Cellular and mitochondrial adaptation mechanisms in the colon of lactating dairy cows during hyperthermia

Heat stress causes barrier dysfunction and inflammation of the small intestine of several species. However, less is known about the molecular and cellular mechanisms underlying the response of the bovine large intestine to hyperthermia. We aimed to identify changes in the colon of dairy cows in response to constant heat stress using a proteomic approach. Eighteen lactating Holstein dairy cows were kept under constant thermo-neutral conditions (16°C and 68% relative humidity (RH); temperature-humidity-index (THI) = 60) for 6 d (period 1) with free access to feed and water. Thereafter, 6 cows were equally allocated to: 1) thermoneutral condition with ad libitum feeding (TNAL; 16°C, RH = 68%, THI = 60), 2) heat-stressed condition (HS; 28°C, RH = 50%, THI = 76) with ad libitum feeding, or 3) pair-feeding at thermoneutrality (TNPF; 16°C, RH = 68%, THI = 60) for another 7 d (period 2). Rectal temperature, milk yield, dry matter and water intake were monitored daily. Then, cows were sacrificed and colon mucosa samples were taken for proteomic analysis. Physiological data were analyzed by ANOVA and colon proteome data was processed using DESeq2 package in R. Rectal temperature was significantly higher in HS than in TNPF and TNAL cows in period 2. Proteomic analysis revealed an enrichment of activated pathways related to colonic barrier function and inflammation, heat shock proteins, amino acid metabolism, reduced overall protein synthesis rate, and post-transcriptional regulation induced by heat stress. Further regulations were found for enzymes of the tricarboxylic acid cycle and components of the mitochondrial electron transport chain, presumably to reduce the generation of reactive oxygen species, maintain cellular ATP levels, and prevent apoptosis in the colon of HS cows. These results highlight the cellular, extracellular, and mitochondrial adaptations of the colon during heat stress and suggest a


INTRODUCTION
According to the Intergovernmental Panel on Climate Change (IPCC, 2022), global temperatures are expected to rise by 1.5 to 2°C in 21th century, even when a rapid, deep and immediate reduction of greenhouse gas emission across all sectors will be achieved.Combined with a growing human population, especially in tropical and subtropical regions of the world (Roser et al., 2013), high ambient temperatures are and will increasingly be a significant challenge for animals, agriculture and food security.Hyperthermia affects several aspects of animal production, including milk production.The magnitude of the heat-related loss in milk production is, besides the climate effect, which often expressed as temperature-humidity-index (THI; Summer et al., 2019), mainly determined by the level of milk production at thermoneutrality.Furthermore, heat stress also negatively affects milk quality (Bernabucci et al., 2002b;Summer et al., 2019) and reproduction of dairy cows (Bernabucci et al., 2010;Wolfenson and Roth, 2019).
Recent advances in understanding the effects of heat stress on animal physiology have revealed that the majority of heat-related adverse consequences are mediated by direct effects of hyperthermia, as opposed to those mediated via reduced feed intake (Schwartz et al., 2009;Rhoads et al., 2010;Wheelock et al., 2010).Heat stress has been reported to impair the intestinal barrier function causing a `leaky gut` and allows the penetration of bacteria and microbial particles through the intestine, leading to local and systemic inflammation in various farm animal species, such as pigs, chicken, and rats (Liu et al., 2016Rostagno et al., 2020;Li et al., 2021).However, the mechanisms through which heat Cellular and mitochondrial adaptation mechanisms in the colon of lactating dairy cows during hyperthermia Mehdi Eslamizad, 1 Dirk Albrecht, 2 Björn Kuhla, 1 and Franziska Koch1* stress alters intestinal function and permeability are not thoroughly understood.
Only few studies described the effects of heat stress on the gastrointestinal tract in bovine species.In midlactating cows experiencing a 4-d heat stress, distinct cellular responses were observed in the jejunum including, among others, the impairment of epithelial barrier function and inflammation (Koch et al., 2019, Koch et al., 2021), while these changes were not observed in the rumen epithelium (Eslamizad et al., 2020).Furthermore, exposure to heat stress induced a subchronic inflammation also in mesenteric lymph nodes and peripheral blood mononuclear cells of dairy cows (Koch et al., 2023a), highlighting the effect of heat stress on multiple organs.Although some studies have focused on the small intestine, little is known about the response of the bovine large intestine to hyperthermia.
In terms of size and area, the colon makes up only 19-21% of the total intestine of cattle (Snipes & Snipes, 1997).The major role of the colon is to absorb water, electrolytes and short-chain fatty acids.The colonic epithelium is a dynamic structure and renews within a few days (Blanchier et al., 2022).Therefore, the energy demand for colonocytes is high and requires a high rate of protein and ATP synthesis (Blanchier et al., 2022).However, during periods of high environmental temperatures, the feed intake is decreased and, at the same time, blood supply is restricted to the internal organs including the intestine, thus affecting nutrient acquisition and partitioning throughout the organism.We hypothesized that heat stress will directly alter colon function through mechanisms apart from the effects of reduced feed intake.These mechanism may include, among others, hypoxia and increased production of reactive oxygen species (Kregel et al., 1988;Lambert., 2008) that are not yet documented for the large intestine.Therefore, we aimed at identifying changes in the expression of proteins in the colon of dairy cows exposed to 7 d of continuous heat stress relative to ad libitum and pair-fed cows using an untargeted proteomics approach.Analyzing protein expression profile may provide hints on behavior of the colon tissue under heat stress and feed restriction and therefore shed light on the mechanisms of hyperthermia-induced disruption of dairy cow performance.

Animals and treatments
Cows of the same parity were used to eliminate the potential impact of parity on the response of the animals.Therefore, 18 primiparous, non-pregnant German Holstein cows (171 ± 12 DIM) were adapted to climate chambers at thermoneutral conditions (TN; constant 16°C and 68% relative humidity; THI = 60) for 6 d (period 1) and received a TMR twice daily to meet the nutritional requirements for energy, protein, vitamins and minerals (NRC, 2001).After the acclimation period, cows were randomly assigned for 7 d (period 2) to the following treatments: 1) TN (n = 6) with ad libitum feed intake (TNAL), 2) heat-stress (HS, n = 6) with constant 28°C and 50% relative humidity (THI = 76) with ad libitum feed intake, and 3) TN conditions with pair-feeding (TNPF).The feed intake of HS cows was calculated as percentage of the daily mean feed intake per kg body weight (BW) and offered to TNPF cows one day later.The actual climatic conditions applied over the course of the experiment are presented in Table 1.Cows were fed TMR twice daily at 0730 h and 1730 h (Koch et al., 2023a).Feed intake was recorded daily for individual cows before morning feeding by collecting and weighing orts from the previous day.The HS cows had free access to water; feed and water were tempered to 28°C.Cows were milked twice daily at 0700 h and 1730 h.Water intake, afternoon-morning milk yield, and rectal temperature were recorded daily.At the end of the treatment period, all cows were sacrificed in the institutional slaughterhouse, and colon mucosa scrapings were taken, frozen in liquid nitrogen, and stored at −80°C for later analysis.The pH of the colon digesta was determined using a pH meter (CG 841, Schott, Mainz, Germany) and samples were centrifuged at 15,700 × g for 10 min at 4°C.The supernatant (digesta fluid) was aliquoted and stored at −20°C.All procedures were approved by the ethics committee of the State Government in Mecklenburg-West Pomerania, Germany (LALLF M-V/TSD/7221.3-1.1-60/19).All methods were in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020).

Colon proteome profiling
Colon mucosa (30 mg) was homogenized in lysis buffer (pH = 7.8) containing 50 mM Tris-HCl (Carl Roth), 1 mM EDTA (GE Healthcare), 10 mM NaF (Thermo Fisher Scientific), 1% (vol/vol) IGEPAL CA-630 (Sigma-Aldrich), 0.1% (vol/vol) Triton X100 (GE Healthcare), 0.5% (vol/vol) DOC (Sigma-Aldrich), 0.1% SDS (USB Corporation), and Roche complete Protease Inhibitor Cocktail tablet (one tablet per 10 mL buffer; Roche Diagnostic, Mannheim, Germany).The homogenized extract was centrifuged at 15,700 × g for 20 min at 4°C.The protein concentration of the supernatant was determined using the Bradford kit (Thermo Fisher Scientific).Equal amounts of protein (25 μg) from each sample were separated on a 15% SDS-PAGE.The resultant gel was stained with Coomassie brilliant blue (Serva Electrophoresis GmbH, Heidelberg, Germany) overnight and washed with distilled water.The gel was sliced in 8 horizontal lines yielding 8 slices per lane (8 slices per animal, 144 slices in total).Each slice was transferred into a 1.5 mL reaction tube and washed twice with 100 μL of a solution with 50% CH 3 OH and 50% 50 mM NH 4 HCO 3 for 30 min, and once with 100 μL of 75% CH 3 CN for 10 min.Samples were dried at 37°C for 20 min and incubated with 4 μg/ml trypsin solution overnight at 37°C.For extraction, gel slices were covered with 60 μL of 0.1% trifluoroacetic acid in 50% CH 3 CN and incubated under shaking for 30 min.The peptide containing supernatant was transferred into a clear glass vial and dried at 45°C for 100 min in a concentrator (Eppendorf, Hamburg, Germany).The dry peptides (non-reduced or alkylated) were re-suspended in 10 μL of CH 3 CN/H 2 O/trifluoroacetic acid (50%/49.5%/0.5%).Peptides were separated and analyzed using a Proxeon easy nLCII-system (Thermo Scientific) coupled to a Thermo Scientific LTQ Orbitrap-XL mass spectrometer.A 0.1 × 200 mm column with C18 Aeris Peptide (Phenomenex, Torrance, CA, USA) and a gradient of 0.5%/min (buffer A = 0.1% formic acid in water, Optima LC/MS; buffer B = 0.1% formic acids in 99.9% CH 3 CN, Optima LC/MS; Fisher Scientific) at a flow rate of 0.3 mL/min was applied.For MS and MS/MS analysis, a full survey scan in the Orbitrap-XL with a mass range (m/z 300-2,000) and a Fourier transform (FT) resolution of 30,000 was followed by data-dependent fragmentation experiments of the 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 FTM scans were acquired with a resolution of 30,000 and a target value of 1.2 × 10 6 in the Orbitrap analyzer.The ion-trap MS scans were acquired with uni mass resolution in the LTQ using 3,000 as target value, 2 as the default charge state,  Each Mascot search included the data from all 8 gel slices per lane and results loaded into Scaffold software (version 5.0.1.,Proteome Software Inc., Portland, OR, USA).The Scaffold viewer was utilized to validate MS/ MS based peptide and protein identifications.Peptide identifications were accepted with 2 peptides characterizing uniquely one protein with 95% probability to achieve a false discovery rate less than 0.1% by the Peptide Prophet algorithm with Scaffold delta-mass correction (Table S1, https: / / doi .org/ 10 .6084/m9 .figshare.24314176).Only proteins identified in at least in 4 of 6 animals per group were considered for further analysis.

Statistics and bioinformatics analysis
Physiological data including DMI, water intake, milk yield, and rectal temperature was analyzed as repeated measures using the MIXED procedure of SAS (version 9.1).The statistical model included treatment, period, and their interaction as fixed effect and individual cow as random effect.Variance-covariance structure was decided for use among autoregressive, toeplitz, unstructured, and compound symmetry based on the smallest Akaike information criterion (AIC), corrected Akaike information criterion (AICC), and Bayesian information criterion (BIC) values.
For the analysis of differential protein expression, raw spectral counts were processed utilizing the DE-Seq2 package of the bioconductor repository in R (www .bioconductor.org).The raw spectral counts and the metadata were used to generate a DESeqDataSet object with DESeqDataSetFromMatrix (Table S1, https: / / doi .org/ 10 .6084/m9 .figshare.24314176).The DESeq function performed estimate size factors, estimate disper-sions, and negative binomial WALD test analysis with the p-value criteria of 0.05.Table S2 contains the list of all proteins with adjusted p-values for all 3 comparisons (TNPF vs. TNAL, HS vs. TNPF, and HS vs. TNAL, https: / / doi .org/ 10 .6084/m9 .figshare.24314176).Proteins that were differentially regulated in each comparison were subjected to functional enrichment analysis using the Cytoscape software version 3.7.0applying database from Gene Ontology.Results of the pathway analysis were visualized with the ClueGO plug-in (version 2.5.8) of the Cytoscape (Bindea et al., 2009).The ontology selection on the basis of biological processes was performed by the right-side hypergeometric statistic test by utilizing the Bonferoni step-down method.

Physiological response to heat stress or reduced feed intake
During period 1, the mean rectal temperature was similar among all experimental groups (Table 2), but mean rectal temperature was significantly higher in HS compared with TNPF and TNAL cows during period 2 (39.98 vs. 38.27and 38.16°C, respectively; P < 0.01).Dry matter intake, as adjusted for kg body weight, was statistically similar among the groups in period 1.During period 2, HS cows reduced DMI/BW by 45% and TNPF cows by 36%, relative to period 1.All cows produced similar volumes of milk during period 1, but HS cows had a production loss of 36% and TNPF cows of 11% in period 2 relative to period 1.During period 2, pair-fed cows reduced their overall water intake, but HS cows maintained similar water intake levels as period 1.This resulted in a significantly greater water intake per kg of DMI in HS cows compared with TNPF and TNAL counterparts in period 2.

Colonic SCFA, metabolites and cytokine concentrations
HS cows tended to have a lower portion of colonic iso-butyric acid than TNAL cows (P = 0.09, Table 3).However, heat stress did not have any effects on other colonic SCFAs, pH, mucosa TNFα, IFNγ, and lactate concentrations or LDH activity (Table 3 and 4).

Proteomic response to hyperthermia
From a total of ~21,000 identified proteins in 18 animals from 3 groups, 1002 proteins were subjected to DESeq2 statistical analysis based on our criteria of being identified as a Bos taurus protein with probability of over 95% in at least 4 out of 6 animals of each group.

Physiological responses to heat stress or pairfeeding
The high ambient temperatures in the present study induced heat stress in dairy cows as indicated by lower milk yield and DMI, while rectal temperatures and water intake normalized to DMI increased during 7 d of continuous 28°C.The magnitude of reduction in milk production in period 2 compared with period 1 was almost 3 times greater in HS cows than in the TNPF group, despite both were maintained at a similar plane of nutrition during period 2. This indicates that the reduced DMI/BW accounted for an average of approx.31% of milk production loss in this study and that the remaining portion (approx.69%) was directly attributable to hyperthermia.However, the latter value was slightly higher than the 50 to 65%, previously reported from other trials with lactating heat-stressed cow (Rhoads et al., 2009;Wheelock et al., 2010;Baumgard et al., 2011).This can be explained by differential degree of DMI loss between HS and TNPF cows in period 2 relative to period 1 in our study (45 vs. 36%, respectively) which might be due to the pre-existing numerically lower DMI of TNPF cows in period 1 (Table 2).In addition, we exposed cows to constant as opposed to cyclic heat stress, providing a further explanation for the greater heat induced production loss in our experiment.Nevertheless, our results confirm the reproducibility of the pair-feeding protocol in differentiating the direct and indirect effects of heat stress on milk production.Heat stress activates multiple compensatory mechanisms to obtain homeostasis at the expense of DMI and milk production.
There is growing evidence of a causal link between high ambient temperatures, heat stress, and intestinal dysfunction.In contrast to the small intestine, the colon plays an important role in absorption of water, electrolytes and SCFAs from the digesta fluid (Blachier et al., 2022).During heat stress, the water and electrolyte absorption is increased to reduce dehydration and facilitate sweating (Burhans et al., 2022).This should alter the concentration of non-absorbed metabolites and potentially the pH in the digesta fluid (Blachier et al., 2022).In this study, the pH of the colonic digesta fluid was not altered in HS cows suggesting the activation of mechanisms maintaining colonic pH homeostasis.Our results are in line with previous findings showing that the pH of jejunal digesta fluid in HS cows was not different compared with the ad libitum fed control group (Koch et al., 2023b).Among SCFA, n-butyric acid is the major energy source for colonocytes and primary produced from dietary fiber and starch (Salvi et al., 2021), while iso-butyric acid is a product of bacterial protein degradation (Lalles et al., 2007).Comparable n-butyric acid concentrations among groups suggest a comparable energy supply for colonocytes, whereas the tending lower iso-butyric acid concentration in the digesta fluid of HS cows suggest reduced protein degradation or increased absorption.In contrast to our results, earlier studies reported reduced concentrations of colonic n-butyric, acetic, and propionic acid concentrations but in line with our results also lower iso-butyric acid concentrations in the colonic digesta of pigs exposed to 7 d of heat stress (Hu et al., 2021).Interestingly, the reduced SCFA concentrations were associated with a lower villus height-crypt depth ratio, lower number of goblet cells, and inflammatory signs in colonic tissue of HS pigs (Hu et al., 2021).
To understand the cellular and molecular adaptation processes in the colon in dairy cows, an untargeted proteomic approach was utilized to identify pathways activated or deactivated during heat stress.The Venn analysis of our proteome data indicates that the majority of changes in colonic protein expression were directly related to hyperthermia and not to reduced feed intake.These results are in accordance with a study in porcine intestines, which reported that 281 proteins were differentially regulated between heat stress vs. thermo-neutral ad libitum, 138 proteins between heat stress vs. thermo-neutral pair-fed, but only 20 proteins between thermo-neutral ad libitum vs. thermo-neutral pair-fed comparisons (Pearce et al., 2015).

Mucosal inflammation and barrier function
Our pathway enrichment analysis showed that several aspects of inflammatory reactions such as the dendritic cell migration and chemotaxis, NF-kB signaling, and the production of TNF-α, IL-1 and INF-γ were positively regulated in the colon of HS cows in comparison to the control groups (Table 5 and Figure 3).High mobility group protein B1 (HMGB1) occurred in almost all terms associated with inflammation, suggesting a central role for this protein in triggering and regulating inflammatory reactions.Recent findings show that HMGB1 is a damage-associated protein with alarmin function activating innate immunity after being actively released from cells or passively released upon cell death (Yang et al., 2020).A distinct molecular conformation enables HMGB1 to bind to the TLR4/MD-2 complex to induce cytokine release from macrophages and initiate an inflammation (Yang et al., 2013).However, when we analyzed colon tissue samples for inflammatory cytokines, we found no difference between HS and control cows regarding TNF-α and INF-γ.This result might be because the analysis was performed in whole colonic mucosa consisting of a mixture of colonic enterocytes,  endocrine cells and immune cells (Newsholm and Carrié, 1994).Nevertheless, our proteomic results provide hints for a compromised mucin synthesis during heat stress, as indicated by the lower expression of mucin-2 protein, a major component of the mucus layer of the small and large intestine.Previous studies have shown a strong link between mucin-2 expression and intestinal function.Muc2-knockout mice had no mucus layer in the colon and evoked a direct contact between luminal bacteria and epithelial cells, causing inflammation (Johannson et al., 2008).Moreover, heat stress induced a reduction in mucin-producing goblet cells and caused lower Muc2 gene expression in mice (Lu et al., 2023) and broilers (Zhang et al., 2017).For mucin production, de novo synthesis of mainly palmitic acids is required to allow for S-palmitoylation and secretion of mucin-2 protein (Wei et al., 2012).In HS cows, we observed not only reduced expression of mucin-2, but also downregulation of fatty acid synthase and enzymes of the pentose phosphate pathway, the latter generating NADPH supporting fatty acid synthesis (Table S2, https: / / doi .org/ 10 .6084/m9 .figshare.24314176).Furthermore, we detected that the expression of several structural proteins (e.g., filamin, spectrin α and β, desmoplasmin, plectin) were lower in the colon of HS cows (Table S2).Among them, plectin is directly implicated in the maintenance of the colon barrier function (Krausova et al., 2021).Altogether, our proteomics data suggest impaired intestinal barrier function in the colon.Based on the cytokine data, however, we obtained no signs for an inflammatory process in the colon, questioning the disruption of the colonic barrier.Therefore, measurement of the colonic permeability should be performed in future experiments.

Cellular translation and protein folding
It is well known that gene transcription costs up to 10% of the total energy; therefore oligonucleotide synthesis is often inhibited to limit endogenous heat production (Storey, 2015).In our proteome analysis, we found indications that also translational processes were downregulated during heat stress.Specifically, the abundances of proteins involved in the ligation of several amino acids to tRNA for protein synthesis were lower in the colon mucosa of HS than TNAL cows.Our data add to the previously reported decrease in the overall protein synthesis rate in other organs such as the mammary gland of heat-stressed dairy cows (Collier et al., 2006;Gao et al., 2019).It was suggested that a large portion of transcribed mRNA is not transported from the nucleus to cytoplasm for translation due to widespread inhibition of mRNA splicing following heat shock (Shalgi et al., 2014).In addition, functional enrichment analysis also revealed that HS cows have partially shifted toward the post-transcriptional control of gene expression by negative regulation of mRNA splicing and activation of mRNA degradation (Table 4).
To maintain the function of translational products, several heat shock proteins (HSPE1, HSPD1, HSPAA1, HSPA1A, HSPH1 and HSPB1) were found to be differentially expressed in the colon of HS compared with control cows, indicating an adaptive mechanism in the colonic mucosa during hyperthermia.Heat shock proteins (HSP) are involved in chaperone-mediated protein folding, protecting proteins against denaturation and loss of cell viability during heat stress (Riezman, 2004).Our findings are consistent with a previous study, in which HSP27, 65, 70, 90-α and 90-β were higher abundant in the ileum of heat stressed pigs after exposure to 37°C for 12 h to prevent protein misfolding and denaturation (Pearce et al., 2015).Another pathway found induced during heat stress is the negative regulation of the oxidative stress-induced intrinsic apoptotic signaling pathway.This finding indicates a prioritization of the cellular nutrient utilization to maintain vital processes and prevent apoptosis.

Intermediate energy metabolism
Several TCA cycle enzymes (OGDH, FH, DLST, PDHA1) were downregulated in the HS compared with Proteins with no mark differed significantly in both HS and TNPF vs. TNAL indicating effect of reduced feed intake.
ϯ differed significantly between HS vs. TNAL but were similar in HS vs. TNPF and TNPF vs. TNAL.
control groups, indicating a blunted ability of the tissue to convert and degrade nutrients and intermediary metabolites for NADH production.These results agree with those of others reporting reduced expression of TCA cycle enzymes, e.g., malate dehydrogenase 2 in the porcine jejunum after 21 d of heat stress (Cui et al., 2015) and reduced citrate synthase activity in the skeletal muscle of poultry upon 14 d of heat stress (Azad et al., 2010).The duration of heat stress might play a critical role in downregulation of the TCA cycle enzymes, as an earlier study did not observe alterations in the expression of jejunal TCA cycle enzymes of 4-d heat stressed cows (Koch et al., 2021).Downregulation of the TCA cycle relative to TNAL cows can be explained by reduced nutrient intake and relative to TNPF cows by intestinal ischemia (Lambert, 2008), resulting in hypoxia and increased oxidative stress (Hall et al., 1999) in HS cows.Under reduced plane of nutrition, acetyl-CoA generated from β-oxidation of free fatty acids and from degradation of ketone bodies seems to replenish the TCA cycle as indicated by the higher expression of mitochondrial 3-ketoacyl-CoA thiolase and succinyl CoA-ketoacid CoA transferase in TNPF relative to TNAL cows, but not in HS cows.While downregulation of TCA cycle enzymes should be accompanied by reduced NADH and ATP production in HS cows, this gap seems not to be filled by anaerobic glycolysis, because lactate concentration and LDH activity in colonic mucosa were comparable among groups.

Mitochondrial electron transport and antioxidative defense
Heat stress increases the blood flow to the periphery to dissipate heat via the skin (Hall et al., 1999).As a result, the blood flow in the intestine decreases leading to hypoxic episodes and concomitantly increased reactive oxygen species (ROS) production (Kregel et al., 1988;Lambert et al., 2008).At the cellular level, the mitochondrial electron transport chain (ETC) is the main site of ROS production.It has been shown that the steady state of ROS flux is disturbed resulting in the accumulation of ROS during heat stress (Lin et al., 2006;Paul et al., 2009;Yu et al., 2013;Cui et al., 2016).In an attempt to reduce ROS production under hyperthermia, compensatory mechanisms are activated to further reduce the entrance of NADH to the ETC via complex I and III, thus lowering ROS production.In addition, the decreased production of reducing equivalents through the TCA cycle may reduce the cellular ATP status in HS cows.However, HS cows might have compensated for decreased ATP production via more efficient functioning of the electron transport chain.First, the expression of cytochrome c and 2 subunits of cytochrome c oxidase (namely the COX4I1 and COX5A) were higher in HS cows compared with both TNPF and TNAL counterparts.Cytochrome c oxidase in complex IV of the respiratory chain is the last and rate-limiting enzyme eliminating electrons from ETC and its activity is an indicator of the oxidative capacity of the cell (Srinivasan and Avadhani, 2012).Specifically, upregulation of cytochrome c subunits, i.e., COX4, under hypoxic conditions has been associated to enhanced efficiency of respiration (Vogt et al., 2011).Second, the expression of ATP5A and ATP5C1 subunits of the ATP synthase complex were similarly higher in HS and TNPF compared with TNAL cows.Although the latter appears to be a response to reduced feed intake, it suggests comparable activity of ATP synthase complex in both groups.A functioning ATP synthase in the colon mucosa is important to maintain Na/K ATPase activity ensuring absorption of water from the lumen of the colon (Blachier et al., 2022), thus preventing dehydration of the organism under heat stress.
Furthermore, lower protein expression of 3 core subunits of the NADH dehydrogenase may have led to reduced NADH dehydrogenase activity in complex I, and subsequent accumulation of NADH.Since NADH is a potent feedback inhibitor of the TCA cycle (Williamson and Cooper, 1980), an increased NADH/NAD + ratio is suggested to signal reduced TCA cycling in a negative feed-back manner.
There is also evidence that chronic heat stress over several days and weeks leads to a depletion of both endogenous and exogenous antioxidant reserves (Sreedhar et al., 2002;Sahin et al., 2004;Zhao et al., 2006;Eslamizad et al., 2020).We observed a hyperthermia-induced reduction in channeling of methionine through the trans-sulfuration pathway and glutathione synthesis, as evidenced by lower expression of adenosylhomocysteinase 3 (AHCYL2) and glutathione synthase (GSS) in HS compared with both TNPF and TNAL cows.This could be considered as a consequence of reduced NADH dehydrogenase activity because defects in the activity of complex I has led to remarkable losses in cellular glutathione levels (Ni et al., 2019;Hargreaves et al., 2005) but not vise versa (Heales et al., 2011).Glutathione synthesis is a redox sensitive process (Deplancke and Gaskins, 2002) meaning that pro-oxidants enhance and anti-oxidants suppress de novo glutathione synthesis (Vitvitsky et al., 2003).Because reduced NADH dehydrogenase activity diminishes ROS production, glutathione synthesis may also be modulated.Increased abundances of enzymatic antioxidants (SOD1, PRDX5) in the colon mucosa of HS suggests activation of compensatory mechanisms for compromised glutathione

Conclusion
Reduced feed intake accounted for the minority of the changes in milk production and colon proteome of heatstressed cow.Heat stress directly downregulated the expression of structural proteins and those accounting for mucus production in the colon.Furthermore, heat stress altered the intermediate energy metabolism by reducing the protein expression of the major TCA cycle enzymes.Increased expression of heat shock proteins likely act to maintain protein function during increased oxidative stress, whereas changes in the mitochondrial ETC are aligned to lower ROS generation while maintaining cellular ATP levels and preventing apoptosis.While the present study provided only information on the abundance of proteins, additional metabolome profiling as well as functional studies on the mitochondrial performance and colonic permeability are warranted to confirm our findings.
and a lower intensity threshold for MS2 of 3,000 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 list are as follows: repeat count = 1, repeat duration = 30 s, exclusion list size = 500, and exclusion duration = 30.The mass spectrometry were deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the data set identifier PXD046580.Data files were searched against the National Center for Biotechnology information Bovine database (http: / / www .ncbi.nlm.nih.gov/ ) using Mascot version 2.6.2 with the common contaminant 'Keratine' specified.The Mascot search was carried out considering the following parameters: parent ion mass tolerance of 10 ppm, fragment ion mass tolerance of 0.80 Da, and Met oxidation (+ 15.99492 Da).
Eslamizad et al.:  Heat stress directly affects colon proteome Table2.Dry matter intake normalized for body weight, water intake, milk yield and rectal temperature as measured in the acclimation period (period 1) and the treatment period (period 2) in heat-stressed (HS), thermoneutral pair-fed (TNPF), and thermoneutral ad libitum fed (TNAL) groups.Data are shown LSmean ±

Figure 1 .
Figure 1.Venn diagram representing the number and percentage of proteins differentially regulated across 3 group comparisons.Note the high overlap between HS vs. TNAL and HS vs. TNPF and the small overlap between HS vs. TNAL and TNPF vs. TNAL.

Figure 2 .
Figure 2. Sparse Partial Least Square-Discriminant Analysis (sPLS-DA) explained 18% of variation 1 and 10% of variation 2 in heat-stressed (HS), thermoneutral pair-fed (TNPF), and thermoneutral ad libitum fed (TNAL) lactating dairy cows.The figure indicates that the heat stress group discriminated significantly from both TNAL and TNPF cows.The majority of the effects of heat stress seem to be directly induced by hyperthermia and not via reduced feed intake.

Figure 3 .
Figure 3. Representative terms and pathways obtained by functional enrichment analysis of upregulated proteins in HS vs. TNAL and TNPF comparison, indicating the direct effect of hyperthemia.The analysis was performed by right-sided hypergeometric test with Bonferroni step down method for P-value correction (P < 0.05).

Figure 4 .
Figure 4. Representative terms and pathways obtained by functional enrichment analysis of downregulated proteins HS vs. TNAL and TNPF comparison, indicating the direct effect of hyperthemia.The analysis was performed by right-sided hypergeometric test with Bonferroni step down method for P-value correction (P < 0.05).
Eslamizad et al.:  Heat stress directly affects colon proteome synthesis to maintain antioxidative defense during heat stress.
Eslamizad et al.: Heat stress directly affects colon proteome

Table 1 .
Eslamizad et al.:Heat stress directly affects colon proteome Climate conditions calculated as means during the adaptation period (period 1) for all animals and during the treatment period (period 2) for heat stress or control conditions at thermoneutrality.Data are shown LSmean ± SEM

Table 3 .
Colonic pH and short-chain fatty acid (SCFA) composition of the digesta fluid of mid-lactation Holstein dairy cows exposed to heat stress (HS), thermoneutral ad libitum feeding (TNAL), or thermoneutral pair-feeding (TNPF).Data are shown LSmean ± SEM Different capital letters indicate a trend P < 0.09.