Yeast extract plus blood plasma in diets for piglets from 36 to 60 days old

Abstract

To evaluate inclusion levels and residual effects of diets containing yeast extract (YE) plus blood plasma (BP) on the performance of 36 to 60-days old piglets, 105 piglets were used (8.75 +/- 0.72kg), and they were distributed under a randomized block experimental design with five treatments (control -2.0% BP; 1.0% BP plus 0.0, 1.0, 2.0, or 3.0% YE), seven repetitions, and three animals per experimental unit. Piglets were given experimental diets from 36 to 49 days of age. From 50 to 60 day of age, all piglets received the same diet. No effects from treatments (P > 0.10) were observed on the performance variables from 36 to 49 day of age. From 50 to 60 day of age, an effect was observed from YE levels on the daily feed consumption (P < 0.03), daily weight gain (p < 0.01), and final weight (P < 0.06), which increased quadratically up to 1.67, 1.67, and 1.55% YE levels, respectively. Piglets which were fed with the diet containing 1.0% BP + 2.0% YE were observed to have better feed conversion ratios (P < 0.01) as compared to the control diet (2.0% BP). As it provides a better residual effect, the inclusion of 1.67% yeast extract may replace 50% of blood plasma in 36 to 49-days old piglet diets.

Full text

Yeast extract plus blood plasma in diets for piglets from 36 to 60 days old.
Ciência Rural, v.46, n.6, jun, 2016.
1107
Yeast extract plus blood plasma in diets
for piglets from 36 to 60 days old
Extrato de levedura associado ao plasma sanguíneo sobre
o desempenho de leitões dos 36 aos 60 dias
Cinthia Maria Carlos PereiraI Juarez Lopes DonzeleII Rita Flávia Miranda de Oliveira DonzeleII
Charles KieferIII Verônica Maria Pereira BernardinoIV Eric Marcio BalbinoII
Gabriel Cipriano RochaII*
ISSN 1678-4596
Ciência Rural, Santa Maria, v.46, n.6, p.1107-1112, jun, 2016
Received 11.10.13 Approved 11.28.15 Returned by the author 03.29.16
CR-2013-1508.R3
http://dx.doi.org/10.1590/0103-8478cr20131508
ABSTRACT
To evaluate inclusion levels and residual effects of
diets containing yeast extract (YE) plus blood plasma (BP) on the
performance of 36 to 60-days old piglets, 105 piglets were used
(8.75±0.72kg), and they were distributed under a randomized
block experimental design with ve treatments (control - 2.0%
BP; 1.0% BP plus 0.0, 1.0, 2.0, or 3.0% YE), seven repetitions,
and three animals per experimental unit. Piglets were given
experimental diets from 36 to 49 days of age. From 50 to 60 day of
age, all piglets received the same diet. No effects from treatments
(P>0.10) were observed on the performance variables from 36 to
49 day of age. From 50 to 60 day of age, an effect was observed
from YE levels on the daily feed consumption (P<0.03), daily
weight gain (p<0.01), and nal weight (P<0.06), which increased
quadratically up to 1.67, 1.67, and 1.55% YE levels, respectively.
Piglets which were fed with the diet containing 1.0% BP + 2.0%
YE were observed to have better feed conversion ratios (P<0.01)
as compared to the control diet (2.0% BP). As it provides a better
residual effect, the inclusion of 1.67% yeast extract may replace
50% of blood plasma in 36 to 49-days old piglet diets.
Key words: performance, residual effect, nutrition, swines.
RESUMO
Para avaliar níveis de inclusão e o efeito residual de
dietas contendo extrato de levedura (ExL) associado ao plasma
sanguíneo (PS) sobre o desempenho de leitões dos 36 aos 60
dias, foram utilizados 105 leitões (8,75±0,72kg), distribuídos em
delineamento experimental de blocos ao acaso com cinco tratamentos
(controle - 2,0% de PS; 1,0% PS, associado ao 0; 1,0; 2,0 ou 3,0%
de ExL), sete repetições e três animais por unidade experimental.
As dietas experimentais foram fornecidas dos 36 aos 49 dias e,
dos 50 aos 60 dias de idade, os animais passaram a receber dieta
única para avaliar o efeito residual dos tratamentos experimentais.
Não foram observados efeitos dos tratamentos (P>0,10) sobre as
variáveis de desempenho dos 36 aos 49 dias de idade. Dos 50 aos
60 dias de idade, houve efeito dos níveis de ExL sobre o consumo de
ração diário (P<0,03), ganho de peso diário (P<0,01) e peso nal
(P<0,06) que aumentaram de forma quadrática até os níveis de 1,67,
1,67 e 1,55% de ExL, respectivamente. Leitões alimentados com a
dieta contendo 1,0% de PS + 2,0% de ExL apresentaram melhor
(P<0,01) conversão alimentar em relação à dieta controle (2% de
PS). Por proporcionar melhor efeito residual, a inclusão de 1,67%
de extrato de levedura pode substituir 50% do plasma sanguíneo na
dieta de leitões dos 36 aos 49 dias de idade.
Palavras-chave: desempenho, efeito residual, nutrição, suínos.
INTRODUCTION
The post-weaning period may be considered
critical for piglets, once they are submitted to several
stress situations (SOBESTIANSKY et al., 2001)
such as environmental changes (new facilities and/
or environmental temperatures), removal from the
sow, replacement of liquid with solid diets, and social
tensions arising from getting animal grouped back
after being separated (FERREIRA et al., 2001). Thus,
aiming to minimize the stress which is observed in this
initial phase, and to avoid sudden feed consumption
reductions, studies have been developed in order
to seek feeds which may be added to piglet diets in
IDepartamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz (UESC), Ilhéus, BA, Brasil.
IIDepartamento de Zootecnia, Universidade Federal de Viçosa (UFV), Avenida Peter Henry Rolfs, s/n, 36570-900, Viçosa, MG, Brasil.
E-mail: gcrocha@ufv.br. *Corresponding author.
IIIFaculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS, Brasil.
IVDepartamento de Zootecnia, Universidade Federal de Lavras (UFLA), Lavras, MG, Brasil.
ANIMAL PRODUCTION

1108 Pereira et al.
Ciência Rural, v.46, n.6, jun, 2016.
their post-weaning period. Use of those feeds intends
to minimize the negative effects which generally take
place on the performance of animals during that period.
Among those feeds, we can mention blood plasma
(BP) and yeast extract (YE).
Blood Plasma which is rich in
immunoglobulins and glutamic acid, and it has been
used in piglet diets during the rst days after they
are weaned, yielding excellent results in regards to
feed consumption, due to its excellent palatability.
However, studies (TOUCHETTE et al., 1996;
BARBOSA et al., 2007) showed that the benecial
effect from BP on animal performance may better
observed when it is fed for longer periods. Therefore,
BP removal from piglet diets can lead to lower feed
intake and, consequently, poorer performance.
Yeast extract, which is obtained from
the internal contents of cells from specic strains of
Saccharomyces cerevisae 1026 yeast, has been used
in post-weaning diets associated with BP to improve
intestinal mucosa and performance during the rst days
after weaning (CARLSON et al., 2005; RIGUEIRA et
al., 2013). However, more studies need to be conducted
in order to evaluate whether said effects linger on
subsequent phases and whether adding YE to BP
provides a residual effect on the performance of piglets.
Therefore, this study aimed to evaluate
the effect from the use of yeast extract plus blood
plasma in 36 to 49-days old piglets, as well as the
possible residual effect on the performances of 50 to
60-days old piglets.
MATERIALS AND METHODS
One hundred and ve 36 to 60-days
old piglets from Large White x Landrace lineage
were used. From weaning (21 days) to 35 days of
age, piglets were fed with a single diet. With 36
days of age, animals with initial average weight of
8.75±0.72kg were distributed under a randomized
block experimental design with ve treatments
(control - 2.0% BP; 1.0% BP plus 0.0. 1.0, 2.0, or
3.0% YE), seven repetitions, and three animals per
experimental unit. The blocks took into account the
weight of animals with 36 days of age. In order to
evaluate the residual effect from the experimental
diets (provided from 36 to 49 days of age), animals
received a single diet from 50 to 60 days of age.
Experimental diets contained the same
levels of energy and amino acids, and they were
prepared so as to meet the animals’ nutritional
requirements, as preconized by ROSTAGNO et al.
(2005). Percentage and nutrient compositions of
experimental diets that were used within the period
from 36 to 49 days are presented in table 1. From 50
to 60 days of age, all piglets received the same diet,
which was based on corn and soy bean meal, with
3,280kcal kg-1 metabolizable energy, 20.8% crude
protein, 1.16% digestible lysine, 0.72% Ca, and
0.40% phosphorus available. Nutrient compositions
of AP 920® blood plasma and NuPro® yeast extract
are presented in table 2.
Animals were conned in suspended cages
with wooden oors, semi-automatic feeders, and
nipple drinkers, inside a masonry warehouse with
concrete oor, wooden ceiling, and ceramic roof tiles.
Thermal variations inside the room were recorded
daily at 4 p.m., through a maximum–minimum
thermometer. Water and feed were provided ad
libitum during the experimental period.
To determine their performance, animals
were weighed at the beginning and at the end of each
experimental phase in order to have their average daily
gain (ADG) estimated. Feed leftovers on the oor were
collected daily, and their amounts were summed to feed
leftover from the feeders at the end of the experimental
period in order to estimate their average daily feed
intake (ADFI) and feed conversion ratio (FC).
Performance variables were statistically
analyzed through Sistema de Análises Estatísticas e
Genéticas (SAEG) software, which was developed by
UNIVERSIDADE FEDERAL DE VIÇOSA (2007).
The data were submitted to analysis of variance.
Averages from the diets containing different YE levels
were submitted to regression analysis. Additionally,
the averages of each YE level that was associated with
BP were compared to the average from the control diet
(2.0% BP), for each period (from 36 to 49 and from 50
to 60 days of age), through contrast analysis. A 10%
signicance level was used for all tests performed.
RESULTS AND DISCUSSION
The temperatures recorded by the
maximum–minimum thermometer inside the
shed during the experimental period remained,
respectively, between 26.1±1.01°C and 29.8±1.32°C.
Considering that the average minimum temperature
recorded during the experimental period was above
the optimal temperature range (18.0 to 26.0°C) that is
proposed for 14 to 40-days old piglets (FERREIRA,
2005), it can be inferred that piglets were raised in a
thermoneutral environment.
Yeast extract levels did not inuence
(P>0.10) piglets weight at 49 days (Table 3).
There was no signicant effect of YE addition to

Yeast extract plus blood plasma in diets for piglets from 36 to 60 days old.
Ciência Rural, v.46, n.6, jun, 2016.
1109
diets, regardless of the levels used, as compared
to the control diet (2.0% BP) on piglets’ weight
at 49 days. There was no effect (P>0.10) of YE
levels on the ADFI of piglets from 36 to 49 days.
Likewise, when the ADFI of piglets fed the control
diet was compared with animals which received
diets containing different YE levels, no differences
were found (P>0.10). Studies such as the one by
RIGUEIRA et al. (2013) have not reported signicant
differences regarding the ADFI of 7 to 49-days old
fed diets containing BP (2.0%) or BP plus YE (1.0:
1.0%). However, CARLSON et al. (2005) reported
improved ADFI for 34 to 47-days old piglets, by
adding 2.5% YE alone in their diet, as compared to
animals fed with the control diet (0.0%).
According to COFFEY & CROMWELL
(1995) and ROSSI et al. (2007), the benecial effects
from BP and YE, respectively, on ADFI, is expected
in situations in which piglets are submitted to
immunological challenges. Considering that facilities
were properly washed, disinfected, and sealed
thereafter, prior to the execution of this study, and that
growth promoters were added to experimental diets, it
can be inferred that animals may have been submitted
to low immunological challenges, which may either
justify the lack of ADFI variation between treatments.
Yeast extract levels did not inuence
(P>0.10) 36 to 49-days old piglets’ ADG. This result
differs from the one that was obtained by CARLSON
et al. (2005) who observed improved ADG for piglets
Table 1 - Percentages and nutritional compositions of the experimental diets for piglets from 36 to 49 days old.
Item (%) 2.0% BP 1.0% BP+ 0.0% YE 1.0% BP+ 1.0% YE 1.0% BP+ 2.0% YE 1.0% BP + 3.0% YE
Corn 50.57 51.10 50.18 49.11 47.94
Dried whey 8.57 8.57 8.57 8.57 8.57
Extruded soybea n 21.28 21.28 21.28 21.39 21.50
Soybean meal 45% 14.00 14.00 14.00 14.00 14.00
Blood plasma 2.00 1.00 1.00 1.00 1.00
Yeast extract 0.00 0.00 1.00 2.00 3.00
Soybeal oil 0.00 0.20 0.20 0.22 0.33
Dical cium phosphat e 1.67 1.76 1.70 1.64 1.57
Limestone 0.73 0.68 0.72 0.75 0.79
Salt 0.37 0.42 0.37 0.33 0.29
L-lysine HCl 0.29 0.37 0.37 0.37 0.37
DL-methionine 0.14 0.16 0.16 0.16 0.17
L-threonine 0.07 0.11 0.11 0.11 0.11
L-trypt ophan 0.01 0.01 0.01 0.01 0.01
L-valine 0.00 0.03 0.03 0.04 0.04
L-isoleucine 0.00 0.01 0.000 0.000 0.01
Vitamin10.12 0.12 0.12 0.12 0.12
Minera l20.08 0.08 0.08 0.08 0.08
Colistin Sulfate 10% 0.10 0.10 0.10 0.10 0.10
---- ------------ ------------ ---- ------------ ----------- ------------ ----Nutr iti onal composition-----------------------------------------------------------------------
ME (kcal kg-1)3,400 3,400 3,400 3,400 3,400
Crude protein (%) 21.57 20.95 21.38 21.83 22.29
Digestible Lysine (%) 1.30 1.30 1.30 1.30 1.30
Digestible met + cys (%) 0.73 0.73 0.73 0.73 0.73
Digesti ble thr eonine (%) 0.82 0.82 0.82 0.82 0.82
Digesti ble try ptophan (%) 0.22 0.22 0.22 0.22 0.22
Digesti ble valine (%) 0.90 0.90 0.90 0.90 0.90
Digestible isoleucine (%) 0.73 0.73 0.73 0.73 0.73
Lacto se (%) 6.00 6.00 6.00 6.00 6.00
Calciu m (%) 0.85 0.85 0.85 0.85 0.85
Availa ble pho sphuros (%) 0.50 0.50 0.50 0.50 0.50
BP - blood plasma YE - yeast extract. 1
Content per kg of feed: 12.000IU- vit A-; 2,250IU - vit. D3; 27mg - vit E; 3mg - vit K; 2.25mg -
thiamine; 6mg - riboflavin; 2.25mg - pyridoxine; 27mcg - vit B12; 400mcg - folic acid; 150mcg - biotin; 22.5mg - pantothenic acid; 45mg -
niacin; 300mcg - Se;
2
Content per kg of feed: 100mg iron; 100mg zinc; 10mg copper; 40mg manganese; 1.5mg iodine; 1mg cobalt.

1110 Pereira et al.
Ciência Rural, v.46, n.6, jun, 2016.
by adding 2.5% YE in the diets. In the present study
no differences were found (P>0.10) for the ADG of
animals which received diet containing 2.0% BP
as compared to those which were fed with the YE
diets. This result is in agreement with RIGUEIRA et
al. (2013) who have not found ADG variation when
comparing diets containing BP (2.0%) and BP plus
YE (1.0; 1.0%). Low exposure of animals to the
immunological challenge may have contributed for
the lack of positive effects from BP and YE on ADG
in this study.
No effects (P>0.10) were observed from YE
levels on the FC of piglets. Similarly, no differences
were found (P>0.10) for the FC of animals which
received the diet containing 2.0% BP as compared to
those which were fed with the YE diets. In the present
study, the absence of effect from YE levels on the FC
is consistent with results of ADFI and ADG. Some
authors reported improved FC for piglet fed YE due
to the lower ADFI associated with similar ADG (de
ANDRADE et al., 2011; HU et al., 2014). The lower
ADFI of piglets fed with YE have been attributed to its
lower palatability. However, in the present study YE
did not affect piglets ADFI and also did not affect FC.
The divergence of performance results
between studies may be attributed to YE processing
procedures (TRCKOVA et al., 2014). Different
production and drying process may affect the quality of
the yeast and its products regarding the composition of
amino acids and peptides (THANISSERY et al., 2010).
A quadratic effect (P<0.06) was found for
YE levels on the nal weight of 60-days old piglets,
which has improved up to the estimated level of
1.55% YE (Table 3). No differences were observed
(P>0.10) on nal weights, when YE levels were
compared with the control diet.
A quadratic effect (P<0.03) was found for
YE levels on the ADFI of 50 to 60-days old piglets,
which has improved up to the estimated level of
1.67% YE. No differences were observed (P>0.10)
for ADFI in that phase, when diets containing BP plus
YE were compared to control group (2.0% BP). Such
result was similar to the one reported by RIGUEIRA
et al. (2013), who have not found a residual effect
on the ADFI 7 to 63-days old piglets which were fed
with diets containing BP or BP plus YE.
Piglets which were fed with diet containing
1.0% BP + 1.0% YE (from 36 to 49 days old) were
observed to have better ADFI in the period from 50 to
60 days, in absolute values, as compared to the ones
given the control diet containing 2.0% BP (1.120 x
1.070). Although the observed ADFI increase was
not signicant, it was enough to provide higher body
weight, in absolute values, for piglets at 60 days of age.
A quadratic effect (P<0.01) was also found
for YE levels on the ADG, which has improved up to
the estimated level of 1.67% YE. Results obtained in
this study differ from CARLSON et al. (2005), who
reported no differences when evaluating the residual
effect on ADG of 48 to 75-days old piglets, after they
had been fed with diets containing YE (0; 2.5%) from
34 to 47 day of age.
No effects were observed (P>0.10)
on the piglets’ ADG comparing control diet and
Table 2 - Nutritional composition of blood plasma AP 920® and yea st extract NuPr o®.
Nutrients Blood plasma AP 920®1 Yeast extract NuPro®2
Metab oliza ble energy (Kcal k g-1)3,906 3,314
Crude protein (%) 78.00 43.70
Calciu m (%) 0.15 0.02
Availa ble pho sphorus (%) 1.30 1.20
Sodium (%) 2.20 0.27
Potassiu m (%) 0.30 1.15
Lysine (%) 6.80 2.65
Methionine (%) 0.70 0.63
Threonine (%) 4.80 1.60
Tryptophan (%) 1.40 0.34
Valine (%) 5.30 2.15
Isoleucine ( %) 2.90 1.64
Gluta mic acid (%) 11.70 4.98
Nucleic acid (%) -5.20 – 6.60
Inosit ol (g kg-1) - 5.83
1APC AnLGlCompany. 2Alltech.

Yeast extract plus blood plasma in diets for piglets from 36 to 60 days old.
Ciência Rural, v.46, n.6, jun, 2016.
1111
diets containing different YE levels. Conversely,
RIGUEIRA et al. (2013) observed higher ADG
for piglets (7 to 63-days old) which received a diet
containing BP plus YE, as compared to the ones fed
with plasma (from 7 to 49 days of age).
Yeast extract levels did not inuence
(P>0.10) the animals’ FC. This result was similar to
RIGUEIRA et al. (2013) who reported no inuences
from YE on FC. However, in the present study the
piglets which were fed with the diet containing 1.0%
BP + 2.0% YE were observed to have better FC
(P<0.01) as compared to the ones given the control
diet (2.0% BP). Once the ADFI of piglets which
received the control diet and the ones which were fed
with diet containing 1.0 BP + 2.0% YE was similar in
absolute values (1070 x 1080), the improved FC that
was observed for piglets fed with diet containing 1.0
BP + 2.0% YE may be justied by the higher usage
efciency of nutrients in the diet, and consequently by
their increased ADG, in absolute values (719 x 658).
Comparing the initial phase (from 36 to
49 days of age) and the phase which evaluated the
residual effect (from 50 to 60 days of age), it was
possible to observe that the differences regarding nal
weights, ADFI and ADG were higher in the residual
period when compared to the initial period. In the
initial period, the diet containing 1.0% BP + 1.0% YE
was observed to provide, in absolute values, higher
weights, ADG, and ADFI at day 49, as compared
to the diet containing 2.0% BP, which correspond
respectively to 1.27, 3.14, and 0.27%.
Likewise, when analyzing the residual
effect, the diet containing 1.0% BP + 1.0% YE was
observed to provide additional 3.45, 7.97, and 4.46%
to nal weights, ADG, and ADFI, as compared to diet
containing 2.0% BP. Those results showed that diets
containing BP alone have a lower residual effect on
performance as compared to the diets containing
BP plus YE. That result is conrmed in the study by
BARBOSA et al. (2007), who observed that BP only
yields positive effects on the piglets’ performances
during the period in which it is being consumed. In
turn, results from the studies by VAN DJIK et al. (2001)
and TORRALARDONA et al. (2002) concluded that
BP provides its maximum benecial effect on piglets’
performances during the rst week post-weaning.
According to TOUCHETTE et al. (1996),
the reduced performances of piglets in the residual
period after BP is removed from their diets takes
place due to the reduced ADFI, among other factors.
That hypothesis is reinforced by the results from
studies by JIANG et al. (2000), who reproduced
the effect from the increased feed consumption that
is provided by BP through the isolated inclusion of
immunoglobulins in diets. These studies suggested
that immunoglobulins may be the main fraction
responsible for the improved performances of piglets.
Besides that, as diets had the same amounts
of nutrients, benets that were observed with YE may
be related to factors that are present in that ingredient,
which are not considered to be essential nutrients in diets
formulation, such as nucleotides, non-essential amino
Table 3 - Piglets performance fed diets with blood plasma and/or yeast extract from 36 to 49-da ys and from 50 to 60-days old.
---- ------------ ------------ ---- ------------ ----------- -Lev els of plasma and yeast ext ract (%)--------------------------------------------------------
Item
2.0 BP 1.0 BP + 0.0 YE 1.0 BP + 1.0 YE 1.0 BP + 2.0 YE 1.0 BP + 3.0 YE Regression CV (%)
---- ------------ ------------ ---- ------------ ----------- ------------ -----------36 to 49-days old------------------------------------------------------------------------------
IBW (kg) 8.79 8.91 8.77 8.74 8.32 ns 4.36
BW49 (kg) 15.70 15.41 15.90 15.42 14.92 ns 5.19
ADFI (g) 726 689 728 686 683 ns 8.35
ADG ( g) 493 464 509 477 471 ns 9.50
FC 1.47 1.48 1.43 1.44 1.45 ns 5.64
---- ------------ ------------ ---- ------------ ----------- ------------ ----------50 to 60 -days old-----------------------------------------------------------------------------
FBW (kg) 22.95 22.38 23.77 23.34 22.69 Q16.38
ADFI (g) 1.070 0.999 1.120 1.080 1.050 Q28.58
ADG ( g) 658 634 715 719 670 Q310.30
FC 1.63a1.58a1.57a1.50b1.57ans 5.87
BP (blood plasma); YE (yeast extract); IBW (initial body weight); BW49 (body weight at 49 days); FBW (final body weight); ADFI
(average daily fe ed intake); ADG (average dail y gain); FC (feed conversion rati o); CV ( coeffi cient o f variati on). Regression: Regr ession
analysis between diets containing 0.0 to 3.0% yeast extract – ns (not significant); Q -1Quadradic effect (P<0.06) Y = 22.46 + 1.58X – 0.51X
2
(r²=0.89); 2Quadradic effect (P<0.03) Y = 1.006 + 0.122X – 0.036X2(r²=0.83); 3
Quadradic effect (P<0.01) Y = 0.635 + 0.109X – 0.032X2
(r²=0.99).
a,b- Mean values of each YE level followed by different superscripts in the same row differ from the control diet, according to
Dunnet´s test.

1112 Pereira et al.
Ciência Rural, v.46, n.6, jun, 2016.
acids, and inositol. Use of exogenous nucleotides leads
to the prevalence of benecial gut microbiota, such as
bidobacteria and lactobacilli, and smaller quantities of
gram-negative ones (UAUY et al., 1994), and it plays an
important role in the recovery of intestinal mucosa after
animals go through to some kind of challenge.
CONCLUSION
As it provides a better residual effect, the
inclusion of 1.67% yeast extract allow ED in 36 to 49-
days old piglet diets, the replacement of 50% plasma,
whose inclusion may be reduced from 2.0 to 1.0%.
BIOETHICS AND BIOSSECURITY
COMMITTEE APPROVAL
Approved by the ethics committee in the use of animals/
Universidade Federal de Viçosa (UFV), under protocol no. 19/2012.
ACKNOWLEDGMENTS
We would like to thank Conselho Nacional de
Desenvolvimento Cientíco e Tecnológico (CNPq) for providing
a research grant, and Alltech do Brasil for the nancial support.
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