Journal Pone 0270350
Journal Pone 0270350
Journal Pone 0270350
RESEARCH ARTICLE
Funding: The author(s) received no specific virginiamycin. Furthermore, CNSL–castor oil acted as a modulator of intestinal microbiota,
funding for this work. reducing the abundance of pathogenic bacteria.
Competing interests: The authors have declared
that no competing interests exist.
Introduction
Coccidiosis is an infection caused by Eimeria spp. that seriously affects poultry farming world-
wide. Recent studies have estimated a worldwide loss of approximately £10.36 billion per year
owing to prophylaxis and production losses in chickens due to coccidiosis [1]. The parasite
affects the intestinal epithelium and immune response of chickens, reduces nutrient digestion,
and consequently has a negative impact on the performance indices of broilers [2]. Addition-
ally, coccidiosis disturbs the diversity and composition of the intestinal microbiota. For exam-
ple, the increased leakage of plasma proteins into the lumen induced by the parasite provides
substrate for the proliferation of Clostridium perfringens [3]. Although this bacterium is part of
the normal microbiota of broiler cecum, when there is a significant increase in its proliferation
in the small intestine, it can cause necrotic enteritis [2, 3].
Over the years, antibiotic growth promoters (AGP) have been used to control pathogens
and preserve intestinal integrity and enhance production indicators for broilers. The perfor-
mance improvement because of AGP is associated with the modification of the intestinal
microbiota. AGP promotes a balance in the microbial population, as it reduces the number of
toxin-producing microorganisms in the intestinal lumen [4], in addition to acting as an anti-
bacterial and direct anti-inflammatory agent [5].
However, some countries have banned the use of AGPs because of the risks to human
health caused by residues in animal products, as well as the possibility of inducing bacterial
resistance [6]. In addition, a meta-analysis showed that the effectiveness of antibiotics as
growth promoters was less evident in recently published studies than that in the 1980s [7].
Thus, the production response to AGP usage could be reduced with good production condi-
tions, such as “hygienic facilities and balanced nutrition [7].
The initiative to reduce the use of antibiotics as growth promoters has stimulated research
into alternative methods to simultaneously minimize the impact of parasites and act as growth
promoters. The balance of intestinal microbiota is crucial for good animal performance, espe-
cially when facing sanitary challenges. In addition, Moraes et al. [8] and Vieira et al. [9] dem-
onstrated that when receiving a commercial product consisting of a mixture of cashew
nutshell liquid and castor oil (CNSL–castor oil), chickens challenged by coccidiosis presented
a better balance in their microbiota, reducing pathogenic bacteria such as Clostridium perfrin-
gens and improving animal performance.
This study aimed to compare the effects of CNSL–castor oil with multiple growth-promot-
ing antibiotics used as anticoccidials in broiler chickens challenged by coccidiosis.
Statistical analysis
Statistical analyses were performed using SAS statistical software (version 9.0; SAS Inst. Inc.,
Cary, NC, United States). The data were subjected to analysis of variance using PROC GLM,
with each box considered as an experimental unit. Differences (p < 0.05) were assessed using
Tukey’s multiple comparison test. Regression analysis was performed to check the effect of the
CNSL–castor oil inclusion level. Injury scores were evaluated using the Student–Newman–
Keuls (SNK) method. The microbiota indices were assessed using the Kruskal–Wallis test, and
each treatment was compared pairwise using the Wilcoxon test corrected by the false discovery
rate. Beta diversity distances were compared using the Adonis test.
Results
Performance data and lesion score
The performance responses, separated into periods including days 1–21, 21–28, 28–42, and
1–42, are listed in Table 1. At 21 d, the broilers that received tylosin presented a statistically sig-
nificant increased weight gain compared with control animals (p < 0.05), and consequently,
tylosin-treated birds had higher live weight and better feed conversion ratio (p < 0.05). The
control group showed the poorest feed conversion (P < 0.05).
After 14 d of challenge by Eimeria spp., at 28 d of age, the animals that received tylosin
had higher feed intake than broilers treated with enramycin (p < 0.05). The other treat-
ments showed no significant differences. Animals treated with the antibiotic tylosin
showed more significant weight gain than those treated with enramycin and the control.
The other treatments exhibited intermediate behaviors. The live weight at 28 d was lower
for the birds in the control, CNSL–Castor oil 0.5 kg/t, enramycin, and virginiamycin
groups compared with those in the tylosin group; and CNSL–Castor oil 0.75 and 1 kg/t
groups behaved as intermediate treatments (p < 0.05). Feed conversion was higher in con-
trol birds than that in other treatment groups (p < 0.05), except for CNSL–Castor oil
(0.5 kg/t). In the last phase, from 28 to 42 d, the broilers showed greater weight gain when
receiving tylosin and enramycin than the control broilers, CNSL–Castor oil 0.5 kg/t, or
CNSL–Castor oil 0.75 kg/t groups (P < 0.05); animals that received other treatments
showed intermediate effects.
Assessing the complete period of 1–42 d, the treatment with tylosin provided a greater
weight gain than the control or any CNSL–Castor oil treatments. Treatment with
Table 1. Live weight (LW), body weight gain (WG), feed intake (FI), and feed conversion ratio (FCR) of challenged broilers from 1 to 42 d of age.
Item Additives SEM2 p-value
1
Control CNSL-CO 0.5 kg/t CNSL-CO 0.75 kg/t CNSL-CO 1.0 kg/t Enramycin Virginiamycin Tylosin
LW (1D), g 0.037 0.037 0.037 0.037 0.037 0.037 0.037 0.00007 0.476
Phase 1 to 21 days
LW, g 773 a 757 ab 749 ab 745 ab 740 ab 739 ab 778 b 0.00369 0.036
b ab ab ab ab ab
BWG, g 33.41 34.3 33.91 33.72 33.51 33.45 35.28 a 0.00018 0.035
FI, g 45.75 46.35 44.94 45.27 45.14 44.44 46.00 0.00025 0.420
FCR, g/g 1.370 a 1.352 ab 1.327 ab 1.343 ab 1.348 ab 1.329 ab 1.303 b 0.00586 0.000
Phase 21 to 28 days
LW, g 1242 b 1289 b 1295 ab 1292 ab 1260 b 1283 b 1355 a 0.00610 0.000
b ab ab ab b ab
BWG, g 72.28 75.98 77.95 78.14 74.25 77.70 82.49 a 0.00070 0.003
FI, g 121.96 ab 121.30 ab 122.05 ab 122.02 ab 117.33 b 120.77 ab 125.78 a 0.00061 0.021
a ab b b b b
FCR, g/g 1.696 1.601 1.572 b 1.567 1.585 1.559 1.527 0.01050 0.000
Phase 28 to 42 days
LW, g 2548 c 2620 bc 2632 bc 2640 bc 2672 ab 2656 abc 2767 a 0.01210 0.000
b b b ab a ab
BWG, g 93.29 95.13 95.50 96.30 100.84 98.08 100.88 a 0.00075 0.037
FI, g 179.79 177.87 179.13 178.18 177.31 177.89 176.54 0.00077 0.951
FCR, g/g 1.932 a 1.872 ab 1.879 ab 1.861 ab 1.759 b 1.816 ab 1.760 b 0.01220 0.000
Phase 1 to 42 days
BWG, g 59.78 c 61.52 bc 61.72 bc 61.98 bc 62.75 ab 62.37 abc 65.02 a 0.00029 0.000
FI, g 103.13 102.68 102.52 102.65 101.23 101.65 102.8 0.00036 0.817
FCR, g/g 1.726 a 1.669 ab 1.660 b 1.654 b 1.613 bc 1.630 bc 1.583 c 0.00697 0.000
Means with different letters differ statically by Tukey, on the row within the same variable. Data are expressed as means of the information collected in 360 broilers per
treatment.
1
CNSL-CO: CNSL-castor oil. Essential (US Patent N˚. 8377,485; Oligo Basics Ind. Ltda., Cascavel, Paraná, Brazil).
2
SEM: standard error of the mean.
https://doi.org/10.1371/journal.pone.0270350.t001
enramycin and virginiamycin did not differ from other treatments (p > 0.05). Conse-
quently, the pattern of live weight at 42 d was identical to that of weight gain. Feed intake
did not differ between the treatments in the last phase or in the total period (p > 0.05). Tylo-
sin showed comparatively better feed conversion, followed by CNSL–Castor oil levels of
0.75 and 1.0 kg/t. However, the CNSL–Castor oil 0.75 and 1.0 kg/t treatments did not signif-
icantly differ from treatments with enramycin and virginiamycin. In turn, neither antibiotic
treatment differed from that with tylosin. As expected, the control treatment resulted in the
lowest feed conversion.
In the first week after infection, broilers subjected to the control treatment had a higher
lesion score for E. acervulina and E. tenella compared with other treatments (p < 0.05). At 28
d, there was no statistical difference in the injury score among the treatments (S1 Fig).
Fig 1. Alpha diversity of broilers cecum samples at 28 and 42 d of trial. The study tested seven feed additives: enramycin (8 ppm), virginiamycin
(16.5 ppm), tylosin (55 ppm), CNSL–castor oil (CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the control diet (without additives).
Comparison over time (28 and 42 d) using Chao-1, Shannon, and Simpson indices.
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Comparing the alpha diversity-calculated Shannon index for each treatment at different times,
we observed that for Control, CNSL–castor oil 0.5 kg/t, CNSL–castor oil 0.75 kg/t, and tylosin
treatments, there was an increase in diversity from 28 to 42 d. The opposite was observed for
virginiamycin, whereof the diversity at 28 d was greater than that at 42 d. No differences were
observed with the additives enramycin and CNSL–castor oil 1.00 kg/t (Fig 2). The same trend
was observed for indices (S2A and S2B Fig).
Comparing the different additives at each time point, we observed statistically significant
differences between them (Kruskal–Wallis, P < 0.05). At 28 d, there were no significant differ-
ences compared with the control (Fig 3). At 42 d, the control differed from treatments with vir-
giniamycin, CNSL–castor oil 1.00 kg/t, and enramycin (S3A and S3B Fig).
Bray–Curtis dissimilarity values were calculated to measure individual differences in taxo-
nomic structure. To visualize the differences between the microbiome profiles, we performed
PCoA for the Bray–Curtis dissimilarity matrix, which did not reveal a clear grouping pattern.
All samples were scattered with overlapping ellipses at 28 and 42 d (Fig 4A). At day 42, the dif-
ferences observed were statistically significant (Adonis, p < 0.05) (Table 2). Fig 4A shows the
distribution of paired beta diversity values between treatments in each period studied; the dis-
tributions were not similar. Comparing each treatment at different times, we found that
CNSL–castor oil 0.5 kg/t, enramycin, virginiamicin, and tylosin treatments differed statistically
from 28 to 42 d (Adonis, p < 0.05) (Fig 4B and Table 2).
Fig 2. Comparison of alpha diversity for each additive between time points using Shannon index of broilers cecum samples at 28 and 42 d of trial. The study tested
seven feed additives: enramycin (8 ppm), virginiamycin (16.5 ppm), tylosin (55 ppm), CNSL-castor oil (CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the
control diet (without additives).
https://doi.org/10.1371/journal.pone.0270350.g002
Fig 3. Comparison of alpha diversity at each time point using Shannon index of broilers cecum samples at 28 and 42 d of trial. The study tested seven feed additives:
enramycin (8 ppm), virginiamycin (16.5 ppm), tylosin (55 ppm), CNSL-castor oil (CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the control diet (without
additives). Probabilities and means with different letters differ statistically by Tukey: 0 ‘��� ’ 0.001 ‘�� ’ 0.01 ‘� ’ 0.05.
https://doi.org/10.1371/journal.pone.0270350.g003
Actinobacteria, and Proteobacteria. Firmicutes was the most abundant phylum in all treat-
ments (> 84%) (Fig 5A). A complete list of the identified sequences (relative abundances) per
treatment is provided in S3 Table. Of the 47 families identified, 19 had a relative
Fig 4. Beta diversity based on Bray–Curtis dissimilarity of broilers cecum samples at 28 and 42 d of trial. The study tested seven feed additives: enramycin (8 ppm),
virginiamycin (16.5 ppm), tylosin (55 ppm), CNSL-castor oil (CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the control diet (without additives). (A)
Comparison between all treatments at each time point, with (top) the principal coordinate analysis (PCoA) plot and (bottom) the differences in within-group Bray–Curtis
distances. (B) Comparison for each additive between time points.
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abundance > 1% (S3 Table and Fig 5B). The phylum Firmicutes was the most abundant,
regardless of treatment and age, with values between 84.33% and 95.16%. Ruminococcaceae,
Lachnospiraceae, and Lactobacillaceae families were predominant in all groups (>15%),
mainly at 42 d.
Discussion
In this study, we compared the effects of increasing CNSL–castor oil levels and growth-pro-
moting antibiotics tylosin, enramycin, and virginiamycin associated with the anticoccidials
semduramicin + nicarbazin and sodium monensin in the initial phase (0 to 28 d) broilers chal-
lenge with coccidiosis. At 21 d of age, the animals that received the antibiotic tylosin showed
comparatively better performance. However, at 42 d, there was no difference in performance
among the antibiotic treatments. The use of CNSL–castor oil 0.75 and 1 kg/t treatments did
not differ from treatments with enramycin and virginiamycin, although these CNSL–castor oil
groups did present better feed conversion.”
Antibiotics and anticoccidials (chemicals and ionophores) are widely used together, alone,
or in combination in coccidiosis prevention programs. When clinical signs become apparent,
it is too late to prevent pathological consequences and decreased performance caused by para-
site infection [17]. Tylosin is an antibiotic with beneficial effects on broiler performance. Hung
Table 2. Adonis test result of the beta diversity based on Bray–Curtis dissimilarity of broilers cecum samples at
28 and 42 d of trial with seven feed additives: Enramycin (8 ppm), virginiamycin (16.5 ppm), tylosin (55 ppm),
CNSL-castor oil (CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the control diet (without additives).
Variable F.model R2 p-value
Additive + Time
Additive 1.1093 0.0400 0.002
Time 2.6391 0.0159 0.001
Time
Time 28D 1.0361 0.0775 0.155
Time 42D 1.0947 0.0786 0.001
Additive/Time
Control 1.098 0.0475 0.065
CNSL-CO_0.5 kg/t 1.4735 0.0628 0.001
CNSL-CO_0.75 kg/t 1.0672 0.0463 0.164
CNSL-CO_1.0 kg/t 1.1944 0.0538 0.057
Enramycin 1.3004 0.0558 0.002
Virginiamycin 1.3845 0.0647 0.001
Tylosin 1.2325 0.0531 0.002
Time 42/Control
CNSL-CO_0.5 kg/t 1.0386 0.0451 0.248
CNSL-CO_0.75 kg/t 1.0012 0.0435 0.432
CNSL-CO_1.0 kg/t 1.0774 0.0467 0.182
Enramycin 1.0789 0.0468 0.079
Virginiamycin 1.1194 0.0484 0.045
Tylosin 1.0959 0.0475 0.080
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Fig 5. Relative abundance of broilers cecum samples at 28 and 42 d of trial. The study tested seven feed additives: enramycin (8 ppm),
virginiamycin (16.5 ppm), tylosin (55 ppm), CNSL–castor oil (CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the control diet
(without additives). Relative abundances are presented in percentage (%) of taxa at (A) Phylum and Class, and (B) Family levels.
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et al. [18] observed that the use of 55 mg/kg tylosin provided beneficial effects on digestion
and intestinal integrity, increasing disaccharidase activity and maintaining intestinal perme-
ability, consequently increasing performance.
Although the advantages of using antibiotics as growth promoters have been extensively
studied and reviewed, consumer pressure to reduce the use of antibiotics and AGP for animal
production is high, as these treatments have been linked to the rise in antibiotic-resistant bac-
teria for humans [19]. Cardinal et al. [20] concluded that AGP withdrawal has a negative
impact, mainly in the initial stages of broiler performance. Corroborating the results of this
study, animals that received tylosin showed better performance in the first phase and remained
until the end of the experiment. Brazil banned the use of tylosin and other antibiotics, such as
tiamulin and lincomycin, in 2020 [21], indicating the importance of studies comparing alter-
native substances.
However, ionophores are considered antibiotics of non-medical importance for human
health [21], and in animal production, they are the primary choice to control coccidiosis. One
important factor to consider is that they do not entirely suppress parasite development, thus
allowing the development of host immunity after initial exposure [17, 22]. Parent et al. [23]
observed that programs that used antibiotics presented similar performance to those that
exclusively used ionophores. However, cases of parasite resistance to ionophores have been
reported owing to the use of these substances for an extended period [22].
Studies with different phytogenics have shown promising results for broiler performance
when the birds are challenged by coccidiosis [24–26]. Moraes et al. [8] compared the effect of
CNSL–Castor oil with that of monensin in broilers challenged with coccidiosis and found sim-
ilar results between the functional oil and the ionophore. The mechanisms of action of CNSL–
castor oil have not been fully elucidated. However, in the face of the challenge caused by coc-
cidiosis, CNSL–Castor oil acts as an immunomodulator [27] and modulator of the intestinal
microbiota [28]. CNSL–Castor oil helps maintain the animal’s immune systems, eliminate par-
asites, maintain intestinal balance, and improve animal performance [8, 28, 29].
This study observed a higher lesion score for E. acervulina and E. tenella in the control
group compared with that in other treatments, demonstrating the beneficial action of CNSL–
Castor oil during coccidiosis challenge (21 d), similar to the effect of antibiotics. However, the
same pattern was not observed at 28 d, where there was no difference in the intestinal lesion
score regardless of treatment. Alternatives to antibiotics can become ineffective in an environ-
ment with compromised biosafety conditions and management errors, which can be a limiting
factor for the exclusive use of alternatives to antibiotics [30, 31]. In the field, combining alter-
native products with anticoccidials is standard practice. In this study, the use of anticoccidials
in the initial phase, followed by CNSL–Castor oil, resulted in a similar performance to that of
antibiotics. This study used lower levels of CNSL–Castor oil (0.75 and 1.0 kg/t) than previous
studies that have demonstrated beneficial effects using a dose of 1.5 kg/t.
Invasion by coccidian parasites into intestinal cells disturbs microbiota homeostasis in the
healthy intestine [32]. Changes in the intestinal environment caused by these parasites include
changes in nutrient absorption and digestibility, increased mucogenesis, membrane perme-
ability, nutrient availability, and the proliferation of pathogenic bacteria [33].
The microbial richness and diversity of the gut are closely related to the health of broilers
[34]. Coccidiosis infection alters the intestinal microbiota profile, reducing the diversity of the
intestinal environment, especially in the cecum, which has the greatest biodiversity [9, 35].
However, microbiota modulators can reduce this impact, making the animals resilient during
coccidiosis [9, 31, 36, 37]. In this study, when animals were evaluated at 28 and 42 d of age, it
was observed that even after treatment with antibiotics, there was an increase in microbial
diversity, except in groups treated with enramycin or 1 kg/t CNSL–castor oil. In a separate
study, Vieira et al. [9] demonstrated that the use of 1.5 kg/t CNSL–castor oil maintained simi-
lar diversity at 7 and 14 d after the coccidiosis challenge, in contrast to the use of 100 ppm of
sodium monensin.
As in other studies, Firmicutes was the most abundant phylum in the cecum, but its abun-
dance decreased with the age of the chickens [38]. Firmicutes, as it comprises a large propor-
tion of commensal bacteria, is associated with the efficiency of energy capture, and
consequently, with better product performance [39]. In this study, at 28 d, except for virginia-
mycin, all treatments showed a greater abundance of this phylum than the control. Corrobo-
rating the results of this study, Vieira et al. [9], working with CNSL–castor oil alone, also
observed an increase in Firmicutes in broilers challenged with coccidiosis, which was not
observed when only monensin was added. Using this same product, an increase in Firmicutes
was observed in newly weaned piglets; this a phase where there is a disturbance in the balance
of the intestinal microbiota [9, 40].
The phylum Actinobacteria also increased in all treatments compared with that in the con-
trol. Bacteria representing this phylum are aerobic and represent a small percentage of the
intestinal microbiota. However, Actinobacteria can maintain intestinal homeostasis and are
negatively correlated with Proteobacteria, including pathogenic species such as Salmonella and
Escherichia. Proteobacteria have been associated with poor performance in broilers [41]. This
fact was also observed in this study, as there was a reduction in Proteobacteria at 28 d—that is,
14 d after the challenge—compared with that in the control treatment. Previous studies have
demonstrated that CNSL–castor oil reduces the abundance of Proteobacteria at times of stress
in different animals, whether under coccidiosis in chickens [9] or at weaning in pigs [28]. Con-
versely, [42] it has been observed that monensin supplementation produced a greater relative
abundance of gram-negative bacteria of the phylum Proteobacteria and class Clostridia. Dan-
zeisen et al. [43] observed that the combination of monensin with virginiamycin or tylosin
increased the presence of this phylum, especially E. coli.
Among these families, two considered pathogenic are important to highlight. Streptococca-
ceae, facultatively anaerobic bacteria, produce lactic acid and are characterized as an indication
of dysbiosis after infection by Eimeria tenella [44]. All treatments in the current study reduced
the abundance of this family compared with the levels seen in the control. The Enterobacteria-
ceae family, also facultative anaerobes and lactic acid producers, presented lower abundance in
the CNSL–Castor oil, tylosin, and virginiamycin treatments relative to that in the control. In
this family, the main pathogenic bacteria are related to the most significant moments of stress
in broiler chickens [9, 45].
The families Erysipelotrichaceae, Lachnospiraceae, Rikenellaceae, and Ruminococcaceae
are anaerobic bacteria associated with the fermentation of structural carbohydrates and the
production of short-chain fatty acids [44, 46]. The families Lachnospiraceae and Ruminococ-
caceae showed greater abundance in treatments with tylosin and enramycin when compared
with the control. All CNSL–Castor treatments decreased the abundance of the Ruminococca-
ceae, Lachnospiraceae, and Rikenellaceae families at 28 d compared with those in the control.
Previous studies have shown that different AGPs can enrich the cecum with butyrate-pro-
ducing bacteria [47, 48]. Robinson et al. [49] observed that tylosin and enramycin increased
the abundance of Ruminococcacea, whereas salinomycin and monensin reduced the abun-
dance of this family. In this study, after the removal of the ionophores, at 42 d, there was an
Conclusions
Tylosin showed better performance indices in chickens challenged with coccidiosis. The
CNSL–castor oil levels of 0.75 and 1 kg/t were more effective than a level of 0.50 kg/t, present-
ing similar results to enramycin and virginiamycin. Furthermore, CNSL–castor oil acted as a
modulator of intestinal microbiota, reducing the abundance of pathogenic bacteria.
Supporting information
S1 Fig. Lesion score in coccidiosis challenged broilers at 21 (A) and 28 (B) days of age—7 and
14 d after challenge, respectively. CNSL–Castor (US Patent N. 8377,485; Oligo Basics Ind.
Ltda., Cascavel, Parana, Brazil). Means with different letters differ statically as per the Student–
Newman–Keuls method.
(PNG)
S2 Fig. A, B. Comparison of alpha diversity for each additive between time points using
CHAO -1 (A) and Simpson (B) index of broilers cecum samples at 28 and 42 d of trial. The
study tested seven feed additives: enramycin (8 ppm), virginiamycin (16.5 ppm), tylosin
(55 ppm), CNSL–castor oil (CNSL-CO) in different doses (0.5, 0.75, and 1.00 kg/t), and the
control diet (without additives).
(PNG)
S3 Fig. A, B. Comparison of alpha diversity at each time point using CHAO -1 (A) and Simp-
son (B) index of broilers cecum samples at 28 and 42 d of trial The study tested seven feed
additives: enramycin (8 ppm), virginiamycin (16.5 ppm), tylosin (55 ppm), CNSL–castor oil
(CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the control diet (without
additives).
(PNG)
S1 Table. Ingredient formulae and chemical composition of experimental diets according
to the rearing period.
(DOCX)
S2 Table. Number of reads that passed through each step of quality control for the experi-
ment testing seven feed additives: Enramycin (8 ppm), virginiamycin (16.5 ppm), tylosin
(55 ppm), CNSL–castor oil (CNSL-CO) at different doses (0.5, 0.75, and 1.00 kg/t), and the
control diet (without additives) of broilers cecum samples at 28 and 42 d of trial with 12
replicates.
(XLSX)
S3 Table. Relative abundance at phylum, family, and genera levels present in the experi-
ment. The experiment tested seven feed additives: enramycin (8 ppm), virginiamycin
(16.5 ppm), tylosin (55 ppm), CNSL–castor oil (CNSL-CO) at different doses (0.5, 0.75, and
1.00 kg/t), and the control diet (without additives) of broilers cecum samples at 28 and 42 d of
trial with 12 replicates.
(XLSX)
Author Contributions
Conceptualization: Lucélia Hauptli, Douglas Haese, Carolina D’ávila Pozzatti, Priscila de Oli-
veira Moraes.
Formal analysis: Tatiany Aparecida Teixeira Soratto, Vilmar Benetti Filho, Carolina D’ávila
Pozzatti, Priscila de Oliveira Moraes.
Investigation: Pedro Torres, Tatiany Aparecida Teixeira Soratto, Douglas Haese, Carolina
D’ávila Pozzatti.
Methodology: Paula Gabriela da Silva Pires, Tatiany Aparecida Teixeira Soratto, Vilmar Bene-
tti Filho, Lucélia Hauptli, Glauber Wagner, Douglas Haese, Carolina D’ávila Pozzatti.
Project administration: Douglas Haese, Carolina D’ávila Pozzatti.
Writing – original draft: Paula Gabriela da Silva Pires, Pedro Torres, Tatiany Aparecida Teix-
eira Soratto, Vilmar Benetti Filho, Lucélia Hauptli, Priscila de Oliveira Moraes.
Writing – review & editing: Glauber Wagner, Priscila de Oliveira Moraes.
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