
ex ac ed, s ained h ough a cheeseclo h and i s pH was
eco ded (Tes o 205, Tes o AG, Ge many). Then, uminal
fluid was sampled o DNA ex ac ion, and de e mina ion o
ammonia-ni ogen (N) and ola ile a y acid (VFA)
concen a ions, and immedia ely ozen on d y ice. Sample
p ese a ion condi ions and analy ical p ocedu es o
ammonia-N and VFA de e mina ion a e de ailed in Cos a-
Rou a e al.(2020).
Ex ac ion and sequencing o DNA
Ex ac ion o DNA was pe o med on eeze-d ied uminal
fluid ( he ini ial amoun o he sample was 60 mg) h ough
physical dis up ion (1 min) by using a bead bea e (Mini-bead
Bea e 1, BioSpec P oduc s, USA) and subsequen DNA
pu ifica ion was pe o med wi h he QIAamp DNA S ool
Mini Ki (ID: 51504; QIAGEN N.V., Ge many), wi h he
modifica ions o g ea e empe a u e (95C) and g ea e
elu ion ime (3 min) o ensu e maximum DNA
concen a ion in he final elu e. Amplifica ion o DNA was
pe o med by using p ime s 341F and 805R, which a ge he
V3 and V4 egions o he bac e ial and a chaeal 16S
RNA. Sequencing was conduc ed on an Illumina MiSeq
2x300 pla o m by E a7 Bioin o ma ics (Spain).
Assembly and fil a ion o sample eads, as well as
ope a ional axonomic uni (OTU) p epa a ion ha e been
de ailed in Cos a-Rou a e al.(2020).
Es ima ion o RFI and clus e ing
Weigh da a we e fi ed o a hi d-deg ee polynomials model in
unc ion o age (Eqn 1) ha allows he es ima ion o he ADG o
each animal a any age.
Weigh i;age ¼P
j¼3
j¼0bBATCH;jþAi;j

agejþei;age ð1Þ
whe e b
BATCH,j
is he ba ch e ec (fixed); A
i,j,
is he j h andom
coe ficien o he i h animal e ec ; age is he age o he animal
(days) and e
i,age
he esidual e m.
The ea e , ADG_de was ob ained as he fi s de i a i e o
Eqn 1 o each mon h by using he mon hly a e age age o each
animal (Eqn 2). The indi idual ADG de ia ion (ADG_de ) will
accoun o hedi e enceo g ow ho heanimalcompa edwi h
he a e age o he ba ch a each age.
ADG de i;age ¼P
j¼3
j¼1j·Ai;jage j-- 1ðÞ ð2Þ
In o al, 86 eco ds (3%) ou o h ee s anda d de ia ions o
he mean we e conside ed as ou lie s and excluded om he
da ase .
Residual eed in ake was modelled (Eqn 3) using he andom
eg ession coe ficien app oach p oposed by Sa ie o e al.
(2014). The model included ba ch, age (mon hs), ADGde ,
me abolic weigh (MW; mon hly mean BW
0.64
) and was
defined as ollows:
FIij ¼B0;animal i þBa ch ·agejþðBa ch þB1;animal iÞ
·MWij þðBa ch þB2;animal iÞ·ADG de ij þeij
ð3Þ
whe e FI
ij
is DM in ake measu ed o Animal iin Mon h jand
B
k
,
animal i
a e he andom coe ficien s o animal e ec
modelled using an uns uc u ed ma ix o a iances be ween
hem.
The inclusion o ba ch e ec in Eqn 1and Eqn 3assu ed
ha he FE calcula ed was no a ec ed by die di e ences.
On he basis o he indi idual coe ficien s o ADG
(B
1, animal i
) and MW (B
2, animal i
), animals we e seg ega ed
in o ou ca ego ies o FE, as ollows:
(1) Animals wi h posi i e coe ficien s o bo h ADG and MW
belonged o ‘low-e ficiency in ADG and low-e ficiency in
MW’ca ego y.
(2) Animals wi h posi i e coe ficien o ADG bu nega i e
coe ficien o MW belonged o ‘low-e ficiency in ADG
and high-e ficiency in MW’ca ego y.
(3) Animals wi h nega i e coe ficien o ADG bu posi i e
coe ficien o MW belonged o ‘high-e ficiency in ADG
and low-e ficiency in MW’ca ego y.
(4) Animals wi h nega i e coe ficien s o bo h ADG and MW
belonged o ‘high-e ficiency in ADG and high-e ficiency in
MW’ca ego y.
Fo he pu pose o he p esen s udy, he wo ex eme
ca ego ies (1 and 4) we e conside ed as high-e ficiency (HE,
posi i e RFI) and low-e ficiency (LE, nega i e RFI) animals,
espec i ely. This clus e ing (HE s LE) was subjec ed o
bioin o ma ic analyses o appa en diges ibili y, uminal
e men a ion and mic obio a da a as explained below.
Bioin o ma ics
Sequence da a we e no malised and abiodi e si y indices
we e calcula ed o measu e he a iabili y o OTUs wi hin a
sample (R Co e Team 2020, Vegan package).
To measu e di e ences in mic obio a composi ion among
samples, bdi e si y was app oached h ough pe o ming a
canonical co espondence analysis, based on log- ans o med
OTU da a (ze os we e eplaced by adding 1 o each alue), and
including FE (HE s LE), pe iod (GRO s FIN) and bo h ADG
and MW coe ficien s as explana o y a iables (R Co e Team
2020, Vegan package).
To ci cum en he composi ional bias p oblem (Tsilimig as
and Fodo 2016; Gloo e al.2017;Calle2019), we applied he
Ai chison’s cen ed log a io (cl ) ans o ma ion o ca y he
da a o a Euclidean space, a e eplacing ze os by adding 1 o
each alue. So as o es he significance o he ollowing
e ec s: FE (HE s LE), pe iod (GRO s FIN) and bo h
ADG and MW coe ficien s on mic obio a composi ion, a
pe mu a ional mul i a ia e analysis o a iance (Adonis)
was conduc ed on he basis o he cl Euclidean dis ance
and calcula ing s a is ical significance a e 10000 andom
pe mu a ions (R Co e Team 2020, Vegan package). So as o
deciphe which gene a abundance we e esponsible o he
di e ences among g oups, an ANOVA-like di e en ial
Ruminal mic obio a and eed e ficiency in bulls Animal P oduc ion Science C
exp ession (ALDEx) analysis was conduc ed o e hose
gene a p esen a leas a 50% o he indi iduals (R Co e
Team 2020, Aldex2 package; Fe nandes e al.2013). Finally,
o desc ibe he in e ac ions wi hin umen mic obial
communi y, we pe o med a ne wo k analysis h ough
spa se co ela ions o composi ional da a (Spa CC)
echnique (R Co e Team 2020, SpiecEasi package;
F iedman and Alm 2012) o e hose gene a p esen a leas
a 50% o he indi iduals. Mic obial ne wo ks we e
g aphically ep esen ed (R Co e Team 2020; ig aph
package) and hei complexi y was desc ibed in e ms o
numbe o nodes (gene a), numbe o edges (significan
posi i e o nega i e co ela ions), node deg ee (numbe o
connec ions ha any node es ablishes wi h o he nodes) and
be weenness (measu e o cen ali y in a g aph based on
sho es pa hs).
Mic obio a unc ional con en was assessed using a opic
model app oach (R Co e Team 2020; heme agenomics
package) ha consis s on (1) cap u ing g oups o co-
occu ing axa e med ‘ opics’, (2) unco e ing wi hin- opic
unc ional po en ial, and (3) linking hese opics and hei
unc ional con en o specific sample ea u es (e.g. FE
pheno ypes; Woloszynek e al.2019).
S a is ical analyses
The models o RFI we e sol ed using MIXED p ocedu e o
SAS s a is ical so wa e (SAS 9.4, Ca y, NC, USA). In ake,
appa en diges ibili y, uminal e men a ion pa ame e s and
mic obial abiodi e si y da a we e analysed wi h a mixed
model, including FE (HE s LE), pe iod (GRO s FIN) and
hei in e ac ion as fixed e ec s and animal as a andom e ec ,
o accoun o epea ed measu emen s (R Co e Team 2020,
lme4 package). Di e ences among leas squa e means we e
assessed using Tukey mul iple-compa ison es (R Co e Team
2020, emmeans package). Indi idual samples ou o h ee
s anda d de ia ions o he mean we e disca ded and no
included in he s a is ical analysis. Resul s a e epo ed as
leas squa e means and s anda d e o o mean. Significan
e ec s we e decla ed a P<0.05 and endency o di e ence a
Pbe ween 0.05 and 0.10.
Resul s
The ou defined FE ca ego ies based on andom eg ession
coe ficien s included be ween 85 and 108 animals each; he
wo ex eme ca ego ies co esponding o HE and LE animals
had s a is ically di e en means o ADG and MW coe ficien s
(Table S1, a ailable as Supplemen a y Ma e ial o his pape ).
The se o 48 bulls ha we e sampled o appa en diges ibili y,
uminal e men a ion and mic obio a cha ac e isa ion we e
equally dis ibu ed wi hin he ou ca ego ies.
The FE by pe iod in e ac ions we e no significan o any
o he esponse a iables measu ed in he p esen s udy; hus,
only he main e ec s means a e p esen ed and discussed.
In ake, appa en diges ibili y and uminal e men a ion
pa ame e s
Da a on DM in ake (Table 2) indica ed ha bulls’concen a e
and o age in akes we e simila be ween FE ca ego ies (HE s
LE). Howe e , animals classified as HE had g ea e appa en
Table 2. D y ma e (DM) in ake, nu ien appa en diges ibili y and uminal e men a ion pa ame e s
Ob ained in in ensi ely ea ed bulls in he ollowing wo pe iods: g owing (GRO: 159 days old and 225 kg bodyweigh ) and finishing (FIN: 266 days old
and 434 kg bodyweigh ). Residual eed in ake was modelled o classi y animals in o wo ca ego ies o eed e ficiency, namely, high e ficiency (HE, n = 12)
and low e ficiency (LE, n = 13). S anda d e o o he mean (s.e.m.) and significance o eed e ficiency and pe iod e ec s a e shown. No eed e ficiency by
pe iod in e ac ion was s a is ically significan (P>0.05) and hese a e no included in he able. A: P, ace a e- o-p opiona e a io; CP, c ude p o ein; N,
ni ogen; OM, o ganic ma e ; VFA, ola ile a y acids. Mean alues wi hin a ow ollowed by di e en le e s di e significan ly (a P=0.05)
Pa ame e Feed e ficiency Pe iod s.e.m. P- alue
HE LE GRO FIN Feed e ficiency Pe iod
In ake
DM (kg/day) 7.52 7.64 5.88b 9.28a 0.235 0.699 <0.001
Concen a e DM 6.40 6.59 5.28b 7.72a 0.191 0.479 <0.001
Fo age DM 1.09 0.93 0.60b 1.42a 0.137 0.397 <0.001
Appa en diges ibili y coe ficien s (%)
DM 75.16a 70.82b 72.88 73.09 0.955 0.002 0.875
OM 76.15a 71.94b 73.84 74.25 0.995 0.003 0.760
CP 71.53a 68.52b 69.64 70.41 1.020 0.043 0.579
Ruminal e men a ion pa ame e s
pH 6.93 6.97 7.16a 6.75b 0.080 0.710 <0.001
Ammonia-N (mg/L) 12.36 19.27 12.03 19.59 3.618 0.181 0.095
VFA (mmol/L) 70.87 74.41 68.22 77.05 6.102 0.676 0.183
VFA (%)
Ace a e 49.63 51.42 49.32 51.73 1.532 0.403 0.093
P opiona e 37.70 35.88 38.89a 34.69b 1.867 0.487 0.015
Bu y a e 8.18 8.38 7.70b 8.86a 0.425 0.737 0.025
B anched-chain VFA 1.93 1.99 1.66b 2.27a 0.128 0.724 <0.001
Ra io A:P 1.42 1.58 1.30b 1.69a 0.148 0.431 0.017
DAnimal P oduc ion Science S. Cos a-Rou a e al.
diges ibili y coe ficien s o DM, OM and CP han did hei LE
coun e pa s.
Da a on uminal e men a ion pa ame e s (Table 2)showed
no di e ences in uminal pH be ween HE and LE bulls.
Ammonia-N concen a ion was low and a iable among
animals; he e o e, no s a is ical di e ences be ween
FE ca ego ies we e ound. Al hough he o al VFA
concen a ion emained una ec ed by FE, nume ical
di e ences we e ound in mola p opo ions o he main
VFA; HE animals had a lowe p opo ion o ace a e and a
highe p opo ion o p opiona e han did LE ones. Bu y a e
p opo ion inc eased wi h ime in he case o LE animals
(7.46% s 9.30% o GRO and FIN pe iods, espec i ely;
P= 0.047) whe eas i emained equal o HE animals (7.95%
s 8.42% o GRO and FIN, espec i ely; P= 0.908). In
con as , b anched-chain VFA p opo ion (isobu y a e and
iso ale a e) inc eased wi h ime only in he case o HE
bulls (1.55% s 2.31% o GRO and FIN, espec i ely;
P=0.006).
Mic obial da ase ea u es
Sequencing p ocedu e yielded an a e age o (mean s.e.m.)
19862 2215 sequences pe sample, esul ing in 973259
sequences in he whole s udy. In o al, 787 OTUs we e
ob ained a he 98% sequence-simila i y cu -o le els, wi h
114 5 as he mean numbe o OTUs pe sample. Good’s
co e age alue was 99.69 0.03%, sugges ing ha mo e han
99% o bac e ial and a chaeal phylo ypes we e iden ified. The
unclassified a e o OTUs a genus le el was 0.75 0.09%.
Sha ed OTUs by all indi iduals in each FE ca ego y and pe iod
we e deemed o be co e bac e ial/a chaeal communi ies. Co e
communi y ga he ed 69.90 2.94% o analysed sequences
and was composed o fi e OTUs, namely, P e o ella
uminicola,unclassified P e o ella (bo h ep esen ing mo e
han 84% o sha ed sequences), unclassified Rosebu ia,
Sha pea azabuensis and unclassified Me hanob e ibac e .
Mic obial communi y biodi e si y
Alpha biodi e si y (Table 3) was ound o be simila among
bulls di e ing in hei FE; howe e , Shannon and Simpson
index alues inc eased wi h ime only in LE animals (Shannon
index 1.51 s 2.13, P= 0.005; Simpson index 0.53 s 0.70,
P= 0.020, o GRO and FIN, espec i ely).
Be a biodi e si y is g aphically ep esen ed in Fig. 1,as
well as he e ec s o explana o y a iables included in
he model, namely, FE, pe iod, and bo h MW and ADG
coe ficien s in he RFI model. Samples a e clea ly clus e ed
by pe iod and FE, wi h he e ec s o MW and ADG
coe ficien s being less g aphically e iden . Adonis es
esul s confi med he o eseen di e ences in uminal
mic obio a composi ion when compa ing sampling pe iods
(GRO s FIN, P<0.001), FE ca ego ies (HE s LE, P=
0.022) and MW coe ficien alues (P= 0.021), bu no in he
case o ADG coe ficien alues (P= 0.276). S a is ical
di e ences in abundance o gene a be ween FE ca ego ies
(HE s LE) could no be de ec ed by ALDEx analysis,
ega dless o he sampling pe iod (Fig. S1, a ailable as
Supplemen a y Ma e ial o his pape ).
Mic obial ne wo k
Mic obial ne wo ks we e buil o es in e ac ions among
bac e ial and a chaeal gene a (Fig. 2). Deg ee o in e ac ion
Table 3. Ruminal mic obial abiodi e si y
Ob ained in in ensi ely ea ed bulls in he ollowing wo pe iods: g owing (GRO: 159 days oldand 225 kg bodyweigh )
andfinishing(FIN:266daysoldand434kg bodyweigh ).Residual eedin ake wasmodelled oclassi yanimalsin o wo
ca ego ies o eed e ficiency, namely, high e ficiency (HE, n = 12) and low e ficiency (LE, n = 13). S anda d e o o he
mean (s.e.m.) and significance o eed e ficiency and pe iod e ec s a e shown. No eed e ficiency by pe iod in e ac ion
was s a is ically significan (P>0.05) and hese a e no included in he able. Mean alues wi hin a ow ollowed by
di e en le e s di e significan ly (a P= 0.05)
Pa ame e Feed e ficiency Pe iod s.e.m. P- alue
HE LE GRO FIN Feed e ficiency Pe iod
Shannon index 1.76 1.82 1.53b 2.05a 0.149 0.760 <0.001
Simpson index 0.61 0.61 0.54b 0.68a 0.050 0.942 0.002
Richness 101.10 107.69 96.87b 111.92a 5.824 0.408 0.072
2
1
0
–1
–2
–2 0 2
CCA1
MW
PERIOD
HE_GRO LE_GRO HE_FIN LE_FIN
ADG
FEED
EFFICIENCY
CCA2
Fig. 1. G aphical ep esen a ion o canonical co espondence analysis
(CCA) on bac e ial and a chaeal ope a ional axonomic uni s (OTUs) in
uminal fluid, ob ained in in ensi ely ea ed bulls in he ollowing wo
pe iods: g owing (GRO: 159 days old and 225 kg bodyweigh ) and
finishing (FIN: 266 days old and 434 kg bodyweigh ). Residual eed
in ake was modelled o classi y animals in o wo ca ego ies o eed
e ficiency, namely, high e ficiency (HE) and low e ficiency (LE). The
analysis included eed e ficiency, pe iod, and bo h a e age daily gain
(ADG) and me abolic weigh (MW) coe ficien s as explana o y a iables.
Ruminal mic obio a and eed e ficiency in bulls Animal P oduc ion Science E

was s udied h ough he numbe o gene a (nodes) ha
es ablished significan in e ac ions (edges) wi h o he gene a,
as well as he numbe o in e ac ions es ablished pe node
(node deg ee). Du ing he g owing pe iod, HE bulls
had simila numbe o nodes aking pa in he mic obial
ne wo k as did LE bulls (26 in HE s 24 in LE), bu highe
numbe o edges(57inHE s28inLE)andahighe a e agenode
deg ee (4.38 in HE s 2.33 in LE). Du ing he finishing pe iod,
mic obial ne wo k a chi ec u e changed; HE bulls con inued
o ha e mo e co ela ing nodes han did LE bulls (30 in HE s
21 in LE), bu LE animals d as ically inc eased hei numbe o
edges (53 in HE s 59 in LE) and he node deg ee (3.53 in HE s
5.62 in LE).
Mo eo e , we in es iga ed mic obial gene a ha ac as
main in o ma ion ga eways in ne wo ks in e ms o
be weenness cen ali y, i.e. he ex en o which one
node lies on pa hs ha connec o he nodes. Ne wo ks o
HE animals p esen ed highe be weenness cen ali y
han did hose o LE animals in g owing (3.88 in HE s
1.04 in LE) bu no in finishing (2.37 in HE s 2.52 in LE)
pe iod.
Mic obial unc ional capabili y
A e p edic ing unc ional con en o uminal mic obio a, wo
pa hways we e ound o be di e en ially exp essed depending
on he animal FE pheno ype. In he g owing pe iod, ABC
anspo e pa hway (ATP-dependen anspo o molecules
ac oss cell memb ane) was mo e ac i e in HE animals han in
LE animals, and, in he finishing pe iod, me hane me abolism
Eubac e ium Psych obac e
T eponema
T eponema
Acine obac e
Pseudo amibac e
Pseudo amibac e
Lac obacillus
B e ibac e ium
A h obac e
Dialis e
Dialis e
Bi idobac e ium
Bi idobac e ium
Bac e oides
Pseudobu y i ib io
P e o ella
Me hanob e ibac e
Jeo ga icoccus
Jeo ga icoccus
Tu icibac e
Tu icibac e
Co ynebac e ium
Co ynebac e ium
Co ynebac e ium
B achybac e ium
S aphylococcus
S aphylococcus
Psych obac e S aphylococcus
Pseudomonas
Bu y i ib io
Clos idium
Eubac e ium
Eubac e ium
Mi suokella
Mi suokella Mi suokella
P e o ella
Olsenella
Lac obacillus
Bi idobac e ium
Bu y i ib io
Bu y i ib io
Dialis e
Dialis e
T eponema
Acidaminococcus
Pseudomonas
Pseudobu y i ib io Pseudobu y i ib io
Selenomonas
Selenomonas
O senella
Rosebu ia
Die zia
P e o ella
P e o ella
Aga hobac e
Aga hobac e
Clos idium
Clos idium
Aga hobac e
Pep oclos idium
T eponema
Rosebu ia
Lachnoclos idium
Allisonella
Sha pea
Sha pea
Rosebu ia
Ruminococcus
Ruminococcus
Ruminococcus
Ruminococcus
Bac e oides
Acine obac e
Fib obac e
Selenomonas
Me hanob e ibac e Me hanob e ibac e
Pseudo amibac e
Pseudo amibac e
Megasphae a Megasphae a
Megasphae a
Mi suokella
Allisonella
Sha pea
Acidaminococcus
Olsenella
Selenomonas
Facklamia
Facklamia
Fib obac e
Fib obac e
Fib obac e
(a)(b)
(c)(d)
Candida us_Phy oplasma
Candida us_Phy oplasma
Ae ococcus
Weissella
Fig. 2. Bac e ial and a chaeal gene a ne wo k in he umen o in ensi ely ea ed a ening bulls: (a–c) GRO: 159 days old and 225 kg
bodyweigh ; (b–d) FIN: 266 days old and 434 kg bodyweigh . Residual eed in ake was modelled o classi y animals in o wo ca ego ies
o eed e ficiency, namely, (a, b) high e ficiency (HE) and (c, d)lowe ficiency (LE). Ne wo ks we e gene a ed on he basis o hose gene a
ha es ablished significan co ela ions ( >0.60 and P<0.05). G een and ed edges indica e posi i e and nega i e co ela ions, espec i ely.
Node size is p opo ional o genus abundance in he uminal fluid.
FAnimal P oduc ion Science S. Cos a-Rou a e al.
was down egula ed in he umen o HE indi iduals when
compa ed wi h LE animals.
Discussion
RFI and mechanisms unde lying he a iabili y o FE
Va ia ions in RFI occu due o po en ial physiological
mechanisms such as diges ion, e men a ion and me abolism
(He d and A hu 2009). Ou findings showed ha HE animals
appa en ly diges ed mo e eed, in e ms o DM, OM and CP,
han did LE ones. These esul s a e in acco dance wi h
p e ious s udies in which mo e e ficien animals showed a
highe nu ien diges ibili y and a smalle nu ien loss h ough
was e and me hane emission (Richa dson e al.1996;
Nk umah e al.2006). Negesse e al.(2017) also obse ed
imp o ed appa en diges ibili y coe ficien s o DM, OM and
CP in HE hei e s; hese animals exc e ed a smalle p opo ion
o N h ough aeces and hei N biological alue (diges ible N
a io) was highe han ha o less e ficien hei e s, sugges ing
ha CP diges ion and me abolism may be enhanced in HE
animals. In compa ison, de Assis Lage e al.(2019)didno
find di e ences in diges ibili y coe ficien s o such nu ien s
bu hey epo ed a endency o HE hei e s o be e diges e he
ex ac ac ion.
Vola ile a y acids a e p oduc s o umen mic obial
e men a ion o ca bohyd a es, cons i u ing he main ene gy
sou ce o uminan s (Be gman 1990). Al hough di e ences
be ween HE and LE bulls did no each significance o any
uminal e men a ion pa ame e , nume ical alues indica ed
ha LE animals had a e men a ion pa e n o ien ed owa ds
he p oduc ion o highe mola p opo ion o ace ic and less
p opionic acids han did HE animals, wi h he consequen
e ec on ace a e- o-p opiona e a io. These obse ed
di e ences in umen e men a ion pa e n may be playing a
ole in he bulls’FE pheno ype, since me abolic hyd ogen
p oduced in he fi s s ep o ace ic acid pa hway is la e aken
up by me hanogens, inc easing ene gy loss h ough gas
emissions (Unge eld 2020).
Du ing he g owing pe iod, mola p opo ions o he majo
VFA we e simila o hose obse ed by Yus e e al.(2020)in
bee hei e s ed a simila ad libi um concen a e plus s aw
die . Howe e , du ing he finishing pe iod, he highe amoun
o o al VFA, concomi an wi h significan ly lowe uminal
pH, we e oo ed in he inc eased DM in ake and, consequen ly,
in he highe ex en o e men a ion p ocess. Howe e ,
p opionic acid showed a di e en end and i was highe in
younge animals. He nandez-U dane a e al.(1976) epo ed
ha o age- o-concen a e a io a ec s he mola p opo ions
o VFA, which o high-concen a e die s changes owa ds
dec eased ace ic and inc eased p opionic acid; he e o e, in ou
expe imen , he lowe o age- o-concen a e a io du ing he
g owing (11%) han in he finishing pe iod (18%) can explain
he obse ed dec eased p opo ion o p opionic acid wi h ime.
RFI and uminal mic obial communi y
In he p esen s udy, Illumina sequencing echnology was used
o analyse bac e ial and a chaeal composi ion, biodi e si y,
connec ance and unc ional capabili y wi hin he umen o
in ensi ely ea ed bulls di e ing in hei FE.
A nega i e co ela ion be ween uminal mic obial a
biodi e si y and FE has been p e iously desc ibed in
milking cows (Shaba e al.2016), sugges ing ha e ficien
mic obio as a e less complex bu mo e specialised in p o iding
highe concen a ions o ele an ou pu me aboli es ha
can be used o mee hos ’s ene gy equi emen s. In a
simila manne , ou esul s showed ha mic obial
adi e si y alues significan ly inc eased wi h ime only in
LE bulls. Mic obial di e si y is also posi i ely co ela ed wi h
communi y s abili y and obus ness, as bo h di e en ial
esponse o a iable condi ions and unc ional edundancy
o species a e enhanced (McCann 2000; Moya and Fe e
2016). Thus, i seems easonable o hypo hesise ha a
biodi e si y o he mic obio a has posi i e and nega i e
coexis ing e ec s on ecosys em obus ness and eed
u ilisa ion e ficiency, espec i ely. The ac ha LE bulls
conside ably inc eased hei gene a ne wo k connec ance
wi h ime, while HE bulls kep i cons an o e en
diminished i , suppo s he hypo hesis ha LE animals’
uminal mic obio a could be mo e obus and ha e an
enhanced abili y o cope wi h possible dis u bances (Dunne
e al.2002).
E en hough bdi e si y ep esen a ion showed clea
clus e ing o bulls’mic obial communi y, no s a is ical
di e ences in he abundance o main gene a could be ound
be ween FE ca ego ies. Conside ing ha some s udies ha e
had success in epo ing a ela ionship be ween ce ain
mic obial axa and animal’sFE(McCanne al.2014;Mye
e al.2015; Pe ea e al.2017;Delgadoe al.2019),
we conside ha he ollowing ac o s could hinde
de ec ion o such ela ionship: (1) he e can be subs an ial
animal- o-animal a ia ion in he umen mic obial communi y,
hus equi ing a g ea e numbe o animals o be able o
obse e a significan associa ion be ween mic obial axa and
FE (B ulc e al.2009; Weime e al.2010), and (2) he lack o
di e ences obse ed be ween FE ca ego ies a he main-
gene a le el may indica e ha he impo an a ia ion in
mic obial communi ies lies a a fine esolu ion (e.g. a
species le el o low-abundance gene a).
Ki elmann e al.(2014) desc ibed he exis ence o h ee
uminal mic obial communi ies linked o di e en me hane
yields in sheep; umino ype H was cha ac e ised by he
highes me hane emissions and ha bou ed he highe
abundance o species belonging o Ruminococcus, o he
Ruminococcaceae, Lachnospi aceae, Ca abac e iaceae,
Cop ococcus, o he Clos idiales, P e o ella, o he
Bac e oidales and Alphap o eobac e ia. In a ecen s udy in
sheep, Ghanba i Maman e al.(2020) also iden ified ce ain
genes om Lachnospi aceae, Ruminococcus,Bu y i ib io and
Selenomonas axa ha can ha e significan e ec s on me hane
p oduc ion pa hway. In acco dance wi h hese s udies, ou co-
abundance analysis showed ha ce ain gene a p e iously
ela ed o high me hane emission (e.g. Me hanob e ibac e ,
Rosebu ia,Aga hobac e ,Bu y i ib io,Pseudobu y i ib io,
Ruminococcus,Selenomonas) ei he we e mo e cen al o
e ol ed o be mo e cen al in LE animal ne wo ks du ing
he ansi ion om g owing o finishing pe iods (Tables S2,
Table S3, a ailable as Supplemen a y Ma e ial o his pape ),
which could a leas pa ially cause hei lowe FE.
Ruminal mic obio a and eed e ficiency in bulls Animal P oduc ion Science G
Recen s udies ha e highligh ed a possible ela ionship
be ween mic obial me abolic unc ions and he animal’sFE,
bu he na u e o such ela ionship is s ill unclea . Li e al.
(2016) obse ed ha HE ca le had a mo e ac i e me abolism
o nucleo ides, as well as o a ious ene gy-gene a ing
molecules (e.g. p opanoa e, glyoxyla e and dica boxyla e,
s a ch and suc ose), hypo hesising ha such inc eased
me abolic ac i i y could enhance eed diges ion and p o ide
he hos wi h mo e nu ien s. Li e al.(2016) and Elolimy e al.
(2020) also epo ed ha umen mic obio a o he mos
e ficien ca le was mo e ac i e in cell p oli e a ion and
su i abili y, inducing cellula g ow h and inc easing
ole ance o i al in ec ion; likewise, ou esul s showed
enhanced cell memb ane anspo unc ions in HE g owing
animals. Finally, he obse ed dec ease o me hane
me abolism ac i i y in HE finishing bulls (Shaba e al.
2016) suppo s he p e ious idea ha high and low
me hane emi e s can ha e a simila abundance o uminal
me hanogens bu di e en ial exp ession and ansc ip ion o
me hanogenesis pa hway genes (Shi e al.2014).
Conclusions
The explo a ion o he ela ionship be ween umen mic obial
communi y and hos FE showed inc eased nu ien
diges ibili y in HE animals. Alpha biodi e si y and gene a
ne wo k connec ance inc eased wi h ime in LE bulls,
highligh ing a possible ade-o be ween FE and uminal
mic obio a obus ness. Mo eo e , ce ain gene a ha ha e
p e iously been ela ed o high me hane emission we e
mo e cen al in LE animals’gene a ne wo ks. Ou esul s
ha e p o ided e idence ha he umen mic obio a could be
one o he biological ac o s associa ed wi h a ia ion in
ca le FE.
Conflic s o in e es
The au ho s decla e no conflic s o in e es .
Acknowledgemen s
Special hanks a e ex ended o J. R. Be olín Pa dos o hei labo a o y
assis ance. This s udy is a pa o GenTORE p ojec and ecei ed
unding om he Eu opean Union H2020 p og am unde g an
ag eemen no727213 and Ins i u o Nacional de In es igación y
Tecnología Ag a ia y Alimen a ia (RTA-14-038-C02). S. Cos a-
Rou a is ecipien o a esea ch aining g an om Spanish Minis y
o Science and Inno a ion (FPU 2016/03761). A. R. Se adj con ac is
financed by GenTORE p ojec .
Re e ences
A hu JPF, He d RM (2008) Residual eed in ake in bee ca le. Re is a
B asilei a de Zoo ecnia 37, 269–279. doi:10.1590/S1516-3598
2008001300031
A hu PF, A che JA, Johns on DJ, He d RM, Richa dson EC, Pa nell PF
(2001) Gene ic and pheno ypic a iance and co a iance componen s
o eed in ake, eed e ficiency, and o he pos weaning ai s in Angus
ca le. Jou nal o Animal Science 79,2805–2811. doi:10.2527/
2001.79112805x
Be gman EN (1990) Ene gy con ibu ions o ola ile a y acids om he
gas oin es inal ac in a ious species. Physiological Re iews 70,
567–590. doi:10.1152/phys e .1990.70.2.567
B ulc JM, An onopoulos DA, Be g Mille ME, Wilson MK, Yanna ell AC,
DinsdaleEA,Edwa dsRE,F ankED,Eme sonJB,WacklinP,Cou inho
PM, Hen issa B, Nelson KE, Whi e BA (2009) Gene-cen ic
me agenomics o he fibe -adhe en bo ine umen mic obiome e eals
o age specific glycoside hyd olases. P oceedings o he Na ional
Academy o Sciences o he Uni ed S a es o Ame ica 106, 1948–1953.
doi:10.1073/pnas.0806191105
Calle ML (2019) S a is ical analysis o me agenomics da a. Genomics &
In o ma ics 17, e6. doi:10.5808/GI.2019.17.1.e6
Cos a-Rou a S, Balcells J, de la Fuen e G, Mo a-Gil J, Llanes N, Villalba D
(2020) Nu ien u iliza ion e ficiency, uminal e men a ion and
mic obial communi y in Hols ein bulls ed concen a e-based die s
wi h di e en o age sou ce. Animal Feed Science and Technology
269, 114662. doi:10.1016/j.ani eedsci.2020.114662
de Assis Lage CF, Ges ei a Coelho S, Diniz Ne o H do C, Rocha Malacco
VM, Pacheco Rod igues JP, Sac amen o JP, Sama ini Machado F,
Ribei o Pe ei a LG, Ribei o Tomich T, Magalhães Campos M (2019)
Rela ionship be ween eed e ficiency indexes and pe o mance, body
measu emen s, diges ibili y, ene gy pa i ioning, and ni ogen
pa i ioning in p e-weaning dai y hei e s. PLoS One 14, e0223368.
doi:10.1371/jou nal.pone.0223368
Delgado B, Bach A, Guasch I, González C, Elcoso G, P yce JE, Gonzalez-
Recio O (2019) Whole umen me agenome sequencing allows
classi ying and p edic ing eed e ficiency and in ake le els in ca le.
Scien ific Repo s 9, 11. doi:10.1038/s41598-018-36673-w
Dunne JA, Williams RJ, Ma inez ND (2002) Ne wo k s uc u e and
biodi e si y loss in ood webs: obus ness inc eases wi h connec ance.
Ecology Le e s 5, 558–567. doi:10.1046/j.1461-0248.2002.00354.x
Elolimy A, Alha hi A, Zeineldin M, Pa ys C, Loo JJ (2020) Residual
eed in ake di e gence du ing he p eweaning pe iod is associa ed wi h
unique hindgu mic obiome and me abolome p ofiles in neona al
Hols ein hei e cal es. Jou nal o Animal Science and
Bio echnology 11, 13. doi:10.1186/s40104-019-0406-x
Fe nandesAD,MacklaimJM,LinnTG,ReidG, Gloo GB (2013)ANOVA-
like di e en ial exp ession (ALDEx) analysis o mixed popula ion
RNA-seq. PLoS One 8, e67019. doi:10.1371/jou nal.pone.0067019
F iedman J, Alm EJ (2012) In e ing co ela ion ne wo ks om genomic
su ey da a. PLoS Compu a ional Biology 8, e1002687. doi:10.1371/
jou nal.pcbi.1002687
Ghanba i Maman L, Palizban F, Fallah A anaki F, Elmi Ghiasi N,
A iaeenejad S, Gha a i MR, Hosseini Salekdeh G, Ka ousi K (2020)
Co-abundance analysis e eals hidden playe s associa ed wi h high
me hane yield pheno ype in sheep umen mic obiome. Scien ific
Repo s 10, 4995. doi:10.1038/s41598-020-61942-y
Gloo GB, Macklaim JM, Pawlowsky-Glahn V, Egozcue JJ (2017)
Mic obiome da ase s a e composi ional: and his is no op ional.
F on ie s in Mic obiology 8, 2224. doi:10.3389/ micb.2017.02224
He d RM, A hu PF (2009) Physiological basis o esidual eed in ake.
Jou nal o Animal Science 87, E64–E71. doi:10.2527/jas.2008-1345
He nandez-U dane a A, Coppock CE, McDowell RE, Gianola D, Smi h NE
(1976) Changes in o age-concen a e a io o comple e eeds o dai y
cows. Jou nal o Dai y Science 59, 695–707. doi:10.3168/jds.
S0022-0302(76)84260-9
Hun ing on G (1990) Ene gy me abolism in he diges i e ac and li e o
ca le: influence o physiological s a e and nu i ion. Rep oduc ion,
Nu i ion, De elopmen 30,35–47. doi:10.1051/ nd:19900103
Ki elmann S, Pina es-Pa iño CS, Seedo H, Ki k MR, Ganesh S, McEwan
JC, Janssen PH (2014) Two di e en bac e ial communi y ypes a e
linked wi h he low-me hane emission ai in sheep. PLoS One 9,
e103171. doi:10.1371/jou nal.pone.0103171
LiF,ZhouM,OminskiK,GuanLL(2016)Does he umenmic obiomeplay
a ole in eed e ficiency o bee ca le? Jou nal o Animal Science 94,
44–48. doi:10.2527/jas.2016-0524
HAnimal P oduc ion Science S. Cos a-Rou a e al.
McCann KS (2000) The di e si y–s abili y deba e. Na u e 405, 228–233.
doi:10.1038/35012234
McCann JC, Wiley LM, Fo bes TD, Rouque e FM, Tedeschi LO (2014)
Rela ionship be ween he umen mic obiome and esidual eed in ake-
e ficiency o B ahman bulls s ocked on be mudag ass pas u es. PLoS
One 9, e91864. doi:10.1371/jou nal.pone.0091864
Moya A, Fe e M (2016) Func ional edundancy-induced s abili y o gu
mic obio a subjec ed o dis u bance. T ends in Mic obiology 24,
402–413. doi:10.1016/j. im.2016.02.002
Mye PR, Smi h TPL, Wells JE, Kuehn LA, F ee ly HC (2015) Rumen
mic obiome om s ee s di e ing in eed e ficiency. PLoS One 10,
e0129174. doi:10.1371/jou nal.pone.0129174
Negesse T, Da C, Kundu SS (2017) Residual eed in ake, diges ibili y o
nu ien s and e ficiency o wa e u iliza ions in Mu ah bu alo hei e s.
Jou nalo Dai y,Ve e ina y&AnimalResea ch5,74–80.doi:10.15406/
jd a .2017.05.00138
Nk umah JD, Okine EK, Ma hison GW, Schmid K, Li C, Basa ab JA, P ice
MA, Wang Z, Moo e SS (2006) Rela ionships o eedlo eed e ficiency,
pe o mance, and eeding beha io wi h me abolic a e, me hane
p oduc ion, and ene gy pa i ioning in bee ca le. Jou nal o Animal
Science 84, 145–153. doi:10.2527/2006.841145x
Owens FN, Hanson CF (1992) Ex e nal and in e nal ma ke s o
app aising si e and ex en o diges ion in uminan s. Jou nal o
Dai y Science 75,2605–2617. doi:10.3168/jds.S0022-0302(92)
78023-0
Pe ea K, Pe z K, Oli o SK, Williams A, Lachman M, Ishaq SL, Thomson
J, Yeoman CJ (2017) Feed e ficiency pheno ypes in lambs in ol e
changes in uminal, colonic, and small-in es ine-loca ed mic obio a.
Jou nal o Animal Science 95,2585–2592. doi:10.2527/jas2016.1222
R Co e Team (2020) ‘R: a language and en i onmen o s a is ical
compu ing.’(R Founda ion o S a is ical Compu ing: Vienna, Aus ia)
Richa dson EC, He d RM, A hu PF, W igh J, Xu G, Dibley K, Oddy VH
(1996) Possible physiological indica o s o ne eed con e sion
e ficiency in bee ca le. P oceedings o he Aus alian Socie y o
Animal P oduc ion 21, 103–106.
Sa ie o D, Be y DP, F iggens NC (2014) Towa ds an imp o ed
es ima ion o he biological componen s o esidual eed in ake in
g owing ca le. Jou nal o Animal Science 92,4
67–476. doi:10.2527/
jas.2013-6894
Schenkel FS, Mille SP, Wil on JW (2004) Gene ic pa ame e s and b eed
di e ences o eed e ficiency, g ow h, and body composi ion ai s o
young bee bulls. Canadian Jou nal o Animal Science 84,177–185.
doi:10.4141/A03-085
Shaba SKB, Sasson G, Do on-Faigenboim A, Du man T, Yaacoby S,
Be g Mille ME, Whi e BA, Sh e ze N, Miz ahi I (2016) Specific
mic obiome-dependen mechanisms unde lie he ene gy ha es
e ficiency o uminan s. The ISME Jou nal 10,2958–2972.
doi:10.1038/ismej.2016.62
Shi W, Moon CD, Leahy SC, Kang D, F oula J, Ki elmann S, Fan C,
Deu sch S, Gagic D, Seedo H, Kelly WJ, A ua R, Sang C, Soni P, Li
D, Pina es-Pa iño CS, McEwan JC, Janssen PH, Chen F, Visel A,
Wang Z, A wood GT, Rubin EM (2014) Me hane yield pheno ypes
linked o di e en ial gene exp ession in he sheep umen mic obiome.
Genome Resea ch 24,1517–1525. doi:10.1101/g .168245.113
Tsilimig as MCB, Fodo AA (2016) Composi ional da a analysis o he
mic obiome: undamen als, ools, and challenges. Annals o
Epidemiology 26,330–335. doi:10.1016/j.annepidem.2016.03.002
Unge eld EM (2020) Me abolic hyd ogen flows in umen e men a ion:
p inciples and possibili ies o in e en ions. F on ie s in Mic obiology
11, 589. doi:10.3389/ micb.2020.00589
Weime PJ, S e enson DM, Man o ani HC, Man SLC (2010) Hos
specifici y o he uminal bac e ial communi y in he dai y cow
ollowing nea - o al exchange o uminal con en s. Jou nal o Dai y
Science 93,5902–5912. doi:10.3168/jds.2010-3500
Woloszynek S, Mell JC, Zhao Z, Simpson G, O’Conno MP, Rosen GL
(2019) Explo ing hema ic s uc u e and p edic ed unc ionali y o 16S
RNA amplicon da a. PLoS One 14, e0219235. doi:10.1371/jou nal.
pone.0219235
Yus e S, Amanzouga ene Z, de Vega A, Fonde ila M, Blanco M, Casasús
I (2020) E ec o p eweaning die on pe o mance, blood me aboli es
and umen e men a ion a ound weaning in cal es o wo bee b eeds.
Animal P oduc ion Science 60,1018–1027. doi:10.1071/AN19152
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