Journal of Animal Science, 2022, 100, 1–10

https://doi.org/10.1093/jas/skac289
Advance access publication 3 September 2022
Ruminant Nutrition

Effects of feeding different probiotic types on metabolic,

performance, and carcass responses of Bos indicus feedlot
cattle offered a high-concentrate diet

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Bruno G. C. Dias,† Flávio A. P. Santos,†,1, Murillo Meschiatti,† Bárbara M. Brixner,†
Alecsander A. Almeida,† Oscar Queiroz,‡ and Bruno I. Cappellozza‡,
†
Departamento de Zootecnia, Universidade de São Paulo, Piracicaba, SP 13418-900, Brazil
‡
Chr. Hansen A/S, Hørsholm 2970, Denmark
Corresponding author: [email protected]
1

Abstract
Two experiments were designed to evaluate the effects of different probiotic combinations on rumen fermentation characteristics, performance,
and carcass characteristics of feedlot Bos indicus beef bulls offered a high-concentrate diet. In experiment 1, 30 rumen-fistulated Nellore steers
were blocked by initial body weight (BW = 350 ± 35.0 kg) and within blocks (n = 10), animals were randomly assigned to receive: 1) high-con-
centrate diet without probiotic supplementation (n = 10; CONT), 2) CONT plus 1 g per head of a probiotic mixture containing three strains of
Enterococcus faecium and one strain of Saccharomyces cerevisiae (3.5 × 109 CFU/g; n = 10; EFSC), and 3) CONT plus 2 g per head of a probiotic
mixture containing Bacillus licheniformis and Bacillus subtilis (3.2 × 109 CFU/g; n = 10; BLBS). The experimental period lasted 35 d, being 28
d of adaptation and 7 d of sampling. From day 34 to day 35 of the experimental period, ruminal fluid and fecal samples were collected every
3 h, starting immediately before feeding (0 h) for rumen fermentation characteristics and apparent nutrient digestibility analysis, respectively.
In experiment 2, 240 Nellore bulls were ranked by initial shrunk BW (375 ± 35.1 kg), assigned to pens (n = 4 bulls per pen), and pens randomly
assigned to receive the same treatments as in experiment 1 (n = 20 pens per treatment). Regardless of treatment, all bulls received the same
step-up and finishing diets throughout the experimental period, which lasted 115 d. In both experiments, data were analyzed as orthogonal
contrasts to partition-specific treatment effects: 1) probiotic effect: CONT vs. PROB and 2) probiotic type: EFSC vs. BLBS (SAS Software Inc.).
In experiment 1, no contrast effects were observed on nutrient intake, overall nutrient digestibility, and rumen fermentation analyses (P ≥ 0.13).
Nonetheless, supplementation of probiotics, regardless of type (P = 0.59), reduced mean acetate:propionate ratio and rumen ammonia-N con-
centration vs. CONT (P ≤ 0.05). In experiment 2, no significant effects were observed for final BW and dry matter intake (P ≥ 0.12), but average
daily gain and feed efficiency tended to improve (P ≤ 0.10) when probiotics were offered to the animals. Probiotic supplementation or type of
probiotic did not affect carcass traits (P ≥ 0.22). In summary, supplementation of probiotics containing a mixture of E. faecium and S. cerevisiae
or a mixture of B. licheniformis and B. subtilis reduced rumen acetate:propionate ratio and rumen ammonia-N levels and tended to improve the
performance of feedlot cattle offered a high-concentrate diet.

Lay Summary
Two experiments were designed to evaluate the effects of different probiotic combinations on rumen fermentation characteristics, performance,
and carcass characteristics of feedlot Bos indicus beef bulls offered a high-concentrate diet. The two probiotics consisted of a mixture containing
three strains of Enterococcus faecium and one strain of Saccharomyces cerevisiae or a mixture of Bacillus licheniformis and Bacillus subtilis.
Supplementation of probiotics, regardless of type, reduced acetate:propionate ratio, and mean rumen ammonia-N concentration and tended to
improve the performance of feedlot cattle offered a high-concentrate diet, demonstrating the potential of this technology to be used as a feed
additive for beef cattle.
Key words: Bacillus, Bos indicus, Enterococcus faecium, feedlot, metabolism, Saccharomyces cerevisiae
Abbreviations: Ac:Pr, acetate:propionate ratio; ADF, acid detergent fiber; ADG, average daily gain; AIC, Akaike Information Criterion; aNDF, ash-corrected NDF;
BFT, backfat thickness; BW, body weight; CP, crude protein; DDBS, dry distiller’s bran with solubles; DE, digestible energy; DM, dry matter; DMI, dry matter
intake; DP, dressing percent; EE, ether extract; EG, energy gain; G:F, feed efficiency; GIT, gastrointestinal tract; HCW, hot carcass weight; ME, metabolizable
energy; N, nitrogen; NDF, neutral detergent fiber; NEg, net energy for gain; NEm, net energy for maintenance; REA, ribeye area; TDN, total digestible nutrients;
VFA, volatile fatty acids

Introduction of rumen fermentation characteristics, promoting the estab-

Probiotics are classified as live microorganisms that, when lishment of beneficial rumen microflora, and/or enhanc-
administered in adequate amounts, confer health benefits ing fiber and overall nutrient digestibility (Krehbiel et al.,
to the host (FAO/WHO, 2001). In cattle, most published 2003). Nonetheless, as its main premise, probiotics improve
studies have attempted to evaluate how probiotics may gastrointestinal tract (GIT) health, including the rumen and
favor nutrient digestion in the rumen through modulation lower GIT.

Received July 9, 2022 Accepted September 1, 2022.

© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions,
please e-mail: [email protected].
2 Journal of Animal Science, 2022, Vol. 100, No. 10

Finishing feedlot cattle are often fed high-concentrate and Table 1. Nutritional profile of the diets offered during experiments 1 and 2
high-starch diets for an extended period (>100 d; Samuelson
et al., 2016; Silvestre and Millen, 2021) so that the mainte- Item Diets1
nance of rumen health is imperative to meet a desirable per- ADAP-1 ADAP-2 ADAP-3 FIN
formance during finishing. Furthermore, flint corn cultivars in
Brazil have a high proportion of vitreous endosperm (Correa Inclusion,2 % DM
et al., 2002) and when fed as whole or ground dry corn, the  Corn silage 50.0 40.0 30.0 20.0
resulting flow of starch to the large intestine (Gouvêa et al.,  Ground corn 15.0 25.0 30.0 33.0
2016; Marques et al., 2016) may challenge its health. Differ-  Dried distiller bran 33.0 33.0 38.0 45.0
ent probiotic strains, such as Enterococcus faecium, Bacillus plus solubles

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spp., and Saccharomyces cerevisiae, can support rumen and  Mineral–vitamin mix3 2.0 2.0 2.0 2.0
lower GIT metabolism through different mechanisms (McAl- Nutritional profile
lister et al., 2011; Chiquette et al., 2015; Luise et al., 2022;
 DM 50.5 54.7 63.0 69.3
Santano et al., 2020). However, to the best of our knowledge,
no other research evaluated the effects of different probiotics  Crude protein, % DM 13.8 13.8 14.9 16.3
on rumen and intestinal metabolism, performance, and car-  Ether extract, % DM 4.5 4.8 5.2 5.7
cass traits of Bos indicus beef bulls offered a high-concentrate  Neutral detergent 51.0 46.3 43.6 41.8
diet. Hence, we hypothesized that combining different pro- fiber, % DM
biotics would improve rumen metabolism and performance  Starch, % DM 29.0 33.0 33.6 33.0
of feedlot B. indicus beef bulls fed a high-concentrate diet.  Total digestible 72.0 74.9 77.8 80.8
Therefore, our objective was to evaluate different probiotic nutrientes,4 % DM
combinations on rumen fermentation characteristics and  Metabolizable 2.60 2.71 2.81 2.92
total tract nutrient digestibility (experiment 1), performance, energy,4 Mcal/kg
and carcass characteristics (experiment 2) of feedlot B. indi-  Net energy for 1.69 1.78 1.87 1.97
cus beef bulls fed a high-concentrate diet. maintenance,4
Mcal/kg
 Net energy for 1.08 1.16 1.24 1.32
Materials and Methods gain,4 Mcal/kg
Experiment 1: metabolism trial
1
ADAP-1, step-up diet fed from day 0 to day 5; ADAP-2, step-up diet fed
This experiment was conducted at the metabolism barn from day 6 to day 10; ADAP-3, step-up diet fed from day 10 to day 15;
facility located at the University of São Paulo (USP), Escola FIN, finishing diet.
Superior de Agricultura Luiz de Queiroz (ESALQ), located
2
DM, dry matter.
3
Composition: 275 g/kg Ca, 20 g/kg Mg, 15 g/kg Na, 550 ppm Cu,
in Piracicaba, São Paulo, Brazil (22°43ʹ31ʺS, 47°38ʹ51ʺW, 1,400 ppm Mn, 2,500 ppm Zn, 15 ppm Co, 25 ppm I, 5 ppm Se, 65,000
and elevation of 546 m) from April to May 2021. The min- IU Vit. A, 14,000 IU Vit. D3, and 500 IU Vit. E.
imum and maximum temperatures during the experimental
4
Estimated with the equations proposed by NASEM (2016) and the
tabular TDN values of dent corn and dry distiller’s grain plus soluble,
period were 7.8 and 29.1 °C, respectively, whereas total respectively, for experimental flint corn and DDBS. The corn silage TDN
rainfall was 31 mm. All animals utilized herein were cared value was obtained according to equation proposed by Weiss et al. (1992)
using its chemical composition.
for in accordance with acceptable practices and experi-
mental protocols reviewed and approved by the ESALQ/
USP Institutional Animal Care and Use Committee (#
6538141220). in Table 1. The nutritional profile of the dry distiller’s bran
with solubles (DDBS) used herein had 27.2% crude pro-
Animals, housing, and diets tein (CP), 3.15% ash, 7.99% ether extract (EE), and 55.3%
A total of 30 rumen-fistulated Nellore (B. indicus) steers neutral detergent fiber (NDF). Corn was processed through
were enrolled in the present experiment (initial body weight a hammer mill (Indústria e Comercial Lucato, Limeira, SP,
[BW] 350 ± 35.0 kg). Throughout the experiment, all steers Brazil) to achieve a mean particle size of 1.84 mm (Table
were housed in individual pens with concrete floor (2 × 2 2), according to procedures described by Yu et al. (1998),
m), in the same covered barn, and with ad libitum access to using sieves with 6.0, 3.5, 2.0, and 1.25 mm square pores
water. (Produtest T model; Telastem Peneiras para Análises Ltda.,
Steers were assigned to treatments in a randomized com- São Paulo, SP, Brazil). Diets were mixed using a feed wagon
plete block design, using initial BW as the blocking factor. (Rotormix-40; Casale Equipamentos, São Carlos, SP, Brazil).
Within blocks (n = 10), animals were randomly assigned to The experimental period lasted 35 d, being 29 d of adaptation
receive one of three treatments. Treatments consisted of 1) and 6 d of sampling, and all steers were fed once a day (1100
high-concentrate diet without probiotic supplementation (n = hours) from day 0 to day 35.
10; CONT), 2) CONT plus daily top-dressing supplementa-
tion of 1 g per head of a probiotic mixture containing three Sampling
strains of E. faecium and one strain of S. cerevisiae (3.5 × 109 At the beginning (day 0) of the experimental period, individ-
CFU/g; Probios Precise; Chr. Hansen A/S, Valinhos, SP, Brazil; ual shrunk BW was recorded after 16 h of feed and water
n = 10; EFSC), and 3) CONT plus daily top-dressing supple- withdrawal to determine animal initial BW and to perform
mentation of 2 g per head of a probiotic mixture containing the randomization of the animals into blocks and treatments.
Bacillus licheniformis and Bacillus subtilis (3.2 × 109 CFU/g; Throughout the experimental period (day 0 to day 35), total
Bovacillus; Chr. Hansen A/S; n = 10; BLBS). The complete dry matter intake (DMI) and nutrient intake were recorded
composition and nutritional profile of the diets are reported daily by collecting and weighing feed refusals approximately
Dias et al. 3

Table 2. Corn grain particle size distribution for experiments 1 and 2 according to procedures described by Van Soest et al.
(1991), using a sodium sulfite for all samples and heat-stable
Pores in the sieve % of total alpha-amylase for corn samples. Following NDF determina-
tion, acid detergent fiber (ADF) was evaluated according to
 >6.0 mm 0.0
procedures described by Goering and Van Soest (1970) in an
 ≤6.00 and >3.5 mm 0.7
Ankom-200 (Ankom Tech. Corp.). Apparent digestibility was
 ≤3.50 and >2.0 mm 30.8 calculated according to the formula: TTAD (%) = {[(DMI ×
 ≤2.00 and >1.25 mm 51.0 NCDM) − (FDM × NCFM)] × 100}/(DMI × NCDM), where
 ≤1.25 mm 17.6 TTAD is the total tract apparent digestibility, DMI is the dry
Mean particle size of corn, mm1 1.84 matter intake, NCDM is the nutrient content of the DMI (%),

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FDM is the fecal dry matter, and NCFM is the nutrient con-
1
Corn retained on the 6 mm screen was determined in 20 randomly tent of the fecal DM (%).
particles using a digital caliper. The residue retained in the bottom was The calculations of observed net energy for maintenance
assumed to have a mean particle size of 0.625 mm. Based on Yu et al.
(1998). (NEm) and gain (NEg) were performed from the TTAD calcu-
lations. The equations used for calculations included NEm =
1.37 × ME − 0.138 × ME2 + 0.0105 × ME3 − 1.12 and for NEg
24 h apart. Moreover, total digestible nutrients (TDN) intake = 1.42 × ME − 0.174 × ME2 + 0.0122 × ME3 − 1.65 (NASEM,
was calculated according to equations proposed by Weiss et 2016), in which metabolizable energy (ME) = 0.82 × digest-
al. (1992). Samples of the offered and non-consumed diet ible energy (DE; NRC, 1984). The DE was obtained from the
were collected daily from each pen and dried for 48 h at assumption that DE = TDN × 4.409 (NASEM, 2016), and
50 ± 5 °C in forced air ovens for dry matter (DM) and, conse- the TDN was calculated according to the equation: TDN =
quently, DMI calculation. DCHO + DCP + DEE × 2.25. To predict expected dietary
From day 34 to day 35 of the experimental period, rumi- NEm and NEg, the respective NASEM (2016) equations were
nal fluid samples were collected (approximately 100 mL) used and the tabular values from NASEM (2016) for dry dent
every 3 h, starting immediately before diet feeding (0 h), by corn and DDGS were assumed for the experimental ground
squeezing the ruminal contents into four layers of cheese- flint corn and DDBS, respectively, while the corn silage TDN
cloth and the ruminal fluid pH was immediately determined was calculated according to the equation proposed by Weiss et
(Digimed-M20; Digimed Instrumentação Analítica; São al. (1992) using its chemical composition. Then, the observed
Paulo, SP, Brazil). Approximately 50 mL of the ruminal fluid expected NEm and NEg ratios were calculated.
were collected and stored (−20 °C) for subsequent analysis of
rumen ammonia and molar proportions of individual volatile Experiment 2: performance trial
fatty acids (VFA; acetate, propionate, and butyrate), acetate:- This experiment was conducted at the experimental feedlot
propionate (Ac:Pr) ratio, and total VFA. Frozen ruminal sam- located at the USP, ESALQ, located in Piracicaba, São Paulo,
ples were prepared for analysis by thawing and centrifuging Brazil (22°43ʹ31ʺS, 47°38ʹ51ʺW, and elevation of 546 m)
(15,000 × g) for 10 min at room temperature. Samples were from May to August 2021. The average temperature within
then analyzed for VFA and rumen ammonia-N according to each month from the experimental period (from May to
procedures described by Ferreira et al. (2016) and Broderick August) was 19.8, 18.5, 17, and 20.9 °C, respectively, whereas
and Kang (1980), respectively. total rainfall was 22.9, 12.8, 23.3, and 12.4 mm, respectively.
All animals utilized herein were cared for in accordance with
Total tract apparent nutrient digestibility acceptable practices and experimental protocols reviewed
From day 30 to day 33, total fecal material was collected from and approved by the ESALQ/USP Institutional Animal Care
the individual pens, weighed, sub-sampled (approximately and Use Committee (# 6538141220).
10% of wet weight), and frozen at −20 °C for DM analysis
and determination of total fecal excretion. From day 34 to Animals, housing, and diets
day 35, approximately 50 g of fecal samples were collected From day −3 to day −1 of the study, all animals were housed
directly from the rectum of each animal, every 3 h, starting in group-pens with ad libitum access to water and a diet con-
immediately before feeding (0 h), and stored at −20 °C for taining (DM basis) 50% corn silage, 13% ground corn, 33%
nutrient analysis and subsequent apparent nutrient digestibil- DDBS, and 2% mineral–vitamin mix, in order to acclimate
ity calculations. The samples collected from the rectum of the animals to the facilities prior to the beginning of the experi-
animals on day 34 were weighed and included in the sum of ment.
total fecal material collected from individual pens. On the morning of day 0, 240 Nellore (Bos taurus indicus)
Frozen samples were thawed and dried in a forced air oven bulls were ranked by initial shrunk BW (after 16 h of feed and
at 55 °C for 72 h. Diet (offer and orts) and fecal samples were water restriction; 375 ± 35.1 kg; initial age = 24 ± 2 mo) and
ground into a 1-mm screen using a Willey mill (Marconi Equi- randomly assigned to treatments in a randomized complete
pamentos Laboratoriais, Piracicaba, SP, Brazil). DM content block design. Within blocks (n = 10), animals were randomly
was determined by drying the samples in an oven at 105 °C for assigned into pens (n = 4 bulls per pen; 4 × 8 m) and pens
24 h and ash content was determined by burning the samples were randomly assigned to receive one of three treatments
in a muffle furnace at 550 °C for 4 h (method 930.15; AOAC, (as reported in experiment 1): 1) CONT (n = 20), 2) EFSC
1986). Total nitrogen (N) determination was performed using (3.5 × 109 CFU/g; Probios Precise; Chr. Hansen A/S; n = 20),
a Leco FP-528 (Leco Corporation; Saint Joseph, MI), accord- and 3) BLBS (3.2 × 109 CFU/g; Bovacillus; Chr. Hansen A/S; n
ing to the methodology proposed by AOAC (1997), whereas = 20). Conversely, to what has been described for experiment
ether extract followed the method 920.85 (AOAC, 1986), and 1, probiotics were added and mixed with the other ingredi-
ash-corrected neutral detergent fiber content was analyzed ents of the diet. Regardless of treatment, all bulls received
4 Journal of Animal Science, 2022, Vol. 100, No. 10

the same step-up and finishing diets throughout the experi- dietary NEm and NEg were predicted as reported in experi-
mental period, which lasted 115 d. The adaptation diet was ment 1.
offered for 15 d and consisted of three step-up diets (5 d each) On day 113 of the experimental period, all bulls were
ranging from 50:50 to 30:70 roughage:concentrate ratio in subjected to ultrasound evaluations (Aloka SSD-500V with
step-up diets 1 and 3, respectively, whereas the finishing diet a 17.2 cm/3.50 MHz convex probe; Hitachi Healthcare
had a 20:80 roughage:concentrate ratio. The treatments were Americas, Twinsburg, OH), performed by the same trained
included in EFSC and BLBS diets since the adaptation period, technician (DGT Brasil, Presidente Prudente, SP, Brazil). Eval-
comprising the entire experimental period. The CONT diet uations were conducted according to procedures described by
was the same as the aforementioned (experiment 1) and was the Ultrasound Guidelines Council (UGC, 2014) and mea-
formulated using NASEM (2016) to provide an average daily surements of the ribeye area, marbling, and backfat thickness

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gain (ADG) of 1.5 kg during the experimental period. were collected on the Longissimus thoracis muscle between
On day 0, all bulls were individually identified with ear the 12th and 13th ribs.
tags, vaccinated against clostridial (Covexin-9; MSD, São All animals were slaughtered on the morning of day 116
Paulo, Brazil) pathogens and dewormed with 1 mL/50 kg BW following a waiting period of approximately 16 h, in a
of an antiparasitic (Evol; Ouro Fino Saúde Animal, Cravin- commercial packing plant (Frigorífico Zanqueta, Bauru, SP,
hos, SP, Brazil). Throughout the experimental period, diets Brazil). Hot carcasses were separated into two symmetrical
were supplied once daily as a total mixed ration using a feed sections, weighed to obtain hot carcass weight (HCW), and
wagon (Rotormix-40; Casale Equipamentos, São Carlos, SP, individually identified. Dressing percent was calculated by
Brazil) with an electronic scale (ez3400VL; Digi Star, Fort dividing the HCW and final BW of each animal.
Atkinson) and offered to ensure ad libitum intake and result
in 3% orts. Between the feeding of EFSC and BLBS, as well Statistical analysis
as following complete feeding of BLBS pens, the feed wagon For both experiments, all data were analyzed using the PROC
was washed with a 4% chloride solution (Barbarex, Nova MIXED procedure of SAS (Version 9.4; SAS Inst. Inc.; Cary,
Odessa, SP, Brazil) to avoid any cross-contamination among NC) and the Satterthwaite approximation to determine the
treatments. Additionally, all animals had full access to water denominator df for the test of fixed effects and block as a ran-
and were maintained in open-sided paved pens with a cover- dom variable. All results are reported as least square means,
all in the feed bunk (4.0 to 5.0 m of linear feed bunk per pen). separated using the PDIFF structure, and adjusted with the
TUKEY option of SAS (for orthogonal contrast analysis only;
Sampling and carcass measurements SAS Inst. Inc.). For all the data, significance was set at P ≤
Individual shrunk BW of bulls was collected on day 0 and day 0.05 and tendencies were denoted if P > 0.05 and P ≤ 0.10.
115 after 16 h of feed and water withdrawal and used to calcu- Moreover, specifically for DM and nutrient intake, results
late the BW change (final minus initial BW) and ADG during are reported according to the main effects if no interactions
the experiment. Total DMI was evaluated daily throughout were significant or according to the highest order interaction
the experimental period by weighing the feed offered and detected.
refused on the following day (approximately 24 h). At the end
of the experiment, total BW gain and total DMI were used for Experiment 1
feed efficiency (G:F) calculation, whereas mean BW was used Animal was considered the experimental unit for all analy-
for the determination of DMI as a percentage of BW. ses performed herein. All data were analyzed as orthogonal
Samples of ingredients were collected weekly throughout contrasts to partition-specific treatment effects: 1) Probiotic
the experimental period, pooled across weeks, and analyzed effect: CONT vs. PROB and 2) Probiotic type: EFSC vs.
for nutrient concentration (ESALQ Lab; Piracicaba, SP, Bra- BLBS. Moreover, rumen VFA (mmol/L and proportion), pH,
zil). All samples were analyzed in duplicates by wet chem- and ammonia-N data were analyzed using the REPEATED
istry procedures for concentrations of CP (method 984.13; statement of SAS, using the fixed effects of treatment, hour,
AOAC, 2006), NDF (Van Soest et al., 1991); modified for use and the resulting interaction. Data were analyzed using the
in an Ankom-200 fiber analyzer (Ankom Technology Corp., animal as the random variable, whereas the specified term
Fairport, NY), and ADF (method 973.18 modified for use for the repeated statement was hour, the subject was animal
in an Ankom-200 fiber analyzer; Ankom Technology Corp.; (treatment), and the covariance structure was first-order
AOAC, 2006). Moreover, TDN concentration was calculated autoregressive, which provided the best fit for these analy-
as reported in experiment 1. ses according to the smallest Akaike Information Criterion
The observed NE for each diet was calculated from the (AIC).
performance data using the equations reported by Zinn and
Shen (1998) based on pen average values. Energy gain (EG) Experiment 2
was calculated as EG = (0.0557 × BW0.75) × ADG1.097 (NRC, The model statement used for all performance and carcass
1984), in which EG is daily energy deposited (Mcal/d) and data contained the fixed effects of treatment. All data were
BW is the mean shrunk BW. The equation used to calculate analyzed using block and pen (treatment) as random vari-
maintenance energy expended (MEx; Mcal/d) was MEx = ables, whereas animal (pen) was also included in the ran-
0.077 × BW0.75 (NRC, 1996). From the calculated amounts dom statement for BW, ADG, and carcass ultrasound data.
of energy required for maintenance (NEm) and gain (NEg), Orthogonal contrasts were used to partition-specific treat-
the NEm of each diet was obtained by the quadratic equa- ment comparisons: 1) Probiotic effect: CONT vs. PROB and
tion NEm = [−b ± (b2 − 4ac)1/2]/2a, in which a = − 0.877 × 2) Probiotic type: EFSC vs. BLBS. Moreover, for daily DM,
DMI, b = 0.877 × MEx + 0.41 × DMI + EG, and c = − 0.42 NEm, and NEg intakes, values were averaged within each wk
× MEx and NEg of each diet was obtained by the equation and analyzed as repeated measures. The specified term for the
NEg = 0.877 × NEm − 0.41 (Zinn and Shen, 1998). Expected repeated statement was wk, the subject was pen (treatment),
Dias et al. 5

and the covariance structure was autoregressive 1, which pro- Table 3. Nutrient intake and digestibility in B. indicus beef steers
vided the best fit for these analyses according to the smallest receiving a high-concentrate diet (CONT; n = 10) with the addition of a
probiotic containing E. faecium and S. cerevisiae (1 g per head per d;
AIC.
EFSC; n = 10) or a mixture of B. licheniformis and B. subtilis (2 g per
head per d; BLBS; n = 10) in experiment 11
Results
Item Treatments SEM Contrasts2
Experiment 1
No contrast effects were observed on any of the nutrient CONT EFSC BLBS 1 2
intake and digestibility analyses reported herein (P ≥ 0.18; Nutrient intake, kg/d 3

Table 3). Similarly, total VFA, rumen pH, and individual

 DM 7.76 7.57 7.72 0.303 0.76 0.72

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proportion of the main VFA were not impacted by probiotic
 CP 0.80 0.82 0.80 0.038 0.87 0.79
supplementation or probiotic type (P ≥ 0.13; Table 4). None-
theless, acetate:propionate ratio and mean rumen ammo-  EE 0.38 0.38 0.38 0.014 0.91 0.93
nia-N concentration was lower for probiotic-supplemented  Carbohydrate 3.82 3.89 3.78 0.168 0.92 0.65
steers vs. CON (P ≤ 0.05; Table 4).  TDN 5.48 5.44 5.56 0.288 0.94 0.72
When rumen fermentation traits were analyzed as DE, Mcal/d 4
24.1 24.5 24.0 1.03 0.94 0.73
repeated measures, no treatment × hour interactions were ME, Mcal/d4 19.8 20.1 19.7 0.84 0.94 0.73
observed for individual and total rumen VFA (concentration NEg, Mcal/d4 8.1 8.5 8.0 0.42 0.73 0.45
and proportion), Ac:Pr ratio, and pH (P ≥ 0.41; data not
Observed NEm, Mcal/kg 1.66 1.74 1.64 0.044 0.55 0.14
shown). On the other hand, the same interaction tended to
Observed NEg, Mcal/kg 1.05 1.12 1.03 0.039 0.55 0.13
be observed (P = 0.08) for rumen ammonia-N. Steers fed
EFSC had lower rumen ammonia-N vs. CONT at 3 and 6 h Observed:expected NEm 0.92 0.96 0.91 0.024 0.55 0.13
post-feeding (P = 0.04), whereas the same results tended to Observed:expected NEg 0.89 0.94 0.87 0.033 0.28 0.14
be observed at 9 h post-feeding (P = 0.10). Similarly, supple- Digestibility, %5
mentation with BLBS reduced ammonia-N at 6 h (P < 0.01)  DM 67.0 69.3 66.2 1.53 0.70 0.17
and tended to reduce the concentration of this metabolite  OM 69.4 72.1 68.8 1.49 0.59 0.14
at 3, 9, and 12 h post-feeding (P ≤ 0.08; Figure 1). Con-  CP 60.8 63.1 60.7 1.85 0.64 0.38
versely, no differences on rumen ammonia-N were observed
 NDF 63.6 65.8 61.8 2.23 0.95 0.22
between EFSC and BLBS at any timepoint of the sampling
 EE 90.3 91.3 90.1 0.63 0.61 0.21
period (P ≥ 0.14; Figure 1).
 Carbohydrate
Experiment 2  Total 69.9 72.7 69.1 1.54 0.61 0.13
At the beginning of the feedlot performance trial, BW did  Non-fiber 81.4 85.5 82.5 1.55 0.18 0.19
not differ among treatments (P ≥ 0.59; Table 5). Similarly,  Starch 94.0 93.5 93.7 0.48 0.52 0.66
contrasts were not significant for final BW (P ≥ 0.12), DMI  Ash 33.9 31.3 30.8 4.25 0.58 0.94
reported as kg/d (P ≥ 0.21) or % of BW (P ≥ 0.18). Nonethe-  TDN 70.9 73.4 70.3 1.95 0.58 0.13
less, ADG and G:F tended to improve (P ≤ 0.10) when probi-
otics were offered to the animals, with no further differences 1
CONT, high-concentrate diet without the addition of probiotics; EFSC,
between the type of probiotic being offered to the feedlot cat- 1 g per head per d of a probiotic containing three strains of E. faecium
and one strain of S. cerevisiae (3.5 × 109 CFU/g; Probios Precise, Chr.
tle herd (P ≥ 0.22; Table 5). When DM, NEm, and NEg intake Hansen A/S, Valinhos, SP, Brazil); BLBS, 2 g per head per d of a probiotic
data were analyzed as repeated measures, no treatment × wk containing B. licheniformis and B. subtilis (3.2 × 109 CFU/g; Bovacillus,
interactions were observed (P ≥ 0.74; data not shown). Pro- Chr. Hansen A/S).
2
Contrast analysis: 1) CONT vs. PROB and 2) EFSC vs. BLBS.
biotic supplementation or type of probiotic did not affect any 3
DM, dry matter; CP, crude protein; EE, ether extract; TDN, total
of the carcass traits measured in the present experiment (P ≥ digestible nutrient.
0.22; Table 5). Regardless of the type (P ≥ 0.28), probiotic
4
DE, digestible energy; ME, metabolizable energy; NEg, net energy for gain.
5
OM, organic matter; NDF, neutral detergent fiber.
supplementation tended to increase diet observed NEm and
NEg and observed to expected ratios of intake of NEm and
NEg (P ≤ 0.10; Table 6). biotics described here for beef cattle and, therefore, data will
be discussed on single-strain or different combinations of the
same strains mostly for lactating dairy cows and/or calves.
Discussion Enterococcus faecium is a lactate-producing bacterium and
The main goal of the present manuscript was to evaluate together with oxygen-scavenging properties of S. cerevisiae
whether 1) probiotic supplementation and 2) supplementa- may help to maintain an adequate rumen environment (Chi-
tion of different probiotic strains would impact rumen fer- quette et al., 2012). Hence, the EFSC mixture used herein may
mentation characteristics, total tract nutrient digestibility, have contributed to a constant and steady tonic level of lactic
performance, and carcass traits of B. indicus animals offered acid in the rumen, likely stimulating the growth and activ-
a high-concentrate diet. The probiotics evaluated in both ity of fibrolytic and lactic acid-utilizing bacteria (Nisbet and
experiments present a different composition of strains (E. Martin, 1991; Newbold et al., 1996; Chaucheyras-Durand et
faecium + S. cerevisiae for EFSC and B. licheniformis + B. al., 2008), resulting in sustained low or undetectable concen-
subtilis for BLBS) with different modes of action, precluding trations of lactic acid in the rumen, which, in turn, would tend
potential differences on rumen and intestinal metabolism, as to increase the pH (Nocek et al., 2003). In fact, dairy cows
well as performance of feedlot beef cattle. To the best of our supplemented with a mixture of E. faecium (two out of three
knowledge, a limited number of research evaluated the pro- strains used herein) and S. cerevisiae had greater mean daily
6 Journal of Animal Science, 2022, Vol. 100, No. 10

Table 4. Rumen volatile fatty acids (VFA; mmol and proportion), pH, Table 6. Energy intake of B. indicus beef bulls receiving a high-
and ammonia concentration of B. indicus beef steers receiving a concentrate diet (CONT; n = 20) with the addition of a probiotic
high-concentrate diet (CONT; n = 10) with the addition of a probiotic containing E. faecium and S. cerevisiae (1 g per head per d; EFSC; n =
containing E. faecium and S. cerevisiae (1 g per head per d; EFSC; n = 20) or a mixture of B. licheniformis and B. subtilis (2 g per head per d;
10) or a mixture of B. licheniformis and B. subtilis (2 g per head per d; BLBS; n = 20) in experiment 21
BLBS; n = 10) in experiment 11
Item Treatments SEM Contrasts2
Item Treatments SEM Contrasts2
CONT EFSC BLBS 1 2
CONT EFSC BLBS 1 2
NEg intake, Mcal/d 13.7 14.3 14.2 0.27 0.13 0.84
VFA, mmol/100 mol Expected3

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 Acetate 46.6 46.2 44.8 1.35 0.49 0.45  NEm 1.94 1.94 1.94 -- -- --
 Propionate 20.2 23.1 24.7 1.56 0.12 0.39  NEg 1.30 1.30 1.30 -- -- --
 Butyrate 24.0 21.8 23.2 1.64 0.61 0.14 Observed4
 Isobutyrate 2.3 2.2 1.7 0.17 0.15 0.04  NEm 1.93 1.95 1.99 0.022 0.10 0.28
 Valerate 2.1 2.0 1.9 0.22 0.55 0.68  NEg 1.28 1.30 1.33 0.019 0.10 0.28
 Isovalerate 4.9 4.6 3.6 0.38 0.10 0.09 Observed:expected
Total VFA 85.3 84.2 96.8 7.32 0.56 0.25  NEm 0.98 0.99 1.01 0.011 0.10 0.32
Acetate:propionate ratio 2.71 2.38 2.10 0.164 0.03 0.25  NEg 0.97 0.99 1.01 0.015 0.09 0.30
pH 6.40 6.41 6.28 0.087 0.59 0.31
Ammonia-N, mg/L 15.7 14.9 14.6 0.43 0.05 0.59 1
CONT, high-concentrate diet without the addition of probiotics; EFSC,
1 g per head per d of a probiotic containing three strains of E. faecium
and one strain of S. cerevisiae (3.5 × 109 CFU/g; Probios Precise, Chr.
1
CONT, high-concentrate diet without the addition of probiotics; EFSC, Hansen A/S, Valinhos, SP, Brazil); BLBS, 2 g per head per d of a probiotic
1 g per head per d of a probiotic containing three strains of E. faecium containing B. licheniformis and B. subtilis (3.2 × 109 CFU/g; Bovacillus,
and one strain of S. cerevisiae (3.5 × 109 CFU/g; Probios Precise, Chr. Chr. Hansen A/S).
Hansen A/S, Valinhos, SP, Brazil); BLBS, 2 g per head per d of a probiotic 2
Contrast analysis: 1) CONT vs. PROB and 2) EFSC vs. BLBS.
containing B. licheniformis and B. subtilis (3.2 × 109 CFU/g; Bovacillus, 3
Estimated with the equations proposed by NASEM (2016) and the
Chr. Hansen A/S). tabular TDN values of dent corn and dry distiller’s grain plus soluble,
2
Contrast analysis: 1) CONT vs. PROB and 2) EFSC vs. BLBS. respectively for experimental flint corn and DDBS. The corn silage TDN
value was obtained according to the equation proposed by Weiss et al.
(1992) using its chemical composition.
Table 5. Feedlot performance of B. indicus beef bulls receiving a
4
Calculated according to Zinn and Shen (1998).
high-concentrate diet (CONT; n = 20) with the addition of a probiotic
containing E. faecium and S. cerevisiae (1 g per head per d; EFSC; n =
20) or a mixture of B. licheniformis and B. subtilis (2 g per head per d; same mixture of probiotics yielded greater milk production
BLBS; n = 20) in experiment 21 vs. non-supplemented cohorts over 70 d post-partum (Nocek
et al., 2003; Nocek and Kautz, 2006). When evaluated during
Item Treatments SEM Contrasts2 a sub-acute ruminal acidosis challenge in lactating dairy
CONT EFSC BLBS 1 2 cows, E. faecium and S. cerevisiae supplementation alleviated
the loss in milk yield, reduced the time that rumen pH was
Performance data < 6.0, and increased maximum rumen pH (Chiquette et al.,
Body weight, kg 2012, 2015), but did not impact total and proportion of indi-
 Initial 374.4 374.1 373.9 11.77 0.59 0.79 vidual VFA (Chiquette et al., 2012). On the other hand and in
 Final 546.7 554.3 554.1 14.00 0.12 0.98 agreement to our results, others also did not report positive
Average daily gain, kg 1.50 1.57 1.57 0.038 0.10 0.98 effects of E. faecium (1, 2, or 3 strains) and S. cerevisiae on
Dry matter intake rumen fermentation characteristics, such as rumen pH during
 kg/d 10.7 11.0 10.7 0.34 0.67 0.21 a regular feeding regime, total, and proportion of individual
 % Body weight 2.32 2.37 2.31 0.300 0.97 0.18 VFA (Beauchemin et al., 2003; Chiquette, 2009; Chiquette et
Feed efficiency, g/kg 140 143 147 3.5 0.07 0.22 al., 2012, 2015), suggesting that effects related to diet compo-
Carcass data sition and/or experimental period length and total DMI were
 Hot carcass weight, kg 311.1 315.0 313.9 8.41 0.22 0.71 not enough to cause ruminal challenges and, possibly, demon-
 Dressing percent, % 56.8 56.8 56.7 0.21 0.96 0.60
strate positive effects of probiotic supplementation to beef
 Ribeye area, cm2 77.8 78.4 78.4 1.17 0.84 0.66
animals. The inclusion of DDBS while reducing the amount of
ground corn in the diet does not necessarily reduce its rumi-
 Backfat thickness, mm 5.12 5.01 5.13 0.210 0.59 0.79
nal challenge. The DDBS is a byproduct resulting from the
1
CONT, high-concentrate diet without the addition of probiotics; EFSC, fiber separation process before fermentation is performed by
1 g per head per d of a probiotic containing three strains of E. faecium the corn ethanol industry (Lima Júnior et al., 2022). Garland
and one strain of S. cerevisiae (3.5 × 109 CFU/g; Probios Precise, Chr. et al. (2019) did not observe differences in ruminal pH of
Hansen A/S, Valinhos, SP, Brazil); BLBS, 2 g per head per d of a probiotic
containing B. licheniformis and B. subtilis (3.2 × 109 CFU/g; Bovacillus, cattle fed either a high-corn diet or high-DDBS diet. In sev-
Chr. Hansen A/S). eral studies, the partial replacement of corn by DDGS did not
2
Contrast analysis: 1) CONT vs. PROB and 2) EFSC vs. BLBS.
affect the ruminal pH of feedlot cattle (Corrigan et al., 2009;
Vander Pol et al., 2009). Cattle in the metabolism study con-
rumen pH, mean nadir pH, DM digestibility of forages, and sumed only 2.06% of BW during the sampling period, being
corn (Nocek et al., 2002a, 2002b; Nocek and Kautz, 2006). lower than the values on performance trial (experiment 2),
In dairy cattle, pre- and post-partum supplementation of the where mean DMI as %BW was roughly 2.33%. This lower
Dias et al. 7

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Figure 1. Rumen ammonia-N concentration (mg/L) of beef B. indicus seers receiving a high-concentrate diet (CONT; n = 10) with the addition of a
probiotic containing E. faecium and S. cerevisiae (1 g per head per d; EFSC; n = 10) or a mixture of B. licheniformis and B. subtilis (2 g per head per d;
BLBS; n = 10) in experiment 1. A treatment × hour interaction tended to be observed (P = 0.08). Within hour, different letters denote differences at P <
0.05. a, CONT vs. EFSC (P = 0.04); b, CONT vs. BLBS (P < 0.01).

DMI in the metabolism trial may not have challenged the et al., 2014). On the other hand, Bacillus spp. are known
ruminal environment to the experimental cattle. Nonetheless, to produce a different set of enzymes, including cellulases,
the acetate:propionate ratio was lower for probiotic-supple- expansin-like proteins, and amylases (Rojo et al., 2005;
mented animals, suggesting an improved energetic efficiency Pech-Cervantes et al., 2019; da Silva et al., 2021; Luise
by feeding probiotics. et al., 2022). Recently, Pan et al. (2022) reported overall
On the other hand, Bacillus spp. are classified as improvement on in vitro DM, NDF, and starch digestibil-
Gram-positive, catalase-positive, spore-forming, aerobic, ity of different forage sources (high- and low-quality) and
and facultative anaerobic bacteria (Luise et al., 2022). To high-starch substrates inoculated with BLBS. Therefore, it
the best of our knowledge, few experiments evaluated the would be logical to speculate that the probiotics fed to feed-
effects of Bacillus spp. on lactate production. Other authors lot beef steers would positively impact nutrient digestibility.
reported the production of d- and l-lactate by B. subtilis However, no differences were observed when probiotics and
in aerobic and anaerobic culture (Ohara and Yahata, 1996; type of probiotics were fed in any nutrient digestibility eval-
Gao et al., 2012; Awasthi et al., 2018), but no such info uated in experiment 1. Similarly, Souza et al. (2017) also did
has been reported in beef or dairy animals and/or rumen not report improvement on nutrient digestibility of lactating
fluid with more acidic conditions (pH ≤ 6.5). In calves, sup- dairy cows offered a corn silage-based diet. One cannot dis-
plementation of B. subtilis promoted rumen development regard that those differences between grain types (flint vs.
mainly due to an altered rumen fermentation pattern (Sun et dent), processing, type, and amount of the fiber included in
al., 2011). Molar proportions of propionate increased and the diet might also lead to differences on nutrient digest-
NDF digestibility was lower when lactating dairy cows were ibility and rumen fermentation characteristics (Owens et
fed B. subtilis in a 50% roughage diet (Sun et al., 2013). al., 1997, 2016; Marques et al., 2016). On the other hand,
Altogether, these data suggest a positive effect of B. subtilis despite the fact that both experimental probiotics did not
strains on rumen metabolism, but it is worth mentioning contribute to the decrease in the load of dietary starch into
that the experiments above used higher dosages of B. sub- the large intestine, they may have acted positively protecting
tilis (1 × 1010, 5 × 1010, and 1 × 1011 CFU per head per d; the large intestinal epithelium in a challenging high-starch
Sun et al., 2011, 2013) than the dose fed herein in combina- environment. Although probiotic supplementation has not
tion with B. licheniformis (6.4 × 109 CFU per head per d). impacted rumen ammonia in previous reports (Ghorbani
As observed with EFSC feeding, cattle fed BLBS had lower et al., 2002; Chiquette et al., 2012), mean rumen ammonia
acetate:propionate ratio, indicating an improved energetic concentration was lower in probiotic-fed steers, a decrease
efficiency of probiotic-fed cattle and a likely reduction in that was mainly observed in the initial 6 h post-feeding,
methane emission (NASEM, 2016). which is concomitant to the greater numerical rumen pH
Probiotics are also known to improve post-ruminal values observed in EFSC and BLBS (data not shown), likely
metabolism, including alteration of gut microbial popula- suggesting a greater growth of cellulolytic bacteria in the
tions, improvement on nutrient digestibility, and improved rumen (Qadis et al., 2014).
immunity (Seo et al., 2010; McAllister et al., 2011). As an Although no changes were observed on rumen individual
example, total tract starch digestibility was improved when proportion of VFA and nutrient digestibility, probiotic sup-
the same EFSC mixture was fed to dairy cows receiving an plementation, regardless of type, tended to improve ADG
18% and 22% starch diet pre- and post-partum, respec- and FE of B. indicus feedlot bulls. This potential improve-
tively, without further effects on fiber digestibility (AlZahal ment on performance could be explained by the reduced
8 Journal of Animal Science, 2022, Vol. 100, No. 10

acetate:propionate ratio observed in experiment 1, demon- Broderick, G. A., and J. H. Kang. 1980. Automated simultaneous de-
strating that energetic efficiency was likely improved by pro- termination of ammonia and total amino acids in ruminal fluid
biotic supplementation. Results of probiotic supplementation and in vitro media. J. Dairy Sci. 63:64–75. doi:10.3168/jds.S0022-
to feedlot beef cattle have been variable, ranging from no 0302(80)82888-8
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herein might be related to the impacts on rumen fermenta- fed to dairy cows during a subacute ruminal acidosis challenge.
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strains, such as against E. coli and. C. perfringens, as well as ci.2009.07.001
potential benefits on leaky gut following a high-concentrate Chiquette, J., M. J. Allison, and M. Rasmussen. 2012. Use of Prevotella
diet feeding (Copani et al., 2020; Segura et al., 2020). bryantii 25A and a commercial probiotic during subacute acidosis
Evaluating the effects of B. subtilis and yeast supplemen- challenge in mid-lactation dairy cows. J. Dairy Sci. 95:5985–5995.
doi:10.3168/jds.2012-5511
tation to receiving cattle, Colombo et al. (2021) did not
Chiquette, J., J. Lagrost, C. L. Girard, G. Talbot, S. Li, J. C. Plaizier,
report benefits on performance over a 45-d period. The and I. K. Hindrichsen. 2015. Efficacy of the direct-fed microbial
number of articles evaluating BLBS is still limited, but Kri- Enterococcus faecium alone or in combination with Saccharo-
tas et al. (2006) reported an improvement on milk produc- myces cerevisiae or Lactococcus lactis during induced subacute
tion, milk fat, and protein in pregnant ewes receiving BLBS ruminal acidosis. J. Dairy Sci. 98:190–203. doi:10.3168/
from 45 d pre- to 75 d post-lambing. Fat-corrected milk jds.2014-8219
production and milk production efficiency were improved Colombo, E. A., R. F. Cooke, A. P. Brandão, J. B. Wiegand, K. M.
when a probiotic mixture containing BLBS was offered to Schubach, C. A. Sowers, G. C. Duff, E. Block, and V. N. Gou-
lactating dairy cows (Oyebade et al., 2021). In Holstein vêa. 2021. Performance, health, and physiological responses
calves, ADG, weaning BW, and starter intake was greater of newly received feedlot cattle supplemented with pre- and
probiotic ingredients. Animal 15:100214. doi:10.1016/j.ani-
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alski et al., 2009). Copani, G., O. C. M. Queiroz, and E. J. Boll. 2020. Lactobacillus ani-
In summary, supplementation of probiotics containing a malis LA51 and Bacillus sp. probiotics confer protection from the
mixture of E. faecium and S. cerevisiae or a mixture of B. damaging effects of pathogenic Clostridium perfringens and Esch-
licheniformis and B. subtilis reduced acetate:propionate ratio erichia coli on the intestinal barrier. J. Dairy Sci. 103(Suppl. 1):103
and rumen ammonia levels, and also tended to improve the (Abstr.).
performance of feedlot cattle offered a high-concentrate Correa, C. E. S., R. D. Shaver, M. N. Pereira, J. G. Lauer, and K. Kohn.
diet. However, no further probiotic effects were observed on 2002. Relationship between corn vitreousness and ruminal in situ
rumen fermentation characteristics, nutrient digestibility, and starch degradability. J. Dairy Sci. 85:3008–3012. doi:10.3168/jds.
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Corrigan, M. E., T. J. Klopfenstein, G. E. Erickson, N. F. Meyer, K. J.
to further understand potential effects of different probiotic
Vander Pol, M. A. Greenquist, M. K. Luebbe, K. K. Karges, and M.
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Conflict of Interest Statement da Silva, R. N., L. F. A. Melo, and C. L. L. Finkler. 2021. Optimization of
the cultivation conditions of Bacillus licheniformis BCLLNF-01 for
The authors declare no conflicts of interest.
cellulase production. Biotechnol. Rep, 29:e00599. doi:10.1016/j.
btre.2021.e00599
Dick, K. J., G. C. Duff, S. W. Limesand, S. P. Cuneo, D. K. Knudson, C.
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