The rumen has several important physiological functions, including absorption, transport, metabolic activity, and host protection (Roh et al., 2007). The rumen is extensively researched due to the importance of ruminants in agriculture and the major role played by rumen microbes in nutrient utilization and health of the ruminant animal. The microbial community of the rumen is altered by diet (McCann et al., 2014), age (Jami et al., 2013), and environment

Aluthge, Nirosh D.

Duffy, Erin M.

Fernando, Samodha C.

Knoell, Allison L.

Petersen, Jessica L.

Schmidt, Ty B.

Tietze, Shauna M.

Yates, Dustin T.

English

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Faculty Papers and Publications in Animal 
Science Animal Science Department 
2018 
Rumen bacterial composition in lambs is affected by β-adrenergic 
agonist supplementation and heat stress at the phylum level 
Erin M. Duffy 
Shauna M. Tietze 
Allison L. Knoell 
Nirosh D. Aluthge 
Samodha C. Fernando 
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This Article is brought to you for free and open access by the Animal Science Department at 
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Lincoln. 
Authors 
Erin M. Duffy, Shauna M. Tietze, Allison L. Knoell, Nirosh D. Aluthge, Samodha C. Fernando, Ty B. Schmidt, 
Dustin T. Yates, and Jessica L. Petersen 
S145
Rumen bacterial composition in lambs is affected by β-adrenergic agonist 
 supplementation and heat stress at the phylum level1
Erin M. Duffy,2 Shauna M. Tietze, Allison L. Knoell, Nirosh D. Aluthge, Samodha C. Fernando,  
Ty S. Schmidt, Dustin T. Yates, and Jessica L. Petersen,2
Department of Animal Science, University of Nebraska–Lincoln, NE 68583-0908
© The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of 
Animal Science.  
This is an Open Access article distributed under the terms of the Creative Commons Attribution 
Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits 
 non-commercial re-use, distribution, and reproduction in any medium, provided the original  
work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.
Transl. Anim. Sci. 2018.2:S145–S148
doi: 10.1093/tas/txy052
INTRODUCTION
The rumen has several important physio-
logical functions, including absorption, trans-
port, metabolic activity, and host protection (Roh 
et al., 2007). The rumen is extensively researched 
due to the importance of ruminants in agriculture 
and the major role played by rumen microbes in 
nutrient utilization and health of the ruminant 
animal. The microbial community of the rumen 
is altered by diet (McCann et al., 2014), age (Jami 
et al., 2013), and environment.
Heat stress is an important environmental 
stressor that can reduce efficiency in ruminant 
livestock. Alternatively, β-adrenergic agonists (β-
AA) increase value due to increased efficiency in 
muscle growth (Buntyn et  al., 2016). Currently, 
there are two β-AA approved for use in livestock 
in the United States: zilpaterol HCl (ZHCl), a 
β2 agonist, and ractopamine HCl (RHCl), a β1 
agonist (Delmore et al., 2010; Boler et al., 2012). 
The β2 agonist increases muscle glucose oxidation 
(Barnes et al. 2017; Cadaret et al. 2017). However, 
the mechanism behind β1 action is poorly under-
stood, and there is a need to understand the im-
pact of β-AA fed during heat stress.
With the profound role of the rumen microbi-
ome in nutrient utilization and animal health, it is 
possible that heat stress events can impact the micro-
biota by decreasing metabolic activity and fermen-
tation, thus decreasing heat produced and energy 
provided to the animal. Additionally, β-AA may 
influence the microbiome to increase nutrient di-
gestion, providing more energy to the animal in the 
form of volatile fatty acids. However, the influence 
of concurrent heat stress and β-AA supplementation 
on the rumen microbiome has not been investigated. 
Therefore, the objective of this study was to deter-
mine the impact that β1 agonists, β2 agonists, and 
heat stress have on the rumen bacterial community.
MATERIALS AND METHODS
Animals and Experimental Design
This study was approved by the Institutional 
Animal Care and Use Committee at the University 
of Nebraska–Lincoln (Project 1300), an AAALAC 
International-accredited institution. Columbia–
Suffolk crossbred lambs were purchased com-
mercially. After a 3-wk acclimation, individually 
penned lambs were fed identical high-energy diets 
for 21 d and housed under either thermoneutral 
(25 °C, 15% relative humidity) or heat stress (40 °C, 
1This project is based on research that was partially sup-
ported by the Nebraska Agricultural Experiment Station with 
funding from the Hatch Multistate Research capacity fund-
ing program (Accession Number 1011055) from the USDA 
National Institute of Food and Agriculture as well as from 
a Layman Award from the University of Nebraska–Lincoln.
2Corresponding author: jessica.petersen@unl.edu
Received March 17, 2018.
Accepted April 14, 2018.
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S146 Duffy et al.
35% relative humidity) conditions. Each lamb 
received one of three dietary supplements: no sup-
plement, RHCl (0.03996 g/hd/d), or ZHCl (0.025 g/
hd/d) delivered in 200-g ground corn added to the 
ration. Each treatment ( environment × supplement) 
contained eight lambs with the exception of an add-
itional lamb in the thermoneutral group fed the con-
trol diet. On day 22, lambs were harvested. Contents 
of the rumen were collected immediately after har-
vest and flash frozen in liquid nitrogen until further 
analysis.
DNA Isolation
DNA was isolated from the rumen contents of 
all lambs in the study using the Mobio PowerSoil 
Soil DNA Isolation Kit (MoBio Laboratories, 
Carlsbad, CA). The V4 region of the 16S rDNA 
gene specific to eubacterial communities was then 
amplified using the Terra PCR Direct Polymerase 
Mix Kit (Takara Bio USA) and 515F and 806R 
primers (Illumina; Kozich et al., 2013). PCR condi-
tions were as follows: 3 min at 98 °C for initial de-
naturation, 25 cycles of 30 s at 98 °C, 30 s at 55 °C, 
and 45 s at 68 °C; the profile was terminated after a 
final 4-min hold at 68 °C. Amplification and product 
size were verified by agarose gel electrophoresis. 
PCR products were normalized to 2  ng/µL using 
the Invitrogen SequalPrep Normalization Plate 
kit (Fredrick, MD), and libraries were prepped, 
normalized, and pooled (Eppendorf epMotiom; 
M5073, Germany). The pooled libraries were puri-
fied as described in the NucleoSpin Gel and PCR 
Clean-Up kit (Clontech Laboratories Inc., CA), 
and size selected (380  bp) using the Pippin Prep 
(Sage Science, Inc.). Finally, the quality and quan-
tity of purified products were verified using Agilent 
2100 Bioanalyzer high sensitivity DNA chips 
(Agilent Technologies). Resulting amplicons were 
sequenced using the Illumina MiSeq platform using 
a V2 500 cycle kit with the dual-index sequencing 
strategy according to Kozich et al. (2013).
Microbial Community Analysis
Raw sequence reads were quality filtered as 
described by Anderson et al. (2016). In Quantitative 
Insights Into Microbial Ecology (QIIME) 
(ver.19.1), sequences with one or more errors in the 
barcode were removed (Caporaso et al., 2010). The 
paired-end reads were processed in Mothur (Schloss 
et  al., 2009) to improve operational taxonomic 
unit (OTU) classification (Edgar, 2013). After re-
verse complementation in Mothur, sequences were 
processed using a custom pipeline (Fernando Lab, 
UNL), chimera identification and removal, and 
OTUs selected based on 97% sequence similarity 
(Paz et al., 2018). Target OTU sequences generated 
previously were aligned, and OTUs aligning out-
side the 16s gene eliminated. Taxonomic classifica-
tion was performed using QIIME via GreenGenes 
database (ver.13_8).
An overall (total) and core measurable micro-
biome were determined based on supplementation 
and environment. The core measurable microbiome 
was defined as OTUs present in at least 50% of the 
samples in each subset.
Statistical Analysis
Sequence reads were analyzed using bioinfor-
matics pipelines from UPARSE (Edgar et  al., 
2011), QIIME, and Mothur. Reads per sample were 
rarefied across samples, and a Good’s coverage test 
was performed. Alpha diversity estimators Chao1 
and observed OTUs were calculated for the overall 
rumen microbiome in QIIME. To select taxa that 
were significantly different in abundance between 
supplementation and environment, the bioinfor-
matics tool linear discriminate analysis effect size 
(LEfSe) (Segata et al., 2011) was used with default 
parameters of P ≤ 0.05 and a linear discriminant 
analysis (LDA) score ≥ 2.
RESULTS
Rumen Bacterial Richness and Bacterial 
Composition
One of the 49 samples was removed due to insuf-
ficient reads (594). Rarefaction to the lowest depth 
(7,000 reads) resulted in 1,565 OTUs. Rarefaction 
curves showed a diminishing rate of OTU identi-
fication at ~6,000 sequences per sample, and the 
Good’s coverage test showed all samples were able 
to characterize ~98% of the bacterial community.
Based on taxonomic analysis of the reads, re-
gardless of environment and supplementation, 
Bacteroidetes and Firmicutes were the predominant 
phyla within the core microbiome accounting for 
48.5% and 33.1% of the total reads, respectively 
(Figure 1).
Bacterial Community Composition Associated with 
Environment
Between the thermoneutral and heat stress 
groups fed the control diet, three phyla were 
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S147Microbiome, β agonists, heat
differentially (P ≤ 0.05) abundant; Bacteroidetes 
were more abundant (P ≤ 0.05) in the ther-
moneutral lambs, while Fibrobacters and 
Actinobacteria  were  more abundant (P ≤ 0.05) in 
heat-stressed lambs. Additionally, among lambs 
supplemented with RHCl, Elusibacteria were more 
abundant (P ≤ 0.05) in heat stress conditions than 
thermoneutral conditions. No differences between 
environmental conditions were observed in lambs 
fed ZHCl.
Bacterial Community Composition Associated with 
Supplementation
Among thermoneutral lambs, Fibrobacteres and 
Bacteroidetes differed (P ≤ 0.05), with Bacteroidetes 
more abundant (P ≤ 0.05) in lambs fed the control 
diet and Fibrobacters more abundant (P ≤ 0.05) 
in lambs fed ZHCl. Also, among thermoneutral 
lambs, the abundance of Bacteroidetes was higher 
(P ≤ 0.05) in the nonsupplemented lambs compared 
with those fed RHCl.
DISCUSSION
The general characterization of the core mi-
crobial communities from this study shows the 
overall microbial composition was similar to those 
seen previously (Petri et al. 2013; Paz et al. 2016). 
Additionally, diet appeared to have a lesser role 
in determining the microbial community compos-
ition than heat stress. The thresholds for making 
changes in community compositions are currently 
unknown, but temperature and β-AA supplemen-
tation may act directly or indirectly via changes in 
the physiology of animals in response to heat stress 
conditions and diet.
At the phylum level, the microbial communities 
differed by treatment. Additionally, Bacteroidetes 
was more abundant in the thermoneutral group fed 
the control diet compared with those supplemented 
with β-AA. This is different than Tajima et  al. 
(2007), who observed no obvious effects of heat 
stress on rumen composition at the phylum level. 
Our findings indicate that supplementation with β-
AA works within the rumen to create an environ-
ment that alters the relative competitive advantage 
of other phyla. Although this study produced sig-
nificant changes at the phylum level of classifica-
tion, more research must be done at the family level 
to determine discrete differences in rumen micro-
bial composition.
IMPLICATIONS
In this study, we show that β-AA supplementa-
tion and heat stress affect the ruminal microbiome 
of wethers. Despite these changes, the two primary 
phyla Bacteroidetes and Firmicutes remained the 
same. Further examination is necessary to iden-
tify the overall impact these changes have on the 
animal and how this information could be uti-
lized to improve production efficiency and animal 
health. Ongoing work will compare transcriptional 
changes of the host rumen across treatments as well 
as to the observed microbial communities.
LITERATURE CITED
Anderson, C. L., C. J.  Schneider, G. E.  Erickson, J. 
C.  MacDonald, and S. C.  Fernando. 2016. Rumen bac-
terial communities can be acclimated faster to high concen-
trate diets than currently implemented feedlot programs. J. 
Appl. Microbiol. 120:588–599. doi:10.1111/jam.13039
Figure 1. Phylum level classification of the bacterial community composition depending on environment and supplementation. 
D
ow
nloaded from
 https://academ
ic.oup.com
/tas/article-abstract/2/suppl_1/S145/5108324 by U
niversity of N
ebraska-Lincoln Libraries user on 16 Septem
ber 2019
Translate basic science to industry innovation
S148 Duffy et al.
Barnes, T. L., R. M. Kubik, C. N. Cadaret, K. A. Beede, E. M. 
Merrick, S. Chung, T. S. Schmidt, J. L. Petersen, and D. T. 
Yates. 2017. WS identifying hyperthermia in heat-stressed 
lambs and its effects on β agonist–stimulated glucose oxi-
dation in muscle. Proc. West. Sec. Am. Soc. Anim. Sci. 
63:25–29. doi:10.2527/asasws.2017.0038
Boler, D. D., A.  L. Shreck, D. B. Faulkner, J. Killefer, F. K. 
McKeith, J. W. Homm, and J. A.  Scanga. 2012. Effect 
of ractopamine hydrochloride (Optaflexx) dose on live 
animal performance, carcass characteristics and tender-
ness in early weaned beef steers. Meat Sci. 92(4):458–463. 
doi:10.1016/j.meatsci.2012.05.011
Buntyn, J. O., N.  C. Burdick Sanchez, T. B. Schmidt, G. E. 
Erickson, S. E. Sieren, S. J. Jones, and J. A. Carroll. 2016. 
The metabolic, stress axis, and hematology response of 
zilpaterol hydrochloride supplemented beef heifers when 
exposed to a dual corticotropin-releasing hormone and 
vasopressin challenge. J. Anim. Sci. 94(7):2798–2810. 
doi:10.2527/jas.2015-0192
Cadaret, C. N., K. A. Beede, H. E. Riley, and D. T. Yates. 2017. 
Acute exposure of primary rat soleus muscle to zilpa-
terol HCl (β2 adrenergic agonist), TNFα, orIL-6 in cul-
ture increases glucose oxidation rates independent of the 
impact on insulin signaling or glucose uptake. Cytokine 
96:107–113. doi:10.1016/j.cyto.2017.03.014
Caporaso, J. G., J. Kuczynski, J. Stombaugh, K. Bittinger, F. 
D.  Bushman, E. K.  Costello, N.  Fierer, A. G.  Peña, J. 
K.  Goodrich, J. I.  Gordon, et  al. 2010. QIIME allows 
analysis of high-throughput community sequencing data. 
Nat. Methods 7:335–336. doi:10.1038/nmeth.f.303
Delmore, R. J., J. M.  Hodgen, and B. J.  Johnson. 2010. 
Perspectives on the application of zilpaterol hydro-
chloride in the United States beef industry. J. Anim. Sci. 
88(8):2825–2828. doi:10.2527/jas.2009-2473
Edgar, R. C. 2013. UPARSE: highly accurate OTU sequences 
from microbial amplicon reads. Nat. Methods 10:996–
998. doi:10.1038/nmeth.2604
Edgar, R. C., B. J.  Haas, J. C.  Clemente, C.  Quince, and 
R.  Knight. 2011. UCHIME improves sensitivity and 
speed of chimera detection. Bioinformatics 27:2194–2200. 
doi:10.1093/bioinformatics/btr381
Jami, E., A.  Israel, A.  Kotser, and I.  Mizrahi. 2013. 
Exploring the bovine rumen bacterial community from 
birth to adulthood. ISME J. 7:1069–1079. doi:10.1038/
ismej.2013.2
Kozich, J. J., S. L. Westcott, N. T. Baxter, S. K. Highlander, and 
P. D. Schloss. 2013. Development of a dual-index sequenc-
ing strategy and curation pipeline for analyzing amplicon 
sequence data on the MiSeq illumina sequencing platform. 
Appl. Environ. Microbiol. 79(17):5112–5120. doi:10.1128/
AEM.01043-13
McCann, J. C., T. A. Wickersham, and J. J. Loor. 2014. High-
throughput methods redefine the rumen microbiome and 
its relationship with nutrition and metabolism. Bioinform. 
Biol. Insights 8:109–125. doi:10.4137/BBI.S15389
Paz, H. A., C. L.  Anderson, M. J.  Muller, P. J.  Kononoff, 
and S. C.  Fernando. 2016. Rumen bacterial commu-
nity composition in Holstein and Jersey cows is differ-
ent under same dietary condition and is not affected by 
sampling method. Front. Microbiol. 7:1206. doi:10.3389/
fmicb.2016.01206
Paz, H. A., K. E. Hales, J. E. Wells, L. A. Kuehn, H. C. Freetly, 
E. D.  Berry, M. D.  Flythe, M. L.  Spangler, and S. 
C.  Fernando. 2018. Rumen bacterial community struc-
ture impacts feed efficiency in beef cattle. J. Anim. Sci. 
96(3):1045–1058. doi: 10.1093/jas/skx081
Petri, R. M., T. Schwaiger, G. B. Penner, K. A. Beauchemin, 
R. J.  Forster, J. J.  McKinnon, and T. A.  McAllister. 
2013. Characterization of  the core rumen micro-
biome in cattle during transition from forage to 
concentrate as well as during and after an acidotic 
challenge. PLoS ONE 8(12):e83424. doi:10.1371/
journal.pone.0083424
Roh, S. G., M. Kuno, D. Hishikawa, Y. H. Hong, K. Katoh, 
Y.  Obara, H.  Hidari, and S.  Sasaki. 2007. Identification 
of differentially expressed transcripts in bovine rumen and 
abomasum using a differential display method. J. Anim. 
Sci. 85:395–403. doi:10.2527/jas.2006-234
Schloss, P. D., S. L.  Westcott, T.  Ryabin, J. R.  Hall, 
M.  Hartmann, E. B.  Hollister, R. A.  Lesniewski, B. 
B.  Oakley, D. H.  Parks, C. J.  Robinson, et  al. 2009. 
Introducing mothur: open-source, platform-inde-
pendent, community-supported software for describ-
ing and comparing microbial communities. Appl. 
Environ. Microbiol. 75:7537–7541. doi:10.1128/
AEM.01541-09
Segata, N., J. Izard, L. Waldron, D. Gevers, L. Miropolsky, W. 
S. Garrett, and C. Huttenhower. 2011. Metagenomic bio-
marker discovery and explanation. Genome Biol. 12:R60. 
doi:10.1186/gb-2011-12-6-r60
Tajima, K., I. Nonaka, K. Higuchi, N. Takusari, M. Kurihara, 
A.  Takenaka, M.  Mitsumori, H.  Kajikawa, and R. 
I.  Aminov. 2007. Influence of high temperature and 
humidity on rumen bacterial diversity in Holstein 
heifers. Anaerobe 13(12):57–64. doi:10.1016/j.
anaerobe.2006.12.001
D
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ic.oup.com
/tas/article-abstract/2/suppl_1/S145/5108324 by U
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ebraska-Lincoln Libraries user on 16 Septem
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Rumen bacterial composition in lambs is affected by β-adrenergic agonist
supplementation
and heat stress at the phylum level

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=2067&context=animalscifacpub

Rumen bacterial composition in lambs is affected by β-adrenergic agonist supplementation and heat stress at the phylum level

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