Feedlot diets containing increasing starch levels and different feed additives changes cecal proteome profile involved on energy metabolism and inflammatory response of Nellore cattle 2

Background: Diets for feedlot cattle require higher energy density, thus contributing to the high rate 13 of fermentable carbohydrate. The use of feed additives is necessary to reduce possible metabolic 14 disorders. The objective of this study was to analyze the post-rumen effects of different levels of starch 15 (25, 35, and 45%) and additives (Monensin, Blend of essential oil + exogenous α -Amylase) in diets 16 for Nellore cattle feedlot. The cecum tissue proteome was separated by two-dimensional 17 polyacrylamide gel electrophoresis (2D-PAGE), and then, with the verification of differentially 18 expressed protein SPOTS, these were characterized by electrospray ionization mass spectrometry 19 (ESI-MS/MS). Results: The expression of nine enzymes participating in the Steps of the glycolysis pathway was 21 verified, such as: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Glyceraldehyde-3- 22 phosphate dehydrogenase like-17 protein, Triosephosphate isomerase (Step 1); Phosphoglycerate 23 mutase and Phosphoglycerate mutase 2 (Step 2); Alpha-enolase (ENO1), Beta-enolase (ENO3) and 24 Fructose-bisphosphate pyruvate: L-lactate dehydrogenase B, L-lactate dehydrogenase A chain and L-lactate dehydrogenase. 27 The ATP synthase subunit beta and ATP synthase subunit beta_mitochondrial participate in the 28 electron transport chain, producing ATP from ADP in the presence of a proton gradient across the 29 membrane. Due to the manipulation of diets, the expression of the Leukocyte elastase inhibitor protein, 30 associated with the inflammatory response. 31 Conclusions: The use of blends of essential oil associated with α -amylase as a feed additive promoted 32 the greater expression of enzymes in the pathway of glycolysis and gluconeogenesis (and the absence 33 of proteins linked to inflammation (Leukocyte elastase inhibitor) in cecum tissues. On the other hand, 34 the increase in starch in the diets promoted a reduction in enzymes linked to carbohydrate degradation 35 with increased responses linked to inflammatory injuries.

PMSF, with ultrapure water in the presence of protease inhibitors). The buffer which showed the beset 137 results for protein extraction was ultrapure water. 138 To extract the protein fraction, the tissue was macerated with a mortar and pestle in the presence 139 of liquid nitrogen. The extracting solution was added in a proportion of 1g/1 mL (tissue/ultrapure 140 water), and then homogenized with an OMMI-BEAD RUPTOR4 cell disruptor (Kennesaw, Georgia, 141 The gels obtained were scanned and their images analyzed using the image processing program 178 ImageMaster 2D Platinum 7.0 (GeneBio, Geneva, Switzerland), which allows the isoelectric points 179 and the molecular masses of the separated proteins to be estimated, and the number of SPOTS obtained 180 in gel electrophoresis to be calculated. Three replicates of each gel run were used to evaluate the 181 reproducibility of each protein SPOT obtained in the replicates of the gels, by overlaying the image 182 from one gel over the other, using the image treatment program [24][25][26][27]. 183

Protein identification by mass spectrometry (ESI MS) 184
The protein spots were characterized by ESI-MS after being subjected to tryptic digestion and 185 the elution of peptides according to the methodology described by [28]. The aliquots of the solutions 186 containing the peptides were analyzed to obtain the mass spectra through the nanoAcquity UPLC 187 system coupled to the Xevo G2 QTof mass spectrometer (Waters, Milford, MA, USA). The 188 identification of proteins was performed by searching in database UniProt (2020)

Image analysis and SPOTS expression 209
In the "Workspace", Classes (Groups) were created to analyze differences in protein 210 expression; for that, the analysis of variance (ANOVA) tests the hypothesis (H θ ) that the expressed 211 SPOTS are identical (as shown in supplementary material). When testing all classes, protein SPOTS 212 were differentially expressed, as described in Table 2. 213 214 215

Proteins characterization by ESI-MS/MS 230
The differentially expressed SPOTS were characterized from mass spectrometry, after the 231 identification was standardized considering the highest Score Protein, pI and molecular mass (MM) 232 closest to the theoretical and experimental results. Among the proteins identified, 15 were addressed 233 as functional for the purpose of the study, which involve energy metabolism and inflammatory 234 response.  (Table 3)

Effects of feed additives and starch level on glucose and energy metabolism 266
The expression values ( 05 . 0 ≤ P ) (Table 4) were grouped from the hierarchical cluster 267 analysis, and ordered by the homogeneity between the treatments tested. Animals fed with identical 268 levels of starch, but submitted to different feed additives, showed differentiation for proteins that 269 exercise functions in energy metabolism.

298
In view of the different feeding strategies, key enzymes were identified in the degradation of 299 carbohydrates in the large intestine of cattle. Fructose-bisphosphate aldolase (ALDOB), an enzyme 300 that converts fructose-1,6-bisphosphate to fructose 6-phosphate catalyzed by Triosephosphate 301 isomerase (TPI), is a precursor of glyceraldehyde-3-phosphate (GA3P), which is acted upon by the 302 glyceral enzyme 3-phosphate dehydrogenase (GAPDH) during glycolysis. Alpha-enolase (ENO1) and 303 beta-enolase (ENO3) are isoforms of enolase that are involved in Step 4 of glycolytic metabolism. 304 Phosphoglycerate mutase (PGM) is a catalytic enzyme that converts 3-phosphoglycerate to 2-305 phosphoglycerate, and finally pyruvate kinase (PKM), which synthesizes pyruvate in the last step of 306 glycolysis. In ruminants, a high concentration of starch enables the fermentation of carbohydrates in 307 the cecum with lactate production, which increases glucose metabolism in the intestine observed 308 expression of the enzyme L-lactate dehydrogenase and its isoforms L-lactate dehydrogenase B and L-309 lactate dehydrogenase A, which are responsible for the synthesis of lactate from pyruvate. 310 311

Inflammatory response 312
In the protein SPOTS of groups 25BEOα and 45BEOα, in relation to those fed MON, there 313 was an absence in the expression of leukocyte elastase inhibitor, which is a serine protease inhibitor 314 that is essential in the regulation of inflammation responses, and which limits the activity of 315 inflammatory caspases [29]. When comparing 25% vs. 45% of starch in the diet, regardless of the type 316 of additive used, there was a greater expression of this protein, corroborating with previous studies, 317 which demonstrate that inflammatory injuries are caused by the increased use of concentrate in diets 318 [30,31]. 319 320

Conclusions 321
To verify the differential expression of the cecal proteome in cattle, our results show that the 322 blend of essential oils associated with α-amylase, incorporated as a feed additive for beef cattle, The datasets used can be made available by the corresponding author on reasonable request. 346 347 Ethics approval and consent to participate 348 The experiment was carried out according to the standards issued by the National Council for 349 Animal Experimentation Control -CONCEA, and approved by the Ethics and Use of Animals 350