Lima et al , 2017 - Macrotyloma

Prévia do material em texto

J Anim Physiol Anim Nutr. 2017;1–8.	 wileyonlinelibrary.com/journal/jpn	 	 | 	1© 2017 Blackwell Verlag GmbH
 
Received:	2	March	2017  |  Accepted:	8	August	2017
DOI: 10.1111/jpn.12810
O R I G I N A L A R T I C L E
Nutritional evaluation of the legume Macrotyloma axillare using 
in vitro and in vivo bioassays in sheep
P. M. T. Lima1  | G. D. Moreira2 | G. Z. Sakita1 | A. S. Natel1  | W. T. Mattos3 |  
F. M. A. Gimenes3 | L. Gerdes3 | C. McManus4 | A. L. Abdalla1 | H. Louvandini1
1Centre	for	Nuclear	Energy	in	
Agriculture,	N.A.P.T.I.S.A.,	University	of	São	
Paulo,	Piracicaba,	São	Paulo,	Brazil
2Faculty	of	Agriculture	and	Veterinary	
Medicine,	University	of	Brasília,	Brasília,	
Distrito	Federal,	Brazil
3Centre	for	Research	and	Development	of	
Animal	Nutrition	and	Pastures,	Institute	of	
Animal	Science,	APTA,	SAA-SP,	Nova	Odessa,	 
São	Paulo,	Brazil
4Institute	of	Biological	Sciences,	University	of	
Brasília,	Brasília,	Distrito	Federal,	Brazil
Correspondence
P.	M.	T.	Lima,	Centre	for	Nuclear	Energy	
in	Agriculture,	University	of	São	Paulo,	
Piracicaba,	São	Paulo,	Brazil.
Email:	pmtlima@cena.usp.br
Funding information
São	Paulo	Research	Foundation—FAPESP,	
Grant/Award	Number:	2013/02814-5;	
Coordination	for	the	Improvement	of	Higher	
Education	Personnel
Summary
This	study	consisted	of	two	experiments	with	the	following	objectives:	to	evaluate	the	
effects	of	tannins	from	the	tropical	legume	macrotiloma	(Macrotyloma axillare)	on	total	
gas	and	methane	(CH4)	production,	as	well	as	on	ruminal	fermentation	parameters	by	
performing	an	in	vitro	bioassay,	with	samples	incubated	with	and	without	polyethyl-
ene	glycol	(PEG)	in	a	semi-	automatic	system;	and	secondly	in	a	17	day	in	vivo	experi-
ment,	to	determine	apparent	total	tract	digestibility	(ATTD)	of	dietary	nutrients	and	
ruminal	fermentation	parameters	of	12	intact	8-		to	9-	month-	old	Santa	Inês	(averaging	
24.95	±	1.8	kg	body	weight)	ewes	fed	tropical	grass	hay	supplemented	with	macroti-
loma	hay.	The	ewes	were	divided	into	two	treatment	groups	depending	on	their	diet:	
chopped	aruana	grass	hay	(Panicum maximum	cv.	Aruana)	(control—CON);	and	aruana	
grass	hay	supplemented	with	chopped	macrotiloma	hay	(macrotiloma—MAC).	The	ani-
mals	were	kept	for	5	consecutive	days	in	metabolic	cages	for	the	ATTD	assay,	and	at	
the	end	of	this	period,	samples	of	rumen	fluid	were	collected	from	each	ewe	to	deter-
mine	ammoniacal	nitrogen	(NH3-	N)	and	short-	chain	fatty	acid	(SCFA)	production,	and	
protozoa	count.	For	the	in	vitro	assay,	a	decrease	in	total	gas	and	CH4	production	was	
observed	for	samples	incubated	without	PEG	(p < .05).	No	differences	were	observed	
for	the	other	parameters	evaluated	(p > .05).	In	the	in	vivo	experiment,	increased	in-
take	and	ATTD	of	crude	protein	were	observed	for	the	animals	fed	MAC	when	com-
pared	to	CON	(p < .05).	For	rumen	fermentation	parameters,	increased	NH3-	N,	total	
SCFA	and	 isobutyrate	 concentrations,	 as	well	 as	 reduced	protozoa	 count	were	ob-
served	for	MAC	when	compared	to	CON	(p < .05).	The	results	observed	here	indicated	
the	potential	of	macrotiloma	for	use	as	a	ruminant	feed,	and	antimethanogenic	poten-
tial	of	this	plant	was	noted.
K E Y W O R D S
digestibility,	fermentation,	methane,	polyethylene	glycol,	protozoa,	tannins
1  | INTRODUCTION
Forage	grasses	are	the	main	feed	resource	for	ruminants	in	many	parts	
of	the	world.	In	tropical	countries	such	as	Brazil,	a	pronounced	oscil-
lation	 is	 observed	 in	 the	 availability	 and	 nutritional	 quality	 of	 these	
plants,	mostly	 in	function	of	soil	nutrients	and	rainfall	patterns,	with	
decreases	in	forage	mass	production	and	nutritional	quality	during	the	
dry	seasons	(Bezabih,	Pellikaan,	Tolera,	Khan,	&	Hendriks,	2014).	Most	
tropical	grasses	present	C4	metabolic	pathway	 for	photorespiration,	
and,	usually	in	periods	of	rain	shortage,	these	plants	increase	their	pro-
portion	of	cell	wall	and	reduce	crude	protein	(CP),	where	 levels	may	
be	below	70	g/kg	(on	dry	matter	[DM]	basis),	factors	that	can	lead	to	
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2  |     LIMA et AL.
reduction	in	voluntary	feed	intake	and	digestibility	(Archimède	et	al.,	
2011;	Sampaio	et	al.,	2010).
Considering	this	scenario,	tropical	legumes	are	important	feed	re-
sources	for	ruminants.	These	plants	are	well	adapted	to	a	wide	variety	
of	climatic	conditions	and	may	be	either	grazed	or	harvested	and	sup-
plemented	to	the	animals.	These	forages	may	lead	to	increased	animal	
performance	when	employed	 in	 ruminant	production	 systems	given	
their	typical	higher	digestibility	and	CP	content	than	grasses,	associ-
ated	with	lower	fibre	content.	In	addition,	legumes	may	also	represent	
a	 significant	 source	 of	 minerals	 (Hammond	 et	al.,	 2011;	 Ramírez-	
Restrepo,	Barry,	López	Villalobos,	Kemp,	&	Harvey,	2005;	Seresinhe,	
Madushika,	Seresinhe,	Lal,	&	Orskov,	2012;	Tiemann	et	al.,	2009).
Commonly	 found	 in	 legumes,	 tannins	are	 secondary	metabolites	
of	these	plants	that	consist	chemically	of	phenolic	compounds	charac-
terised	by	their	ability	to	bind	to	proteins	and	carbohydrates.	Based	on	
their	molecular	structure,	tannins	are	qualified	into	two	classes,	hydro-
lysable	and	condensed.	The	effects	of	these	molecules	on	the	metab-
olism	of	ruminants	vary	according	to	their	origin,	chemical	nature	and	
concentration	 in	diet	 (Rodríguez,	de	 la	Fuente,	Gómez,	&	Fondevila,	
2014).	In	ruminant	nutrition,	tannins	are	seen	as	capable	of	generating	
detrimental	 effects	 in	 the	 animals,	 such	 as	 reduction	 in	 digestibility	
and	palatability	of	diets,	 as	well	 as	beneficial	 effects;	 as	 included	 in	
diets	in	concentrations	of	up	to	40	g/kg	of	DM,	these	molecules	may	
increase	the	absorption	of	dietary	amino	acids	in	the	small	intestine	by	
binding	to	proteins	and	decreasing	their	 ruminal	degradation	 (Hoste	
et	al.,	2012)	or	reduce	methane	(CH4)	production	during	enteric	fer-
mentation	(Jayanegara,	Leiber,	&	Kreuzer,	2012).
The	 tropical	 legume	Macrotyloma axillare	 has	 been	 described	 in	
several	regions	worldwide	and	presents	potential	for	being	used	as	a	
ruminant	feed	(Blumenthal	&	Staples,	1993)	despite	little	data	being	
available	in	the	literature.	This	plant	is	reported	as	having	low	levels	of	
tannins	(i.e.,	total	tannins	=	19	g/kg	DM	and	condensed	tannins	=	5	g/
kg	DM—Valarini	&	Possenti,	2006),	thus	reducing	chances	of	antinu-
tritional	effects	due	to	its	consumption	as	these	effects	are	described	
when	tannins	are	 included	 in	concentrations	of	60–120	g/kg	DM	in	
total	diet	(Frutos,	Hervás,	Ramos,	Giráldez,	&	Mantecón,	2002).	Under	
intercropping	with	 aruana	 grass	 (Panicum maximum	 cv.	Aruana),	 the	
Accession	NO	279	 of	 this	 legume	 showed	 higher	 forage	mass	 pro-
duction	and	provided	greater	CP	increase	(i.e.,	N	transfer	through	the	
soil)	to	the	associated	grass	species	when	compared	to	other	legume	
species	and	accessions	(Gerdes	et	al.,	2009).
Performing	 in	vitro	 bioassays	 using	 polyethylene	 glycol	 (PEG),	 a	
tannin	binding	agent,	is	a	simple	and	useful	method	for	studying	the	ef-
fects	of	tannins	on	ruminal	fermentation	processes	(Makkar,	Blümmel,	
&	Becker,	1995;	Rodríguez	et	al.,	2014).	We	hypothesised	that	tannins	
from	the	tropical	legume	macrotiloma	(Macrotyloma axillare,	Accession	
NO	279)	may	affect	rumen	fermentation	parameters,	and	that	these	
molecules	could	also	affect	the	apparent	total	tract	digestibility	(ATTD)	
of	nutrients	in	ruminants	fed	this	plant.
Therefore,	this	study	aimed	to	evaluate	the	effects	of	tannins	from	
the	tropical	legume	macrotiloma	on	in	vitro	gas	and	CH4	production	as	
well	as	on	fermentation	parameters,	using	substrates	incubated	with	
and	without	PEG;	and	also	in	an	in	vivo	experiment,	we	aimed	to	eval-
uate	the	ATTD	of	dietary	nutrients	and	ruminal	fermentation	parame-
ters	of	Santa	Inês	ewes	supplemented	with	macrotiloma.
2  | MATERIAL AND METHODS
All	the	procedures	involving	the	use	of	animalsthat	took	place	in	this	
study	were	approved	by	Ethics	Committee	on	Use	of	Animals	of	the	
Escola	Superior	de	Agricultura	“Luiz	de	Queiroz”—CEUA-	ESALQ/USP	
(Protocol	Number	005061).	The	experiments	were	carried	out	at	the	
Laboratory	of	Animal	Nutrition	of	 the	Centre	 for	Nuclear	Energy	 in	
Agriculture	from	the	University	of	São	Paulo	 (LANA/CENA/USP),	 in	
the	city	of	Piracicaba,	São	Paulo.
2.1 | In vitro bioassay
The	substrates	used	for	this	assay	consisted	of	macrotiloma	hay,	pro-
duced	at	the	Institute	of	Animal	Science	(IZ/APTA/SAA),	in	the	city	of	
Nova	Odessa,	São	Paulo	state.	For	hay	production,	macrotiloma	was	
harvested	during	the	flowering	stage,	108	days	after	seeding,	present-
ing	an	estimated	biomass	production	of	2.6	t/ha	and	540	g/kg	of	DM	
content.	After	harvest,	the	material	was	dried	in	the	shade	during	10	
consecutive	days.	 Immediately	after	 the	hay	was	prepared,	a	 repre-
sentative	sample	(1.0	kg)	of	the	material	was	collected	and	ground	in	
a	Wiley	mill	through	a	1-	mm	sieve	for	performing	the	in	vitro	gas	pro-
duction	assay	and	chemical	composition	analyses	(Table	1).	Chemical	
composition	was	expressed	on	a	DM	basis;	CP	(ID	number:	2001.11),	
ether	extract	 (EE;	 ID	number:	2003.5)	and	ash	 fraction	 (ID	number:	
942.05)	were	 determined	 according	 to	AOAC	 (2011),	while	 neutral	
detergent	fibre	(NDF)	and	acid	detergent	fibre	(ADF)	were	determined	
TABLE  1 Chemical	composition	of	forages	used	in	this	study	
(values	expressed	on	dry	matter	basis).	For	the	in	vitro	assay,	only	
macrotiloma	hay	was	used
Parameter Aruana grass hay Macrotiloma hay
Dry	matter	(g/kg)a 953.85 873.87
Crude	protein	(g/kg)a 91.10 154.02
Ether	extract	(g/kg)a 24.15 30.18
Neutral	detergent	fibre	
(g/kg)b
731.06 626.58
Acid	detergent	fibre	(g/
kg)b
456.10 446.36
Ash	fractiona 61.75 77.65
Total	phenolic	
compoundsc,d
– 29.64
Total	tanninsc,d – 23.23
Condensed	tanninsc,e – 2.14
aAnalyses	performed	according	to	AOAC	(2011).
bAnalyses	performed	according	to	Van	Soest	et	al.	(1991)	and	adapted	by	
Mertens	(2002).
cAnalyses	performed	according	to	Makkar	(2000).
dValues	expressed	in	equivalent	gram	of	tannic	acid/kg	dry	matter.
eValues	expressed	in	equivalent	gram	of	leucocyanidin/kg	dry	matter.
     |  3LIMA et AL.
using	a	fibre	analyser	(Tecnal	TE-	149,	Piracicaba,	Brazil)	and	Ankom	
filter	bags	(Ankom	F-	57,	Macedon,	NY,	USA)	according	to	Van	Soest,	
Robertson,	 and	Lewis	 (1991)	 and	adapted	by	Mertens	 (2002).	Also,	
in	 the	 case	of	macrotiloma	 total	 phenolic	 compounds,	 total	 tannins	
and	condensed	tannins	were	determined	according	to	methodologies	
described	in	Makkar	(2000—p.4	and	p.6	respectively).	A	pool	of	differ-
ent	Schinus terebinthifolius	(aroeira)	samples	was	used	as	a	reference	
standard	for	these	analyses.
For	the	in	vitro	gas	production	assay,	inocula	were	prepared	with	
ruminal	content	from	three	adult	rumen-	cannulated	Santa	Inês	male	
sheep	(averaging	65.00	±	2.30	kg	of	body	weight	[BW])	collected	be-
fore	the	morning	feed.	The	animals	were	fed	ad	libitum	tropical	grass	
hay	 (Tifton	85—Cynodon	 spp.—DM:	891.38	g/kg;	NDF:	876.40	g/kg	
DM;	ADF:	 491.86	g/kg	DM;	 CP:	 78.31	g/kg;	 ash	 fraction:	 62.13	g/
kg	DM),	water	and	mineral	salt.	Liquid	and	solid	fractions	of	ruminal	
content	from	the	animals	were	collected	into	thermal	containers	using	
a	silicone	tube	adapted	to	a	60-	ml	syringe	(Becton-	Dickson	Indústria	
Cirúrgica,	Curitiba,	Brazil)	and	a	crucible	tong,	respectively,	and	used	
for	preparation	of	the	three	inocula	utilised	in	this	assay.	Inocula	were	
prepared	adopting	a	50:50	solid:liquid	ratio	(on	a	volume	basis)	(Bueno	
et	al.,	2005).	 Incubation	was	carried	out	according	to	methodologies	
described	 by	 Theodorou,	 Williams,	 Dhanoa,	 McAllan,	 and	 France	
(1994)	 and	Mauricio	 et	al.	 (1999),	 with	 adaptations	 of	 Bueno	 et	al.	
(2005)	and	Longo	et	al.	(2006).	A	half	gram	of	ground	(1	mm)	macroti-
loma	hay	was	transferred	into	160-	ml	bottles	together	with	50	ml	of	
incubation	medium	(Menke’s	buffered	medium)	and	25	ml	of	inoculum.
Macrotiloma	 substrates	 (0.5	g)	 were	 incubated	 with	 and	 with-
out	PEG	 (PEG	6000	LabSynth,	Diadema,	Brazil),	 using	 a	1:1	macro-
tiloma:PEG	 (on	 mass	 basis)	 ratio	 for	 samples	 incubated	 with	 PEG,	
to	 observe	 the	 effects	 of	 tannins	 on	 fermentation	 parameters.	Two	
bottles	 per	 treatment	 (two	 with	 PEG	 (+PEG);	 two	 without	 PEG	 
(−PEG))	were	incubated	with	each	one	of	the	three	inocula	(i.e.,	3	in-
ocula	×	2	+	PEG	×	2	–PEG	=	12	bottles—experimental	units).	 In	addi-
tion,	for	each	inoculum,	four	blank	bottles	(two	+PEG	and	two	−PEG)	
containing	 only	 inoculum	 and	 incubation	 medium,	 and	 four	 bottles	
(two	+PEG	and	two	−PEG)	with	internal	standard	samples	(Tifton	85	
Cynodon	spp.)	were	incubated,	totalling	36	bottles.	After	that,	bottles	
were	 sealed	 and	 incubated	 in	 a	 forced	ventilation	oven	at	39°C	 for	
24 hr.
At	4,	8,	12	and	24	hr	after	start	of	incubation,	the	internal	pressure	
of	each	bottle	was	measured	using	a	pressure	transducer	and	a	data	
logger	(Pressure	Press	800,	LANA,	CENA/USP,	Piracicaba,	Brazil).	Total	
volume	of	gas	produced	in	each	bottle	was	determined	following	the	
equation	V =	(7.365	*	P)	where:	V =	gas	volume	(ml)	and	P =	measured	
pressure	(psi)	(Araujo,	Pires,	Mourão,	Abdalla,	&	Sallam,	2011).	Inocula	
from	three	different	animals	were	used	in	the	in	vitro	assay	to	avoid	
any	 inoculum-	induced	bias	 in	our	 results.	Based	on	 that,	we	used	a	
single	incubation	period	of	24	hr.	All	results	were	corrected	using	val-
ues	from	blank	bottles,	and	the	total	gas	production	(GP)	at	the	end	of	
incubation	was	calculated	by	summing	the	gas	volume	values	obtained	
in	each	measurement	event.
After	the	measurement	of	internal	pressure,	2.5	ml	of	gas	samples	
were	 collected	 from	each	bottle	 in	 10-	ml	vacuum	 tubes	 using	5-	ml	
syringes	(Becton-	Dickson	Indústria	Cirúrgica,	Curitiba,	Brazil)	for	de-
termination	of	CH4	concentration	in	a	gas	chromatograph	(Shimadzu	
GC-	2010,	Tokyo,	Japan)	equipped	with	flame	ionization	detector	(FID)	
and	a	capillary	HP-	Molesieve	column	(GC	30	m	×	0.53	mm	×	25	μm). 
Chromatograph	conditions	were	as	follows:	column	temperature	(iso-
thermal)	at	60°C,	injector	at	200°C,	detector	at	240°C	and	carrier	gas	
(Helium—He)	 in	 constant	 flux	 at	 10	ml/min.	Methane	 concentration	
was	 determined	 using	 a	 calibration	 curve	 (0,	 30,	 90	 and	 120	ml/L)	
prepared	with	 a	 commercial	CH4	 standard	 (Praxair	 Industrial	Gases,	
Osasco,	Brazil;	995	ml/L	purity).	Following	the	gas	sampling,	the	pres-
sure	of	each	bottle	was	relieved,	their	content	was	homogenised	and	
they	were	returned	to	the	oven.
After	24	hr	of	incubation	and	the	last	pressure	measurement/gas	
sampling	procedure,	the	content	of	each	bottle	was	used	for:	pH	mea-
surement,	by	means	of	a	pH	meter	(model	TEC-	2,	Tecnal,	Piracicaba,	
Brazil);	determination	of	ammoniacal-	N	(NH3-	N)	concentration	using	
micro-	Kjeldahl	 steam	 distillation	 with	 sodium	 tetraborate	 solution	
(5%)	 according	 to	Preston	 (1995);	 and	determination	of	 short-	chain	
fatty	acid	(SCFA)	concentration,	using	methodology	of	Palmquist	and	
Conrad	(1971)	(adapted).
For	 the	 determination	 of	 SCFA,	 1.6	ml	 of	 the	 content	 of	 each	
bottle	was	 centrifuged	 (RC	5B	PLUS,	 Sorval,	Wilmington,	DE,	USA)	
at	10,400	g	for	40	min	at	4°C,	and	800	μl	of	the	supernatant	was	col-
lected	in	microtubes	and	added	100	μl	of	2-	ethyl-	butyric	acid	(internal	
standard	 MW=116.16;	 Sigma	 Chemie	 Gmbh,	 Steinheim,	 Germany)	
and 200 μl	 formic	acid	 (85%).	After	 that,	1	μl	 aliquot	of	 the	samples	
was	 injected	 in	 a	 gas	 chromatograph	 (GC	 2014	 Shimadzu,	 Tokyo,	
Japan)	equipped	with	FID	and	with	 the	column	GP	10%	SP-	1200/1	
H3PO4	80/100	Chromosorb	WAW	(Cat.	no.	11965,	6′	×	1/8′’stainless	
steel,	Supelco,	Bellefonte,	PA,	USA).	Column	temperature	(isothermal)	
was	at	115°C,	injector	at	200°C	and	detector	at	260°C.	The	carrier	gas	
was	He	at	25	ml/min.	Detector	hydrogen	and	synthetic	air	werekept	
at	40	and	400	ml/min	respectively.	Total	analysis	time	was	10.55	min.
A	calibration	curve	was	prepared	with	standards	of	known	concen-
trations	(acetate	99.5%,	CAS	64-	19-	97;	propionate	99%,	CAS	04-	09-	
79;	 isobutyrate	99%,	CAS	79-	31-	2;	butyrate	98.7%,	CAS	107-	92-	6;	
isovalerate	 99%,	 CAS	 503-	74-	2	 and	 valerate	 99%,	 CAS	 109-	52-	4,	
Chem	Service,	West	Chester,	PA,	USA).
2.2 | Apparent total tract digestibility assay
During	 a	 17-	day	 experimental	 period	 (12	days—adaptation	 to	 diets	
and	5	days—collection	period),	 twelve	8-		 to	9-	month-	old	Santa	 Inês	
ewes	 averaging	 24.95	±	1.80	kg	 of	 initial	 body	 weight	 (BW)	 were	
used.	Initially,	the	animals	were	randomly	divided	into	two	treatment	
groups	(two	different	diets)	allocated	in	collective	pens	(six	animals	in	
each	pen)	for	10	days,	to	adapt	to	experimental	diets,	and	they	were	
then	 transferred	 to	metabolic	 cages	 for	 a	2	days	adaptation	period.	
Then	a	5-	day	collection	period	for	the	ATTD	assay	was	carried	out.
Diets	 used	 for	 this	 assay	 were	 as	 follows:	 (Table	1):	 (control—
CON)—ad	libitum	chopped	aruana	grass	hay;	(macrotiloma—MAC)—ad	
libitum	chopped	aruana	grass	hay	supplemented	with	chopped	mac-
rotiloma	hay.	Animals	were	 fed	 twice	 a	day	 (8:00	 and	16:00)	 based	
4  |     LIMA et AL.
on	a	DM	 intake	 (DMI)	 of	3%	of	BW	and	 leftovers	 equal	 to	10%	of	
total	offer	 (on	DM	basis).	For	 the	MAC	group,	 the	animals	were	fed	
following	a	75:25	grass:legume	ratio	(as	fed	basis).	Both	forages	were	
fed	separately	so	that	the	intake	of	each	one	could	be	precisely	calcu-
lated.	Macrotiloma	supplementation	was	offered	in	the	morning	feed,	
prior	 to	 aruana	 hay.	After	 one	 hour,	 animals	 had	 already	 consumed	
the	legume,	leftovers	were	collected,	and	then	aruana	grass	hay	was	
offered.	During	the	adaptation	period,	as	well	as	in	collections,	water	
and	mineral	salt	were	available	ad	libitum.
Feed	offer	samples	 (equivalent	 to	10%	of	 total	 individual	offer—
DM	basis),	 individual	feed	leftovers	and	faeces	were	completely	col-
lected	on	a	daily	basis	during	the	assay.	Feed	leftovers	and	faeces	were	
weighed,	and	10%	(on	weight	basis)	samples	of	these	materials	were	
randomly	collected	(for	each	animal)	and	stored	in	plastic	bags	under	
refrigeration	 at	 −2°C	 for	 chemical	 analyses,	which	were	 carried	 out	
at	 the	end	of	 the	assay.	 Immediately	after	 the	end	of	 the	collection	
period,	feed	leftovers	and	faeces	samples	were	removed	from	the	re-
frigeration	and	thawed	for	12	hr	under	ambient	conditions.	Feed	offer	
samples	(total	sampled	material),	leftovers	and	faeces	(thawed	mate-
rial)	were	then	dried	in	a	forced	ventilation	oven	at	40°C	for	5	consec-
utive	days	and	were	ground	in	a	Wiley	mill	through	a	1-	mm	sieve	for	
chemical	composition	analyses	(organic	matter	(OM),	DM,	NDF,	ADF	
and	CP)	 following	procedures	previously	described.	All	 the	 analyses	
were	performed	in	duplicate.
Rumen	fluid	samples	(100	ml)	were	collected	from	each	animal	at	
end	of	the	ATTD	assay	using	an	oesophageal	tube,	three	hours	after	
feed	was	offered.	These	samples	were	used	for	performing	protozoa	
count,	 according	 to	Dehority,	Damron,	 and	McLaren	 (1983)	 and	 for	
determining	 the	 concentrations	of	NH3-	N	 and	SCFA	using	method-
ologies	previously	described.	For	NH3-	N	and	SCFA	analyses,	samples	
(15	ml	 for	 each	 analysis/animal)	 were	 stored	 under	 refrigeration	 at	
−20°C	until	the	moment	of	the	analysis;	while	for	protozoa	count,	2	ml	
of	each	sample	was	mixed	with	4	ml	of	methyl	green	formalin	 (35%	
formaldehyde)	saline	solution	(MFS)	and	preserved	from	light	at	room	
temperature	until	counting.
2.3 | Data analysis and statistics
Statistical	 analysis	 of	 the	 data	 was	 carried	 out	 using	 SAS	 v.	 9.4	
(Statistical	Analysis	System	Institute,	Cary	NC,	USA).	For	the	in	vitro	
bioassay	data,	analysis	of	variance	was	performed	using	PROC	MIXED,	
considering	the	PEG	inclusion	as	fixed	effect	and	inocula	as	random.	
In	 the	case	of	 the	ATTD	assay,	 analysis	of	 variance	was	performed	
by	PROC	GLM,	and	comparison	of	means	was	by	Tukey’s	 test.	The	
significance	level	adopted	for	all	the	analysis	was	5%.
3  | RESULTS
The	addition	of	PEG	to	substrates	showed	an	effect	on	GP	and	CH4 
production	(Table	2).	In	both	cases,	−PEG	samples	presented	reduced	
(p < .05)	GP	and	CH4	production	when	compared	 to	+PEG	samples.	
Methane	 proportion	 was	 the	 parameter	 considered	 as	 the	 volume	
of	GP	(in	percentage—%)	represented	by	CH4.	The	presence	of	PEG	
caused	a	tendency	towards	the	reduction	in	CH4	proportion	(p < .10). 
The	inclusion	of	PEG	in	substrates	showed	no	effect	on	in	vitro	fer-
mentation	pH	nor	on	NH3-	N	and	SCFA	concentration	(p > .05).
In	the	ATTD	assay	(Table	3),	ewes	fed	MAC	presented	higher	CP	
intake	(p < .05)	when	compared	to	those	fed	CON,	while	no	difference	
(p > .05)	was	observed	between	treatments	for	the	other	dietary	nu-
trients.	When	considering	ATTD	(Table	4),	animals	fed	MAC	showed	
higher	CP	digestibility	than	the	animals	fed	CON	(p < .05).
In	the	evaluation	of	rumen	fermentation	parameters	from	animals	
on	the	ATTD	assay	(Table	4),	increased	NH3-	N,	total	SCFA	and	isobu-
tyrate	concentrations	were	determined	for	MAC	in	comparison	with	
CON	(p < .05).	The	opposite	was	seen	for	the	protozoa	count,	as	CON	
showed	higher	counts	than	MAC	(p < .05).
4  | DISCUSSION
In	the	present	study,	tannins	from	macrotiloma	affected	GP	and	CH4 
production	 under	 in	 vitro	 fermentation	 conditions.	 Total	 gas	 pro-
duction	may	be	used	as	 a	parameter	 indicative	of	 the	degradability	
TABLE  2 Effect	of	the	presence	(+)	and	absence	of	PEG	(−) on in 
vitro	gas	production	and	fermentation	parameters	of	macrotiloma	
substrates.	Results	presented	are	arithmetic	means
Parameter
PEG
SEM p value(−) (+)
Experimental	units	
(n)
6 6 – –
GP	(ml/g	DM) 117.73 125.57 3.00 .04
CH4	(ml/g	DM) 5.55 7.82 0.90 .04
CH4	proportion	
(%)
4.72 6.22 0.53 .09
NH3-	N	(mg/dl) 12.75 11.61 1.79 n.s.a
pH 6.85 6.89 0.03 n.s.
SCFA
Total	(mmol/L) 47.16 48.11 0.55 n.s.
Acetate	(%) 75.56 75.77 0.21 n.s.
Propionate	(%) 15.56 15.48 0.10 n.s.
Butyrate	(%) 5.97 5.84 0.09 n.s.
Valerate	(%) 1.00 1.02 0.02 n.s.
Isobutyrate	(%) 0.44 0.42 0.01 n.s.
Isovalerate	(%) 1.48 1.47 0.02 n.s.
Acetate/
propionate
4.86 4.90 0.04 n.s.
GP,	total	gas	production	per	dry	matter	incubated	(v/m);	CH4/DM,	meth-
ane	production	per	dry	matter	incubated	(v/m);	CH4	proportion,	percent-
age	(%)	of	methane	in	relation	to	total	gas	produced;	NH3-	N,	ammoniacal	
nitrogen;	 SCFA,	 short-	chain	 fatty	 acids;	 PEG,	 polyethylene	 glycol;	 SEM,	
standard	error	of	the	mean.
an.s.,	p > .05.
     |  5LIMA et AL.
of	substrates	 (Sallam	et	al.,	2010).	Tannins	are	molecules	capable	of	
reducing	the	breakdown	of	nutrients,	such	as	fibre	and	protein	(Goel	
&	Makkar,	2012;	Tiemann	et	al.,	2009).	Therefore,	the	decreased	GP	
observed	for	the	−PEG	samples	led	us	to	initially	suggest	that	the	deg-
radability	of	those	was	reduced	as	they	were	subjected	to	the	effects	
of	tannins	from	macrotiloma.
The	 increase	 in	GP	caused	by	 the	 addition	of	PEG	corroborates	
the	 fact	 that	 effects	of	 tannins	on	 ruminal	 fermentation	depend	on	
their	source	and	chemical	nature	and	not	only	on	their	concentrations	
(Goel	&	Makkar,	2012;	Rodríguez	et	al.,	2014).	Soltan	et	al.	(2013)	per-
formed	an	 in	vitro	 gas	production	 assay	using	 the	 legume	Leucaena 
leucocephala	and	determined	34.80	and	23.30	g/kg	DM	of	total	and	
condensed	tannins,	respectively,	and	the	presence	of	PEG	did	not	lead	
to	 increased	 total	 gas	 production,	 showing	 that	 tannins	 from	 L. leu-
cocephala	were	not	capable	of	affecting	this	parameter.	On	the	other	
hand,	macrotiloma	had	total	and	condensed	tannin	concentrations	of	
23.23	and	2.14	g/kg	DM,	respectively,	lower	than	values	from	L. leuco-
cephala	and	even	so,	reduced	GP	was	observed	here.
During	 ruminal	 fermentation,	 tannins	 affect	 CH4	 production	 by	
both	 direct	 and	 indirect	 mechanisms.The	 direct	 mechanism	 is	 at-
tributed	 to	 the	 toxic	effect	 that	 tannins	may	have	on	methanogens,	
strict	 anaerobic	microbes	 of	 the	Archaea domain and Euryarchaeota 
phylum,	which	 are	 responsible	 for	methanogenesis	 in	 ruminant	me-
tabolism.	Meanwhile,	 indirect	mechanisms	may	be	explained	by	 the	
reduction	 in	 degradability	 of	 nutrients,	 which	 is	 directly	 related	 to	
CH4	production,	and	by	the	decrease	in	ruminal	protozoa	counts,	as	
these	unicellular	eukaryotic	organisms	may	live	physically	associated	
to	methanogens,	 thus	 contributing	 towards	 CH4	 production	 (Hook,	
Wright,	&	McBride,	2010;	Jayanegara	et	al.,	2012).
In	 cases	where	 methanogenesis	 is	 directly	 affected	 (e.g.,	 meth-
anogens	 inhibition)	and	not	only	a	consequence	of	 reduced	degrad-
ability,	 the	proportion	of	CH4	 in	 total	gas	production	 is	expected	to	
be	reduced	(Bhatta,	Saravanan,	Baruah,	&	Prasad,	2015).	Our	results	
regarding	CH4	proportion	showed	no	significant	statistical	difference	
(p > .05);	however,	 a	 tendency	 towards	 reduction	 in	CH4	proportion	
was	observed,	indicating	that	possibly	another	CH4	inhibition	mecha-
nism,	which	could	not	be	identified	in	our	in	vitro	assay	has	occurred,	
also	contributing	to	the	reduction	in	CH4	production.
The	 results	observed	 in	our	analyses	of	 rumen	 fermentation	pa-
rameters	 also	 corroborate	 this	 argument.	 Ammoniacal-	N	 and	 SCFA	
production	 are	 parameters	 that	 may	 be	 used	 as	 indicators	 of	 the	
breakdown	 of	 proteins	 and	 carbohydrates.	 Decreased	 NH3-	N	 pro-
duction	 is	 commonly	 observed	 in	 cases	 of	 reduced	 degradability	 of	
proteins,	while	 total	SCFA,	acetate	and	 iso-	SCFA	production	may	all	
be	decreased	when	fibre	and	protein	are	poorly	degraded	(Jayanegara	
et	al.,	2012).	As	no	signs	of	decreased	degradability	of	these	nutrients	
were	 observed	 when	 analysing	 fermentative	 parameters,	 and	 con-
sidering	the	tendency	for	a	reduced	CH4	proportion	verified	in	−PEG	
samples,	our	hypothesis	 that	 a	 factor	other	 than	decreased	degrad-
ability	was	responsible	for	reducing	methanogenesis	was	reinforced.
During	 the	ATTD	 assay,	 feed	 intake	 from	 ewes	 from	 CON	 and	
MAC	was	all	 in	 accordance	with	 individual	DMI	 levels	described	by	
the	NRC	(2007),	which	varies	in	the	range	of	600–1000	g/day	for	an-
imals	between	20	and	30	kg	BW.	The	similar	DMI	observed	for	both	
treatments	 showed	 that	macrotiloma	 supplementation	did	not	 have	
TABLE  3 Average	individual	daily	intake	of	dietary	nutrients	of	
ewes	fed	control	(CON)	and	macrotiloma	(MAC)	diets.	Results	
presented	are	arithmetic	means
Nutrient intake CON MAC SEM p value
Animals	per	
treatment	(n)
6 6 – –
DMI	g/d 822.38 815.88 19.86 n.s.a
OMI	g/d 771.60 762.33 18.60 n.s.
NDFI	g/d 601.21 575.61 14.33 n.s.
ADFI	g/d 375.09 370.18 9.04 n.s.
CPI	g/d 74.92 86.88 1.92 .0014
DMI,	dry	matter	intake;	OMI,	organic	matter	intake;	NDFI,	neutral	deter-
gent	fibre	intake;	ADFI,	acid	detergent	fibre	intake;	CPI,	crude	protein	in-
take;	SEM,	standard	error	of	the	mean.
an.s.,	p > .05.
TABLE  4 Apparent	total	tract	digestibility	(ATTD)	of	dietary	
nutrients	and	rumen	fermentation	parameters	of	Santa	Inês	ewes	fed	
control	(CON)	and	macrotiloma	(MAC)	diets.	Results	presented	are	
arithmetic	means
ATTD CON MAC SEM p value
Animals	per	
treatment	(n)
6 6 – –
DM	(%) 54.62 55.62 10.87 n.s.a
OM	(%) 57.56 58.61 9.64 n.s.
NDF	(%) 56.62 56.46 11.59 n.s.
ADF	(%) 53.58 53.34 14.31 n.s.
CP	(%) 58.27 64.74 10.63 .0016
Ruminal	parameters
NH3-	N	(mg/dl) 9.06 11.61 0.78 .04
Protozoa	
(×105/ml)
6.05 4.13 0.58 .04
SCFA
Total	(mmol/L) 70.01 82.28 3.74 .03
Acetate	(%) 78.08 77.98 0.59 n.s.
Propionate	(%) 14.51 13.80 0.37 n.s.
Butyrate	(%) 5.73 6.09 0.32 n.s.
Valerate	(%) 0.51 0.45 0.02 n.s.
Isobutyrate	(%) 0.15 0.52 0.05 .0016
Isovalerate	(%) 0.97 1.24 0.14 n.s.
Acetate/
propionate
5.41 5.66 0.18 n.s.
DM,	dry	matter;	OM,	organic	matter;	NDF,	neutral	detergent	fibre;	ADF,	
acid	detergent	 fibre;	CP,	crude	protein;	ATTD	values	are	presented	as	a	
percentage	 in	 relation	 to	 the	 amount	 ingested	 of	 each	 dietary	 nutrient;	
NH3-	N,	ammoniacal	nitrogen;	SCFA,	short-	chain	fatty	acids;	SEM,	stand-
ard	error	of	the	mean.
an.s.,	p > .05.
6  |     LIMA et AL.
a	negative	 impact	on	 feed	 intake.	 In	 this	 experiment,	 no	 reductions	
in	ATTD	of	dietary	nutrients	were	verified	for	the	animals	fed	MAC,	
and	feeding	this	 legume	 increased	not	only	 intake	but	also	ATTD	of	
CP,	corroborating	the	elevated	nutritional	value	and	digestibility	which	
are	 generally	 attributed	 to	 these	 plants	when	 compared	 to	 grasses	
(Hammond	et	al.,	2011).
Despite	usually	presenting	greater	nutritional	quality	than	tropical	
grasses,	including	legumes	in	diets	of	ruminants	may	not	always	lead	
to	improvements	in	animal	performance,	as	the	presence	of	second-
ary	metabolites	such	as	tannins	may	limit	the	utilisation	of	nutrients	
thus	possibly	impairing	productive	parameters.	Tiemann	et	al.	(2008)	
evaluated	 the	 effect	 of	 including	 two	 different	 tannin-	rich	 legumes	
(Calliandra calothyrsus and Flemingia macrphylla	 that	 contributed,	 re-
spectively,	with	18.8	and	8.6	g/kg	DM	of	condensed	tannins	when	in-
cluded	at	300	g/kg	DM	in	total	diet)	in	the	diet	of	lambs	fed	different	
proportions	of	grass	and	legume	mixtures	and	observed	pronounced	
reduction	 in	CP	ATTD	for	the	animals	fed	the	highest	 levels	of	con-
densed	tannins.
The	increased	production	of	NH3-	N	of	animals	fed	MAC	was	likely	
a	consequence	of	 their	higher	CP	 intake.	 In	 the	 rumen,	 the	protein-	
binding	 ability	 of	 tannins	 usually	 leads	 to	 decreases	 in	 deamination	
reactions	 and	 consequently	 lessens	 NH3-	N	 production	 (Aufrère,	
Dudilieu,	Andueza,	Poncet,	&	Baumont,	2013;	Wang,	Berg,	Barbieri,	
Veira,	&	McAllister,	2006).	As	observed	in	our	 in	vitro	assay,	tannins	
from	macrotiloma	apparently	did	not	 reduce	 ruminal	degradation	of	
proteins	 in	 our	 in	vivo	ATTD	 assay.	 For	 the	 proper	 development	 of	
microbes,	NH3-	N	levels	should	be	in	the	range	of	5–20	mg/dl	(Leng,	
1990;	 Pimentel	 et	al.,	 2012).	 In	 both	 experiments	 here,	 the	 levels	
observed	in	the	presence	of	macrotiloma	remained	between	11	and	
13	mg/dl,	 showing	 that	 this	 legume	was	capable	of	meeting	 the	 re-
quirements	of	rumen	microbes	for	this	parameter.	On	the	other	hand,	
it	is	important	to	note	that	if	tannins	from	macrotiloma	were	binding	
to	dietary	proteins	and	reducing	the	NH3-	N	production	in	rumen,	an-
imal	performance	parameters	may	be	increased,	as	this	mechanism	is	
likely	to	increase	intestinal	absorption	of	dietary	amino	acids	(Ramírez-	
Restrepo	et	al.,	2005).
Similarly,	SCFA	production	of	animals	fed	MAC	was	affected	by	the	
CP	 intake.	Total	SCFA	and	 isobutyrate	production	were	 increased	 in	
animals	fed	MAC.	The	iso-SCFA	is	originated	from	ruminal	degradation	
of	proteins	(Bhatta	et	al.,	2013),	and	the	results	observed	here,	as	with	
the	NH3-	N	analysis,	were	a	consequence	of	the	higher	CP	intake	for	
the	animals	fed	MAC,	as	evidenced	by	the	elevated	isobutyrate	values,	
factor	that	also	led	to	the	higher	total	SCFA	production	observed	for	
these	animals.
Although	tannins	from	macrotiloma	apparently	did	not	show	any	
negative	effect	on	ATTD	of	nutrients	and	ruminal	degradability	of	diet	
in	this	experiment,	protozoa	counts	seemed	to	be	affected	by	these	
molecules.	 Tannins	 are	 pointed	 out	 as	 capable	 of	 reducing	 ruminal	
protozoa	 populations	 (Jayanegara	 et	al.,	 2012),	 and	 despite	 some	
controversy,	reducing	protozoa	count	is	considered	a	CH4	mitigation	
strategy,	 thus	 characterising	 another	 possible	 benefit	 regarding	 the	
use	of	macrotiloma	(Hristov	et	al.,	2013;	Nguyen,	Bremner,	Cameron,	
&	Hegarty,	2016).
In	 the	 in	 vitro	 experiment,	 reduced	 CH4	 production	 was	 ob-
served	for	the	−PEG	samples,	but	mechanisms	of	action	that	led	to	
this	could	not	be	precisely	identified.	In	this	case,	the	CH4 propor-
tion	for	−PEG	samples	suggested	that	otherthan	a	consequence	of	
reduced	substrate	degradability,	another	CH4	inhibition	mechanism	
was	also	taking	place.	Considering	our	findings	in	the	ATTD	assay,	
we	 inferred	that	probably	protozoa	counts	were	also	reduced	 in	−
PEG	samples,	thus	leading	to	decreased	CH4	production	during	the	
in	vitro	incubation.
Even	though	in	vitro	assays	are	a	very	reliable	tool	to	provide	an	
indication	regarding	the	nutritional	value	of	feed	for	ruminants	(Bueno	
et	al.,	 2005),	 the	 findings	 obtained	 from	 these	 should	 be	 validated	
under	in	vivo	conditions.	Our	ATTD	was	conducted	in	relatively	short	
experimental	period,	but	showed	us	some	evidence	that	 the	250	g/
kg	 DM	 inclusion	 of	 macrotiloma	 in	 the	 diet	 did	 not	 cause	 any	 im-
pairment	 in	 the	ATTD	of	any	dietary	nutrient.	Tiemann	et	al.	 (2008)	
worked	with	castrated	male	 lambs	and	observed	reduction	 in	ATTD	
of	CP	when	a	300	g/kg	DM	inclusion	of	tannin-	rich	legumes	was	used	
in	a	6	×	6	Latin-	square	design	trial,	with	28	days	per	experimental	pe-
riod	(14	days	of	adaptation;	7	days	of	collection	and	7	days	of	resting	
period).
Considering	 the	 results	 from	 the	 above-	mentioned	 author,	 it	
may	 be	 inferred	 that	 the	 experimental	 period	 adopted	 here	 was	
enough	 for	 finding	effects	of	 legume	 supplementation	on	 the	pa-
rameters	 evaluated.	 However,	 the	 relatively	 low	 levels	 of	 tannins	
from	MAC,	 together	with	 the	ATTD	and	ruminal	 fermentation	pa-
rameters	 results	observed	here,	 indicated	 that	 the	 legume	macro-
tiloma	may	be	used	in	the	diet	of	lambs	respecting	a	250	g/kg	DM	
inclusion	level,	with	no	detrimental	effects	on	the	ATTD	of	dietary	
nutrients.
The	 results	 obtained	 in	 our	 experiments	 provided	us	 an	over-
view	about	 the	value	of	 this	 legume	and	 indicated	 that	 it	may	be	
used	as	a	source	of	nutrients	for	ruminants.	Antimethanogenic	po-
tential	was	verified	for	this	plant	as	shown	by	the	in	vitro	results	and	
by	the	reduced	protozoa	counts	observed	in	ewes	fed	MAC;	how-
ever,	further	research	is	needed	to	consolidate	our	findings.	Longer	
experimental	periods	and	increased	inclusion	levels	of	macrotiloma	
should	provide	more	accurate	and	 reliable	data	 regarding	 the	use	
of	this	plant.
ACKNOWLEDGEMENTS
The	 authors	 thank	 São	 Paulo	 Research	 Foundation—FAPESP	 (grant	
number:	2013/02814-	5)	and	the	Coordination	for	the	Improvement	
of	 Higher	 Education	 Personnel	 (CAPES)	 for	 financial	 support	 and	
scholarships;	and	the	Instituto	de	Zootecnia	(IZ/APTA/SAA)	(Institute	
of	Animal	Science),	for	providing	technical	support	and	study	material	
(macrotiloma).
ORCID
P. M. T. Lima http://orcid.org/0000-0002-6354-3139
A. S. Natel http://orcid.org/0000-0002-8252-1090
http://orcid.org/0000-0002-6354-3139
http://orcid.org/0000-0002-6354-3139
http://orcid.org/0000-0002-8252-1090
http://orcid.org/0000-0002-8252-1090
     |  7LIMA et AL.
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How to cite this article:	Lima	PMT,	Moreira	GD,	Sakita	GZ,	
et	al.	Nutritional	evaluation	of	the	legume	Macrotyloma axillare 
using	in	vitro	and	in	vivo	bioassays	in	sheep.	J Anim Physiol 
Anim Nutr. 2017;00:1–8. https://doi.org/10.1111/jpn.12810
https://doi.org/10.1111/jpn.12810