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DOI: 10.55905/rdelosv18.n64-058
ISSN: 1988-5245
Originals received: 1/10/2025
Acceptance for publication: 2/5/2025
Revista DELOS, Curitiba, v.18, n.64, p. 01-29, 2025
jan. 2021
Oregano essential oils, castor vegetable oil, and Baccharis hydroethanolic
extract on growth inhibition of rumen gram-positive and gram-negative
bactéria
Óleos essenciais de orégano, óleo vegetal de mamona e extrato hidroetanólico
de Baccharis na inibição do crescimento de bactérias gram-positivas e gram-
negativas do rúmen
Aceites esenciales de orégano, aceite de ricino y extracto hidroetanólico de
Baccharis en la inhibición del crecimiento de bacterias grampositivas y
gramnegativas en el rumen
Rodrigo Augusto Cortez Passetti
PhD in Animal Science
Institution: Universidade Estadual de Maringá
Address: Maringá – Paraná, Brazil
E-mail: racpassetti@gmail.com
Venício Macêdo Carvalho
PhD in Animal Science
Institution: Universidade Estadual de Maringá
Address: Maringá – Paraná, Brazil
E-mail: venicio.mcarv@gmail.com
Vicente Alfonso Díaz Ávila
PhD in Animal Science
Institution: Universidade Estadual de Maringá
Address: Maringá – Paraná, Brazil
E-mail: vadiaza@ut.edu.com
Jéssica Geralda Ferracini
Master in Animal Science
Institution: Universidade Estadual de Maringá
Address: Maringá – Paraná, Brazil
E-mail: jess.ferracini@gmail.com
Rodolpho Martin do Prado
PhD in Animal Science
Institution: Universidade Estadual de Maringá
Address: Maringá – Paraná, Brazil
E-mail: rodolphoprado@hotmail.com
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Mariana Garcia Ornaghi
PhD in Animal Science
Institution: Universidade Estadual de Maringá
Address: Maringá – Paraná, Brazil
E-mail: mariana.ornaghi@safeeds.com.br
Hilário Cuquetto Mantovani
PhD in Animal Science
Institution: Universidade Federal de Viçosa
Address: Viçosa – Minas Gerais, Brazil
E-mail: hcmantovani@gmail.com
Ivanor Nunes do Prado
PhD in Animal Science
Institution: University of Rennes
Address: Rennes – Brittany, France
E-mail: inprado@uem.br
ABSTRACT
Natural additives are a promising tool in modulating rumen fermentation due to the recent
concerns regarding microbial resistance to antibiotics. We evaluate the antimicrobial capacity of
different levels (10, 20, 50 and 100 mg/L) of monensin, oregano essential oil (Origanum vulgare
L), castor oil (Ricinus communis) and Baccharis dracunculifolia (BD) hydroethanolic extract
against gram-negative (Prevotella albensis, Prevotella bryantii and Treponema saccharophilum)
and gram-positive (Ruminococcus albus, Ruminococcus flavefaciens and Streptococcus bovis)
bacteria. Optical density (600 nm) was measured at 8, 12 and 24 h. Monensin inhibited gram-
positive bacterial growth (P < 0.05). However, higher dosages were also effective against gram-
negative bacteria, possibly due to a toxicological effect. Oregano essential oil inhibited (P < 0.05)
both gram-negative and gram-positive bacteria. Castor oil had marginal-to-no effect on gram-
negative bacteria, but inhibited the growth of cellulolytic bacteria (Ruminococcus albus) at 12 h.
Concentrations of Baccharis dracunculifolia used in this study (up to 100 mg/L) had no
inhibitory effect on rumen bacteria. Natural additives are a promising tool to modulate rumen
fermentation, which highlights the importance of further studies to evaluate the potential of new
products.
Keywords: Baccharis, microbiome, natural additives, ruminal modulation.
RESUMO
Os aditivos naturais são uma ferramenta promissora na modulação da fermentação ruminal
devido às preocupações recentes relacionadas à resistência microbiana aos antibióticos.
Avaliamos a capacidade antimicrobiana de diferentes níveis (10, 20, 50 e 100 mg/L) de
monensina, óleo essencial de orégano (Origanum vulgare L), óleo de mamona (Ricinus
communis) e extrato hidroetanólico de Baccharis dracunculifolia (BD) contra bactérias gram-
negativas. microrganismos (Prevotella albensis, Prevotella bryantii e Treponema
saccharophilum) e bactérias gram-positivas (Ruminococcus albus, Ruminococcus flavefaciens e
Streptococcus bovis). A densidade óptica (600 nm) foi medida em 8, 12 e 24 h. A monensina
inibiu o crescimento bacteriano gram-positivo (P < 0,05). Entretanto, dosagens mais altas
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também foram eficazes contra bactérias gram-negativas, possivelmente devido a um efeito
toxicológico. O óleo essencial de orégano inibiu (P < 0,05) bactérias gram-negativas e gram-
positivas. O óleo de rícino teve efeito marginal ou nenhum sobre bactérias gram-negativas, mas
inibiu o crescimento de bactérias celulolíticas (Ruminococcus albus) em 12 h. As concentrações
de Baccharis dracunculifolia usadas neste estudo (até 100 mg/L) não tiveram efeito inibitório
sobre as bactérias do rúmen. Os aditivos naturais são uma ferramenta promissora para modular a
fermentação ruminal, o que destaca a importância de mais estudos para avaliar o potencial de
novos produtos.
Palavras-chave: Baccharis, microbioma, aditivos naturais, modulação ruminal.
RESUMEN
Los aditivos naturales son una herramienta prometedora para modular la fermentación ruminal
debido a las preocupaciones recientes relacionadas con la resistencia microbiana a los
antibióticos. Se evaluó la capacidad antimicrobiana de diferentes niveles (10, 20, 50 y 100 mg/L)
de monensina, aceite esencial de orégano (Origanum vulgare L), aceite de ricino (Ricinus
communis) y extracto hidroetanólico de Baccharis dracunculifolia (BD) frente a bacterias gram
-negativo microorganismos (Prevotella albensis, Prevotella bryantii y Treponema
saccharophilum) y bacterias grampositivas (Ruminococcus albus, Ruminococcus flavefaciens y
Streptococcus bovis). La densidad óptica (600 nm) se midió a las 8, 12 y 24 h. La monensina
inhibió el crecimiento de bacterias grampositivas (P < 0,05). Sin embargo, dosis más altas
también fueron eficaces contra bacterias gramnegativas, posiblemente debido a un efecto
toxicológico. El aceite esencial de orégano inhibió (P < 0,05) bacterias gramnegativas y
grampositivas. El aceite de ricino tuvo un efecto marginal o nulo sobre las bacterias
gramnegativas, pero inhibió el crecimiento de bacterias celulolíticas (Ruminococcus albus) en 12
h. Las concentraciones de Baccharis dracunculifolia utilizadas en este estudio (hasta 100 mg/L)
no tuvieron efecto inhibitorio sobre las bacterias del rumen. Los aditivos naturales son una
herramienta prometedora para modular la fermentación ruminal, lo que resalta la importancia de
realizar más estudios para evaluar el potencial de nuevos productos.
Palabras clave: Baccharis, microbioma, aditivos naturales, modulación ruminal.
1 INTRODUCTION
Brazil’s cattle herd has approximately 215 million animals, its majority being raised on
pasture and low input production systems (ANUALPEC, 2024). Extensive production systems
increase the slaughter age, which promotes high methane (CH4) emissions per kg of product
(Cardoso et al., 2016). However, the number of cattle finished in feedlots in Brazil have doubled
in the last 10 years (ANUALPEC, 2024), and it has been estimated that intensification practices
could reduce up to 48% of the greenhouse gas emissions per kg of carcass, and reduce the total
land area used per kg carcass 7-fold (Cardoso et al., 2016).
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In more intensive production systems, antibiotics, or ionophores like monensin, are
frequently added into ruminants’ diets as a strategy to modulate rumen fermentation and improve
animal efficiency (Zawadzki et al., 2011). In contrast, routine usage of antibiotics as growth-
promoters in feed has generated public health concerns due to the emergence of antibiotic-
resistant bacteria that could represent risks to human health (Schäberle and Hack, 2014).
Consequently, considerable efforts have been employed towards the development of natural
alternatives to antibiotics and ionophores (Ornaghi et al., 2017; Souza et al., 2019; Ornaghi et
al., 2020). Among these substances, plants extracts and essential oils have attracted the most
attention (Monteschio et al., 2017; Rivaroli et al., 2017; Fugita et al., 2018; Ornaghi et al., 2020).
Oregano essential oil has monoterpenes, like carvacrol and thymol, that are capable of
affecting the cellular membrane permeability of gram-negative bacteria (Dutra et al., 2019).
Castor oil, a co-product of castor seed, is composed by 90% ricinoleic acid, which is commonly
known for its antimicrobial properties (Morales et al., 2012; Mottin et al., 2022). Other studies
have been focusing on a plant commonly found in South America, Baccharis dracunculifolia,
which is the raw material used by bees (Apis melifera) to produce green propolis (Maróstica
Junior et al., 2008), which could be used to modulate rumen fermentation due to its richness in
secondary metabolites, like flavonoids that have antioxidant and antimicrobial properties (Bonin
et al., 2020; Carvalho et al., 2021).
Considerable scientific information has been generated to show that these alternative
additives can alter rumen fermentation, feed digestion and bacterial and archaeal communities
(Cobellis et al., 2016). However, due to the large variety of plants, as well as extraction and
processing methods, results in literature are still divergent. This highlights not only the
importance, but also the potential of new studies. While animal studies are costly and time
consuming, in vitro techniques in testing additives against the growth of pure bacterial cultures
could be used for the screening the selection of new products (Aguiar et al., 2013) to be further
tested on animals.
This study was realized to evaluate alternative natural additives (oregano essential oil,
castor oil, and B. dracunculifolia extracts) in comparison to monensin in the growth of pure
bacterial cultures.
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2 MATERIALS AND METHODS
2.1. MONENSIN AND NATURAL ADDITIVES
Sodium monensin (Elanco®) was purchased in a feeding mill supply shop (AB Araújo –
Maringá, Paraná Brazil). Oregano essential oil was obtained from FERQUIMA® (Vargem
Grande Paulista, São Paulo, Brazil). Castor vegetable oil was obtained from SAFEEDS®
(Cascavel, Paraná, Brazil) and stored at ± 4º C.
Oregano essential oil has previously been analyzed and shows to have a great amount of
gama-terpinene and ortho-cymene (8.0% and 9.4%, respectively), while carvacrol was found to
be its major compound (68.3%) (Biondo et al., 2017). Baccharis dracunculifolia samples were
collected in Maringá (Paraná, southern Brazil), latitude 23°27’S and longitude 51°59’W.
Climatic conditions of the region include an annual average temperature of 18° C and annual
average rainfall of 1,114 mm. Whole plants collected were weighed and dried in a forced air
circulation oven (TECNAL – TE-394/2 – Piracicaba, São Paulo, Brazil) at a temperature of 40°
C. The dry material was then, milled using a 1 mm sieve and a knife mill (WILLEY). Then, 10
g of the material was mixed with a hydroethanolic solution (70:30, v/v) and placed in agitation
(20 min) with rest (15 min) for 2 h. The extract was kept in a water bath (35° C) for 24 h. It was
then filtered and concentrated using a rotary evaporator (FISATOM – São Paulo, Brazil) at
ambient temperature, until the solvent was completely evaporated. The remaining crude extract
was lyophilized and stored at 4° C.
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Figure 1. Chromatographic extract profile of Baccharis draunculifolia extract
Source: Author.
Figure 2. Chromatographic profile of castor oil.
Source: Author.
Lyophilized Baccharis dracunculifolia samples were diluted in acetonitrile at a 1:1 ratio
and analyzed by UHPLC-HRMS using a Nexera X2 ultra-high performance liquid
chromatography system, as described by Bonin et al. (2020). MS and MS/MS spectra were
visualized using Software Data Analysis 4.3, then compared to the existing literature and
analyzed using a free-access mass spectrometry database such as the Human Metabolome
Database (HMDB) (Wishart et al., 2012). Twelve compounds were identified: germacrene B;
spathulenol; naringenin; kaempferol; artepillin C; alpha-pinene; hydroxycinnamic acid;
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apigenin; kaempferide; limonene; phenylethanol and β-caryophyllene (Figure 1), as described by
Bonin et al. (2020). The same procedure was used for castor oil, where ricinoleic acid was
identified as the main compound (Figure 2).
2.2. BACTERIA STRAIN AND CULTURE CONDITIONS
Three gram-negative bacteria: Prevotella albensis (DSMZ 11370); Prevotella bryantii
(DSMZ 11371) and Treponema saccharophilum (DSMZ 2985), and three gram-positive
bacteria: Ruminococcus albus (DSMZ 20455); Ruminococcus flavefaciens (DSMZ 25089) and
Streptococcus bovis (DSMZ 20480) were purchased (DSMZ – Brunswick, Germany).
Lyophilized bacteria were activated according to the manufacturer’s recommendations, and then
replicates were frozen in Hungate tubes with Hobson M2 media (Hobson & Stewart, 2012), plus
glycerol (20%) and stored at -80º C. The medium consisted of 2 g glucose, 2 g maltose, 4 g
sodium bicarbonate, 10 g bacto casitone, 2.5 g yeast, 2 g cellobiose, 150 mL mineral solution I,
150 mL mineral solution II, 200 mL clarified ruminal fluid, 10 mL 60% (w/v) sodium lactate
solution and 1 mL 0.1% resazurin solution, in 1 L of distilled water. Mineral solution I consists
of 3 g of dipotassium phosphate in 1 L of distilled water. Mineral solution II consists of 3 g of
monopotassium phosphate, 6 g of ammonium sulfate, 6 g of sodium chloride, 6 g of magnesium
sulfate and 0.6 g of calcium chloride in 1 L of distilled water. The medium was prepared under
anaerobic conditions by boiling, addition of a reducing agent, and continued flushing of O2 and
free CO2 into the flask and tubes using the Hungate technique (Hungate, 1966). After the culture
medium reduction, the tubes were sealed with butyl stoppers and autoclaved, before inoculation.
2.3. EFFECT OF ADDITIVES ON GROWTH OF PURE BACTERIAL CULTURE
Hungate tubes of each bacteria were thawed overnight and subcultures (3 stepwise
repetitions) were grown in Hobson M2 (Hobson & Stewart, 2012) media at 39º C, for 24 h, to
washout glycerol before the start of the assay. The assay was carried out in duplicate tubes
containing 9 mL of the culture medium and 0.5 mL of cultured medium containing bacteria and
0.5 mL of each additive working solution. For the working solution, additives (monensin,
oregano essential oil, castor oil, and B. dracunculifolia extract) were solubilized in Tween 5%,
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at the concentrations of 200, 400 1.000 and 2.000 mg/L. The tubes were inoculated under
anaerobic conditions (stream of O2-free CO2 while the tube is open) and incubated at 39o C.
Preliminary, data indicated that 12 h of growth corresponded to the early stationary phase. Then,
bacterial growth was assessed at 0, 8, 12 and 24 h at 39º C by using optical density (OD) at 600
nm. The incubation time 0 was used with the only purpose of a baseline as at this time there is
no action of the compounds over the bacterial growth. The absorbance of culture medium tubes
containing the additives, but not inoculated, was measured and subtracted from the absorbance
of the assay tube. The antimicrobial activity was assessed in the Hungate tubes using the final
concentration of 10, 20, 50 and 100 mg/L for the plant additives and monensin. Tubes containing
only culture medium were also inoculated and used as controls (0 mg/L).
2.4. STATISTICAL ANALYSES
Optical density was interpreted by analysis of variance using the GLM from IBM
Statistical Package for the Social Sciences (SPSS version 22), with the effect of incubation time
analyzed as a repeated measurement. Differences among means were then identified using the
Bonferroni procedure with significance declared at P ≤ 0.05. Regression was performed to
analyze the effect of the concentrations (0, 10, 20, 50 and 100 mg/L) and the equation used to
estimate the additive (mg/L) amount necessary to inhibit 50% of bacterial growth (MICe).
3 RESULTS
Considering the three gram-negative bacteria (Prevotella albensis [DSMZ 11370,
proteolytic and amylolytic], Prevotella bryantii [DSMZ 11371, proteolytic and amylolytic] and
Treponema saccharophilum [DSMZ 2985, pectinolytic]), and three gram-positive bacteria
(Ruminococcus albus [DSMZ 20455, fibrolytic], Ruminococcus flavefaciens [DSMZ 25089,
fibrolytic], Streptococcus bovis [DSMZ 20480, proteolytic and amylolytic]), when no additive
was added (0 mg/L), the growth of most bacteria used in our study reached their stationary phase
between 8 and 12 h, with the exception of Ruminococcus albus, which continued growing until
24 h.
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3.1. MONENSIN
P. albensis growth plateaued at 8 h of incubation, with OD values ranging from 1.45 to
1.50, and no differences (P > 0.05) were observed over time (Table 1). In contrast, monensin
lightly reduced (P < 0.05) the growth of P. albensis at 8 h (P < 0.05). At high concentrations (20,
50 and 100 mg) there was still bacterial growth over time, with the highest OD values at 12 h,
which was reduced at 24 h. This could be caused by the bactericidal effect of the long exposure
to sodium monensin. However, this bacterium presented high tolerance to monensin (MICe > 200
mg/L). Prevotella bryantii continued to grow until 12 h, but a slightly reduction in the OD was
observed at 24 h when no additive was added (Table 1).
A quadratic effect showed that monensin reduced growth of this species, but
concentrations higher than 50 mg/L had no detrimental effects in the OD values. This species
was less resistant to monensin than P. albensis, as this inhibitory effect continued at 24 h, with
MICe values at around 45 mg/L.
Table 1. Influence of monensin in the anaerobic gram-negative bacteria growth
Monensin1 concentration, mg/L
Time (h)
Repeated
measures
8
12
24
SEM
P < Value
Prevotella albensis
0
1.45
1.50
1.48
0.023
0.886
10
1.55
1.55
1.42
0.042
0.244
20
1.43AB
1.55A
1.38B
0.038
0.023
50
1.20
1.44
1.24
0.038
0.128
100
1.23B
1.49A
1.25B
0.046
0.003
SEM
0.040
0.027
0.025
L
0.003
0.480
0.001
Q
0.006
0.719
0.001
MICe (mg/L)
251
-
347
Prevotella bryantii
0
1.30B
1.39A
1.32B
0.027
0.038
10
0.98A
0.92B
0.71C
0.036
0.004
20
0.80B
0.85A
0.60B
0.032
0.001
50
0.32B
0.35A
0.28B
0.010
0.023
100
0.31B
0.38A
0.33B
0.011
0.004
SEM
0.088
0.089
0.086
L
0.001
0.001
0.001
Q
0.001
0.001
0.001
MICe (mg/L)
47
45
33
Treponema saccharophilum
0
1.35
1.37
1.37
0.022
0.898
10
1.06A
1.04B
0.75C
0.042
<0.001
20
0.92A
0.94B
0.71C
0.032
0.004
50
0.55A
0.53A
0.43B
0.016
<0.001
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100
0.54B
0.61A
0.50C
0.014
<0.001
SEM
0.071
0.070
0.077
L
0.001
0.001
0.030
Q
0.001
0.001
0.001
MICe (mg/L)
67
65
49
1Monensin = 20% of sodium monensin; A, B = Different uppercase letters mean difference in the same line at
Bonferroni (P < 0.05). L = linear effect; Q = quadratic effect; MICe = estimated amount of additive concentration
(mg/L) necessary to reduce 50% of optical density.
Source: Author.
Treponema saccharophilum growth plateaued at 8 h of incubation and no differences (P
> 0.05) were observed over time (Table 1). In contrast, when monensin was added, these bacteria
presented a reduction in the OD values that continued over time. Like P. albensis, a quadratic
effect of monensin concentration was also observed. However, Treponema sacharophilum was
more resistant, as MICe values were at around 65 mg/L.
Monensin successfully inhibited the gram-positive bacterial growth, like Ruminoccocus
albus (Table 2). However, the normal growth of these bacteria was slower, as OD values started
at 0.51 and continued to increase between 12 and 24 h (P < 0.05). Similarly, when these bacteria
were incubated with monensin, it also presented a slight increase in OD (P < 0.05) over time,
with exception to the higher concentrations, in which OD values remained at around 0.10.
Ruminoccocus albus was the most sensitive bacteria to Monensin in our study, with a MICe value
of 2 mg/L between 8 and 12 h of incubation.
Ruminoccocus flavefaciens growth plateaued at 8 h with an OD of 1.33 and no differences
were observed over time (Table 2). Monensin concentration showed both linear and quadratic
effects (P < 0.05), resulting in a reduction in the OD. Optical density increased at 12 h but
resumed decreasing at 24 h of incubation. Surprisingly, this species was much more resistant
than Ruminoccocus albus, with MICe values at around 45 mg/L.
Table 2. Influence of monensin in the anaerobic gram-positive bacteria growth
Monensin1 concentration mg/L
Time (h)
Repeated
measures
8
12
24
SEM
P < Value
Ruminococcus albus
0
0.51C
0.84B
0.98A
0.085
0.052
10
0.09C
0.13B
0.34A
0.035
0.001
20
0.11C
0.13B
0.26A
0.022
0.001
50
0.07
0.11
0.09
0.011
0.164
100
0.05
0.08
0.13
0.012
0.098
SEM
0.047
0.071
0.074
L
0.030
0.020
0.002
Q
0.008
0.002
0.001
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MICe (mg/L)
20
20
18
Ruminococcus flavefaciens
0
1.33
1.41
1.38
0.043
0.762
10
1.03A
0.97B
0.92C
0.014
0.016
20
0.82A
0.84A
0.67B
0.022
0.005
50
0.38B
0.40A
0.40A
0.007
0.019
100
0.30B
0.40A
0.34B
0.012
0.001
SEM
0.091
0.088
0.088
L
0.001
0.001
0.001
Q
0.001
0.001
0.001
MICe (mg/L)
45
46
41
Streptococcus bovis
0
1.34A
1.29A
1.25B
0.021
0.019
10
0.94A
0.94A
0.79B
0.019
0.001
20
0.79A
080A
0.63B
0.023
0.001
50
0.31A
0.33A
0.26B
0.009
0.004
100
0.29B
0.34A
0.28B
0.007
0.005
SEM
0.091
0.085
0.083
L
0.001
0.001
0.001
Q
0.001
0.001
0.001
MICe (mg/L)
41
45
45
1Monensin = 20% of sodium monensin A, B = Different uppercase letters mean difference in the same line at
Bonferroni (P < 0.05); L = linear effect; Q = Quadratic effect; MICe = estimated amount of additive concentration
(mg/L) necessary to reduce 50% of optical density.
Source: Author.
Streptococcus bovis growth plateaued between 8 and 12 h, but a slight reduction in the
OD was observed at 24 h (Table 2). Similar effects were observed over time when monensin was
added. Monensin reduced (P < 0.05) the OD of S. bovis, but a quadratic effect showed that
concentrations higher than 50 mg/L had no improvements on the inhibitory capacity. In addition,
the MICe observed in Streptococcus bovis was also around 45 mg/L.
3.2. OREGANO ESSENTIAL OIL
Oregano essential oil reduced (P < 0.05) Prevotella albensis growth at 8 h, and these
values remained at around 0.50, in spite of the concentration tested (Table 3). However, these
were temporary effects, as bacterial growth returned to normal levels at 12 and 24 h of incubation.
Oregano essential oil was more effective in inhibiting the initial P. albensis growth in
comparison to monensin, as its MICe was lower than 50 mg/L at 8 h. Similarly, the growth of the
other two gram-negative bacteria, P bryantii and T. saccharophilum (Table 3), was also inhibited
at 8 h by oregano essential oil (MICe of 37 and 48 mg/L, respectively), but returned to normal
levels at 12 and 24 h (P < 0.05).
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Table 3. Influence of oregano essential oil in the anaerobic gram-negative bacteria growth
Oregano essential oil concentration, mg/L
Time (h)
Repeated Measures
8
12
24
SEM
P < Value
Prevotella albensis
10
NE
NE
NE
NE
NE
20
0.51B
1.33A
1.55A
0.137
0.010
50
0.45C
1.27B
1.62A
0.150
0.001
100
0.53B
1.44A
1.50A
0.135
0.001
SEM
0.108
0.027
0.032
L
0.010
0.829
0.919
Q
0.001
0.001*
0.233
MICe (mg/L)
43
46
48
Prevotella bryantii
0
1.30B
1.39A
1.32B
0.027
0.038
10
NE
NE
NE
NE
NE
20
0.40B
1.17A
1.57A
0.148
0.006
50
0.39C
1.14B
1.59A
0.152
0.009
100
0.42B
1.22A
1.61A
0.152
0.008
SEM
0.100
0.029
0.048
L
0.009
0.130
0.066
Q
0.001
0.001*
0.059
MICe (mg/L)
37
50
45
Treponema saccharophilum
0
1.35
1.37
1.37
0.022
0.898
10
NE
NE
NE
NE
NE
20
0.49C
1.38B
1.60A
0.145
0.004
50
0.47B
1.34A
1.57A
0.146
0.004
100
0.51B
1.37A
1.46A
0.132
0.009
SEM
0.095
0.020
0.038
L
0.008
0.942
0.764
Q
0.001
0.946
0.103
MICe (mg/L)
48
56
55
A, B = Different uppercase letters mean difference in the same line at Bonferroni (P < 0.05). L = linear effect; Q =
quadratic effect MICe = estimated amount of additive concentration (mg/L) necessary to reduce 50% of optical
density; * = equation to calculate MICe does not have real square roots; NE = not evaluated.
Source: Author.
Ruminoccocus albus growth was inhibited (P < 0.05) at 8 and 12 h, but these bacteria
resumed growing at 24 h (Table 4). Interestingly, the MICe values were 48 and 69 mg/L at 8 and
12 h of incubation, showing that concentrations higher than 50 mg/L of oregano essential oil are
necessary to extend the inhibitory effect on these bacteria. Oregano essential oil reduced
Ruminoccocus flavefaciens growth at 8 h (P < 0.05), but bacteria resumed growing at 12 and 24
h. A quadratic effect was observed at 12 h, possibly indicating a slight OD reduction and a weak
inhibitory capacity at this time point. However, when compared at 8 h, it was more susceptible
(MICe of 28 mg/L) at 8 h than R. albus. Streptococcus bovis (Table 4) was the only bacteria that
was not affected by oregano essential oil.
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3.3. CASTOR OIL
Castor oil had marginal effects over gram-negative bacteria (Table 5). Prevotella albensis
growth was slightly reduced at 8 h of incubation, while Prevotella bryantii was not affected by
concentration nor incubation time. The OD of T. saccharophilum was linearly reduced at 8 h, but
this vegetable oil presented a MICe of 660 mg/L for these bacteria.
Castor oil showed a tendency (P < 0.10) to reduce the OD of R. albus at 8 h, but the
bacteria resumed growth after 12 h (Table 6). However, castor oil continued to inhibit growth (P
< 0.05) of R. albus, with a MICe value of 88 mg/L at 12 h. In contrast, castor oil only showed a
tendency to slightly inhibit R. flavefaciens growth at 12 h. Streptococcus bovis growth was not
affected by castor oil concentrations, but a tendency to reduce the OD over time was observed.
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Table 4. Influence of oregano essential oil in the anaerobic gram-positive bacteria growth
Oregano essential oil concentration, mg/L
Time (h)
Repeated measures
8
12
24
SEM
P < Value
Ruminococcus albus
0
0.51C
0.84B
0.98A
0.085
0.052
10
NE
NE
NE
NE
NE
20
0.22C
0.50B
1.38A
0.151
0.003
50
0.23C
0.40B
0.58A
0.045
0.005
100
0.12C
0.36B
1.16A
0.135
0.001
SEM
0.050
0.057
0.079
L
0.009
0.003
0.913
Q
0.016
0.001
0.291
MICe (mg/L)
48
69
52
Ruminococcus flavefaciens
0
1.33
1.41
1.38
0.043
0.762
10
NE
NE
NE
NE
NE
20
0.25C
0.98B
1.52A
0.160
0.003
50
0.30C
1.12B
1.52A
0.155
0.003
100
0.34C
1.23B
1.51A
0.152
0.001
SEM
0.119
0.044
0.036
L
0.018
0.657
0.357
Q
0.001
0.014*
0.375
MICe (mg/L)
28
32
36
Streptococcus bovis
0
1.34A
1.29A
1.25B
0.021
0.019
10
NE
NE
NE
NE
NE
20
1.34
1.35
1.38
0.014
0.584
50
1.31
1.43
1.33
0.031
0.294
100
1.32
1.36
1.33
0.023
0.747
SEM
0.024
0.020
0.016
L
0.746
0.289
0.428
Q
0.909
0.052
0.195
A, B = Different uppercase letters mean difference in the same line at Bonferroni (P < 0.05). L = linear effect; Q =
quadratic effect; MICe = estimated amount of additive concentration (mg/L) necessary to reduce 50% of optical
density, * = equation to calculate MICe does not have real square roots; NE = not evaluated.
Source: Author.
Table 5. Influence of castor oil in the anaerobic gram-negative bacteria growth
Castor oil concentration, mg/L
Time (h)
Repeated measures
8
12
24
SEM
P < Value
Prevotella albensis
0
1.45
1.50
1.48
0.023
0.886
10
1.33B
1.45A
1.46A
0.030
0.032
20
1.24
1.53
1.54
0.050
0.087
50
1.25
1.49
1.30
0.039
0.277
100
1.30B
1.48A
1.45A
0.033
0.030
SEM
0.023
0.020
0.026
L
0.189
0.702
0.308
Q
0.011*
0.875
0.132
Prevotella bryantii
0
1.30B
1.39A
1.32B
0.027
0.038
10
1.46
1.48
1.39
0.020
0.089
20
1.39
1.38
1.37
0.029
0.929
50
1.49
1.52
1.42
0.035
0.458
100
1.45
1.38
1.40
0.038
0.320
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SEM
0.024
0.030
0.019
L
0.113
0.889
0.260
Q
0.071
0.494
0.261
Treponema saccharophilum
0
1.35
1.37
1.37
0.022
0.898
10
1.40
1.47
1.44
0.022
0.536
20
1.27
1.37
1.36
0.021
0.239
50
1.22B
1.43A
1.44A
0.039
0.015
100
1.26
1.41
1.35
0.028
0.117
SEM
0.021
0.018
0.019
L
0.044
0.751
0.604
Q
0.022
0.805
0.461
A, B = Different uppercase letters mean difference in the same line at Bonferroni (P < 0.05). L = linear effect (P <
0.05); Q = Quadratic effect; MICe = estimated amount of additive concentration (mg/L) necessary to reduce 50%
of optical density; * = equation to calculate MICe does not have real square roots.
Source: Author.
Table 6. Influence of castor oil in the anaerobic gram-positive bacteria growth
Castor oil concentration mg/L
Time (h)
Repeated measures
8
12
24
SEM
P < Value
Ruminococcus albus
0
0.51C
0.84B
0.98A
0.085
0.052
10
0.17C
1.07B
1.31A
0158
0.001
20
0.16C
0.29B
0.93A
0.106
0.001
50
0.20B
0.51A
0.42A
0.041
0.006
100
0.16
0.48B
0.99A
0.110
0.001
SEM
0.040
0.069
0.069
L
0.101
0.041
0.289
Q
0.064
0.024
0.005*
MICe (mg/L)
86
88
85
Ruminococcus flavefaciens
0
1.33
1.41
1.38
0.043
0.762
10
1.28
1.46
1.32
0.028
0.061
20
1.28
1.39
1.32
0.020
0.291
50
1.22
1.41
1.35
0.035
0.161
100
1.19B
1.36A
1.35A
0.037
0.038
SEM
0.025
0.025
0.017
L
0.060
0.367
0.942
Q
0.147
0.670
0.826
Streptococcus bovis
0
1.34A
1.29A
1.25B
0.021
0.019
10
1.37
1.29
1.21
0.025
0.164
20
1.37A
1.30A
1.20B
0.026
0.007
50
1.39
1.29
1.22
0.029
0.114
100
1.33
1.33
1.26
0.018
0.165
SEM
0.015
0.014
0.013
L
0.599
0.396
0.316
Q
0.287
0.648
0.332
A, B = Different uppercase letters mean difference in the same line at Bonferroni (P < 0.05). L = linear effect; Q =
quadratic effect; MICe = estimated amount of additive concentration (mg/L) necessary to reduce 50% of optical
density; * = equation to calculate MICe does not have real square roots.
Source: Author.
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Table 7. Influence of Baccharis dracunculifolia hydroethanolic extract in the anaerobic gram-negative bacteria
growth
Baccharis dracunculifolia extract concentration, mg/L
Time (h)
Repeated Measures
8
12
24
SEM
P < Value
Prevotella albensis
0
1.45
1.50
1.48
0.023
0.886
10
1.45
1.52
1.54
0.038
0.176
20
1.46
1.49
1.42
0.029
0.454
50
1.48
1.44
1.49
0.040
0.723
100
1.54
1.65
1.44
0.043
0.057
SEM
0.029
0.032
0.019
L
0.261
0.132
0.441
Q
0.528
0.093
0.750
Prevotella bryantii
0
1.30B
1.39A
1.32B
0.027
0.038
10
1.54
1.53
1.43
0.049
0.433
20
1.55
1.55
1.35
0.048
0.081
50
1.51A
1.51A
1.37B
0.047
0.030
100
1.46
1.46
1.39
0.040
0.509
SEM
0.043
0.028
0.025
L
0.497
0.944
0.634
Q
0.767
0.440
0.889
Treponema saccharophilum
0
1.35
1.37
1.37
0.022
0.898
10
1.49
1.49
1.40
0.034
0.349
20
1.38
1.38
1.37
0.028
0.985
50
1.33
1.33
1.41
0.030
0.175
100
1.40
1.40
1.37
0.018
0.887
SEM
0.021
0.026
0.014
L
0.844
0.681
0.883
Q
0.911
0.710
0.707
A, B = Different uppercase letters mean difference in the same line at Bonferroni (P < 0.05). L = linear effect; Q =
quadratic effect; MICe = estimated amount of additive concentration (mg/L) necessary to reduce 50% of optical
density; * = equation to calculate MICe does not have real square roots.
Source: Author.
3.4. BACCHARIS DRACUNCULIFOLIA EXTRACT
Baccharis dracunculifolia concentration did not inhibit gram-negative and gram-positive
bacterial growth (Table 7 and 8). Surprisingly, when incubated with B. dracunculifolia extract,
R. albus presented a higher OD than control.
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4 DISCUSSION
4.1. GRAM-NEGATIVE BACTERIA
Modulating rumen fermentation using additives for a more favorable volatile fatty acid
ratio, and reduced deamination and methane production, has always been the objective of
ruminant nutritionists (Zawadzki et al., 2011; Aguiar et al., 2013; Valero et al., 2014). There are
two major groups of bacteria found in the rumen that are responsible for the production of
ammonia. The first is present in low numbers but with very high specific activity (hiper-ammonia
producing bacteria or HAP), and the second in high numbers with low specific activity, like
Prevotella sp (Hobson & Stewart, 2012). The HAP are generally gram-positive and represent
less than 1% of the total ruminal microbiota; thus, considering that monensin inhibits around
30% of the ammonia-forming activity in the rumen, it is theorized that other monensin-
insensitive bacteria play a bigger role (Wallace et al., 2002).
Bacteria from the genus Prevotella are some of the most predominant bacteria in the
rumen and play an important role in peptide degradation (Hobson, 1969). These bacteria are also
known for their high activity of the enzyme dipeptidyl peptidase, which breaks dipeptides from
proteins (Wallace & McKain, 1991). Thus, a reduction in the total number of proteolytic bacteria
could lead to an increase in amino acids escaping the rumen, which could benefit the animal
through a higher efficiency of nitrogen utilization (Aguiar et al., 2013).
Surprisingly, in our study, the gram-negative P. bryantii and T. saccharophilum bacteria
were susceptible to the action of monensin in high dosages. The potential of monensin in
modulating rumen fermentation is related to its ability to selectively inhibit gram-positive over
gram-negative bacteria, promoting a shift in the acetate to propionate ratio towards more
propionate (McGuffey et al., 2001; Appuhamy et al., 2013). As monensin had 20% of sodium
monensin the corresponding concentrations used in our study were of 0, 0.5, 1.0, 2.5 and 5 mg/L
of monensin. Newbold et al. (1988) showed that some species of gram-negative bacteria were
more sensitive (Bacteroides succinogenes) to monensin (0.023 mg/L) than others (Bacteroides
ruminicola, 2.702 mg/L). In this sense, the reduced OD observed for monensin dosages greater
than 20 mg/L may be explained by a toxic concentration of monensin (>2.5 mg/L of monensin),
rather than its ability to interact with the outer membrane.
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Oregano essential oil reduced the OD of gram-negative bacteria. Similar results were
observed by Zhou et al. (2019), which evaluated a blend of essential oils (thymol, eugenol,
vanillin, and limonene) against a wide range of rumen bacteria. Cobellis et al. (2016), using the
quantitative PCR technique, observed that oregano essential oil (1.125 mL/L) decreased the total
bacterial count, especially of Prevotella spp and archaea. Generally, gram-positive bacteria are
more sensitive to essential oils than gram-negative bacteria, but small compounds such as
carvacrol and thymol are able to interact with the cell membrane of gram-negative bacteria,
leading to cell content loss and cell lysis (Benchaar & Greathead, 2011). Previous in vitro studies
demonstrated that these compounds affect rumen fermentation. As an example, carvacrol in low
dosages (2.2 mg/L) inhibited proteolysis or stimulated peptide lyses of bacteria (Busquet et al.,
2005). In contrast, in greater dosages (300 mg/L), it increased pH and butyrate, and decreased
acetate, propionate, and total VFA concentration (Busquet et al., 2005). Low thymol dosages (50
mg/L) had no effect on rumen fermentation but, at greater dosages (500 mg/L), it reduced total
VFA (Castillejos et al., 2006).
Table 8. Influence of Baccharis dracunculifolia hydroethanolic extract in the anaerobic gram-positive bacteria
growth
Baccharis dracunculifolia extract
concentration, mg/L
Time (h)
Repeated
measures
8
12
24
SEM
P < Value
Ruminococcus albus
0
0.51C
0.84B
0.98A
0.085
0.052
10
0.73B
1.15A
0.97AB
0.059
0.008
20
0.73B
1.19A
0.92AB
0.059
0.005
50
0.82B
1.14A
1.12AB
0.046
0.007
100
0.90
1.16
1.14
0.039
0.063
SEM
0.040
0.039
0.029
L
0.003
0.118
0.005
Q
0.004*
0.047
0.019
Ruminococcus flavefaciens
0
1.33
1.41
1.38
0.043
0.762
10
1.40
1.36
1.33
0.022
0.590
20
1.40
1.43
1.27
0.033
0.279
50
1.39
1.49
1.35
0.027
0.118
100
1.40
1.40
1.38
0.025
0.070
SEM
0.026
0.021
0.020
L
0.742
0.720
0.495
Q
0.864
0.348
0.497
Streptococcus bovis
0
1.34A
1.29A
1.25B
0.021
0.019
10
1.41A
1.30B
1.19B
0.030
0.035
20
1.36
1.33
1.29
0.026
0.344
50
1.31
1.40
1.34
0.0333
0.572
100
1.38
1.44
1.33
0.018
0.213
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SEM
0.010
0.025
0.021
L
0.927
0.024
0.052
Q
0.291
0.071
0.088
A, B = Different uppercase letters mean difference in the same line at Bonferroni (P < 0.05). L = linear
effect; Q = quadratic effect; MICe = estimated amount of additive concentration (mg/L) necessary to reduce 50%
of optical density * = equation to calculate MICe does not have real square roots.
Source: Author.
The toxic effect of high monensin dosages, or the lower selectivity of oregano essential
oil, is a concern in modulating rumen fermentation, as other bacteria of interest may be
compromised, like T. saccharophilum. There is a large variety of microorganisms capable of
fermenting pectin. However, the growth of T. saccharophilum is often dependent on the
abundance of pectin as a substrate (Liu et al., 2014). Pectin is a structural nonfibrous carbohydrate
that is normally found in plant feedstuffs (Paiva et al., 2009). Pectin is rapidly degraded in the
rumen but, different from starch, acetate is its major product. Thus, favoring the growth of pectin
users’ bacteria could be beneficial for the animal, reducing the risk of acidosis and other
metabolic disorders caused by diets rich in starch (Hatfield & Weimer, 1995).
4.2. GRAM-POSITIVE BACTERIA
Ruminoccocus flavefaciens and Ruminoccocus albus are important gram-positive
cellulolytic bacteria found in the rumen (Hobson & Stewart, 2012; Oyama et al., 2017). This
group of bacteria are capable of hydrolyzing cellulose using the enzyme cellulase (Pell &
Schofield, 1993) and forming a vast range of end products like acetate, butyrate succinate and
formate, CO2, H2 ethanol and lactate (Hungate, 1966). As expected, monensin successfully
inhibited the growth of gram-positive bacteria. Interestingly, R. flavefaciens showed lower
susceptibility to monensin than R. albus. Similar results were observed by Newbold et al. (2013),
in which after a series of stepwise adaptations to monensin, R. flavefaciens presented an increase
in the MICe (0.388 to 0.588 mg/L), while R. albus remained the same (0.064 mg/L).
Ruminal acidosis generally occurs with VFA accumulation when cattle are fed high
concentrate diets. Streptococcus bovis is an important proteolytic and amylolytic bacterium often
associated with this metabolic disorder (Russell & Hino, 1985). In normal conditions, the end
products of S. bovis fermentation are formate, acetate and ethanol. However, when pH is lower
than 5.5 it changes to lactate, which boosts the decrease in pH even more (Russell & Hino, 1985).
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Thus, controlling S. bovis growth has been of interest to research due to its role in ruminal
acidosis (Fernando et al., 2010; Belanche et al., 2012). Monensin inhibited S. bovis, especially
after 24 h of incubation. However, our lowest concentration (10 mg/L) was higher (<1.0 mg/L)
than those observed by other authors (Newbold et al., 1988; Newbold, 1995; Newbold et al.,
2013).
Oregano essential oil affected cellulolytic bacteria (R. albus and R. flavefasciens). This
was expected, since oregano essential oil is rich in carvacrol and thymol, which are monoterpenes
with strong antimicrobial activity against a wide range of gram-positive bacteria (Calsamiglia et
al., 2007). However, Cobellis et al. (2016) only observed changes in the relative abundance of R.
albus when oregano essential oil was mixed with cinnamon and rosemary essential oil, which is
explained by their synergistic effect. More recently, Castañeda-Correa et al. (2019) observed that
thymol was more efficient than carvacrol in reducing methane production, possibly due to an
indirect effect of EO on microorganisms that produce substrates such as hydrogen or formate,
which are used by methanogenic bacteria. S. bovis growth was not affected by oregano essential
oil, which corroborates the findings of Evans & Martin (2000), who tested different thymol
concentrations, in which only those > 100 mg/L reduced the OD.
Castor oil (Ricinus communis) is one of the most important crops in developing countries
because of its potential to be used in the biodiesel industry. It can be obtained by pressing the
castor seeds, which are rich in ricinoleic acid (Vaisman et al., 2008). Past studies showed its
antimicrobial potential against a wide range of anaerobic bacteria (Novak et al., 1961). Because
of that, castor oil has been theorized in modulating rumen fermentation and improving animal
performance. In fact, some in vivo trials reported improvements on animal performance of young
bulls fed with ricinoleic acid (2 g/animal day) (Gandra et al., 2012) or a mix of cashew and castor
oil (3 g/animal day) (Valero et al., 2014).
In our study, high castor oil concentrations affected R. albus growth, which is an
important bacterium responsible for fiber degradation (Latham & Wolin, 1977). It is well known
that the inclusion of plant oils in diets can reduce DMI and NDF digestibility (Vargas et al.,
2020). More recently, Ibrahim et al. (2021) reported that the inclusion of oils (palm, olive and
sunflower) in the diet of goats at 6% of DM basis significantly reduced the population of R. albus,
but no differences were observed in other cellulolytic bacteria like Fibrobacter succinogenes and
R. flavefaciens. Baccharis dracunculifolia, popularly known as "Alecrim-do-campo" (field
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rosemary), is the main raw material used by bees (A. mellifera) to produce green propolis, whose
benefits to human health have been widely studied (Maróstica Junior et al., 2008). More recently,
studies revealed that propolis had antimicrobial activity against ruminal gram-positive and gram-
negative bacteria (Aguiar et al., 2013), and that feeding propolis (3 g/animal day) improved the
performance of young bulls (Valero et al., 2014). The antimicrobial properties of propolis could
be explain by its composition, which is 50% resin (where flavonoids and phenolic acids are
found), 30% wax, 10% essential oils, 5% pollen and 5% other organic matter (Gómez-Caravaca
et al., 2006). To the best of our knowledge, no studies with ruminal microorganisms have been
conducted with B. dracunculifolia yet. However, due to its similar chromatography profile to
propolis (Maróstica Junior et al., 2008), it has been theorized that this plant could have the same
benefits as propolis, with the advantage of reduced costs. Our research group evaluated the
addition of B. dracunculifolia leaves in the diet (5, 10 and 15 mg/day) of Nellore steers, but no
differences on blood parameters, final body weight, average daily gain, dry matter intake or feed
efficiency (Souza et al., 2020; Carvalho et al., 2021).
Baccharis dracunculifolia did not affect gram-negative and gram-positive bacterial
growth. In fact, it increased the OD in some cases (R. albus). This bias may be caused by the
lower growth rate of these bacteria. On the other hand, when tested against aerobic
microorganisms, B. dracunculifolia essential oil (up to 10 μL/ disc) presented antimicrobial
activity against gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-
positive (Staphylococcus aureus) (Ferronatto et al., 2007) bacteria. More recently, Bonin et al.
(2020) evaluated the antimicrobial activity of B. dracunculifolia extracts and observed better
antimicrobial action against gram-positive bacteria, in which the MIC was 125 mg/L for both the
bacteria Staphylococcus aureus and Bacillus subtilis, and 250 mg/L for Bacillus cereus. The
same plant extracts used by Bonin et al. (2020) were used in our study. However, the final
concentrations tested were lower than by Bonin et al. (2020), which could explain this marginal
response. Furthermore, different methods of extraction (hydroethanolic extract vs. essential oil)
may affect composition and, consequently, the concentration necessary to inhibit the growth of
ruminal microorganisms.
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5 CONCLUSIONS
As expected, monensin showed great antimicrobial capacity against gram-positive
bacteria. However, high doses (≥ 20 mg/L) were also toxic for gram-negative bacteria. Oregano
essential oil showed great antimicrobial activity against both gram-positive and gram-negative
bacteria, meaning that, despite of its antimicrobial potential, its inability to select gram-positive
over gram-negative organisms limits its usage in modulating rumen fermentation. Castor oil
showed more selectivity against gram-positive bacteria; however, it reduced the growth of R.
albus, which is an important fiber degrader, rather than S. bovis, which is a bacterium often
associated with ruminal acidosis. B. dracunculifolia extract concentrations (up to 100mg/L) used
in this trial were ineffective against all rumen bacteria tested. Future studies with higher
concentrations and different extraction methods may be necessary to fully understand the
potential of B. dracunculifolia.
5.1 FUTURE IMPLICATIONS
This was the first study conducted in our research group with ruminal anaerobic
microorganisms. The technique was satisfactory to antimicrobial capacity of natural additives
against rumen bacteria of research interest, especially those of proteolytic, amylolytic,
cellulolytic and pectinolytic activity. Other microorganisms of interest that were not evaluated in
this study are the hyper ammonia-producing bacteria and archaea methanogens, which could be
addressed in future studies. In addition, complimentary techniques like batch culture, in vitro
rumen fermentation, or RUSITEC could also be used to further support the findings.
South America is rich in biodiversity, and castor oil and B. dracunculifolia are only two
additives from a vast array of plants with potential to be explored. Furthermore, natural additives
are dependent on the parts of the plants (root, stem, leaf and flower), season of harvest (summer,
autumn, winter or spring) and extraction methods (ethanolic, methanolic etc.). This highlights
the importance and also the potential of future studies. Thus, this technique could be applied as
the first step in screening potential additives, targeting a more accurate range of concentrations
with a greater spectrum of action, and reducing cost and time spent on future animal trials.
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ACKNOWLEDGEMENTS
This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) via a scholarship, the Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, 400375/2014-1) and the company Safeeds Nutrição Animal (safeeds@safeeds.com.br).
The authors gratefully acknowledge the company for financing and providing the products used
in this research, which made it possible to develop this work. The mention of trade names or
commercial products in this publication is solely for the purpose of providing specific
information and does not imply recommendations or endorsement by the Department of Animal
Science of the State University of Maringá, Paraná, Brazil.
AUTHOR CONTRIBUTION
Conceptual idea: Prado, I. N.; Ornahi, M G. Methodology design: Passetti, R. C. A.; Prado, R.
M. Data collection: Passetti, R. A. C.; Prado, I. N.; Prado, R. M. Carvalho, V. M. Data analysis
and interpretation: Passetti, R. A. C.; Prado, I. N.; Ferracini, J. G. Writing and editing: Passetti,
R. A. C.; Ferreacini, J. G.; Mantovani, H. C.; Prado, I. N.
FUNDING
This study was partially funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brasil (CAPES) and the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), the company Safeeds (safeeds@safeeds.com.br) financed and provided the
supplies used in this research, which made it possible to develop this work.
ETHICS COMMITTEE
All experimental procedures were conducted under the surveillance of the Animal Care and Use
Committee of the State University of Maringá, Paraná, Brazil (protocol no. 1103290719), and
met the requirements of the National Council for the Control of Animal Experimentation
(CONCEA).
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INFORMED CONSENT
All authors have given their consent that this work is valid and represent their views on this study,
and all authors have given their consent for this work to be published.
CONFLICTS OF INTEREST
The authors declare no conflict of interest. The authors declare that they have no competing
interests. The trade names or commercial products in this publication are mentioned solely for
the purpose of providing specific information and do not imply recommendations or endorsement
by the Department of Animal Science, State University of Maringá, Paraná, Brazil.
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