Small Ruminant Research 154 (2017) 70–77

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Small Ruminant Research

journal homepage: www.elsevier.com/locate/smallrumres

Diets based on plants from Brazilian Caatinga altering ruminal parameters, MARK
microbial community and meat fatty acids of Santa Inês lambs
⁎
A.L. Abdalla Filhoa, , P.S. Corrêaa, L.N. Lemosa, D. Dineshkumara, J. Issakowicza, E.H. Iedaa,
P.M.T. Limaa, M. Barrealb, C. McManusc, T.S. Muia, A.L. Abdallaa, H. Louvandinia
a
Centre for Nuclear Energy in Agriculture, University of São Paulo, 13400-970 Piracicaba, SP, Brazil
b
Montpellier SupAgro, Département MPRS (Milieux, Productions, Ressources et Systèmes), 2 Place Pierre Viala, Bâtiment 22, 34060 Montpellier, France
c
University of Brasilia, Faculty of Agriculture and Veterinary Medicine, 70910-900 Distrito Federal, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Strategies for the sustainable intensification of ruminant production suggest breeding animals adapted to the
Babassu environment in which they live using local fodders to maximize efficient ruminal fermentation. The purpose of
Finishing performance this study was to explore the effects of using Orbignya phalerata (Babassu) and Combretum leprosum (Mofumbo)
Lambs leaves in ruminal short chain fatty acids (SCFA) and microbial community, as well as finishing performance,
Meat
carcass characteristics and meat fatty acid profile of Santa Inês hair lambs. The experimental treatments were
Microbial community
Mofumbo
diets with 50:50 forage:concentrate ratios, using the leaves of the experimental plants as a 33 g/100 g of dry
Tannins matter (DM) replacement of Cynodon dactylon (Tifton-85) hay, with three groups: Control (no hay replacement),
Babassu and Mofumbo. Twenty-four Santa Inês lambs (body weight = 20.0 ± 5.2 kg) were used in a rando-
mized experimental design with eight repetitions (5 males and 3 female) per treatment. Ruminal fluid samples of
each animal were collected to determine SCFA and microbial community. The male animals were evaluated for
finishing performance and carcass characteristics while longissimus lumborum muscle samples were used for
determination of fatty acid profile. Interaction between treatment and sex was observed for total SCFA
(P < 0.05). Treatment affected (P < 0.05) propionate, isobutyric, isovaleric and valeric fatty acids. Mofumbo
showed a greater relative abundance of rumen fungi and Ruminococcus flavefaciens, and lower Archaea. There
was no difference between treatments (P > 0.05) for finishing performance and carcass characteristics but meat
fatty acid characteristics were affected by treatments. A redundancy analysis based on microbial abundance
profile demonstrated two clusters, one cluster with Babassu and Control treatments, and other cluster with
Mofumbo, with some variables associated to dissimilarity between clusters. These results indicated that the
inclusion of these plants in lamb diets affects ruminal short chain fatty acids and microbial population, without
compromising the production potential, carcass characteristics and meat fatty acid profile.

1. Introduction
Feeding is a key point for the sheep industry and it is regarded as the
most important aspect related to the success of lamb production
(Sepúlveda et al., 2011). In addition to factors such as freshness and
meat flavour there is a growing concern by a large segment of con-
sumers about the sustainability of meat production, its impacts on an-
imal welfare and potential harms to the environment and human health
as well (Troy and Kerry, 2010).
Strategies for the sustainable intensification of ruminant production
suggest breeding animals adapted to the environment in which they live
using local fodders in order to maximize the efficient ruminal fermen-
tation (Eisler et al., 2014). Including leaves of locally available plants
other than grasses in small ruminants’ diets can be an important less
expensive alternative feed resource, contributing to the sustainability of
the livestock activity (Mlambo and Mapiye, 2015) and also food se-
curity, since generaly grasses are grown on arable land which could be
used for direct food production (Schader et al., 2015). However, many
of these plants usually contain some levels of secondary plant com-
pounds, such as tannins (Estell, 2010) whose biological properties are
greatly dependent on their chemical structure (Soltan et al., 2013),
which can be even more important than concentration (Waghorn and
McNabb, 2003).
Even though some antihelmintical (Cenci et al., 2007) and anti-
oxidative activities are described in ruminants, positively affecting
animal welfare as well as meat and milk quality (Luciano et al., 2011),

⁎
Corresponding author.
E-mail address: adibefilho@cena.usp.br (A.L. Abdalla Filho).

http://dx.doi.org/10.1016/j.smallrumres.2017.07.005
Received 2 May 2017; Received in revised form 14 July 2017; Accepted 15 July 2017
Available online 18 July 2017
0921-4488/ © 2017 Elsevier B.V. All rights reserved.
A.L. Abdalla Filho et al. Small Ruminant Research 154 (2017) 70–77

tannins can influence different physiological aspects such as N meta- Table 1

bolism, ruminal microbial population, feed intake and performance of Ingredient proportions, chemical analyses (g/kg DM) and total phenolic content, total
tannin and condensed tannins (CT) of the different diets fed to Santa Inês lambs.
animals (Patra and Saxena, 2011). In addition, they may reduce the
cellulolytic bacteria population, contributing in a positive way to re- Parameters Treatments
duce methanogenesis (Hristov et al., 2013) and may also affect meat
quality characteristics, including traits related to its colour, shelf-life C B M
and fatty acid composition, which has implications for human health
Dry matter (DM, g/kg) 944.3 944.1 944.3
(Raes et al., 2004).
Ruminant products often present high saturated fatty acids (SFA) Ingredients (g/100 g of DM)
Tifton-85 hay 51 34 34
and low polyunsaturated fatty acids (PUFA) content (Vasta and Plant leaves 0 16 16
Luciano, 2011). While the consumption of SFA is associated with in- Corn grain 34 34 34
creasing risk of cardiovascular diseases (Jenkins et al., 2008), PUFA Soybean meal 13 14 14
have the ability to moderate cancer, immune system and cardiovascular Mineral mix 2 2 2
disorders, also presenting others benefits to human health (Pelliccia Chemical composition (g/kg of DM)
et al., 2013). Meat from ruminant livestock contains conjugated linoleic Organic matter 945.1 945.2 943.5
Crude protein 119.7 124.2 124.2
acids (CLA), a family of fatty acids that possess beneficial biological
Neutral detergent fibre 701.8 687.9 673.3
activities in mammals (Vasta et al., 2009a,b). All these mentioned Acid detergent fibre 364.6 369.2 359.2
factors are strongly related to the ruminal biohydrogenation of fatty Hemicelullose 337.2 318.7 314.1
acids, which, may be affected by the inclusion of tannins in the diet Celullose 176.5 183.0 155.8
(Morales and Ungerfeld, 2015). Lignin 179.6 176.0 194.9
Ether extract 33.4 34.9 39.4
A wide variety of tanniniferous plants found in Brazil can be used as
Mineral matter 54.9 54.8 56.5
ruminant feed. Among them, Babassu (Orbignya phalerata) and Total phenolsa 2.20 10.50 26.80
Mofumbo (Combretum leprosum), are plants eaten by sheep (Meier et al., Total tanninsa 1.20 8.00 20.80
2014), but with no information in the literature regarding their possible Condensed tannins (CT)b 0.10 9.30 28.50
effects on ruminal parameters, productive performance and meat a
Total phenols and total tannins expressed in grams of tannic acid/kg DM.
quality of the animals fed on them. This study aimed to evaluate b
Condensed tannins expressed in equivalent gram leucocyanidin/kg DM. C – Control;
ruminal microbial community, short chain fatty acids, finishing per- B – Babassu and M – Mofumbo treatments.
formance, carcass and meat characteristics as well as meat fatty acid
profile of lambs fed on the tanniniferous legumes Babassu and Mo- methodology proposed by Van Soest et al. (1991), as recommended by
fumbo. Udén et al. (2005). Hemicellulose (HEMI) and cellulose (CEL) were
calculated by the difference between NDF, ADF and LIG. Total phenols
2. Materials & methods (TP), total tannins (TT) and condensed tannins (CT) were evaluated
using the methodology proposed by Makkar (2000).
The present experiment was developed at the Animal Nutrition
Laboratory at the Centre for Nuclear Energy in Agriculture of University
2.2. Ruminal samples collection and short-chain fatty acid determination
of São Paulo, located in Piracicaba – SP, Brazil (LANA-CENA/USP). All
the procedures performed were approved by the Ethics Committee on
Rumen fluid samples were collected at the end of the experimental
Animal Use of the Escola Superior de Agricultura “Luiz de Queiroz” –
period by intubation of each animal 3 h after they were fed. The sam-
CEUA-ESALQ/USP (Protocol No. 2013-7).
ples were kept in pre-warmed thermos containers (39 °C) conserving
the temperature and transported to the laboratory to determine SCFA
2.1. Experimental animals and diet formulation
according to Palmquist and Conrad (1971). Rumen fluid was cen-
trifuged (2 mL at 11.000 rpm for 40 min at 4 °C) to remove large feed
During 48 days, 24 Santa Inês lambs (body weight (BW)
particles; 800 μL of the supernatant was added to 100 μL of 2-ethyl-
= 20.0 ± 5.2 kg; 149 ± 4.7 days old) were used, in a randomized
butyric acid and 200 μL of formic acid 98–100% (internal standard,
experimental design with eight repetitions for each treatment (5 males
MW = 116.16; Sigma Chemie Gmbh, Steinheim, Germany). Afterwards
and 3 female). The lambs received experimental diets (Table 1) in in-
800 μL (at the same quantity of the sampling) of mixture of known
dividual pens (1.0 m × 1.5 m). Babassu and Mofumbo leaves were
short-chain fatty acid concentration was added as the external standard
obtained from the experimental farm of the Federal University of Piauí
for integrator calibration and transferred to the chromatographic vial.
(UFPI), dried in the shade and chopped to a maximum length of 1 cm.
One μL was injected onto the gas chromatograph (GC 2014 Shimadzu,
The diets consisted of 33 g/100 g replacement (on DM basis) of Tifton-
Tokyo, Japan) equipped with FID and GP 10% SP-1200/1 H3PO4 80/
85 hay (Cynodon dactylon), by the plant to be evaluated, resulting in
100 Chromosorb WAW column (Cat. no. 11965, 6′ × 1/8″ stainless
three treatments: Control (without replacement), Babassu and Mo-
steel, Supelco, Bellefonte, PA, USA). The column isothermal tempera-
fumbo.
ture was at 120 °C, injector at 160 °C and detector at 190 °C. Helium
In addition, to meet the requirements of weaned lambs with 20 kg of
was used as carrier gas at 25 mL/min and the detector hydrogen and
BW and average daily gain of 100–150 g/d (NRC, 2007), each animal
synthetic air were kept at 40 and 400 mL/min, respectively.
received approximately 335 g/d of concentrate mixture composed of
69 g/100 g ground corn, 29 g/100 g soybean meal and 2 g/100 g mi-
neral mixture. The diets were isoproteic and a 50:50 forage:concentrate 2.3. Ruminal microbial community
ratio was used. Animals had ad libitum access to water.
Chemical analyses of diets (Table 1) were expressed on DM basis, For microbial community evaluation, DNA extraction of the ruminal
and as well as organic matter (OM; ID number 934.01), crude protein samples was carried out using the PowerLyzer™ Power Soil kit (MoBio
(CP; ID number 2001.11) and ether extract (EE; ID number 2003.5) Laboratories, Carlsbad, CA, USA) and the concentration and purity
were performed according to AOAC (2011). Neutral detergent fibre were determined using a Nanodrop ND-1000 spectrophotometer
(NDF; ID number 2002.04) were analyzed according to Mertens (2002); (Thermo Scientific, Ahmadabad, India). The relative abundance of
acid detergent fibre (ADF; ID number 973.18) and lignin (LIG; ID Fibrobacter succionogenes, Ruminococcus flavefaciens, total anaerobic
number 973.18) were determined sequentially following the rumen fungi and Archaea methanogens was measured by real-time

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A.L. Abdalla Filho et al. Small Ruminant Research 154 (2017) 70–77

quantitative PCR (qPCR) assay using primers set of 16S rRNA genes Table 2
(Denman and McSweeney, 2006; Denman et al., 2007). Fatty acid profiles (g/100 g of total fat) in three experimental diets fed to Santa Inês
lambs.
The qPCR was performed using StepOnePlusTM Real Time PCR
System (Life Thecnologies) with fluorescence detection of SYBR green Fatty acids profilea Treatments
dye. The total volume of PCR mixture was 10.0 μL consisted of SYBR
Green Rox (Invitrogen) 5.0 μL, forward primer 1.0 μL (10 μmol L−1 C B M
concentration) reverse primer 1.0 μL (10.0 μmol L−1 concentration),
C4:0 0.12 0.11 0.12
DNA template 1.0 μL and sterile distilled water 2.0 μL. The amplifica- C6:0 0.22 0.15 0.14
tion conditions were as follows: on cycle at 95 °C for 30 s (initial de- C12:0 0.72 0.76 0.46
naturation), followed by 45 cycles of 95 °C for 5 s and 60 °C for 20 s. C13:0 iso 1.46 0.58 1.27
The specificity of amplified products was confirmed by melting tem- C14:0 iso 0.12 0.10 0.09
C14:0 0.82 1.47 1.33
perature and dissociation curve after amplification. The relative po-
C15:0 iso 0.10 0.08 0.08
pulation sizes of those microbial groups was expressed as proportion of C15:0 0.27 0.17 0.17
total rumen bacteria (16S rRNA), calculated from the ΔCt values by C16:0 31.71 29.96 29.68
subtracting the Ct (threshold cycle) value of the target gene from the Ct C16:1 cis-9 0.15 0.12 0.12
value of the reference gene (16S rRNA of BACT) using the 2−ΔΔCt C17:0 0.44 0.79 0.69
C18:0 4.26 4.74 7.48
method (Schmittgen and Livak, 2008). The shifts in microbial com- C18:1 cis-9 18.76 20.98 20.21
munities due to the different diets (Babassu and Mofumbo) in relation C18:1 cis-11 1.82 1.84 1.80
to Control were determined by considering the relative abundance of C18:1 cis-12 0.38 0.43 0.40
target population in Control as 1. C18:2 cis-9 cis-12 27.49 26.85 25.71
C20:0 1.21 0.69 1.27
C18:3 n-3 6.33 6.23 4.08
2.4. Finishing performance, carcass characteristics and meat fatty acids C20:1 0.40 0.51 0.31
profile C22:0 0.71 0.49 0.92
C23:0 0.46 0.35 0.41
C24:0 1.09 1.61 1.06
The 15 Santa Inês male lambs (149 days old and 20.4 ± 2.68 kg
SFA 43.95 42.28 45.48
BW) were selected for the evaluations of finishing performance, carcass UFA 55.47 57.19 53.12
characteristics and meat fatty acid profile. MUFA 21.56 24.05 22.90
During 57 days, feed leftovers were daily collected and weighed in PUFA 33.91 33.14 30.23
order to calculate daily DM intake. Weekly, the animals were weighed n-6 27.58 26.89 25.78
n-3 6.33 6.23 4.13
before morning feeding, to determine average daily body weight gain
n-6:n-3 4.35 4.31 6.23
(calculated by linear regression of the individual animal weights on UFA:SFA 1.26 1.35 1.17
experiment length) and the total weight gain throughout the experi- PUFA:SFA 0.77 0.78 0.66
mental period. Feed conversion ratio was calculated by the ratio be-
a
tween total consumption and total weight gain of the animals. Only the fatty acids with levels greater than 0.1 g/100 g are presented. SFA: saturated
fatty acids; UFA: unsaturated fatty acids; MUFA: monounsaturated fatty acids; PUFA:
At the end of the experiment, the animals were slaughtered to
polyunsaturated fatty acids; n-6: n-6 fatty acids; n-3: n-3 fatty acids; n-6:n-3: n-6 divided
evaluate carcass characteristics. Before slaughter, the animals were by n-3; UFA:SFA: unsaturated divided by saturated fatty acids; PUFA:SFA: poly-
fasted for 24 h and then weighed to determine the fasting body weight unsaturated divided by saturated fatty acids. C – Control; B – Babassu and M – Mofumbo
(FBW). Slaughter was performed according to the current Brazilian laws treatments.
on the Regulation of Industrial and Sanitary Inspection of Animal
Products (RIISPOA, 1952), through stunning by electric shock and then followed the procedures of Hara and Radin (1978) and these were
bleeding out. methylated according to Christie (1982). For the fatty acid profile
After bleeding, skinning and evisceration, the skin, heart, lung, liver analyses of feeds (Table 2), extraction and methylation were performed
and scrotum were weighed. Hot carcass weight (HCW) and hot carcass according to Rodríguez-Ruiz et al. (1998). The samples were analyzed
yield (HCY) were determined using the equation: HCY = HCW/ in a gas chromatograph (GC-Finnigan Focus, Thermo Finnigan, San
FBW × 100. The carcasses were then stored in a cold chamber (4 °C) Jose, CA) with a flame-ionization detector and capillary column (CP-Sil
for 24 h and weighed to determine the cold carcass weight (CCW). The 88; Varian, Palo Alto, CA) measuring 100 m in length × 0.25 mm i.d.,
cold carcass yield (CCY) was calculated according to the equation: with a thickness of 0.20 μm, as described by Paim et al. (2014): Hy-
CCY = CCW/FBW × 100. drogen was used as the carrier gas at a flow rate of 1.8 mL/min. The
With the use of a tape measure, morphometric measurements of the initial temperature of the oven was 70 °C and was increased by 13 °C/
internal length of the carcass, hip circumference, as well as cir- min to 175 °C, where it was maintained for 27 min. The temperature
cumference and length of the shank were made. A subjective evaluation was then increased by 4 °C/min to 215 °C, where it was maintained for
of fat cover score was performed by adopting a scale from 1 for ex- 9 min, followed by another increase by 7 °C/min to 230 °C, where it
cessively thin and devoid of fat to 5 for excessively fat, with 0.25 point remained for 5 min. The temperature of the injector was 250 °C, and the
intervals. Carcass conformation score was also analyzed subjectively on temperature of the detector was 300 °C.
a scale of 1–5, with the value 1 for very poor conformation and the The identification of fatty acids was performed by comparison of
value 5 for excellent conformation. Leg, as well as skin, heart, lung, retention times of the samples using the analytical standards Supelco®
liver and scrotum were weighed. 37 Component FAME Mix (Supelco TM FAME Mix Component Ref No.
After cooling, meat colour was measured at the longissimus lum- 18919; Sigma–Aldrich, Bellefonte, PA, USA) and CLA isomers (Linoleic
borum muscle with a portable spectrophotometer (Konica Minolta – acid, conjugated methyl ester Ref No. O5632; Sigma–Aldrich, Saint
Spectro Photometer CM – 600d) at three different points on the external Louis, MO, USA). Fatty acids were quantified using the Chromquest 4.1
surface to obtain the average value and then expressed in colorimetric software (Thermo Electron, Milan, Italy) and expressed in g/100 g of
values according to the CIE system (CIE, 1986), considering three total fat.
fundamental aspects: L* (lightness), a* (red intensity) and b* (yellow The total amount of desirable fatty acids (DFA) was determined
intensity). according to Landim et al. (2011) as the sum of monounsaturated fatty
Samples of longissimus lumborum muscle were collected for the de- acids (MUFA), polyunsaturated fatty acids (PUFA) and stearic acid
termination of meat fatty acid profile. Lipid extraction of the samples (C18:0): DFA = MUFA + PUFA + C18:0. The activity of Δ9-desaturase

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A.L. Abdalla Filho et al. Small Ruminant Research 154 (2017) 70–77

(C14, C16 and C18) and elongase were estimated as described by Lock and valeric fatty acids, for which Mofumbo showed lower results than
and Garnsworthy (2003) and Raes et al. (2004): ELONG- Control. Acetate:propionate ratio in Mofumbo treatment was higher
ASE = 100 × (C18:0 + C18:1cis-9)/(C16:0 + C16:1cis-9 + C18:0 + (P < 0.05) than Control. Significant negative linear regression was
C18:1cis-9); Δ9-desaturase C14 = 100 × (C14:1cis-9)/(C14:1cis- found for propionate (y = −0.09x + 18.78; R2 = 0.436; P < 0.05),
9 + C14:0); Δ9-desaturase C16 = 100 × (C16:1cis-9)/(C16:1cis- isovaleric (y = −0.02x + 1.11; R2 = 0.382; P < 0.05) and valeric
9 + C16:0); Δ9-desaturase C18 = 100 × (C18:1cis-9)/(C18:1cis- (y = −0.01x + 0.74; R2 = 0.587; P < 0.01) when analysing the ef-
9 + C18:0). Aterogenecity index (ATHERO), considered an indicator of fects of the different CT concentrations on these parameters. For acet-
risk of cardiovascular disease, was calculated according to Ulbricht and ate:propionate ratio a positive linear regression was observed
Southgate (1991): ATHERO = (C12:0 + (4 × C14:0) + C16:0)/ (y = 0.03x + 3.79; R2 = 0.423; P < 0.05).
∑SFA + ∑PUFA. Concerning relative abundance of the microbial community (Fig. 1),
significant positive linear regression was found for the relative abun-
2.5. Statistical analysis dance of fungi (y = −0.32x + 0.34; R2 = 0.536; P < 0.05) and R.
flavefaciens (y = 0.94x + 0.05; R2 = 0.460; P < 0.05) when analysing
Statistical analysis of the data was performed using the SAS® v.9.2 the effects of the different CT concentrations on these parameters. For
program (Statistical Analysis System Institute, Cary, NC, USA 2009). Archaea methanogens relative abundance, a negative linear regression
The values obtained were submitted to analysis of variance using the was observed (y = 1.07x − 0.02; R2 = 0.372; P < 0.05). The fungi
GLM procedure. Fixed effects of treatment (T), sex (S) and their inter- and R. flavefaciens microbial populations were more abundant for Mo-
action (T*S) were tested. PROC REG was used to analyze the effects of fumbo than for Control and Babassu (P < 0.05). The abundance of
the CT concentrations of diets on all variables. For all procedures Archaea methanogens decreased in Mofumbo when compared to Con-
P < 0.05 level of significance was used. Redundancy analysis (RDA) trol (P < 0.05). The Fibrobacter succinogenes abundance was not af-
was used to determine the most influence variables among the com- fected by the different treatments.
parison of the treatments based on similarity profiles. Apparent di- There were no significant differences among treatments (P > 0.05)
gestibility variables, nitrogen balance and enteric production of me- for finishing performance, carcass traits and meat colour (Table 4).
thane from the previous study (Abdalla Filho et al., 2017), as well as The Control group showed lower (P < 0.05) amount of Lauric fatty
ruminal short chain fatty acids, finishing performance, carcass char- acid (C12:0) than Babassu and Mofumbo treatments (Table 5). No dif-
acteristics and meat fatty acids were used as environmental variables ference was observed for any of the other fatty acids (P > 0.05). Si-
normalized into a common range scale and combined with microbial milarly, there were no treatment effects (P > 0.05) for total SFA, UFA,
population (qPCR values). The comparison for microbial abundance DFA, MUFA, PUFA, n-6 and n-3 and in the ratios of n-6:n-3, UFA:SFA
profile and metadata was carried out using R programming language and PUFA:SFA, ELONGASE enzyme and ATHERO index.
and vegan package. When regression analysis was performed, a positive linear effect was
observed only between Δ9-desaturase C18 estimated enzyme activity
3. Results and CT concentrations in diets (y = 0.55x + 7.34, R2 = 0.37 and
P = 0.01).
The animals in this study were all from the same breed (Santa Inês) The RDA based on microbial abundance profile highlighted two
and the nutrient input provided by the diets was similar for all the clusters, one with Babassu and Control treatments, and other with
treatments, differing only in CT levels (Control = 0; Babassu = 9 and Mofumbo treatment. Mofumbo treatment presented nutrient apparent
Mofumbo = 28 g CT/kg DM) and the average daily intake was 663.03, digestibility variables (Fig. 2A), acetate:propionate ratio and acetate
738.73 and 728.56 g DM for Control, Babassu and Mofumbo, respec- values (Fig. 2B) as related to the dissimilarity between the two clusters.
tively. Finishing performance, carcass characteristics and meat fatty acids
An interaction between treatment and sex was observed for Total profile were not associated with any of the clusters (figure not shown).
SCFA (mmol mL−1), where females from the Control and Mofumbo
treatments showed higher values than males from Mofumbo 4. Discussion
(P < 0.05). Data are reported in Table 3. Also, both males and females
of Babassu differed from females of Control (P < 0.05). Treatment Despite the fact that the CT levels used here may be considered as
effect (P < 0.05) was observed for propionate, isobutyric, isovaleric low (Archimède et al., 2011), the results of SCFA and RDA in this study
were consistent with the findings of others authors (Bhatta et al., 2013;
Table 3 Bueno et al., 2015), which showed effect of CT reducing SCFA. As result
Ruminal short chain fatty acids of Santa Inês hair lambs receiving the different treat- of tannin–protein and tannin–fibre binding, changes in ruminal de-
ments. gradation usually occur with subsequent adverse effects in the fer-
Parameters Treatments SEM P value
mentation process and SCFA production (Rodríguez et al., 2014).
Considering the results of ADF apparent digestibility from the previous
C B M T S T*S REG study (Abdalla Filho et al., 2017), SCFA reduction likely induced by the
a b ab * ** *
presence of CT in diets probably occurred due the inhibition of fibre
Total SCFA 84.7 77.15 79.83 1.788 ns
(mmol mL−1)
degradation.
Acetate (%) 70.9 71.74 74.66 1.031 ns ns ns ns Concerning the effects found in microbial population, according to
Propionate (%) 18.7a 17.77ab 16.18b 0.614 *
ns ns *
Patra and Saxena (2011), the sensitivity of microorganisms to tannins
Butyrate (%) 7.9 8.51 7.83 0.439 ns ns ns ns can vary substantially, being dependent on the microbial species and
Isobutyric (%) 0.49a 0.35ab 0.25b 0.059 *
ns ns ns
the type or source of tannin used. Still, these authors affirm that the
Isovaleric (%) 1.20a 1.04ab 0.58b 0.150 *
ns ns *

Valeric (%) 0.77a 0.57b 0.47b 0.037 **

ns ns ** effects of tannins on microbial growth are due to their interactions with
Acetate:propionate ratio 3.82b 4.05ab 4.64a 0.202 *
ns ns * extracellular enzymes and bacteria cell walls, causing morphological
changes in these structures, acting directly on the microbial metabo-
a,b
Different letters in the same row indicate statistical difference. SCFA – short chain fatty lism. Kok et al. (2013) also observed effects of feeding tannins in fungi
acids; T – treatment effect; S – sex effect; T*S – interaction between T and S; REG – linear
population. In their study the population was inhibited during the first
regression; SEM – standard error of the means; ns – non significant (P > 0.05). C –
Control; B – Babassu and M – Mofumbo treatments.
10 days of feeding, followed by a tremendous increase after 15 days and
* P < 0.05. finally decreased at 30 days. So far, there has been no study on the
** P < 0.01. effect of tannin on the anaerobic rumen fungal growth pattern during

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A.L. Abdalla Filho et al.
Fig. 1. Relative abundance of rumen fungi, Archaea methanogens, Ruminococcus flavefaciens and Fibrobacter succinogenes on ruminal samples from each treatment.
a,b
Different letters indicate statistical difference (P < 0.05); bars = standard error of the mean; C – Control; B – Babassu and M – Mofumbo treatments.
different periods of supplementation to compare.
Furthermore, the RDA showed that fungi population was correlated
with apparent digestibility variables, presumably because they have a
broad range of potent polysaccharide-degrading enzymes and are active
fibre degraders, particularly when low-quality diets are consumed
(Hobson and Steart, 1997). Moreover, the microbial population in
rumen was not only dependent on the availability of nutrients, but also
on their tolerance to tannins in the feed (McSweeney et al., 2001a).
However, the mechanism by which they adapt to tannins is still unclear.
It has been reported that some microbes secrete extracellular poly-
saccharides, which have high affinity for tannins, preventing adverse
effects on their population (Brooker et al., 2000).
McSweeney et al. (2001b) observed reduction in cellulolytic rumen
bacteria populations (R. flavefaciens and F. succinogenes) by approxi-
mately 6% when sheep were fed with the tanniniferous tropical forage
Calliandra calothyrsus. In their study, the effect on fungi was less clear as
the results were inconsistent. Also differently from our results, Abdalla
et al. (2012) found that tanniniferous plants such as Mimosa caesoniphila
and Leucaena leuceophalla increased the populations of Archaea me-
thanogens by 151 and 63%, respectively. But these authors suggest that
increase in populations was due to the free-living methanogens.
In our previous study (Abdalla Filho et al., 2017) we found a lower
intensity of enteric methane production (g CH4/kg average daily gain)
in lambs fed Mofumbo when compared to Babassu treatment and this
was also numerically lower (23.4%) in relation to the lambs fed Control
diet. One possible explanation for the effect of tannin, even at moderate
levels, on the reduction in methane production is usually related to its
direct action, by inhibiting Archaea methanogens (Patra and Saxena,
2011). In the present experiment, results obtained in the microbial
community analysis corroborate this hypothesis.
There were no negative effects on productive parameters such as
finishing performance and carcass characteristics, what leads us to in-
vestigate in further research, higher inclusion levels of these plants in
diets.
Colour is the most important sensory attribute affecting consumer-
purchasing decisions of red meats (Mlambo and Mapiye, 2015). Bright
red lean colour is associated with freshness, while brownish with stale
or spoiled meat (Mancini and Hunt, 2005), but the way consumers
usually associate meat colour and brightness with meat freshness varies
74
Small Ruminant Research 154 (2017) 70–77
according to their cultural background (Vasta et al., 2008). Regarding
the effects on meat colour, tannins from different plant species increase
L* and therefore the meat from animals fed these molecules are lighter,
possibly due to a reduced microbial biosynthesis of vitamin B12 (Priolo
and Vasta, 2007). Also, increased a* were observed when feeding CT to
sheep (Luciano et al., 2009) and cattle (Mapiye et al., 2010), which
could be related to haemoglobin and myoglobin levels and iron utili-
zation (Vasta and Luciano, 2011). In the present study, there was no
difference in meat colour between treatments, indicating that at this
level of inclusion, Babassu and Mofumbo did not alter or compromise
this sensory attribute.
Some factors such as diet, genetic group and age at slaughter can
influence the composition of meat fatty acids (Schmid et al., 2006).
When using animals of the same breed and slaughtered at similar ages,
any differences found between treatments in meat fatty acids profile
may be attributed to the diets which animals were fed. Breed is an
important factor regarding carcass quality, but less important than
feeding management (Ramírez-Retamal and Morales, 2014).
It has been shown that feeding tannins to ruminants can favourably
alter ruminal biohydrogenation and thereby the content of some human
health promoting fatty acids, such as Vaccenic (C18:1 cis-11) and
Rumenic (C18:2 cis-9, trans-11), the most important CLA from ruminant
food products (Toral et al., 2011). In addition to different CT levels, the
experimental diets also had different fatty acid profiles for Capronic
(C6:0), Lauric (C12:0), Tridecylic (C13:0 iso), Pentadecylic (C15:0),
Margaric (C17:0) and Arachidic (C20:0) fatty acids (Table 2). However,
meat characteristics regarding its fatty acid composition, such as total
SFA, UFA, DFA, MUFA, PUFA, n-6 and n-3, n-6:n-3, UFA:SFA, PU-
FA:SFA, ELONGASE enzyme and ATHERO index were not affected,
indicating that the inclusion Babassu and Mofumbo also did not com-
promise these meat quality parameters. Other authors also found no
effect of CT on ruminant meat fatty acid profile (Toral et al., 2011).
Discrepancies across studies, showing or not the effects of tannins could
be due to different concentrations or chemical structure, structure-ac-
tivity relation and biological activity of the tannins used (Mueller-
Harvey, 2006).
Although generally considered as unhealthy, some SFA could have
positive effects for humans, which is the case of Lauric acid (C12:0),
found in higher proportion on Babassu and Mofumbo treatments.
A.L. Abdalla Filho et al. Small Ruminant Research 154 (2017) 70–77

Table 4 Table 5
Animal finishing performance, carcass characteristics, non-carcass components and col- Longissimus lumborum fatty acids profile (g/100 g of total fat) and fatty acids index from
orimetric values obtained in the meat from Santa Inês lambs fed the experimental diets. Santa Inês lambs fed experimental diets.

Parameters Treatments SEM P value Parameters Treatments SEM P value

C B M T REG C B M T REG

Animal performance C10:0 0.22 0.18 0.18 0.024 ns ns

Initial body weight (kg) 20.50 20.80 20.10 2.68 ns ns C12:0 0.20b 0.43a 0.33 a 0.049 *
ns
Daily dry matter intake (g DM) 693.84 785.63 745.80 82.484 ns ns C14:0 iso 0.05 0.05 0.05 0.016 ns ns
Daily dry matter intake (% LW) 2.93 3.09 3.19 0.176 ns ns C14:0 3.19 4.55 4.45 0.430 ns ns
Total dry matter intake (kg DM) 31.22 35.35 33.56 3.710 ns ns C15:0 iso 0.26 0.23 0.20 0.024 ns ns
Average daily gain (g/d) 137.77 153.33 151.11 17.576 ns ns C15:0 anteiso 0.32 0.32 0.26 0.045 ns ns
Total weight gain (kg) 6.20 6.90 6.80 0.790 ns ns C14:1 cis-9 0.08 0.14 0.12 0.018 ns ns
Feed conversion 5.49 5.22 4.96 0.422 ns ns C15:0 0.54 0.60 0.49 0.053 ns ns
C16:0 iso 0.18 0.15 0.13 0.023 ns ns
Carcass characteristics
C16:0 23.24 22.47 22.07 1.251 ns ns
FBW (kg) 28.10 28.70 27.60 3.615 ns ns
C17:0 iso 0.31 0.30 0.23 0.027 ns ns
HCW (kg) 11.36 11.48 11.32 1.792 ns ns
C16:1 cis-9 1.93 1.79 2.21 0.119 ns ns
CCW (kg) 11.19 11.31 11.15 1.765 ns ns
C17:0 1.20 1.08 1.17 0.092 ns ns
HCY (%) 40.01 39.65 40.15 1.640 ns ns
C17:1 0.49 0.33 0.61 0.088 ns ns
CCY (%) 39.41 39.06 39.55 1.615 ns ns
C18:0 22.47 22.79 17.29 2.029 ns ns
Inner carcass circumference (cm) 49.00 49.20 48.20 1.740 ns ns
C18:1 t6-t7-t8-t9 0.66 0.66 0.51 0.132 ns ns
Hip circumference (cm) 69.40 70.00 68.40 2.451 ns ns
C18:1 t10-t11-t12 2.04 2.01 2.40 0.388 ns ns
Leg circumference (cm) 33.40 34.20 34.00 1.871 ns ns
C18:1 cis-9 33.92 31.83 37.93 1.718 ns ns
Leg length (cm) 31.40 31.80 30.80 1.175 ns ns
C18:1 cis-11 2.51 2.92 3.30 0.210 ns ns
Leg weight (kg) 1.59 1.66 1.79 0.254 ns ns
C18:1 cis-12 1.01 0.77 1.05 0.136 ns ns
Carcass conformationa 3.00 2.90 3.00 0.346 ns ns
C18:1 cis-13 0.36 0.34 0.52 0.110 ns ns
Fat coverb 1.90 2.30 2.50 0.474 ns ns
C18:1 cis-15 0.11 0.12 0.11 0.023 ns ns
Non-carcass components (kg) C18:2 cis-9 cis-12 n-6 2.35 3.08 2.53 0.294 ns ns
Skin 1.99 1.79 1.85 0.287 ns ns C18:3 n-3 0.22 0.22 0.19 0.019 ns ns
Heart 0.13 0.13 0.13 0.010 ns ns C20:1 0.09 0.10 0.09 0.008 ns ns
Lung 0.31 0.31 0.30 0.042 ns ns C18:2 cis-9 t11 0.42 0.48 0.58 0.081 ns ns
Liver 0.38 0.36 0.36 0.044 ns ns C20:3 n-6 0.04 0.06 0.02 0.018 ns ns
Scrotum 0.29 0.31 0.27 0.055 ns ns C20:4 n-6 0.54 0.82 0.23 0.250 ns ns
C22:5 n-3 0.09 0.17 0.06 0.038 ns ns
Meat colour (longissimus lumborum muscle)
SFA 52.32 53.30 49.01 2.424 ns ns
L* 44.19 43.62 43.36 1.484 ns ns
UFA 47.05 46.05 50.45 2.456 ns ns
a* 8.55 8.59 9.12 0.684 ns ns
MUFA 43.28 41.10 46.58 2.802 ns ns
b* 11.04 10.67 10.86 0.939 ns ns
PUFA 3.76 4.95 3.86 0.536 ns ns
n-6 2.96 4.00 2.98 0.527 ns ns
FBW: fasting body weight; HCW: hot carcass weight; CCW: cold carcass weight; HCY: hot
n-3 0.34 0.44 0.33 0.076 ns ns
carcass yield; CCY: cold carcass yield. L* – colorimetric values of luminosity; a* – col- n-6:n-3 8.42 9.22 9.69 0.944 ns ns
orimetric values of red; b* – colorimetric values of yellow. SEM: standard error of the UFA:SFA 0.92 0.86 1.04 0.095 ns ns
means. T – treatment effect; REG – linear regression; ns – non significant (P > 0.05). C – PUFA:SFA 0.07 0.09 0.07 0.008 ns ns
Control; B – Babassu and M – Mofumbo treatments. DFA 69.53 68.85 69.82 1.390 ns ns
a
Carcass conformation: subjective scale of 5 points, value 1 (very poor carcass) and the Δ9-desaturase C14 2.67 2.96 2.97 0.266 ns ns
value 5 (excellent conformation). Δ9-desaturase C16 7.69 7.31 8.92 0.495 ns ns
b
Fat cover: subjective scale of 5 points, value 1 (excessively thin and devoid of fat Δ9-desaturase C18 59.96 58.51 65.33 3.965 ns *

carcass) and the value 5 (excessively fat). ELONGASE 69.00 69.07 69.40 1.574 ns ns
ATHERO 0.65 0.71 0.75 0.059 ns ns

According to Mensink et al. (2003), the effects of C12:0 rich fats on
cardio-vascular diseases remain uncertain. This fatty acid is considered
to have a major cholesterol-raising effect, however much of this due to
high-density lipoproteins (HDL) cholesterol (German and Dillard,
2004), which presents reduced fat accumulation potential (Delany
et al., 2000; Dayrit, 2015). In addition, C12:0 can be rapidly metabo-
lized into important energy sources (ketone bodys) for many organs,
also having the strongest antimicrobial activity, impairing gram-posi-
* Only the fatty acids with levels greater than 0.05 g/100 g are presented. SFA: satu-
rated fatty acids; UFA: unsaturated fatty acids; MUFA: monounsaturated fatty acids;
PUFA: polyunsaturated fatty acids; n-6: n-6 fatty acids; n-3: n-3 fatty acids; n-6:n-3: n-6
divided by n-3; UFA:SFA: unsaturated divided by saturated fatty acids; PUFA:SFA:
polyunsaturated divided by saturated fatty acids; DFA: desirable fatty acids; Δ9-desaturase
(C14, C16 and C18) – estimated Δ9-desaturase enzyme activity; ELONGASE – estimated
elongase enzyme activity; ATHERO – atherogenecity index. Means in the same row fol-
lowed by a different superscript letter differ significantly (P < 0.05); SEM – standard
error of the means; T – treatment effect; REG – linear regression; ns – non significant
(P > 0.05). C – Control; B – Babassu and M – Mofumbo treatments.

tive bacteria and some viruses and fungi (Dayrit, 2015), being em-
ployed against infections in animals (Kelsey et al., 2006) and presenting
potential use for the control of skin disorders (Nakatsuji et al., 2009).
When analysing the estimated activity of Δ9-desaturase C18 enzyme
against CT levels in diets, significant positive linear effect was observed
as reported by several studies (Vasta et al., 2009a,b; Whitney et al.,
2011; Rana et al., 2012). This fact occurs due to changes induced in the
profile of absorbed fatty acids.
parameters, carcass characteristics and meat quality variables.
Therefore, these leaves may be used as ingredient in lambs feeding,
providing a locally available alternative source of nutrients and con-
tributing to the sustainability of ruminant production. Moreover, fur-
ther research with higher inclusion of these feedstuffs may confirm, or
generate more consistent, results regarding potential changes in meat
fatty acids profile.

5. Conclusions
Conflict of interest
Given the results obtained in this study, including C. leprosum and O.
phalerata leaves in diet of lambs affects some ruminal SCFA and mi- The authors declare that here are no conflicts of interest.
crobial population, without compromising their productivity

75
A.L. Abdalla Filho et al.
Fig. 2. Distance-based redundancy analysis (RDA) of rumen microbial communities: rumen fungi, Archaea methanogens, Ruminococcus flavefaciens and Fibrobacter succinogenes: (A)
superimposed onto the ordination are correlations with the different treatments (Mofumbo, Babassu and Control) and digestibility variables, nitrogen balance (Nitrogen) and enteric
production of methane (Methane) data from the previous study Abdalla Filho et al. (2017); (B) superimposed onto the ordination are correlations with the different treatments (Mofumbo,
Babassu and Control) and ruminal short chain fatty acids.
Acknowledgements
The authors would like to thank the staff of the Animal Nutrition
Laboratory (LANA/CENA-USP) and Profa Dra Vania Rodrigues
Vasconcelos (UFPI) for providing the tested plants. This research was
financially supported by Comissão Nacional de Energia Nuclear (CNEN)
and Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq).
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