J. Dairy Sci.
103:8601–8614
https://doi.org/10.3168/jds.2020-18417
© 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The effect of chronic, mild heat stress on metabolic changes of nutrition
and adaptations in rumen papillae of lactating dairy cows
Mehdi Eslamizad,1 Dirk Albrecht,2 and Björn Kuhla1*
1
Institute of Nutritional Physiology “Oskar Kellner,” Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf,
Germany
2
Institute of Microbiology, Ernst-Moritz-Arndt-University, Felix-Hausdorff-Straße 8, 17487 Greifswald, Germany
ABSTRACT
Global warming and accompanying high ambi-
ent temperatures reduce feed intake of dairy cows
and shift the blood flow from the core of the body
to the periphery. As a result, hypoxia may occur in
the digestive tract accompanied by disruption of the
intestinal barrier, local endotoxemia and inflammation,
and altered nutrient absorption. However, whether the
barrier of the rumen, like the intestine, is affected by
ambient heat has not been studied so far. Lactating
Holstein dairy cows were subjected to heat stress at
28°C (temperature-humidity index = 76; n = 5) with
ad libitum feed intake or to thermoneutral conditions
at 15°C (temperature-humidity index = 60; n = 5) and
pair-feeding to heat-stressed animals for a total of 4
d. Gas exchange and feed intake behavior were mea-
sured in a respiration chamber, and rumen epithelia
were taken after slaughter. Heat stress significantly
reduced meal size and whole-body fat oxidation but
increased meal frequency and carbohydrate oxidation.
The mRNA expression of toll-like receptor 4 (TLR4)
and tight junction proteins and the phosphorylation
of TLR4 downstream targets (interleukin-1 receptor-
associated kinase 4, stress-activated protein kinase,
p38 mitogen-activated protein kinase, and nuclear
factor k-B) in the rumen epithelium were not affected
by heat. The proteomics approach revealed increased
expression of rumen epithelium proteins involved in
the AMP-activated protein kinase (AMPK) and insulin
signaling pathways in heat-stressed cows. Also, proteins
involved in chaperone-mediated folding of proteins were
upregulated, whereas those involved in antioxidant de-
fense system were downregulated. Further, we found
evidence for increased carbohydrate phosphorylation
accompanied with an increased flux of carbohydrates
through the hexosamine biosynthetic pathway, provid-
Received February 24, 2020.
Accepted April 18, 2020.
*Corresponding author: b.kuhla@​fbn​-dummerstorf​.de
8601
ing substrates for protein glycosylation. In conclusion,
the mild heat stress did not induce barrier dysfunction
or inflammatory responses in the rumen epithelium of
dairy cows, probably because of adaptations in feed
intake behavior and defense mechanisms at the tissue
level.
Key words: heat stress, rumen, feeding behavior, tight
junctions, toll-like receptor
INTRODUCTION
Heat stress is a major concern for dairy cows, espe-
cially in tropic and subtropic areas, because it compro-
mises animal health, welfare, and milk production, all
of which cause significant economic losses (St-Pierre
et al., 2003). Heat stress is becoming even more of an
issue with the continuous elevation of the mean globe
temperature (IPCC, 2007). The effect of heat stress on
performance, behavioral and physiological adaptation,
metabolism, and immune system of cattle has been
extensively reported. For example, heat-stressed cows
significantly reduce their feed intake to avoid extra heat
load generated from digestion and metabolic process-
ing of nutrients (Baumgard and Rhoads, 2012). Tra-
ditionally, the decline in milk production during high
environmental temperatures has been attributed to the
reduction of feed intake (Beede and Collier, 1986; West,
1999). However, it was recently stated that the decline
in feed intake is not fully responsible for the reduction
in milk yield of heat-stressed dairy cows (Rhoads et al.,
2009a; Wheelock et. al., 2010; Baumgard et al., 2011).
By the use of pair-feeding trials, in which ad libitum fed
heat-stressed cows were compared with counterparts
kept under thermoneutral conditions while receiving the
same amount of feed as heat-stressed animals ingested,
it was shown that heat stress exerts direct effects on the
metabolism and the endocrine systems of cows beyond
the concomitant effect of energy intake restriction.
Those changes may include an increase in basal and
stimulated insulin concentrations (O’Brien et al., 2010;
Wheelock et al., 2010) and alterations in the macronu-
 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT
trient metabolism favoring the utilization of carbohy-
drates and proteins at the expense of fat (Lamp et al.,
2015). Increased carbohydrate utilization is evident by
lower blood glucose levels, greater glucose disposal af-
ter insulin injection, increased hepatic gluconeogenesis,
augmented depletion of liver glycogen stores, and im-
mune cell activation under heat stress (Baumgard and
Rhoads, 2013). However, the biochemical mechanisms
accounting for the increased carbohydrate utilization
are still not fully understood. For example, it is still
unclear if the absorption of the primary gluconeogenic
precursor propionate via the rumen wall is affected by
heat stress.
Early studies reported the alteration of the rumen-
intestinal epithelium during heat stress. Under condi-
tions of high ambient temperatures, blood flow is di-
verted from the viscera to the periphery (Kregel, 2002;
Lambert et al., 2002), leading to a local oxygen and
nutrient deprivation at the intestinal epithelium and
an ultimate reduction of the intestinal barrier function
(Pearce et al., 2013). As a consequence of an increased
intestinal epithelial permeability, the transmission of
microbial components from the intestinal tract into the
circulation may increase (Lim et al., 2007; Pearce et
al., 2013), suggesting a central role of the intestine, and
potentially the rumen, in endotoxemia and heat stroke
pathophysiology (Leon, 2007; Rhoads et al., 2009b).
The intestinal toll-like receptor 4 (TLR4) is activated
in response to injury of the gut (i.e., after damage of the
intestinal epithelial lining; Abreu, 2010). Recognition of
LPS by TLR4 triggers cellular signaling through in-
terleukin-1-receptor-associated kinases (IRAK), which
ultimately results in the activation of nuclear factor-
kB (NF-κB) and mitogen-activated protein kinases
(MAPK). Whether the activation of the TLR4 path-
way in the rumen papillae is also involved in heat-stress
induced endotoxemia and which molecular mechanisms
in the rumen epithelium contribute to increased whole-
body carbohydrate utilization in heat-stressed dairy
cows have not been studied so far. We hypothesized
that (1) heat stress would cause loosening of the tight
junctions in the rumen epithelium of dairy cows and
that the resulting exposure to luminal bacterial com-
pounds would activate local inflammatory responses via
the TLR4 signaling pathway and (2) heat stress affects
global expression of rumen papillae proteins including
those regulating absorption and facilitating whole-body
carbohydrate utilization. To pursue these hypotheses,
we exposed mid-lactation dairy cows for 4 d to either
continuous heat stress or pair-feeding at thermoneu-
trality, sampled the rumen papillae, and performed
targeted Western blot and PCR analyses to evaluate
expression of tight junction and TLR4 pathway pro-
teins and RNA, as well as untargeted proteomics; the
Journal of Dairy Science Vol. 103 No. 9, 2020
8602
latter approach can be useful for the search of welfare
and stress biomarkers in cattle.
MATERIALS AND METHODS
Animals and Treatments
All procedures were approved by the ethics com-
mittee of the state government in Mecklenburg-West
Pomerania, Germany (LALLF M-V/TSD/7221.3–
1.1–074/12), and the data described herein are part
of a recently published study (Vanselow et al., 2016).
More specifically, 10 German Holstein dairy cows in
established second lactation (192 ± 20 DIM) were kept
under the same normal environmental conditions in a
freestall barn at the Leibniz Institute for Farm Animal
Biology (FBN) with daily ad libitum feeding. The ra-
tion consisted on the DM bases of 6.12 kg of corn silage,
6.6 kg of grass silage, 0.9 kg of wheat straw, 1.7 kg of
hay, 2.1 kg of concentrate, 1.1 kg of corn meal, 0.8 kg
of extracted canola meal, 0.3 kg of extracted soybean
meal, 0.3 kg of wheat, and 0.2 kg of minerals, with a
total ME = 10.6 MJ/kg of DM.
Cows were transported to a climate-controlled cham-
ber and kept at thermoneutral conditions [15°C, 64%
relative humidity, and temperature-humidity index
(THI) = 60] for 6 d to measure ad libitum feed intake.
Thereafter, 5 randomly selected cows were exposed to
28°C (52% relative humidity, THI = 76) with ad libitum
feeding (heat stress group; HS). The ad libitum feed in-
take of 1 HS cow during heat exposure was calculated as
percentage of the mean daily intake determined for the
6-d ad libitum feeding period at THI = 60. The remain-
ing 5 cows were kept continuously at 15°C (THI = 60)
but, with a 1-d time lag, were pair-fed (PF) to allocate
the same level of feed intake per kilogram of BW as
HS cows consumed. This experimental design allowed
us to differentiate between feed intake and heat-stress
effects. Cows were housed in the climate chamber for
2.5 d, immediately transferred to a climate-controlled
respiration chamber, and kept for another 36 h under
the same climatic and feeding conditions, amounting to
a total of 4 d of PF or HS challenge. The stay in the
respiration chamber included a 12-h gas-equilibration
period, followed by a 24-h gas-exchange measuring pe-
riod. In climate and respiration chambers, cows were
fed at 0700 and 1600 h and milked at 0630 and 1630 h.
In parallel to the milking in respiration chamber, the
rectal temperature was measured.
Macronutrient Metabolism and Feed Intake Behavior
The dynamics of feed and water intake, gas exchange,
and physical activity was recorded for 24 h as previous-
 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT
ly described by (Derno et al., 2009). Briefly, individual
feed intake was measured every 6 min by feed disap-
pearance from the feeding bin located on a scale and
connected to an electronic registration device. A meal
was defined when feed intake was greater than 200 g/6
min and lasted for more than 2 consecutive measuring
intervals. Water intake was measured by a water meter
connected to an electronic registration device. Physical
activities were detected by a modified infrared-based
motion detector (IS 120, Steinel, Herzebrock-Klarholz,
Germany) by converting movements of the animal to
the impulses. Concentrations of CO2 and CH4 in the
chamber were analyzed by infrared absorption (UNOR
610, Maihak, Hamburg, Germany), and the concentra-
tion of O2 was analyzed paramagnetically (OXYGOR
610, Maihak) every 6 min. Subsequently, based on the
amount of O2 consumed and CO2 and CH4 produced,
metabolic heat production, fat oxidation, and carbohy-
drate oxidation were calculated (Eslamizad et al., 2015).
Immediately after the stay in the respiration chamber,
cows were transported to the institute’s slaughterhouse
(300 m) within 10 min. After captive bolt stunning and
exsanguination of the cows, tissue samples from the
rumen papillae (~30 cm lateral from the esophageal
entrance) were obtained within 15 min, snap-frozen in
liquid nitrogen, and stored at −80°C.
Western Blot
Whole proteins from rumen tissue were extracted
in a lysis buffer containing 50 mM Tris-HCl (pH 7.8),
1 mM EDTA, 10 mM NaF, 1% IGEPAL CA-630
(Sigma-Aldrich, St. Louis, MO), 0.1% Triton X100,
0.5% deoxycholic acid, 0.1% SDS, and cocktail protease
inhibitor (Roche, Basel, Switzerland). Protein concen-
trations were measured immediately after extraction
using a Bradford assay kit (Thermo Fisher Scientific,
Waltham, MA), and the extracts were stored at −80°C
for later use. Equal protein amounts (50 µg) of boiled
samples were separated by SDS-PAGE and electro-
transferred onto nitrocellulose membranes (Whatman
Protran BA 83, Dassel, Germany). Membranes were
stained with Ponceau S to confirm equal loading.
Membranes were blocked for 1 h in Tris-buffered saline-
Tween 20 (TBST) solution (20 mM Tris, 0.9% NaCl,
0.1% Tween 20, pH 7.4) containing 3% BSA before
incubation at 4°C with primary antibody in the same
solution overnight. The following rabbit antibodies and
dilutions were used: interleukin-1 receptor-associated
kinase 4 (IRAK4; #4363; 1:500) and phosphorylated
(p)-IRAK (p-IRAK4; #11927; 1:300); p38 mitogen-
activated protein kinase (p38MAPK; #9212; 1:1,000)
and p-p38MAPK (#9211; 1:500); stress-activated
protein kinase (SAPK)/c-Jun-N-terminal kinase (JNK)
Journal of Dairy Science Vol. 103 No. 9, 2020
8603
(#9252; 1:1,000) and p-SAPK/JNK (#9251; 1:300);
and NF-κB (#4764; 1:500) and p-NF-κB (#3033;
1:300; all from Cell Signaling Technology, Cambridge,
UK). The membranes were washed 3 times with TBST
and incubated with horseradish peroxidase-conjugated
goat anti-rabbit secondary antibody (1:10,000; BD
Pharmingen Inc., San Diego, CA) for 1 h. Finally, blots
were washed again 3 times in TBST followed by incuba-
tion with chemiluminescent reagent for 1 min (Thermo
Fisher Scientific) and final exposure to hyperfilms (GE
Healthcare, Chalfont St Giles, UK). After detection of
bands, hyperfilms were scanned and signal intensities
were quantified using ImageJ 1.49 software (https:​/​/​
imagej​.nih​.gov/​ij/​download​.html). The ratio between
phosphorylated and nonphosphorylated forms of above-
mentioned proteins was calculated.
Quantitative Reverse Transcription-PCR Analysis
Tissue was crushed with a mortar and pestle in liquid
N2 and total RNA was extracted from 65 mg of tissue
powder with TriFast Reagent (Peqlab, Erlangen, Ger-
many) and incubated with DNaseI digest (innu PREP,
Analytik Jena, Jena, Germany) according to the manu-
facturer’s instructions. The RNA quality was assessed
using an Agilent 2100 Bioanalyzer (Santa Clara, CA),
yielding RNA integrity number factors between 7.1
and 9.1 (mean 8.6). Complementary DNA was synthe-
sized (375 ng of total RNA) using Revert Aid Reverse
Transcriptase (Thermo Fisher Scientific, Dreieich,
Germany) and random Hexamer primers (Metabion
International, Planegg/Steinkirchen, Germany) and
stored at −80°C until use. Intron overspanning primers
were designed using Primer3 Plus software (Untergas-
ser et al., 2012) or PrimerBLAST (Ye et al., 2012).
Complementary DNA was amplified in triplicate on a
Light Cycler 2.0 (Roche) using specific primers (Sup-
plemental Table S1, https:​/​/​doi​.org/​10​.3168/​jds​.2020​
-18417) and Light Cycler FAST DNA Master PLUS
SYBR Green I Reaction Mix (Roche). Amplicons were
sequenced on an ABI 3130 Genetic Analyzer (Life
Technologies GmbH, Darmstadt, Germany) and correct
sequence was confirmed. The efficiency of amplifica-
tion was calculated using LinRegPCR software, version
2014.4.1.1 (Ruijter et al., 2013). The gene expression
stability of 3 candidate reference genes—CKLF-like
MARVEL transmembrane domain-containing protein 6
(CMTM6), 60S acidic ribosomal protein P0 (RPLP0),
and ELKS/Rab6-interacting/CAST family member 1
(ERC1)—was determined in geNorm, and RPLP0 and
ERC1 were used as references genes for final analysis.
Efficiency-corrected data were analyzed as the ratio
between the target gene mRNA abundance and the
geometric mean of the reference genes using qbase Plus
 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT
software (Biogazelle, Gent, Belgium), and the ratio is
presented as relative expression.
Proteome Analysis
A total of 50 µg of protein from tissue extracts was
loaded on a 1-dimensional 12% SDS-PAGE gel and the
protein bands were stained with colloidal Coomassie
(Roti-Blue, Carl Roth, Germany). Fourteen slices per
lane (1 lane per animal) were excised using a sterile
scalpel. Stained gel slices were transferred into 1.5-mL
Eppendorf tubes (Hamburg, Germany) and washed
twice with 100 µL of a solution of 50% CH3OH and
50% 50 mM NH4HCO3 for 30 min, and once with 100
µL of 75% CH3CN for 10 min. After drying at 37°C for
20 min, 10 µL of trypsin solution containing 4 µg/mL
porcine trypsin (Promega, Madison, WI) was added
and incubated overnight at 37°C. For extraction, gel
bands were covered with 60 µL of 0.1% trifluoroacetic
acid in 50% CH3CN and incubated for 30 min under
shaking. The peptide-containing supernatant was
transferred into a clear glass vial and dried at 45°C for
100 min in a concentrator (Eppendorf). The dry pep-
tides (nonreduced or alkylated) were resuspended in
10 µL of 50%/49.5%/0.5% (vol/vol/vol) CH3CN/H2O/
trifluoroacetic acid) and separated and measured online
by electrospray ionization (ESI)-mass spectrometry us-
ing a Proxeon Easy nLCII- system (Thermo Scientific)
coupled to a Thermo Scientific LTQ Orbitrap-XL mass
spectrometer. A gradient of 0.5%/min (buffer A =
0.1% formic acid in water, Optima LC/MS; buffer B
= 0.1% formic acid in 99.9% acetonitrile, Optima LC/
MS; Fisher Scientific) was used. Data were acquired on
an LTQ Orbitrap-XL mass spectrometer using a 0.1 ×
200 mm column with C18 Aeris Peptide (Phenomenex,
Torrance, CA) at a flow rate of 0.3 µL/min. For MS and
MS/MS analyses, a full survey scan in the Orbitrap-XL
with a mass range (m/z 300–2,000) and a Fourier trans-
form (FT) resolution of 30,000 was followed by data-
dependent fragmentation experiments of the 5 most
intense ions. Data were acquired in a data-dependent
“top 5” format, selecting the most abundant precursor
ions from the FTMS scan (mass range 300–2,000 Da).
The FTMS scans were acquired with a resolution of
30,000 and a target value of 1.2 × 106 in the Orbitrap
analyzer. The ion-trap MS scans were acquired with
unit mass resolution in the LTQ using 3,000 as target
value, 2 as the default charge state, and a lower inten-
sity threshold for MS2 of 1,750 counts. The normalized
collision energy in the collision-induced dissociation
was 35 eV and a dynamic exclusion was defined by
a list size of 500 with exclusion duration of 30 s. The
spectra were acquired in the LTQ via collision-induced
dissociation. The parameters for the dynamic exclusion
Journal of Dairy Science Vol. 103 No. 9, 2020
8604
list are as follows: repeat count = 1, repeat duration =
30 s, exclusion list size = 500, and exclusion duration =
30. Data files were searched against the National Center
for Biotechnology Information Bovine database (www​
.ncbi​.nlm​.nih​.gov/​) using SEQUEST with the common
contaminant ‘kreatin’ specified. The Sequest search was
carried out considering the following parameters: parent
ion mass tolerance of 10 ppm, fragment ion mass toler-
ance of 0.50 Da, and Met oxidation (+15.99492 Da).
Each Sequest search included the data from all 14 gel
slices per lane and results loaded into Scaffold software
(version 4.8.7, Proteome Software Inc., Portland, OR).
The Scaffold software was used to validate MS/MS
based peptide and protein identifications. Peptide iden-
tifications were accepted if they could be established
at greater than 93.0% probability to achieve a false
discovery rate less than 0.1% by the Peptide Prophet
algorithm (Keller et al., 2002) with Scaffold delta-mass
correction. Protein identifications were accepted if
they could be established at greater than 70.0% prob-
ability to achieve a false discovery rate less than 1.0%
and contained at least 2 identified peptides. Protein
probabilities were assigned by the Protein Prophet
algorithm (Nesvizhskii et al., 2003). Proteins that
contained similar peptides and could not be differenti-
ated based on MS/MS analysis alone were grouped to
satisfy the principles of parsimony. Relative quantifi-
cation was performed using the “Normalized NSAF”
tool, and the t-test feature of the Scaffold software was
used to identify differentially expressed proteins at P
< 0.05. Results were exported as a Microsoft Excel file
(Supplemental Table S2, https:​/​/​doi​.org/​10​.3168/​jds​
.2020​-18417).
Statistics and Bioinformatics Approach
For Western blot, PCR, and respiration chamber
data, differences between HS and PF cows were tested
using unpaired t-tests of SAS (SAS Institute Inc., Cary,
NC). For proteome analysis, differentially expressed
proteins were submitted to the ClueGo App of the Cy-
toscape software version 3.7.0 (Shannon et al., 2003).
Upregulated and downregulated proteins were assigned
to the software as different clusters. The protein list
was loaded to the Gene Ontology and Kyoto Encyclo-
pedia of Genes and Genomes (KEGG; www​.genome​
.jp/​kegg/​?sess​=​ebfe2ad23e021e38540f798c803dd061)
database. The ontology selection on the base of biologi-
cal processes, immune system activation, and KEGG
was performed by the right-side hypergeometric statis-
tic test and its probability was corrected by the Bonfer-
roni stepdown method. The MS proteomics data has
been uploaded to the ProteomeXchange Consortium
(http:​/​/​www​.proteomexchange​.org/​) via the PRIDE
 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT 8605

repository with the data set identifier PXD016510 and
10.6019/PXD016510.
RESULTS
Milk Yield and Feed Intake During Ad Libitum
Feeding at Thermoneutrality
Daily milk yield during ad libitum feeding at ther-
moneutral conditions (HS: 29.5 ± 1.7 L, PF: 26.0 ±
1.7 L, P = 0.18), as well as during the challenge period
(HS: 22.1 ± 1.6 L, PF: 22.4 ± 1.9 L, P = 0.88), did
not differ between PF and HS cows. The percentage
decrease in milk yield amounted to 25% for HS and
14% for PF cows. During the ad libitum feeding period
at THI = 60, DMI was comparable between HS (17.2
± 1.6 kg/d) and PF (16.7 ± 3.6 kg/d) cows (P = 0.05).
Additionally, DMI normalized to BW was not different
between groups (HS: 3.20 ± 0.37 kg/d vs. PF: 2.40 ±
0.64 kg/d, P = 0.32). However, BW was greater in PF
than HS cows (Table 1).
Feeding Behavior, Macronutrient Oxidation,
and Physical Activity During Challenge
During the challenge period, DMI was again greater
in PF than HS cows, but according to the experimental
design, DMI normalized to BW did not differ between
HS and PF cows (Table 1). Accordingly, metabolic heat
production was comparable between groups (P = 0.27),
but the mean rectal temperature was greater for HS
than PF cows (40.2 ± 0.3°C vs. 38.4 ± 0.1°C, P <
0.05). The HS cows consumed DM at slower rate than
PF cows (P < 0.05). This was associated with more fre-
quent meals, smaller meal sizes, and shorter duration of
meals in HS relative to PF cows (P < 0.05). The mean
time interval between feeding bouts was also shorter in
HS than PF cows (P < 0.05), while the total time spent
eating or daily water intake was not affected between
challenges. However, HS cows had a higher drinking
frequency and consumed more water per unit of BW
(P < 0.05) and tended to oxidize higher amounts of
carbohydrates per kilogram of DMI than PF cows (P
= 0.07). According to the feeding regimen, PF cows
consumed their last meal, which was 2.0 to 17.9 kg of
OM, 12 to 15 h before slaughter. In contrast, HS cows
consumed their last meal, which was 0.6 to 11.0 kg, 2.5
to 17 h before slaughter.
The TLR4 Pathway and TJ Proteins
To examine if there is heat stress–specific activation
of the TLR4 pathway in the rumen papillae, RT-PCR
and Western blot analyses were performed. There was
no significant difference in abundance of TLR4 mRNA
or in phosphorylation of the downstream targets of
TLR4 involving IRAK4, p38MAPK, SAPK/JNK, and
NF-κB (Figure 1), indicating lack of a heat stress–
specific inflammatory response in the rumen papillae.
Comparably, the mRNA abundances of tight junction
proteins, including CLDN3, OCLN, TJP1, TJP2, were
also not different between HS and PF cows (Figure 2).

Table 1. Dry matter intake, feeding behavior, macronutrient oxidation, and activity of mid-lactation dairy
cows after 4 d of continuous heat stress (HS) compared with pair-fed (PF) cows

Treatment

Item1 HS PF P-value
BW, kg 546 ± 24 688 ± 29 0.01
DMI, kg/d 8.7 ± 1.1 13.0 ± 1.0 0.02
DMI·BW−1·100 1.59 ± 0.16 1.75 ± 0.13 0.49
Meal size, kg of DM·meal−1 0.76 ± 0.12 4.14 ± 1.37 0.03
Meal duration, min·meal−1 28.2 ± 2.8 57.1 ± 9.1 0.01
Meal frequency, d−1 11.6 ± 0.9 4.5 ± 1.9 0.01
Eating rate, kg of DM·min−1 0.032 ± 0.004 0.067 ± 0.017 0.06
Eating time, min·d−1 241.2 ± 18.9 175.5 ± 36.2 0.13
Inter-meal interval, min 96.9 ± 10.6 429.2 ± 133.3 0.03
Water intake, mL·kg of BW−1·d−1 91.7 ± 5.8 69.7 ± 7.0 0.04
Water intake per event, L 2.45 ± 0.17 3.46 ± 0.59 0.11
Drinking frequency, d−1 20.6 ± 0.8 14.3 ± 2.0 0.02
COX, g·kg−0.75·DMI−1·d−1 3.99 ± 0.29 2.97 ± 0.41 0.07
FOX, g·kg−0.75·d−1 10.56 ± 1.26 9.04 ± 0.64 0.36
Activity, 1,000 pulses·d−1 139.6 ± 29.6 336.1 ± 36.6 <0.01
Prandial HPR, kJ·BW−0.75·meal−1 37.9 ± 3.2 192.7 ± 68.7 0.04
mHP, kJ·BW−0.75·d−1 990 ± 26 931 ± 39 0.27
1
COX = carbohydrate oxidation; FOX = fat oxidation; HPR = heat production rise; mHP = metabolic heat
production.

Journal of Dairy Science Vol. 103 No. 9, 2020

 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT 8606

Figure 1. Effects of 4 d of continuous heat stress (28°C; temperature-humidity index, THI = 76) and thermoneutrality (15°C; THI = 60) on
the activation of the TLR4 signaling pathway in rumen epithelium of dairy cows. The mRNA abundance of TLR4 was normalized to 2 reference
genes, RPLP0 and ERC1 (A). Western blot analysis of the abundance of phosphorylated (p) stress-activated protein kinase (SAPK; B), inter-
leukin-1 receptor-associated kinase 4 (IRAK4; C), p38 mitogen-activated protein kinase (MAPK; D), and nuclear factor k-B (NF-kB; E) were
normalized to the corresponding nonphosphorylated form of each protein. Data are represented as the mean and SE from 5 heat stress (HS) and
5 pair-fed (PF) cows and P-values were from Student’s t-test.

Proteome Analysis comparison using the Scaffold software revealed 154

differentially (P < 0.05) expressed proteins between
A total of 1,086 to 1,642 rumen papillae proteins groups, with 92 upregulated and 62 downregulated
were identified for each animal by MS/MS. The t-test proteins in HS relative to PF cows (Supplemental Table

Journal of Dairy Science Vol. 103 No. 9, 2020

 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT 8607

Figure 2. The mRNA abundances of rumen tight junction proteins, involving CLDN3 (A), OCLN (B), TJP1 (C), and TJP2 (D) in dairy
cows exposed to continuous heat stress (HS; 28°C; temperature-humidity index, THI = 76) or pair-fed thermoneutral conditions (PF; 15°C; THI
= 60) for 4 d. Data are presented as the mean and SE from 5 HS and 5 PF cows and P-values were from Student’s t-test.

S2, https:​/​/​doi​.org/​10​.3168/​jds​.2020​-18417). Submis-
sion of the list comprising differentially expressed pro-
teins to Gene Ontology Biological Process and KEGG
pathway analysis extracted 55 proteins, from which
33 activated and deactivated pathways were identified
based on the P < 0.05 criterion (Figure 3). We did not
find any term or pathway associated with either the
immune system or the transport of SCFA or minerals
although some of those proteins were identified (Sup-
plemental Table S2). Rather, the majority of differ-
entially expressed proteins were found to be involved
in carbohydrate and protein catabolism, as well as in
the cellular heat-shock response, cellular trafficking,
and antioxidant defense. More specifically, enzymes
involved in glycolysis and gluconeogenesis, particularly
in the phosphorylation and activation of single sugars
such as xylulose kinase, fucokinase, and ribokinase,
were upregulated in HS relative to PF cows (Supple-
mental Table S2). In addition, the enzyme glutamine:​
fructose​-6​-phosphate transaminase 1 (GFPT1), which
controls the flux of glucose into the hexosamine path-
way and thus protein glycosylation, was more highly
expressed in HS than PF cows (Supplemental Table
S2). Increased glycolysis, together with increased ex-
pression of ATP citrate synthase (ACLY), acyl-CoA
synthetase short chain family member 2 (ACSS2), and
dihydrolipoyl dehydrogenase (DLD; Table 2), should
increase acetyl-CoA (Ac-CoA) biosynthesis (Table 2,
Figure 3). On the other hand, enzymes of the fatty
acid β-oxidation pathway producing Ac-CoA, such as
enoyl-CoA hydratase and 3-hydroxy acetyl-CoA dehy-
drogenase, were downregulated under heat stress com-
pared with isoenergetic conditions at thermoneutrality
(Supplemental Table S2). Reduced expression of the
mitochondrial respiratory chain enzymes cytochrome
C oxidase (subunit 2) and ATP synthase (subunit B1)
diminish the ATP tone and support the activation
of the 5′ adenosine monophosphate-activated protein
kinase (AMPK) and the insulin signaling pathways of
HS cows via upregulation of calcium-binding protein
39 (CAB39), calcium-binding protein 39-like (CA-
B39L), ELAV-like RNA binding protein 1 (ELAVL1),
fructose-bisphosphatase 1 (FBP1), protein phospha-
tase 2 regulatory subunit B α (PPP2R2A), protein
kinase AMP-activated noncatalytic subunit gamma 1
(PRKAG1), Ras-related protein Rab-11B (RAB11B),

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 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT 8608

Ras-related protein Rab-14 (RAB14), and the adapter
molecules crk (CRK), FBP1, flotillin 2 (FLOT2),
PRKAG1, AMP-dependent protein kinase type II-α
(PRKAR2A), and type II-β (PRKAR2B) proteins,
respectively (Table 2). Moreover, upregulation of pro-
teins such as cullin 1 through 4 (CUL1, CUL2, CUL3,
CUL4A), eukaryotic translation initiation factor 2
subunit 1 (EIF2S1), heat shock protein (HSP) 105
kDa (HSPH1), protein OS-9 (OS9), phospholipase A-
2-activating protein (PLAA), protein transport protein
Sec23A (SEC23A) and 61A1 (SEC61A1), phosducin-
like protein 3 (PDCL3), chaperonin containing TCP1
subunit 4 (CCT4), cysteine and histidine-rich domain-
containing protein 1 (CHORDC1), HSPH1, heat
shock protein β-1 (HSPB1), axin interactor (AIDA),
CHORDC1, and PRKAR2A support protein process-
ing in the endoplasmic reticulum, protein ubiquitina-
tion and protein degradation, chaperone-mediated
protein folding, as well as the inhibition of serine and
threonine kinase activity in rumen epithelium of HS
relative to PF cows (Figure 3). Vice versa, downregu-
lation of dynactin subunit 1 (DCTN1), heterogeneous
nuclear ribonucleoprotein U (HNRNPU), nuclear
mitotic apparatus protein 1 (NUMA1), myosin 5A
(MYO5A), RAB11B, aminopeptidase N (ANPEP),
glutathione S-transferase A5 (GSTA5), glutathione S-
transferase Mu 1 (GSTM1), and P1 (GSTP1) reduces
mitotic spindle organization, melanosome transport,
while compromising keratin and pigment metabolism
and glutathione metabolism (Table 2, Figure 3). Al-
though the regulation of oxidative stress–induced cell
death was classified as deactivated in HS compared
with PF cows, the process involved increased expres-
sion of HSPH1 and SFPQ, but reduced abundance of

Figure 3. Functional classification of differentially expressed proteins in rumen papillae of dairy cows after 4 d of continuous heat stress based
on Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes pathway analyses using ClueGo software (Shannon et al.,
2003; P < 0.05). Thirty-three activated and deactivated pathways (number of nodes) were identified based on the P < 0.05 criterion. Circles in
red represent pathways or processes with upregulated proteins (cluster 1) and blue circles indicate processes with downregulated proteins (cluster
2). The node size refers to the number of differentially regulated proteins involved in each process or pathway, and the intensity of the node color
represents the degree of specificity of the process or pathway to clusters. Gray nodes represent relatively low specificity of regulated processes
because up- and downregulated proteins are involved in these processes. AMPK = AMP-activated protein kinase.

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 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT 8609
Table 2. Leading terms of the enriched Gene Ontology biological process or Kyoto Encyclopedia of Genes and Genomes pathway analysis and
differentially expressed proteins involved in the rumen epithelium of dairy cows after 4 d of heat stress compared with pair-fed thermoneutral
counterparts

Pathway or process Count1   Protein name2 P-value3

Activated4      
 Glycolysis/Gluconeogenesis 4 ACSS2, ALDH3A2, DLD, FBP1 2.9 E-3
  Acetyl-CoA biosynthetic process 3 ACLY, ACSS2, DLD 1.0 E-2
  AMP-activated protein kinase signaling 8 CAB39, CAB39L, ELAVL1, FBP1, PPP2R2A, 1.0 E-3
PRKAG1, RAB11B↓, RAB14↓
  Insulin signaling 6 CRK, FBP1, FLOT2, PRKAG1, PRKAR2A, 1.6 E-3
PRKAR2B
  Protein processing in the endoplasmic reticulum 8 CUL1, EIF2S1, HSPH1, OS9, PLAA, SEC23A, 7.7 E-4
SEC61A1, RRBP1↓
  Negative regulation of protein serine/threonine kinase 5 AIDA, CHORDC1, PRKAR2A, PRKAR2B↓, 3.5 E-2
  activity HSPB1↓
  Chaperone-mediated protein folding 4 CCT4, CHORDC1, HSPH1, HSPB1↓ 2.3 E-2
  Protein ubiquitination involved in ubiquitin-mediated 6 CUL1, CUL2, CUL3, CUL4A, OS9, PDCL3 2.0 E-2
   protein catabolic process
Deactivated5      
  Regulation of mitotic spindle organization 4 DCTN1, HNRNPU, NUMA1, VPS4B↑ 4.1 E-3
  Melanosome transport 3 DCTN1, MYO5A, RAB11B 1.0 E-2
  Glutathione metabolism 4 ANPEP, GSTA5, GSTM1, GSTP1 1.7 E-4
  Regulation of oxidative stress-induced cell death 5 HDAC6, HSPB1, PYCR1, HSPH1↑, SFPQ↑ 5.4 E-5
1
The number of proteins involved in the pathway or process.
2
ACLY = ATP citrate synthase; ACSS2 = acyl-CoA synthetase short chain family member 2; DLD = dihydrolipoyl dehydrogenase; AIDA = axin
interactor, dorsalization associated; CHORDC1 = cysteine and histidine-rich domain-containing protein 1; HSPB1 = heat shock protein β-1;
PRKAR2A = cAMP-dependent protein kinase type II-α regulatory subunit; PRKAR2B = cAMP-dependent protein kinase type II-β regulatory
subunit; CCT4 = t-complex protein 1 subunit delta; HSPH1 = heat shock protein 105 kDa; CUL1 = cullin 1; CUL2 = cullin 2; CUL3 = cullin
3; CUL4A = cullin 4A; OS9 = protein OS-9; PDCL3 = phosducin-like protein 3; DCTN1 = dynactin subunit 1; HNRNPU = heterogeneous
nuclear ribonucleoprotein U; NUMA1 = nuclear mitotic apparatus protein 1; VPS4B = vacuolar protein sorting-associated protein 4B; MYO5A
= myosin VA; RAB11B = ras-related protein Rab-11B; CD109 = CD109 molecule; CAB39 = calcium-binding protein 39 (MO25alpha) (protein
Mo25); CAB39L = calcium-binding protein 39 like; ELAVL1 = ELAV-like protein; FBP1 = fructose-1,6-bisphosphatase 1; PPP2R2A = serine/
threonine-protein phosphatase 2A 55 kDa regulatory subunit B, PRKAG1 = 5′-AMP-activated protein kinase subunit gamma-1; EIF2S1 =
eukaryotic translation initiation factor 2 subunit 1 (eukaryotic translation initiation factor 2 subunit α); PLAA = phospholipase A2 activating
protein; SEC23A = protein transport protein SEC23; SEC61A1 = protein transport protein Sec61 subunit α isoform 1; CRK = CRK proto-
oncogene adaptor protein; FLOT2 = flotillin-2; ALDH3A2 = aldehyde dehydrogenase; SFPQ = splicing factor proline- and glutamine-rich;
HDAC6 = histone deacetylase; PYCR1 = pyrroline-5-carboxylate reductase 1, mitochondrial.
3
P-value calculated according to a hypergeometric test and corrected with Bonferroni stepdown using ClueGo software (Shannon et al., 2003).
4
Pathway or processes with corresponding proteins upregulated by heat stress unless indicated by an arrow (↓) representing downregulated
proteins.
5
Pathway or processes with corresponding proteins downregulated by heat stress unless indicated by an arrow (↑) representing upregulated
proteins.

HDAC6, PYCR1, and HSPB1 (Table 1). Accordingly,
the regulation of oxidative stress–induced cell death is
not unidirectional, as illustrated in gray in Figure 3.
DISCUSSION
The aim of the present study was to test the hy-
pothesis that heat stress would affect tight junctions,
inflammatory responses via the TLR4 signaling path-
way, and global expression of rumen papillae proteins.
We compared HS and PF cows, but did not include ad
libitum–fed cows at thermal neutral conditions. The
latter group would allow identification of the adapta-
tion response from ad libitum intake at thermoneutral-
ity to reduced feed intake at heat stress; however, the
chosen experimental design was sufficient to identify
heat stress–specific effects at isoenergetic and isonutri-
ent feeding levels.
As designed in the experiment, animals in both
groups consumed same amount of DM per kilogram
of BW (Table 1). However, the meals (feeding bouts)
were distributed more evenly throughout the day in HS
compared with PF cows. Accordingly, HS cows ingested
feed more frequently but had reduced duration and
size of each meal compared with PF counterparts. The
reduction in feed intake along with reduced physical
activity appears to be associated with the old concept
that feed intake and activity are mechanisms for ther-
mal regulation (Brobeck, 1948). Analysis of dynamics
of periprandial heat production mainly influenced by
diet-induced thermogenesis revealed that the average
rise during a single meal was less pronounced in HS
than PF cows. However, daily metabolic heat produc-
tion was comparable between groups, indicating that
an altered feeding behavior avoids excessive heat load
during and after meals. Also, a 4-d heat-stress period

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 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT
caused a significant increase in water intake via more
frequent drinking, most likely to replace the evapora-
tive water loss (Sawka et al., 2001).
Feeding behavior affects rumen fermentation and thus
short-chain fatty acid concentrations. Accordingly, the
expression of genes associated with short-chain fatty
acid transport and acid-base balance may change with
feeding; however, we did not observe differences in pro-
tein expression of monocarboxylate transporter 1, for
example, indicating that the different feeding behavior
of PF and HS cows had no effect on these transporters.
In spite of adaptations in feeding behavior and physi-
cal activity, rectal temperature was significantly higher
in HS than PF cows, indicating that these animals
experienced chronic heat stress. Numerous studies have
demonstrated that severe heat stress produces a rapid
increase in intestinal permeability in several species in-
cluding primates, rats, pigs, and cows (Gathiram et al.,
1988; Hall et al., 2001; Sawka et al., 2001; Koch et al.,
2019). An increased intestinal permeability could po-
tentially expose immune cells of the intestine to patho-
gen-associated molecular patterns of luminal microbes,
and thereby trigger immune responses. In contrast to
the monolayer epithelium of the intestine, the stratified
squamous epithelium of the rumen is composed of 4
distinct strata (Steele et al., 2016). Directly in contact
with the lumen site is the cornified keratinocyte layer,
containing many transporters. (e.g., for short-chain
fatty acid absorption). Underneath the keratinocytes
is the granulosum layer, which is characterized by tight
junctions. Adjacent to the granulosum is the spinosum
layer and the stratum basale, both rich in mitochondria
that facilitate metabolic properties (Steele et al., 2016).
Integrity of the 4 strata of the rumen seems to be more
robust than that in the intestine; however, it can also
be damaged, for instance, by pathogenic infections
that result in the intraepithelial invasion of leukocytes
and activation of T lymphocytes and macrophages in
the basal membrane (Fuertes et al., 2015). We did not
directly measure impairment in terms of permeability
in our study. However, the lack of TLR4 pathway ac-
tivation indicated no difference in the rumen epithelial
barrier function of HS and PF cows. This conclusion
was drawn based on the finding that TLR activation
upon microbial exposure induces secretion of secretory
IgA into the lumen, produces mucins and antimicrobial
peptides, and stimulates epithelial cell proliferation,
thereby maintaining intestinal barrier function (John-
ston and Corr, 2016). In addition, analysis of tight
junction protein expression revealed no significant dif-
ferences between HS and PF cows, indicating no heat
stress–specific alteration of the rumen epithelial bar-
rier function. Results of the effects of ambient heat on
Journal of Dairy Science Vol. 103 No. 9, 2020
8610
the expression of intestinal tight junction proteins are
contradictory in literature. Exposure of caco-2 cells to
39 or 41°C increased occludin expression (Dokladny et
al., 2008), and pigs exposed to 35°C for 24 h showed
increased expression of claudin 3 and occludin (Pearce
et al., 2013), yet incubation of intestinal epithelial
(IEC-6) cells at 42°C for 6 h decreased the expression
of the tight junction proteins occludin, ZO1, and E-
cadherin (Xiao et al., 2013; He et al., 2016). In studies
performed on anesthetized primates, exposure to 41°C
did not result in significant changes in the intestinal
permeability until a rectal temperature of 42°C was
reached (Gathiram et al., 1987). The response of tight
junction protein expression to heat exposure has been
reported to be time and temperature dependent (Xiao
et al., 2013; He et al., 2016). We exposed mid-lactation
dairy cows continuously to mild ambient heat (28°C)
for a relatively long period of 4 d without analyzing
changes before the 4-d-period. We speculated that
molecular adaptations occurring in the long term main-
tain rumen epithelial barrier function. It is known that
HSP interact with tight junction proteins to maintain
their structure (Koch et al., 2019). The expression of
HSPH1 was upregulated (P < 0.05) and that of HSP70
tended to be higher (P = 0.05) in HS compared with
PF cows (Supplemental Table S2, https:​/​/​doi​.org/​10​
.3168/​jds​.2020​-18417). Particularly, these 2 HSP are
implicated in maintaining or protecting intestinal tight
junction barrier function (Dokladny et al., 2006; Yang
et al., 2007). Proteome analysis also revealed numerous
upregulated proteins significantly enriched in “chap-
erone mediated protein folding” in HS cows. Another
mechanism that may contribute to the protection of
tight junction proteins is protein glycosylation. Protein
glycosylation is essential and complementary to the
role of classical HSP, and simultaneous appearance of
both stress-induced glycoproteins and enhanced HSP
accumulation after heat stress (i.e., 15–30 min at 45°C)
is supported by numerous studies (Piper, 1993; Henle
et al., 1995). It has been hypothesized that protein gly-
cosylation may have direct antiaggregation functions or
mediates the interaction of proteins with chaperones,
which is particularly important when the cellular ATP
level is low (Henle et al., 1995). Results of our proteome
study indicated some degree of protein glycosylation in
rumen papillae of HS cows. The abundance of the rate-
limiting enzyme of the hexoseamine pathway, GFPT1,
was more highly expressed in HS cows. This enzyme
catalyzes the transfer of the amine group from gluta-
mine to fructose-6-phosphate to form D-glucoseamine-
6-phosphate and, with several subsequent enzymatic
steps, results in the formation of UDP-GlcNAc, which
directly glycosylates proteins (Oki et al., 1999). An-
 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT
other substrate required for glycosylation is Ac-CoA,
whose synthesis is also upregulated in HS cows, further
supporting a role of glycosylation of rumen papillae
proteins to cope with heat stress. Thus, it appears that
HS cows develop a protective mechanism involving
HSP, protein glycosylation, and Ac-CoA biosynthesis
to maintain epithelial barrier function, all preventing
the activation of the TLR4 pathway and subsequent
inflammation. Moreover, the maintained epithelial bar-
rier function and absence of activated inflammatory
pathways suggest no enhanced immune cell infiltration
into papillae tissue. Therefore, the cell population of the
rumen papillae, predominantly consisting of epithelial
cells and keratinocytes, is likely not different between
HS and PF cows.
A shift from fat toward carbohydrate oxidation after
heat stress has been reported previously (Baumgard
and Rhoads, 2013; Lamp et al., 2015). In most of these
studies, each animal was compared with itself in 2 pe-
riods of thermoneutrality followed by either HS or PF
treatment. For instance, Lamp et al. (2015) reported
that the reduced feed intake of late-pregnant cows di-
minished carbohydrate and increased fat oxidation un-
der thermoneutral, but not heat-stress conditions, for
7 d at 28°C. When whole-body carbohydrate oxidation
was normalized to metabolic BW and DMI, we found
that carbohydrate oxidation tended to be higher in HS
and PF cows. This result confirms increased preference
of body tissues for utilizing carbohydrates over fat for
energy production in heat-stressed mid-lactation cows.
Moreover, our proteome data suggests that the expres-
sion of 2 enzymes involved in β-oxidation of fatty acids
(enoyl-CoA hydratase and 3-hydroxy acyl-CoA dehydro-
genase) was lower in the rumen epithelium of HS than
PF cows, which further point to a reduced preference of
fat oxidation under heat-stress condition. Because heat
stress is closely associated with oxidative stress (Oki et
al., 1999: Slimen et al., 2014), the shift in fuel oxidation
is likely an adaptation strategy to reduce formation of
reactive oxygen species in the mitochondria (Goto et
al., 2015; Lamp et al., 2015). Heat-induced oxidative
stress is largely dependent on the intensity and dura-
tion of heat stress (Slimen et al., 2014). The effect of
acute (short and rapid temperature increase) or chronic
(high ambient temperature over days to weeks) heat
stress on oxidative stress and the antioxidant defense
system of broiler chicken was reviewed by Akbarian et
al. (2016). The authors described a typical increase in
the activities of antioxidant enzymes such as catalase,
glutathione peroxidase, and superoxide dismutase, af-
ter acute heat stress and further showed that chronic
(long-term) heat stress decreases the abundance and
activities of antioxidant enzymes. In agreement with
the latter study (Akbarian et al., 2016), we observed
Journal of Dairy Science Vol. 103 No. 9, 2020
8611
reduced expression of several enzymes involved in glu-
tathione metabolism and enzymes regulating cell death
in response to oxidative stress, suggesting that HS cows
experiencing mild and chronic heat stress combat the
formation of reactive oxygen species in the rumen epi-
thelial cells.
We observed increased expression of several proteins
involved in the AMPK signaling pathway in the ru-
men epithelium of HS cows. This agrees with increased
phosphorylation of AMPK in skeletal muscle of either
dairy cows exposed to 28°C for 4 d (Koch et al., 2016)
or rats challenged for 30 min at 42°C (Goto et al.,
2015). Min et al. (2015) also reported higher serum
AMPK concentrations in dairy cows kept at a THI of
81.7 relative to counterparts kept at a THI of 53.4.
The AMPK is a central regulator of cellular and or-
ganismal metabolism in eukaryotes, which is activated
when intracellular ATP levels are low (Mihaylova and
Shaw, 2011). The level of energy intake between HS
and PF cows was comparable, and therefore should
not account for the activation of AMPK signaling in
HS cow. Instead, this effect is seemingly due to direct
effects of heat stress. Impaired mitochondrial function
due to heat stress has been reported in several studies
(White et al., 2012; Huang et al., 2015). This may have
led to ATP deprivation in HS animals beyond the level
induced by energy intake reduction. In agreement with
this, increased expression of ATP citrate lyase enzyme
and reduced expression of mitochondrial respiratory
chain enzymes (cytochrome C oxidase and ATP syn-
thase) indicate reduced tricarboxylic acid cycling and
mitochondrial energy production in HS cows. Evidence
exists that activated AMPK induces the conservation
of intracellular ATP levels via multiple downstream
pathways, including autophagy (Pease et al., 2009; Tsai
et al., 2016), a process in which regions of cytoplasm,
insoluble protein aggregates, and damaged mitochon-
dria are degraded in lysosomes (Hardie, 2011). It seems
that AMPK-mediated autophagy was activated in HS
cows, at least in part, to remove damaged mitochondria
and other misfolded proteins. Increased degradation of
macromolecules and cellular components is supported
by the fact that several proteins involved in ubiquitin-
mediated protein degradation, endoplasmic reticulum
protein processing, and serine threonine kinase inhibi-
tion were more highly expressed in HS cows.
It is generally known that chronic heat stress in-
creases the plasma concentration of insulin (O’Brien
et al., 2010; Wheelock et al., 2010; Min et al., 2015)
as a result of increased insulin secretion (Itoh et al.,
1998). Although we did not measure serum insulin
concentration, the highly expressed proteins CRK,
FBP1, FLOT2, PRKAG1, PRKAR2A, and PRKAR2B
in HS cows were significantly enriched in the insulin
 Eslamizad et al.: RUMEN PAPILLAE ADAPTATION TO AMBIENT HEAT
signaling pathway, indicating higher insulin action in
HS cows. Several lines of evidence link heat stress to
endotoxemia (Hall et al., 2001; Lim et al., 2007; Pearce
et al., 2013) and show that LPS causes a rise in cir-
culating insulin concentration (Waldron et al., 2006;
Leon, 2007). Whether increased insulin signaling in
the rumen epithelium is a result of increased circulat-
ing insulin concentration due to the leakage in lower
parts of the digestive tract needs additional studies.
However, AMPK phosphorylates the insulin receptor,
providing a direct link between AMPK and the insulin
signaling pathway (Chopra et al., 2012). Results of
an earlier study on rats suggest that even acute heat
stress acts as a rapid stimulator of insulin-independent
glucose transport in skeletal muscle, at least in part by
stimulating AMPK (Goto et al., 2015). It seems that
insulin and AMPK signaling pathways act in synergy
to ensure the transport function of the rumen during
stress conditions. Accordingly, Habegger et al. (2012)
demonstrated that AMPK enhances insulin-dependent
glucose transporter 4 (GLUT4) regulation, at least in
myotubes.
CONCLUSIONS
Behavioral and tissue-level adaptations were success-
fully implemented in dairy cows to reduce heat stress.
There was no heat stress–specific change in the integrity
of the rumen epithelium as evidenced by comparable
expression of tight junction protein and activation of
TLR4 signaling in HS and PF cows. Further, and in
contrast to our hypothesis, transport of short-chain
fatty acids was not specifically affected in HS relative
to isoenergetically fed counterparts. Increased ambient
heat relative to pair-feeding increased the expression
of heat shock proteins along with the activation of the
protein glycosylation machinery, activated AMPK, and
insulin signaling pathways, and increased carbohydrate
oxidation at the expense of fat oxidation. These mecha-
nisms contribute to reduce heat strain and protect ru-
men tissue from injury and subsequent inflammation.
However, whether changes in whole-body macronutrient
oxidation and metabolic pathways in the rumen epithe-
lium are caused by endotoxemia eventually induced by
the leakage of lower parts of the digestive tract or are
gross physiological adaptations to heat stress needs to
be investigated in future studies.
ACKNOWLEDGMENTS
The authors thank the Alexander-von-Humboldt
Foundation (Bonn, Germany) for financial support.
The authors have not stated any conflicts of interest.
Journal of Dairy Science Vol. 103 No. 9, 2020
8612
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ORCIDS
Mehdi Eslamizad https:​/​/​orcid​.org/​0000​-0002​-1071​-3683
Björn Kuhla https:​/​/​orcid​.org/​0000​-0002​-2032​-5502