Feedlot diets containing different starch levels and additives change the cecal proteome involved in cattle’s energy metabolism and inflammatory response

Diets for feedlot cattle must be a higher energy density, entailing high fermentable carbohydrate content. Feed additives are needed to reduce possible metabolic disorders. This study aimed to analyze the post-rumen effects of different levels of starch (25%, 35%, and 45%) and additives (monensin or a blend of essential oils and exogenous α-amylase) in diets for Nellore feedlot cattle. The cecum tissue proteome was analyzed via two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and then differentially expressed protein spots were identified with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The use of blends of essential oils associated with α-amylase as a feed additive promoted the upregulation of enzymes such as triosephosphate isomerase, phosphoglycerate mutase, alpha-enolase, beta-enolase, fructose-bisphosphate aldolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), l-lactate dehydrogenase B, l-lactate dehydrogenase A chain, l-lactate dehydrogenase, and ATP synthase subunit beta, which promote the degradation of carbohydrates in the glycolysis and gluconeogenesis pathways and oxidative phosphorylation, support pyruvate metabolism through the synthesis of lactate from pyruvate, and participate in the electron transport chain, producing ATP from ADP in the presence of a proton gradient across the membrane. The absence of proteins related to inflammation processes (leukocyte elastase inhibitors) in the cecum tissues of animals fed essential oils and amylase may be because feed enzymes can remain active in the intestine and aid in the digestion of nutrients that escape rumen fermentation; conversely, the effect of monensin is more evident in the rumen and less than 10% results in post-ruminal action, corroborating the hypothesis that ionophore antibiotics have a limited effect on the microbiota and intestinal fermentation of ruminants. However, the increase in starch in these diets promoted a downregulation of enzymes linked to carbohydrate degradation, probably caused by damage to the cecum epithelium due to increased responses linked to inflammatory injuries.


Results
Image analysis and protein expression. In the workspace, classes were created to analyze differences in protein expression; an analysis of variance (ANOVA) was used to test the hypothesis (H θ ) that the expressed spots are identical. When testing all classes, protein spots were differentially expressed, as shown in Table 1.
Supplemental Figure S2 shows the distribution of proteins and their biological processes, molecular functions, and cellular components.
Proteins associated with glucose metabolism and energy synthesis ( Table 2) and macromolecules involved in the degradation of carbohydrates through the glycolytic pathway, gluconeogenesis, and oxidative phosphorylation were detected in cecal tissue. The expression of seven enzymes participating in the glycolysis and gluconeogenesis pathways was verified: triosephosphate isomerase (Step 1); phosphoglycerate mutase (Step 2); alpha-enolase (ENO1), beta-enolase (ENO3), and fructose-bisphosphate aldolase (ALDOB) (Step 4); pyruvate kinase (PKM) (Step 5); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Three enzymes linked to pyruvate metabolism or catalytic activities participating in the synthesis of lactate from pyruvate were verified as well: l-lactate dehydrogenase B, l-lactate dehydrogenase A chain, and l-lactate dehydrogenase. ATP synthase subunit beta participated in the electron transport chain, producing ATP from ADP in the presence of a proton gradient across the membrane.
Pathways enrichment and Reactome analysis. The pathway enrichment and Reactome analysis yielded similar results showing that specific pathways were affected. The differential expression found in all groups displayed changes in metabolic pathways as carbohydrate metabolism, pyruvate metabolism, the citric Table 1. Differentially expressed spots in Nellore beef cattle cecum fed with diets containing increasing starch levels (25,35, and 45%) and additives (Monensin, Blend of essential oil + exogenous α-amylase). UP up-regulated spot, Down down-regulated spot, + spot present in the first group in relation to the second, ∅ spot absent in the first group in relation to the second. *P ≤ 0.05.  Up  9  3  7  14  3  8  5  0  1   Down  11  16  5  6  28  4  10  6  13   +  10  59  14  22  65  35  34  16  27   ∅  37  11  14  81  19  42  18  8  16   Total  67  89  40  125  115  89  67  30  www.nature.com/scientificreports/ acid (TCA) cycle, respiratory electron transport, innate immune system, and the immune system were affected in cecum tissues by different feeding strategies (Fig. 1). The data from the Reactome pathway analysis have been provided in Supplemental Table S1. Additionally, the differential expression indicates similar encoding enzymes in the glycolysis and gluconeogenesis pathways in cattle's large intestines under different feeding strategies (Fig. 2).

SPOT (n)
The expression values ( P ≤ 0.05 ) ( Table 3) were grouped with hierarchical cluster analysis (Fig. 3) and ordered by homogeneity between the treatments tested. Animals fed with identical levels of starch but subjected to different feed additives showed differentiation in proteins that contribute to energy metabolism.     www.nature.com/scientificreports/

Discussion
When evaluating the tested additives, we found that the use of BEOα with intermediate levels of starch (35%) resulted in greater expression of glycolysis intermediates, thus, this additive may have a greater effect on the post-rumen tract. A companion study (data under review) reported that the optimum level of dietary starch for cattle fed MON was 25%; however, the optimum level of dietary starch for cattle receiving BEO was 35%, commensurate with protein expression synthesis. In summary, feed intake decreased when MON-and BEO-fed cattle were fed more than 25% and 35% starch, respectively, which resulted in decreased average daily gain and proteins linked with carbohydrate degradation. The diets with 45% starch may have caused excessive ruminal fermentation that may have resulted in increased inflammation of the ruminal epithelium, agreeing with the proteins leukocyte elastase inhibitor. The literature reports that increasing levels of starch play an important role in reducing feed intake (observed in a companion study); however, this effect was more evident in cattle that were fed MON as it is a feed additive that depresses intake.
In the protein spots of groups 25BEOα and 45BEOα compared with those fed MON, leukocyte elastase inhibitor, a serine protease inhibitor that is essential for the regulation of inflammatory responses and limits the activity of inflammatory caspases, was not expressed 22 aligning with the results of the above-mentioned authors, who reported reduced ionophore effects in the hindgut. When comparing diets with 25% or 45% starch, regardless of the additive used, leukocyte elastase inhibitor was expressed more, corroborating previous studies demonstrating that inflammatory injuries are caused by the increased use of concentrates in the diet 23,24 . Additionally, we observed reduced expression of proteins that participate in energy metabolism in animals on high-starch diets, which can damage the epithelium of the cecum.
In a similar study, Toseti et al. 9 observed a reduction in fecal starch using BEOα, suggesting a greater degradation of carbohydrates because the feed enzymes can remain active in the intestine and aid in the digestion of nutrients that escape rumen fermentation 25 . As Thomas et al. 26 demonstrated, the effect of monensin is more evident in the rumen, mainly in the diversity of microorganisms, but less than 10% results in post-ruminal action, corroborating the hypothesis that ionophore antibiotics have a limited effect on ruminants' microbiota and intestinal fermentation.
Protein expression differs depending on the dietary starch level ( P ≤ 0.05 ); the cluster analysis shows differentiation in the profile of the identified proteins involved in energy metabolism ( Fig. 3) but the effect is greater when contrasting 25% and 35% starch or 35% and 45%, mainly when using monensin as a feed additive. In summary, high concentrations of starch may result in inflammatory responses due to the greater supply of undegradable starch in the rumen, thus decreasing the expression of proteins linked to the glycolytic pathway through tissue damage and inflammation. Higher concentrations of carbohydrates (starch) in the intestine along with the low effects of monensin on the cecum may contribute to a greater accumulation of organic acids. Additionally, the intestinal epithelium is much more vulnerable to pH variation than the rumen 21,27 , corroborating our identification of proteins linked to immune responses. Notably, this was not observed when assessing the full range of starch levels (25% vs. 45%) but the proteins involved in inflammatory responses were expressed more (Fig. 3). We attribute this to the greater increase in dietary carbohydrate, which may have increased epithelial injury (indicative damage) 21,28 and upregulated inflammatory response, subsequently reducing the expression of proteins associated with energy metabolism.
Fructose-bisphosphate aldolase (ALDOB), an enzyme that converts fructose-1,6-bisphosphate to fructose 6-phosphate, catalyzed by triosephosphate isomerase (TPI), is a precursor of glyceraldehyde-3-phosphate (GA3P), which is acted upon by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during glycolysis. Alpha-enolase (ENO1) and beta-enolase (ENO3) are isoforms of enolase that are involved in Step 4 of glycolytic metabolism. Phosphoglycerate mutase (PGM) is a catalytic enzyme that converts 3-phosphoglycerate to 2-phosphoglycerate and, finally, pyruvate kinase (PKM) synthesizes pyruvate in the last step of glycolysis (UniProt 29 ). In ruminants, a high concentration of starch enables the fermentation of carbohydrates in the cecum with lactate production, which increases glucose metabolism in the intestine and leads to the observed expression of the enzyme l-lactate dehydrogenase and its isoforms l-lactate dehydrogenase B and l-lactate dehydrogenase A, which synthesize lactate from pyruvate (UniProt 29 ).
The dietary manipulation verified the expression of the leukocyte elastase inhibitor protein, associated with the inflammatory response ( Table 2); this plays an essential role in regulating the innate immune response, inflammation, and cellular homeostasis, and mainly acts to protect cell proteases released into the cytoplasm during stress or infection 29 .

Methods
The experiment was conducted according to the standards issued by the National Council for Animal Experi- where Yijk is the dependent variable; µ is the overall mean; Bk is the block effect; Ci is concentrate; AJ is additive; (C × A)ij is the interaction between concentrate and additive effects; and εijk is the residual error.

Diets and their chemical composition.
The experimental diets were composed of natural bagasse sugarcane, ground corn, soybean hulls, cottonseed, soybeans, core minerals and vitamins, urea, and additives. The transition to the finishing diet was managed as follows: for 14 days, two diets with 65% and 75% concentrate were provided for 7 days each. From the 15th day of the experiment until slaughter, a finishing diet containing 85% concentrate was provided (Table 4). Dietary energy content was calculated according to the LRNS system 31 and Total digestible nutrients (TDN) were determined by the equation: TDN = digestible CP + (digestible EE × 2.25) + digestible NDF + digestible nonstructural carbohydrate (NSC). Crude protein was determined by assessing the nitrogen content of the samples with the Kjeldahl method 33 . The NDF concentration was assessed with the methodology described by Van Soest et al. 34 and corrected for CP and ashes. Starch was determined by the equation: NSC = 100 − CP − EE − NDF − as h, where ash content was determined by incinerating samples at 550 °C for 2 h in a muffle furnace 35 . Physically effective neutral detergent fiber (peNFD) was determined according to Kononoff et al. 's methods 36 . Samples of diets were collected to determine particle-size distribution by sieving with the Penn State particle-size separator and reported on an as-fed basis.
Proteomics sample collection and preparation. The animals were transported to a commercial slaughterhouse where they were stunned by brain concussions with a captive dart gun. After bleeding hide www.nature.com/scientificreports/ removal and evisceration, cecum samples about 4 cm square were collected and washed with phosphate-buffered saline (PBS), transferred to 15 mL polypropylene bottles, and placed in liquid nitrogen (− 196 °C) for later protein extraction. Each pen was considered an experimental unit, so a pool of samples was made by homogenizing cecal tissue from animals given the same treatment; three animals per experimental unit (N = 5) were used, i.e., 15 animals/group or 90 animals total (15 animals from each of six groups).

Extraction, precipitation and quantification of proteins.
To extract the protein fraction, the tissue was macerated with a mortar and pestle in the presence of liquid nitrogen. The extracting solution was added at a rate of 1 mL ultrapure water per 1 g tissue and then the samples were homogenized with an OMMI-BEAD RUPTOR4 cell disruptor (Kennesaw, Georgia, United States) over three 30-s cycles. The samples were then separated into protein extracts and the supernatant was collected after refrigerated centrifugation (− 4 °C) with a UNIVERSAL 320R HETTICH (Tuttlingen, Baden-Württemberg, Germany). The proteins were precipitated in 80% (v/v) acetone (J.T. Baker, Phillipsburg, New Jersey, United States), using 300 μL of supernatant and 600 μL of 80% acetone. The samples were stored at 2 °C for 1.5 h and then centrifuged at 14,000 rpm for 30 min; the supernatant was discarded and the protein pellet was solubilized in 1 mL of 0.50 mol/L NaOH (Merck, Darmstadt, Germany). The protein concentrations were determined by the Biuret method 37  Approximately 900 µL of mineral oil was added at room temperature for 12 h to rehydrate the strips and prevent evaporation and urea crystals. After this period, the strips were added to the EttanTMIPGphorTM3 isoelectric focusing system (IEF) (GE Healthcare, Uppsala, Sweden). The electrical voltage used was established by the protocol described by Braga et al. 38 . At the end of the focusing period, the strip was balanced in two 15-min stages. First, 10 mL of a solution containing 6 mol/L urea, 2% SDS (w/v), 30% glycerol (v/v), 50 mmol/L Tris-HCl (pH 8.8), 0.002% bromophenol blue (w/v), and 2% DTT (w/v) was used to keep the proteins in their reduced forms 38,37 .
In the second stage, a solution in which DTT was replaced with 2.5% (w/v) iodoacetamide was used to alkylate the thiol groups of the proteins and prevent possible reoxidation. After strip balancing, the second portion of the electrophoretic process (SDS-PAGE) occurred. The strip was applied to a 12.5% (w/v) polyacrylamide gel previously prepared on a glass plate (180 × 160 × 1.5 mm). The gel was placed next to the strip with a piece of filter paper containing 6 µL of a molecular mass standard (GE Healthcare, Uppsala, Sweden), with proteins of different molecular masses . The strip and filter paper were sealed with 0.5% agarose solution (w/v) to ensure contact with the polyacrylamide gel. The run program was then applied at 100 V for 30 min and a further 250 V for 2 h. After the run period, the gels were immersed in a fixative solution containing10% acetic acid (v/v) and 40% ethanol (v/v) for 30 min. Then, the proteins were revealed with colloidal Coomassie G-250 (USB, Cleveland, Ohio, United States) for 72 h and removed by washing with ultrapure water [38][39][40][41] .
The gels obtained (Supplemental Fig. S1) were scanned and their images analyzed with the image processing program ImageMaster 2D Platinum 7.0 (GeneBio, Geneva, Switzerland; www. gelif escie nce. com), which allows the estimation of the isoelectric points and molecular masses of the separated proteins and calculation of the number of spots obtained via gel electrophoresis. Three replicates of each gel were used to evaluate the reproducibility of each protein spot obtained in the replicates of the gels by overlaying the image from one gel over the other in the image treatment program 39-42 . Protein identification by mass spectrometry (LC-MS/MS). The differentially expressed spots were characterized via mass spectrometry after the identification was standardized according to the highest protein score, pI, and molecular mass (MM) closest to the theoretical and experimental results. Among the proteins identified, 12 were classified as functional for this study as they are related to energy metabolism and inflammatory responses.
The protein spots were characterized with LC-MS/MS after being subjected to tryptic digestion and peptide elution according to the methodology Shevchenko et al. 43 described. The aliquots of the solutions containing the eluted peptides were analyzed to obtain the mass spectra with the nanoAcquity UPLC system coupled to the Xevo G2 QTof mass spectrometer (Waters, Milford, MA, United States). Proteins were identified by searching in the UniProt database (www. unipr ot. org) within the Bos taurus species. Proteins were considered depending on their theoretical and experimental isoelectric points, molecular masses, and scores (> 60). After identifying FASTA sequences in the proteins, their sequences were analyzed with OMICSBOX software (BLAST2GO) 44 and they were categorized by their molecular function, biological processes, and biochemical activities with gene ontology (GO).
Proteomic statistical analysis. The starch level and additive were the fixed effects analyzed in a factorial design; thus, the groups were compared through contrasts to verify differentially expressed protein spots. Only proteins with significantly altered levels were selected for identification by MS. The images were analyzed with www.nature.com/scientificreports/ ImageMaster Platinum software version 7.0, which establishes correlations (matching) between groups. For this correlation, three gel replicates were compared for volume, distribution, relative intensity, isoelectric point, and molecular mass in an analysis of variance (ANOVA) with a t-test to determine the significance of differentially expressed protein spots.
Following the average mode of background subtraction, individual spot intensity volume was normalized with total intensity volume (the summation of the intensity volumes obtained from all spots in the same 2-DE gel). The normalized intensity volume values of individual protein spots were then used to determine differential protein expression among experimental groups. A heatmap showed the correlation coefficient of the spot expression values and, after checking the differentially expressed spots (t-test, P < 0.05), the log2 FC values were used for hierarchical cluster analysis.
Pathways enrichment analysis. The same KEGG-IDs were used to analyze metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes function (KEGG pathways) [45][46][47] and Reactome pathway enrichment analysis yielded similar results about the specific pathways affected, allowing the expressions of proteins encoding enzymes found in the database to be mapped.

Conclusions
In verifying the differential expression of the cecal proteome in cattle, our results show that the blend of essential oils associated with α-amylase incorporated as a feed additive for beef cattle increased the expression of enzymes at dietary starch levels of 25%, 35%, and 45% compared with monensin. The higher expression of proteins related to carbohydrate degradation that participate in glycolysis and gluconeogenesis depended on increased feed intake and reduced protein synthesis expression. The optimum starch level was 35% for both feed additives; higher concentrations of starch (45%) increased the expression of inflammatory responses and reduced the expression of proteins involved in energy metabolism, probably due to damage to the cecum epithelium. www.nature.com/scientificreports/