Understanding the regulatory mechanisms of milk production using integrative transcriptomic and proteomic analyses: improving inefficient utilization of crop by-products as forage in dairy industry

Understanding the regulatory mechanisms of milk production using integrative transcriptomic and... Background: Bovine milk is an important nutrient source for humans. Forage plays a vital role in dairy husbandry via affecting milk quality and quantity. However, the differences in mammary metabolism of dairy cows fed different forages remain elucidated. In this study, we utilized transcriptomic RNA-seq and iTRAQ proteomic techniques to investigate and integrate the differences of molecular pathways and biological processes in the mammary tissues collected from 12 lactating cows fed corn stover (CS, low-quality, n = 6) and alfalfa hay (AH, high-quality, n =6). Results: A total of 1631 differentially expressed genes (DEGs; 1046 up-regulated and 585 down-regulated) and 346 differentially expressed proteins (DEPs; 138 increased and 208 decreased) were detected in the mammary glands between the CS- and AH-fed animals. Expression patterns of 33 DEPs (18 increased and 15 decreased) were consistent with the expression of their mRNAs. Compared with the mammary gland of AH-fed cows, the marked expression changes found in the mammary gland of CS group were for genes involved in reduced mammary growth/development (COL4A2, MAPK3, IKBKB, LGALS3), less oxidative phosphorylation (ATPsynGL, ATP6VOA1, ATP5H, ATP6VOD1, NDUFC1), enhanced lipid uptake/metabolism (SLC27A6, FABP4, SOD2,ACADM,ACAT1, IDH1,SCP2,ECHDC1),more active fatty acid beta-oxidation (HMGCS1),less amino acid/ protein transport (SLC38A2, SLC7A8, RAB5a, VPS18), reduced protein translation (RPS6, RPS12, RPS16, RPS19, RPS20, RPS27), more proteasome- (PSMC2, PSMC6, PSMD14, PSMA2, PSMA3) and ubiquitin-mediated protein degradation (UBE2B, UBE2H, KLHL9, HSPH1, DNAJA1 and CACYBP), and more protein disassembly-related enzymes (SEC63, DNAJC3, DNAJB1, DNAJB11 and DNAJC12). Conclusion: Our results indicate that the lower milk production in the CS-fed dairy cows compared with the AH-fed cows was associated with a network of mammary gene expression changes, importantly, the prime factors include decreased energy metabolism, attenuated protein synthesis, enhanced protein degradation, and the lower mammary cell growth. The present study provides insights into the effects of the varying quality of forages on mammary metabolisms, which can help the improvement of strategies in feeding dairy cows with CS-based diet. Keywords: Dairy cow, Forage source, Mammary gland, Milk protein production, Proteomics, Transcriptomics * Correspondence: hyliu@zju.edu.cn Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hang Zhou 310058, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dai et al. BMC Genomics (2018) 19:403 Page 2 of 18 Background proteome (expressed protein set from genome) [9]. To Forage accounts for 50% or more of the diet of dairy date, iTRAQ-based quantitative proteomics has exten- cows [1]. Therefore, the quality of forage has a large ef- sively improved protein identification coverage, thus fect on bovine milk production in the dairy industry. In providing more comprehensive linking of proteins to China, alfalfa hay (AH) is widely used as a high-quality their metabolic functions. Recently, integrating tran- forage, but approximately 230 million kilogram of AH scriptomic and quantitative proteomic analyses have has to be imported from other countries annually be- been widely used to promote a better understanding of cause of a shortage [2], substantially increasing milk pro- the molecular mechanisms driving biological process in duction cost. Furthermore, the gap between supply and cells and tissues [11–15]. Here our objective was to elu- demand for high-quality forage is increasing by approxi- cidate a more complete understanding of molecular mately 10% annually [3]. However, China produces ap- mechanisms underlying mammary gland adaptation to proximately 100 million tons of corn stover (CS; a main the alternative nutrient supplies provided by two rations crop byproduct produced in North China) each year, with different forage sources (AH and CS). We per- and most of this crop byproduct is disposed or burned formed transcriptomic and proteomic analyses of the [4]. The use of CS in dairy feed has been limited due to its mammary gland tissue samples collected from 12 lactat- low nutritional value. From an environmental prospective ing cows fed either AH) or CS). and to reduce the dependence on imported AH, it is stra- tegically important for China to explore how to efficiently Methods utilize these low quality forages in dairy production. Experiment design and sample collection Therefore, it is our goal to investigate the regulatory All animal care and procedures in this study were ap- mechanism through which various forages exert their ef- proved by the Animal Care Committee, Zhejiang Uni- fects on dairy production, which will help us to develop versity (Hangzhou, P. R. China) and were in accordance new methods to improve the efficiency of utilizing with the Zhejiang University’s guidelines for animal re- low-quality forages (such as CS) in dairy feeding. search. Twelve multiparous Chinese Holstein dairy cows Compared with AH, CS has lower protein content were randomly assigned into two blocks, among which (crude protein, rumen degradable protein, and rumen six cows with the milk yield of 30.3 ± 5.1 kg/d (mean ± ungradable protein) and non-fiber carbohydrates [5]. In SD) and the body weight of 604 ± 37.4 kg were fed CS as recent studies from our group, cows fed AH-based diets forage, and the other six cows with the milk yield of had better production performance in milk yield, milk 29.2 ± 4.4 kg/d and the body weight of 608 ± 41.4 kg protein content, and milk efficiency (milk yield/dry mat- were fed AH as forage. The two blocks of cows were fed ter intake) compared to cows fed CS-based diets [6]. To 55% concentrate (dry matter, DM), 15% corn silage, and explore the mechanisms underlying the low production either 30% CS (CS group; n = 6) or 23% AH and 7% performance of cows fed a CS diet, we performed the Chinese wild rye hay (AH group; n = 6). The dietary following studies. First, we found that the dairy cows fed compositions and nutritional values of both diets were an AH-based diet had enhanced metabolisms of several reported previously [6]. The experiment was conducted amino acids, including phenylalanine, serine, threonine, over a 14-wk period, with the first 2 wk. for dietary tyrosine, arginine and proline, in the mammary gland adaptation, in Hangjiang Dairy Farm (Hangzhou, China). compared to cows consuming CS-based diets [7]. Sec- The cows were slaughtered at the end of experiment, ond, we demonstrated that low feed and nitrogen effi- and mammary tissue samples were collected from each ciency may play a vital role in contributing to low milk cow, placed in sterile tubes, immediately snap-frozen in protein production in the mammary glands of cows con- liquid N , and subsequently stored at − 80 °Cfor extrac- suming low-quality forage of CS and rice straw [8]. tion of RNA and protein. However, the regulatory mechanisms by which the different-quality forages affect bovine mammary metabo- RNA preparation, cDNA synthesis and RNA sequencing lisms and the subsequent milk production remained RNA was extracted from mammary tissue with Trizol re- largely unknown. agent (Invitrogen, Carlsbad, CA, USA) and purified with Sustained developments in the nucleotide sequencing Qiagen RNeasy kit (Qiagen, Valencia, CA, USA). RNA technology, especially RNA-sequencing, has resulted in quality was examined with the Agilent 2100 Bioanalyzer an explosive growth in the number and quality of tran- (Agilent Technologies, Palo Alto, CA), and RNA quantity scriptome sequenced for various tissues in both prokary- was measured with a NanoDrop (NanoDrop Technologies, otes and eukaryotes [9, 10] and their potential changes Inc. Wilmington, DE, USA). The RNA integrity number under different conditions. Also, a certain of dynamicity (RIN) [16] was more than 7.0. Approximately 5 μgof high by post-transcriptional and translational regulations of quality RNA was used for cDNA library construction ac- gene expression can be estimated by measuring its cording to the Illumina RNA ligation-based method [17]. Dai et al. BMC Genomics (2018) 19:403 Page 3 of 18 2.5 μg RNA from the mammary gland of two cows in each 7500 real-time PCR system (Applied Biosystems, Foster group were combined (three biological replicates per City,CA, USA).Each20 μL reaction included 50 ng of re- group; Fig. 1), and RNA sequencing was performed using a verse transcription product, 40 nM of each forward and re- HISeq 2000 sequencing system (Illumina, San Diego, CA, verse primer [Additional file 1:Table S1,designedby USA) from LC Sciences (Houston, TX, USA). Reads were Primer 5 software (Premier Biosoft International, Palo Alto, processed with Bowtie version 0.12.7 (http://bowtie-bio.- CA, USA)], and SYBR Premix Taq (Takara). The PCR pro- sourceforge.net), aligned in Tophat version 1.3.2 (http:// gram was one cycle of 95 °C for 30 s plus 40 cycles of amp- ccb.jhu.edu/software/tophat/index.shtml), and then mapped lification at 95 °C for 5 s and 58 °C for 30 s, followed by an to bovine reference genome UMD3.1 (ftp://ftp.ensem- additional 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C bl.org/pub/release-79/fasta/bos_taurus/dna/)[18]. Gene to generate melt curves. The relative gene expression −ΔΔCt abundance was estimated as reads per kilobase of exon values were calculated by the 2 method [19]. The model per million mapped reads (RPKM) with Cufflinks gene expression levels were normalized against the internal version 1.2.1 as previously described [17, 18]. Genes with a control genes β-actin and GAPDH. cut-off of 1.5-fold expression changes and p-value less than 0.05 between CS and AH groups were defined as differen- Protein preparation and digestion tially expressed. Protein preparation and digestion were performed as in the previous studies [20, 21]. Briefly, 500 mg mammary The qRT-PCR analysis tissue was ground to a fine powder in liquid N , lysed Total RNA extracted from the mammary gland was reverse with the lysis buffer A (7 M Urea, 2 M Thiourea, 4% RT transcribed for cDNA synthesis using a PrimeScript Re- CHAPS (3-[(3-Cholamidopropy) dimethylammonio] agent Kit with gDNA Eraser (Takara, Tokyo, Ostu, Japan) propane-sulfonate), 40 mM Tris-HCl, pH 8.5), and re- following the manufacturer’s instructions. The qRT-PCR duced with 10 mM DTT at 56 °C for 1 h, followed by al- was performed in triplicate using the Applied Biosystems kylation with 55 mM IAM (Iodoacetamide) in a Fig. 1 An overview of the transcriptomic and proteomic experiment. Schematic diagram of workflow of the RNA-seq transcriptomic and iTRAQ-based proteomic experiments. Six cows were fed either corn stover (CS) or alfalfa hay (AH) as forage for 14 weeks. RNA from two cows and protein samples from three cows within each group were pooled for transcriptomic (3 replicates/group) and proteomic (2 replicates/group) analyses. For transcriptomic assay, the pooled RNA was sequenced on the Illumina platform, and subsequently the reads were aligned and mapped to the Bos taurus genome. For the proteomic assay, the extracted proteins were digested with trypsin, and the peptides are labeled with different iTRAQ reagents, which contain reporter groups of different masses (114, 115, 116, 117), balance groups of different masses (191, 190, 189, 188), and a reactive group (R). The labeled peptides are then mixed equivalently and fractionated by strong cation exchange (SCX) chromatography. Fractions were separated by liquid chromatography (LC) and analyzed by two-step mass spectrometry (MS) Dai et al. BMC Genomics (2018) 19:403 Page 4 of 18 darkroom for 1 h. The reduced and alkylated protein selected mass range of 350–2000 mass/charge (m/z). mixtures were precipitated by adding 4 × volume of The electrospray voltage applied was 1.6 kV. For MS chilled acetone and incubating at − 20 °C overnight. scans, the m/z scan range was 350 to 2000 Da. For After centrifugation at 4 °C and 30,000×g, the pellet was MS/MS scans, the m/z scan range was 100–1800. dissolved in 0.5 M TEAB (Triethylamine borane; Ap- MS/MS data was acquired using the top 15 most plied Biosystems, Milan, Italy) and sonicated on ice. abundant precursor ions with the ion count more After centrifugation at 30,000×g at 4 °C again, an aliquot than 20,000 in the MS scan. These were selected with of the supernatant was assigned for determination of an isolation window of 2 m/z and were fragmented protein concentration by the Bradford method [22]. The via high energy collisional dissociation under normal- proteins in the supernatant were kept at − 80 °C for fur- ized collision energies of 30 eV. For the MS scan, the ther analysis. resolving power was set to 70,000 at m/z 200, the maximal ion injection time was 10 ms and, dynamic The iTRAQ labeling and strong cationic exchange (SCX) exclusion of the selected precursor ions was 15 s. fractionation Automatic gain control (AGC) was used to optimize Total protein (150 μg obtained by mixing 50 μg protein the spectra generated from the orbitrap, and the AGC from the mammary glands of three cows in each group, target value was 3,000,000 for full MS and 100,000 two biological replicates per group; Fig. 1) was digested for MS , respectively. For the MS/MS scans, the re- with Trypsin Gold (Promega, Madison, WI, USA) at 37 ° solving power was set to 17,500 at m/z 200; max- C for 16 h with the ratio of protein: trypsin = 30: 1. After imum ion injection times for MS/MS scans were at digestion, peptides were dried by vacuum centrifugation. 60 ms; and the underfill ratio was defined as 0.1%. Peptides were then reconstituted in 0.5 M TEAB and processed following the manufacturer’sprotocolfor Protein identification and quantification 4-plex iTRAQ reagent (Applied Biosystems) [21]. The The raw files were first merged and transformed to an protein samples from the CS and AH groups were labeled MGF file with Proteome Discoverer ver. 1.2 (Thermo with iTRAQ reagents 114, 115, 116 and 117. Strong cat- Fisher Scientific, San Jose, CA, USA) and were then ionic exchange chromatography was performed with an probed on the Mascot search engine (ver. 2.3.02; Matrix LC-20AB HPLC pump system (Shimadzu, Kyoto, Japan). Science, London, UK) of the Uniprot database of bovine The procedures for SCX fractionation including the elu- (Bos tauru) with 31,661 entries. For protein identifica- tion were essentially the same as in the study of Meng et tion, the parameters were set the same as in the study by al. [20]. Finally, the eluted peptides were pooled into 20 Yang et al. [23] with some minor changes: carbamido- fractions, desalted with a Strata X C18 column (Phenom- methyl (C), iTRAQ 4-plex (N-term), and iTRAQ 4-plex enex, Torrance, CA, USA), and vacuum-dried. (K) were defined as fixed modifications; Gln→pyro-Glu (N-term Q), oxidation (M), deamidated (NQ) were the Liquid chromatography–tandem mass spectrometry (LC/MS) possible variable modifications. The decoy database pat- analysis tern was considered as the reverse of the target database. The sample fractions described above were further The method of peptide identification by false discovery separated and identified on an LC-20 AD nano-HPLC rate (FDR) was performed as Sheng et al. described [24]. system (Shimadzu) loaded with Q-Extractive mass Relative quantification of the identified proteins was spectrometer (Thermo Fisher Scientific, San Jose, CA, performed with the Proteome Discoverer software de- USA). Buffer C consisted of 2% acetonitrile (ACN) scribed above and calculated by the weighted and and 0.1% formic acid (FA) in Milli-Q water; buffer D normalized ratios of uniquely identified peptides that consisted of 98% ACN and 0.1% FA. After resuspen- belong to the specific individual protein. The integra- sion with buffer C, 10 μL sample supernatant was tion window tolerance of the peak was set to 20 ppm. loaded by the auto-sampler onto a C18 trap column Statistics analysis was conducted using Fisher’stest. (2 cm × 100 μm, 5 μm) and then separated on the Proteins with a cutoff of 1.2-fold change between CS reverse-phase analytical C18 column (100 mm × and AH samples and p < 0.05 were determined as sig- 75 μm, 3 μm). The samples were loaded at 8 μL/min nificantly differentially expressed proteins. for 4 min, then a 44 min gradient was run at 300 nL/min starting from 2 to 35% buffer D, followed by 2 min linear gradient to 80% buffer D, and then Bioinformatics analysis maintenance at 80% buffer D for 4 min, and finally Functional annotations were performed using Blast2GO return to 5% buffer D in 1 min. program against the non-redundant protein database (NR; Peptide analysis was performed with a Q-Exactive NCBI). Metabolic pathway analysis was conducted using mass spectrometer in a positive ion mode with a R software (R version 3.2.3) according to the Kyoto Dai et al. BMC Genomics (2018) 19:403 Page 5 of 18 Encyclopedia of Genes and Genomes (KEGG) pathway through one-way ANOVA in SAS (SAS 9.0). The statis- database (http://www.genome.jp/kegg/). The pathway en- tical significance was declared at p ≤ 0.05. richment statistics were performed by Fisher’s exact test with a p-value ≤ 0.05 considered as significant. Results Overview of transcriptomic and quantitative proteomic analyses Western blot analysis Figure 1 shows the workflow of our integrative RNA-seq Approximately 40 μg protein per sample was separated transcriptomic and iTRAQ-based proteomic experi- on 12% SDS (sodium dodecyl sulfate) polyacrylamide ments. In the transcriptomic analysis, 78,138,798 and gels. Proteins were transferred onto 0.45 μm PVDF 90,522,588 raw/clean reads were detected in the CS and membranes (IPVH00010; Millipore, Boston, Massachu- AH groups, respectively (Additional file 2: Table S2), setts, USA) and blocked with blocking buffer (Beyotime, and in proteomic analysis, 62,367 unique spectra were Jiangsu, China). The membranes were incubated with strictly matched to 24,606 unique peptides and further primary antibodies to SCP2 (non-specific lipid-transfer mapped to 3744 unique proteins (Additional file 2: Table protein 2, ab140126; Abcam, Cambridge, MA, USA), S3). With a cutoff of 1.5-fold change and a p-value < IDH2 (isocitrate dehydrogenase 2; ab131263, Abcam), 0.05, a total of 1631 differentially expressed genes SLC7A8 [(solute carrier family 7 (amino acid transporter, (DEGs) were identified between CS and AH groups in L system), member 8, ab75610, Abcam)], COL4A2 (col- transcriptomic analysis, among which 1046 DEGs were lagen, type IV, alpha 2, sc-70,243, Santa Cruz biotechnol- up-regulated and 585 were down-regulated (Fig. 2 and ogy; Cambridge, MA, USA), and β-actin (Beyotime). Additional file 3: Table S4). Using a threshold of 1.2-fold After washing with TBST [tris-buffered saline containing change and p < 0.05, 346 differentially expressed proteins 0.02% (v/v) Tween-20] three times, the membranes were (DEPs; 138 up-regulated and 208 down-regulated) were incubated with goat anti-rabbit IgG or goat anti-mouse detected between CS and AH groups in proteomic ana- IgG secondary antibodies conjugated with horseradish lysis (Fig. 2 and Additional file 3: Table S5). By compar- peroxidase (Beyotime), incubated with ECL (electroche- ing the RNA-seq data with the proteomic data (Fig. 2 miluminescence) Western Blotting Substrate Kits (Beyo- and Additional file 3: Table S6), 40 genes displayed dif- time), and finally visualized with a Kodak Image Station ferential expression at both mRNA and protein levels, of 2000MM (Kodak Molecular Imaging Systems, New which 18 and 15 genes were consistently up- or Haven, USA). The relative intensities of bands were cal- down-regulated, respectively, whereas the remaining 7 culated with ImagePro Plus 6.0 software (Media Cyber- genes had inconsistent expression in mRNA and protein netics, Washington, MD, USA) using β-actin as the levels, which may result from post-translational reference protein. modifications. Statistical analysis The gene ontology (GO) analysis of DEGs and DEPs The data on relative mRNA expression by qRT-PCR and The GO analysis of all the DEGs and DEPs in cows fed protein expression by Western blot were analyzed CS versus AH is shown in Fig. 3. In the cellular Fig. 2 The venn diagram of the differentially expressed genes (DEGs) and proteins (DEPs) in the mammary gland of cows fed either corn stover (CS) or alfalfa hay (AH). The cut-off of differential expression of mRNA is set at 1.5-fold change and p < 0.05, whereas the cutoff of differential expression of protein is set at 1.2-fold and p < 0.05 Dai et al. BMC Genomics (2018) 19:403 Page 6 of 18 Fig. 3 Gene ontology (GO) categories assigned to the differentially expressed genes (DEGs, inner cycle) and proteins (DEPs, outer cycle) in the mammary gland of cows fed either corn stover (CS) or alfalfa hay (AH). The differentially expressed genes were classified into cellular component, biological process, and molecular function by WEGO (Web Gene Ontology Annotation Plot) according to the GO terms component category, most of the DEGs and DEPs were predominant functions of the DEGs and DEPs, and a mainly assigned to cell (25.8% genes and 24% proteins), relatively low proportion of DEGs and DEPs were associ- cell part (25.8% genes and 24% proteins), organelle ated with various biological activities such as molecular (18.9% genes and 19.2% proteins) and organelle part transducer (3.3% genes and 3.2% proteins), transcription (9.8% genes and 10.2% proteins). Notably, a small num- regulator (2.9% genes and 1.4% proteins), transporter ber of the DEGs and DEPs were located in the extracel- (2.2% genes and 7.8% proteins), structural molecule lular region (3.9% genes and 5.4% proteins), (1.6% genes and 6.0% proteins) and electron carrier membrane-enclosed lumen (3.9% genes and 3.0% pro- (0.4% genes and 1.1% proteins). In general, the DEGs teins) and macromolecular complexes (6.5% genes and and DEPs displayed similar GO annotation patterns. 6.9% proteins). In the biological process category, a large We also performed further functional analysis of the number of DEGs and DEPs were involved in cellular up- and down-regulated genes and proteins using the process (16.9% genes and 17.2% proteins), metabolic UniProt knowledgebase and GO database. The increased process (13.0% genes and 12.6% proteins) and biological genes (Fig. 4) in the CS group were significantly and regulation (10.9% genes and 9.0% proteins). Noticeably, abundantly enriched for genes involved in the positive some DEGs and DEPs were assigned to response to regulation of apoptosis, unfolded protein binding, nega- stimulus (4.8% genes and 7.3% proteins), immune system tive regulation of protein ubiquitination and response to process (2.3% genes and 2.4% proteins), cellular compo- stress. In addition, we found that some genes with in- nent biogenesis (2.3% genes and 2.3% proteins) and cell creased abundance were enriched in the negative regula- growth (0.8% genes and 0.9% proteins). For the molecu- tions of translation, DNA binding, NF-κB transcription lar function category, the GO terms including binding factor activity, phosphorylation, cell cycle and in (43.1% genes and 41.7% proteins), enzyme regulator ac- proteasome-mediated/ubiquitin-dependent protein cata- tivity (29.1% genes and 5.1% proteins) and catalytic ac- bolic processes. Notably, nine genes with up-regulated tivity (16.6% genes and 30.6% proteins) were the expression were enriched in the GO term GDP binding. Dai et al. BMC Genomics (2018) 19:403 Page 7 of 18 Fig. 4 Functional characterization of the increased or decreased transcripts in the mammary gland of cows fed alfalfa hay (AH) vs. corn stover (CS) by gene ontology analysis. DEG indicates differentially expressed genes. The x-axis shows the functional categories of the increased or decreased genes, the left y-axis shows the value of –Log (p-value) and the right y-axis shows the number of increased/decreased genes In contrast, the decreased genes (Fig. 4) were principally pathway. Notably, four decreased genes were significantly enriched in cell adhesion, positive regulation of cell pro- enriched in the GO term “cellular response to amino acid liferation, binding activities of actin/receptor/carbohy- stimulus”. Furthermore, the GO term “ATPase activity” drate, structural molecule activity and protein binding was significantly enriched in six down-regulated genes. (295 decreased genes enriched). Additionally, some The up-regulated proteins (Fig. 5) were primarily down-regulated genes were related to the positive regu- enriched in negative regulation of multicellular organis- lation of several transcriptional factors (Stat3 phosphor- mal and developmental processes. In addition, we ob- ylation and NF-κB signaling) and cell-growth associated served a small proportion of increased proteins enriched processes (cell proliferation, multicellular organism in the GO terms related to lipid translocation including growth, and cell growth). Importantly, a small number of lipid transport and long-chain fatty acid binding, and in decreased genes were associated with the cytoskeleton/ GO terms involved in cation homeostasis and ion chan- extracellular matrix and cytokine-mediated signaling nel inhibitor activity. Specifically, eight up-regulated Dai et al. BMC Genomics (2018) 19:403 Page 8 of 18 Fig. 5 Functional characterization of the increased or decreased proteins in the mammary gland of cows fed alfalfa hay (AH) vs. corn stover (CS) by gene ontology analysis. DEP indicates differentially expressed proteins. The x-axis shows the functional categories of increased or decreased proteins, the left y-axis shows the value of –Log (p-value) and the right y-axis shows the number of increased/decreased proteins proteins were enriched in enzyme inhibitor activity, biosynthetic process and three decreased proteins were which may suggest a general reduction of the related to ATPase activity. enzyme-mediated metabolism. The GO annotation ana- lysis of the down-regulated proteins is shown in Fig. 5. KEGG pathway analysis of DEGs and DEPs Notably, the most abundant GO term was extracellular Table 1 shows the KEGG pathway enrichment analysis matrix organization enriched in ten down-regulated pro- of DEGs. In the analysis, a total of 7 decreased DEGs teins. In addition, the body defense-related GO terms, were significantly enriched in the pathway of protein di- such as response to external stimulus, cellular response gestion and absorption (p = 0.0269). In contrast, 10 in- to amino acid stimulus, and defense response, were sig- creased DEGs were significantly enriched in protein nificantly enriched with some decreased proteins. Some processing in endoplasmic reticulum (p = 0.045). Add- proteins of down-regulation were involved in tissue de- itionally, we found that 4, 11, and 7 increased genes were velopment, organ morphogenesis, biological adhesion significantly enriched in protein degradation-related pro- and response to transforming growth factor beta. Im- cesses ubiquitin mediated proteolysis, spliceosome and portantly, 16 down-regulated proteins were involved in proteasome, respectively. In particular, the significant substrate-specific transporter activity. Furthermore, six pathways (p ≤ 0.05) enriched by some decreased DEGs decreased proteins were enriched in glycosyl-compound were related to protein synthesis (such as ribosome Dai et al. BMC Genomics (2018) 19:403 Page 9 of 18 Table 1 The KEGG pathway enrichment by up−/down-regulated genes in the mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH) KEGG ID Pathway Name P-value of No. of ID of Increased Genes Gene Symbol of No. of ID of Decreased Genes Gene Symbol of Fisher’ Increased Increased Genes Decreased Decreased Genes exact Test Genes Genes ko04974 Protein digestion 0.0269 0 7 XLOC_022008;XLOC_001311; SLC38A2; SLC7A8; and absorption XLOC_000568;XLOC_027248; BT.23508; COL12A1; XLOC_000569;XLOC_017584; COL6A2; COL17A1; XLOC_020771 COL6A3 ko04141 Protein processing 0.0450 10 XLOC_000859;XLOC_003872;XLOC_006584; HSPH1;DNAJB1;DNAJA1; 0 in endoplasmic XLOC_009297;XLOC_020319;XLOC_022100; DDIT3;CRYAB;SAR1B;BT.59327; reticulum XLOC_024455;XLOC_025681;XLOC_026233; MAN1A2;SEC63;DNAJB11 XLOC_027001 ko04120 Ubiquitin mediated 0.0277 4 XLOC_024865;XLOC_018298;XLOC_021649; UBE2B;HERC4;UBE2H; 1 XLOC_025357 KEAP1 proteolysis XLOC_025981 BT.19212 ko0970 Aminoacyl-tRNA 0.0312 1 XLOC_025065 RARS biosynthesis ko03010 Ribosome 0.0201 1 XLOC_028013 RPS23 ko03008 Ribosome biogenesis 0.0219 2 XLOC_001726; XLOC_010292 FCF1;UTP6 3 XLOC_025000;XLOC_023340; TCOF1;RRP7A;NOL6 in eukaryotes XLOC_026685 ko03040 Spliceosome 0.0388 11 XLOC_000309;XLOC_004289;XLOC_007964; SNRPB2;TRA2B;SMNDC1;TRA2A; 1 XLOC_012196 SFRS4 XLOC_012182;XLOC_012566;XLOC_017604; BCAS2;SF3B1;PHF5A;BT.59135; XLOC_019531;XLOC_021421;XLOC_023325; SLU7;BT.91058;BT.91058;PLRG1 XLOC_025841; XLOC_028252 ko03050 Proteasome 0.0142 7 XLOC_001624;XLOC_001843;XLOC_003577; BT.22570;PSMD14;PSMA3; XLOC_004136;XLOC_012473;XLOC_020934; BT.56882;PSMA2;POMP;PSMC2 XLOC_021072 ko04142 Lysosome 0.0394 0 5 XLOC_001832;XLOC_006153; BT.35140; LAPTM5; XLOC_010442;XLOC_012190; ARSB; CD68; CTSH XLOC_013979 ko04150 mTOR signaling 0.0300 1 XLOC_018127 DDIT4 1 XLOC_013054; RICTOR pathway ko04350 TGF-beta signaling 0.0190 2 XLOC_003540;XLOC_018178 ID2;BT.48514 3 XLOC_004021;XLOC_004591; FST;ID1;TFDP1 pathway XLOC_013257 ko04115 p53 signaling 0.0247 5 XLOC_007111;XLOC_012771;XLOC_025054; GADD45G;SESN1;BT.36413; 2 XLOC_021046;XLOC_018555 IGFBP3;BT.33239 pathway XLOC_026769;XLOC_026996 SESN2;CCNG1 ko04310 Wnt signaling 0.0458 1 XLOC_007394 CACYBP 1 XLOC_007966 SFRP2 pathway ko03050 Proteasome 0.0142 7 XLOC_001624;XLOC_001843;XLOC_003577; BT.22570;PSMD14;PSMA3; XLOC_004136;XLOC_012473;XLOC_020934; BT.56882;PSMA2;POMP;PSMC2 XLOC_021072 Dai et al. BMC Genomics (2018) 19:403 Page 10 of 18 biogenesis in eukaryotes, aminoacyl-tRNA biosynthesis VPS18 protein (VPS18), and the uncharacterized protein and lysosome). Also, a small number of DEGs were ORAI1] were involved in the processes of “protein significantly enriched in the pathways of mTOR sig- localization” and “transport”. Intriguingly, two naling pathway, p53 signaling pathway, and two up-regulated genes DHFR and KRT15 at mRNA and cell-growth-associated signaling pathways (the protein levels were related to one carbon metabolism TGF-beta signaling and Wnt signaling). and staphylococcus aureus infection, respectively. Add- The KEGG pathway enrichment analysis of the DEPs itionally, the 3 collagens (COL1A1, COL1A2 and COL4A2) is shown in Table 2, and a total of 29 KEGG pathways and mitogen-activated protein kinase 3 (MAPK3) were significantly enriched. Importantly, 7 decreased down-regulated at both mRNA and protein levels were in- DEPs were significantly enriched (p = 0.0101) in the volved in the protein synthesis-related pathways of “pro- “protein digestion and absorption” pathway, while 7 in- tein digestion and absorption” and “ECM-receptor creased DEPs were significantly enriched (p = 0.037) in interaction”. the “protein processing in endoplasmic and reticulum” pathway. Several DEPs were involved in pathways associ- Verification of DEGs by qRT-PCR and DEPs by western ated with energy metabolism― glycolysis/gluconeogene- blot analysis sis (p = 0.0302), citrate cycle (p = 0.0399), pentose Nineteen genes involved in energy metabolism, transcrip- phosphate pathway (p = 0.0352), and PI3K-Akt signaling tion/translation, protein processing/transport, protein (p = 0.0375). Additionally, the KEGG pathways enriched degradation, amino acid metabolism/ transport, fatty acid by a small number of DEPs were related to the protein oxidation, and mammary gland growth and development synthesis/processing (including ribosome, p = 0.0069; were selected for qRT-PCR analysis (Additional file 5: aminoacyl-tRNA biosynthesis, p = 0.0236; mTOR signal- Table S8). Among these genes (Fig. 6), expression levels of ing pathway, p = 0.035; lysosome, p = 0.0350; protein 15 genes were significantly altered between CS- and AH- processing in endoplasmic reticulum, p = 0.0370; and groups, and the abundance of 11 of the 15 gene abun- spliceosome, p = 0.0232) and the metabolisms of several dance were consistent with expression patterns measured amino acids. Noticeably, we also found that a certain of by RNA-seq. Four proteins, including IDH2, SLC7A8, proteins were significantly enriched in two SCP2,and COL4A2 were selected for Western blot ana- cell-growth-signaling pathways (Wnt and TGF-beta sig- lysis (Fig. 7 and Additional file 5: Table S8). Western blot naling pathways; p = 0.0375 and 0.0272, respectively). analysis showed that the protein levels of IDH2, SLC7A8, and COL4A2 in the mammary glands of cows fed the Functional analysis of the common DEGs and DEPs CS-based diet were lower than those cows fed the AH The 40 common expressed genes at mRNA and protein diet, whereas the level of SCP2 was higher. All the immu- levels were further analyzed by the GO and KEGG path- noblot results were consistent with the findings from way (Additional file 4: Table S7). Importantly, 3 in- proteomic analysis. creased genes at mRNA and protein levels (DnaJ homolog subfamily B members—DNAJB11, DNAJB1, Relationship between the DEGs/DEPs and mammary and DNAJC12) were related to protein unfolding and in- metabolism volved in the pathway of “protein processing in endo- A comprehensive view of the molecular mechanisms plasmic reticulum”. Also, two common genes of underlying milk production was summarized based on up-regulation (dihydrolipoamide branched chain transa- the proteomic and transcriptomic data collected (Fig. 8 cylase, DBT and hydroxymethylglutaryl-CoA synthase, and Additional file 6: Table S9). The regulatory subsec- HMGCS1) were involved in the process of “valine, leu- tions proposed include: energy metabolism, amino acid cine and isoleucine degradation”. Noticeably, the de- (AA) /fatty acid metabolism, protein degradation, pro- creased genes IDH2 and ATPase 2 (ATP2B4) at mRNA tein synthesis, protein processing, AA/protein transport, and protein levels were involved in TCA cycle and ATP and cell growth and development. Each of these regula- synthesis, respectively; in contrast, 4 common genes of tory functions is involved in the regulation of mammary up-regulated expression [HMGCS1, SCP2, DBT and metabolism, and the collective analysis shows how the isopentenyl-diphosphate delta-isomerase 1 (IDI1))] were mammary gland adapts to the low nutrient availability of all associated with fatty-acyl-CoA metabolic process. In the CS ration compared with the higher nutrient avail- particular, the commonly decreased gene dimethylgly- ability on the AH ration. Despite that the gene- and cine dehydrogenase (DMGDH) was associated with “gly- protein-level responses are not always consistent, the cine, serine and threonine metabolism”. In addition, relatively higher degree of agreement between analyses several commonly expressed genes of down-regulation of DEGs and DEPs at the functional level suggests that [such as erythrocyte membrane protein (EPB41L3), transcriptomic analysis of the mammary gland might be odorant-binding protein (MGC151921), DMGDH, sufficient to characterize tissue functional responses to Dai et al. BMC Genomics (2018) 19:403 Page 11 of 18 Table 2 The KEGG pathway enrichment by up−/down-regulated proteins in the mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH) KEGG ID Pathway Name P-value of No. of ID of Increased Proteins Gene Symbol of Increased Proteins No. of ID of Decreased Proteins Gene Symbol of Fisher’ exact Increased Decreased Decreased Proteins Test Transcripts Proteins ko03040 Spliceosome 0.0232 4 IPI00687479;IPI00715218; SNRNP40;LSM3; SNRPB;PRPF8 6 IPI00690232;IPI00699558; MAGOHB;SNRPD3; IPI00702381; IPI00687395 IPI00687560;IPI00687015; PCBP1;SNRPD2;SF3B4;BUD31 IPI00717302; IPI00688521 ko04141 Protein 0.0370 7 IPI00702891;IPI00699038; ERP29; TXNDC5; DNAJC3;DNAJB11; 1 IPI00692963 SEC23 processing in IPI00693007; IPI00699107; CALR; SSR2; DNAJB1 endoplasmic IPI00691963; IPI00696616; reticulum IPI00688461 ko04974 Protein digestion 0.0101 0 7 IPI00707857;IPI00708244; COL4A2; COL3A1; and absorption IPI00711933;IPI00712524; COL5A2; COL1A1; IPI00731432;IPI00826022; COL1A2;COL11A1; IPI00905045 COL18A1 ko04142 Lysosome 0.0314 2 IPI00711862; IPI00706203 NPC2; HEXB 4 IPI00697314;IPI00699372; CTSC; ATP6V0A1; IPI00717554;IPI00716195 NAGLU; ATP6V0D1 ko03010 Ribosome 0.0069 0 6 IPI00695732;IPI00699146; RPS2; RPS16; RPS20; IPI00707431;IPI00713536; RPS19; RPS12; RPS27 IPI00714445;IPI00715091 ko00260 Glycine, serine 0.0090 1 IPI00698589 PGAM1 3 IPI00698059;IPI00707303; SARDH; DMGDH; MAOA and threonine IPI00715285 metabolism ko04150 mTOR signaling 0.0350 1 IPI00700182 EIF4B 2 IPI00903663; IPI00732002 IKBKB; MAPK3 pathway ko03008 Ribosome 0.0374 2 IPI00705941;IPI00708018 REXO2; RAN 1 IPI00852474 NAT10 biogenesis in eukaryotes ko00280 Valine, leucine 0.0468 3 IPI00711918;IPI00717256; DBT; ACAT1; HMGCS1 and isoleucine IPI00968674 degradation ko00380 Tryptophan 0.0381 1 IPI00711918 ACAT1 1 IPI00698059 MAOA metabolism ko00330 Arginine and 0.0370 0 2 IPI00698059;IPI00838420 P4HA2; MAOA proline metabolism ko00970 Aminoacyl-tRNA 0.0236 0 2 IPI00689365;IPI00703906 TARS2; AARS2 biosynthesis ko00360 Phenylalanine 0.0473 0 1 IPI00698059 MAOA metabolism ko00340 Histidine 0.0461 0 1 IPI00698059 MAOA metabolism ko00350 Tyrosine 0.0365 0 1 IPI00698059 MAOA metabolism ko00270 0.0432 1 IPI00694739 APIP Dai et al. BMC Genomics (2018) 19:403 Page 12 of 18 Table 2 The KEGG pathway enrichment by up−/down-regulated proteins in the mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH) (Continued) KEGG ID Pathway Name P-value of No. of ID of Increased Proteins Gene Symbol of Increased Proteins No. of ID of Decreased Proteins Gene Symbol of Fisher’ exact Increased Decreased Decreased Proteins Test Transcripts Proteins Cysteine and methionine metabolism ko00010 Glycolysis / 0.0302 3 IPI00696912; IPI00698589; ACSS1; PGAM1; GALM 2 IPI00687211;IPI00715799 HK1; GAPDHS Gluconeogenesis IPI00712164 ko00190 Oxidative 0.0287 0 5 IPI00697768;IPI00699372; ATPsynGL;ATP6V0A1; phosphorylation IPI00712252;IPI00716163; ATP5H;NDUFC1; IPI00716195 ATP6V0D1 ko00020 Citrate cycle 0.0399 2 IPI00702781;IPI00708438 IDH1; SUCLG1 1 IPI00714468 IDH2 (TCA cycle) ko00640 Propanoate 0.0423 3 IPI00696912;IPI00708438; ACSS1; ACAT1; SUCLG1 metabolism IPI00711918 ko00030 Pentose phosphate 0.0352 2 IPI00728589;IPI00904104 TKT; RBKS pathway ko04146 Peroxisome 0.0169 4 IPI00686601;IPI00702781; SOD2; SCP2; IDH1; ECH1 1 IPI00714468 IDH2 IPI00704382;IPI00714468 ko03320 PPAR signaling 0.0263 4 IPI00686601;IPI00699355; SCP2; FABP4; APOA1; PPARD pathway IPI00715548;IPI00839653 ko04975 Fat digestion 0.0370 2 IPI00695965;IPI00715548 APOA4; APOA1 1 IPI00710056 APOB and absorption ko04540 Gap junction 0.0352 0 2 IPI00695917;IPI00732002 MAPK3; GNAS ko04210 Apoptosis 0.0261 1 IPI00704835 DFFA 2 IPI00709124; IPI00903663 ENDOG; IKBKB ko04151 PI3K-Akt signaling 0.0301 1 IPI00700182 EIF4B 9 IPI00697595;IPI00707857; COL4A2; IKBKB; pathway IPI00708244;IPI00712524; COL3A1; COL5A2; IPI00731432;IPI00732002; COL1A1; COL1A2; IPI00826022;IPI00903663; COL11A1; MAPK3; ITGA1 IPI00905045 ko04310 Wnt signaling 0.0375 1 IPI00708311 CACYBP 1 IPI00699355 PPARD pathway ko04350 TGF-beta signaling 0.0272 0 1 IPI00732002 MAPK3 pathway Dai et al. BMC Genomics (2018) 19:403 Page 13 of 18 Fig. 6 Real time PCR analysis of mRNA expression changes of genes involved in mammary metabolism of cows fed corn stover (CS) and alfalfa hay (AH). Relative mRNA expression levels were normalized by the levels of β-actin. Error bars represent the standard deviation. ** and * indicate that the difference in gene expression between CS and AH groups reached p < 0.01, and 0.01 < p < 0.05, respectively altered states but might not always be reflective of shifts milk production in the mammary gland. In this study, in specific proteins. we detected 1046 up-regulated and 585 down-regulated genes by RNA-seq transcriptomics as well as 138 in- Discussion creased and 208 decreased proteins by iTRAQ proteo- Roughage is one of the most important components in mics in the mammary gland of dairy cows fed CS- ruminant diets, the quality of which has a large impact versus AH-based diets. There were only 33 genes with on milk performance in dairy cows [25]. Our previous consistent expression patterns measured by both tran- study showed that compared to cows fed AH as the for- scriptomic and proteomic analyses, suggesting that the age source, cows fed CS had lower yields of milk (kg/d; post-transcriptional regulation may play an important 23.5 vs. 19.4), milk fat (kg/d; 0.98 vs. 0.82), milk protein role in gene expression. The relatively limited overlap of (kg/d; 0.77 vs. 0.62) and lactose (kg/d; 1.15 vs. 0.94) (P < proteins and mRNAs was also seen in other studies in 0.01) [6]. In addition, as a major crop byproduct, a large human [14], mouse [11], fish [26] and bacteria [27]. quantity of corn stover is burned by humans, and this The GO term annotation can help to characterize inefficient utilization of crop residues subsequently pro- physiological and functional changes associated with the duces heavy environmental pollution. Therefore, it is ur- changes in mRNA and protein expression in cells and gent for us to determine strategies to improve the tissues [28]. In up-regulated genes in CS-fed cows versus utilization of low-quality forage (CS) in the dairy indus- AH-fed cows, the GO term “ubiquitin-dependent/pro- try and reduce damage to the environment caused by teasome-mediated protein catabolic process” was signifi- burning CS. However, little is known about the effects of cantly enriched, which may indicate enhanced mammary roughage consumption on the molecular mechanisms in protein catabolism in cows fed CS. Enrichment in the Fig. 7 Western blot analysis of expression of IDH2, SLC7A8, SCP2, and COL4A2 proteins in the mammary gland of cows fed corn stover (CS) and alfalfa hay (AH). β-Actin was used as a sample loading control. ** and * indicate p < 0.01 and 0.01 < p < 0.05, respectively. IDH2: Isocitrate dehydrogenase 2; SLC7A8: also referred as LAT2, L type amino acid transporter 2; SCP2: sterol carrier protein 2; COL4A2: collagen type IV alpha 2 Dai et al. BMC Genomics (2018) 19:403 Page 14 of 18 Fig. 8 An overview of possible biological changes that might contribute to low milk production in cows fed corn stover-based diet vs. cows fed alfalfa hay-based diet. The color coding for the individual genes is as follows: black letters and pink background represents the up-regulated genes, yellow letters and pink background represents increased proteins, black letters and green background represents the down-regulated genes, and blue letters and green background represents decreased proteins. The half blue/half black letters with green background represents genes that were both down-regulated at mRNA and protein levels. The half black/half yellow letters with pink background represent the genes that were up-regulated at both protein and gene levels. The full name of each protein is listed in Additional file 6: Table S9 GO terms of negative regulation of translation, NF-κB associated with positive regulation of Stat3 phosphoryl- transcription factor activity, and DNA binding activity ation and NF-κB signaling pathway in CS-fed cows was suggested inhibited protein synthesis in the mammary consistent with the reduced protein synthesis in these gland, consistent with the reduced milk protein yield in animals. Meanwhile, the lower expression levels of the CS-fed cows [6]. In addition, several proteins that were proteins involved in biological adhesion, extracellular up-regulated in CS-fed cows were enriched in the GO matrix organization, tissue development, and organ mor- term “enzyme inhibitor activity”, which is also consistent phogenesis were consistent with possible reduced mam- with an overall reduced mammary metabolism in these mary cell growth and mammary tissue development in cows. Furthermore, some increased proteins were in- CS-fed cows. Furthermore, the proteins that were lower volved in positive regulation of apoptosis and negative in abundance were enriched in the GO term “response regulations of multicellular organismal processes and de- to transforming growth factor beta”, indicating that velopmental processes, in line with the reduced cell TGF-β may be a major signaling molecule in the regula- growth in the CS group as shown in our previous micro- tion of mammary cell growth in cows fed CS. Collect- RNAome analysis [8]. Interestingly, the fact that the GO ively, GO analysis indicated that the reduced cell growth terms of lipid transport and long-fatty acid binding were and metabolism, attenuated protein synthesis, and en- enriched in the up-regulated proteins in CS-fed cows sup- hanced protein degradation may play major roles leading ported the idea that the mammary gland of these cows to the low milk production in CS-fed dairy cows. may take up more fatty acids from the blood for mam- The KEGG pathway analysis has also been widely used mary metabolism as these cows had a lower acetate supply for systematic understanding of the gene functions in to the mammary gland than AH-fed cows [6]. cells or organisms from large-scale molecular data sets Consistently, the genes that were expressed lower in [29, 30]. In this study, the up-regulated genes were CS-fed cows were enriched for the GO terms of cell ad- enriched in ubiquitin mediated proteolysis, and the hesion, positive regulation of cell proliferation, and genes with lower abundance were enriched in ribosome multicellular organism growth, which also indicated the (the main machinery for protein synthesis), suggesting attenuated cell growth in the mammary gland of CS-fed that these pathways may play an integrative role in low- animals. In addition, down-regulation of genes ering milk protein production in the mammary gland of Dai et al. BMC Genomics (2018) 19:403 Page 15 of 18 CS-fed cows [6]. Additionally, some down-regulated SLC7A8 genes was 0.38- fold lower in CS-fed cows com- proteins from CS-fed cows were enriched in the mTOR pared to the AH-fed cows, indicating a reduced system signaling pathway and metabolisms of several amino L amino acid transporter activity for neutral amino acids acids, including glycine, serine, threonine, arginine, pro- (including isoleucine, leucine, methionine, phenylalanine, line, phenylalanine, histidine, and tyrosine, which is also threonine, alanine and serine) in the mammary gland of consistent with the lower milk protein in the CS group CS-fed cows [41]. This finding was in consistent with [6, 8]. In particular, three down-regulated proteins in our previous observations of amino acid uptake in these CS-fed cows were associated with valine, leucine and cows [6, 31]. isoleucine degradation, indicating a reduction of Ribosomes consist of a small 40S and a large 60S sub- branched-amino acids production in milk [31]. More- units, and ribosomal proteins are required for different over, the pathways enriched among differentially stages of ribosome biogenesis and/or for distinct steps of expressed proteins included the Wnt and TGF-β signal- the translation process [42]. All six ribosomal protein sub- ing pathways. These pathways are known to play roles in units (RPS2, RPS12, RPS16, RPS19, RPS20 and RPS27) regulating mammary growth and differentiation [32–34]. expressed at lower levels in CS-fed cows were essential Milk production is a highly energy-dependent process components of the small ribosomal subunit (40S). The and requires sufficient ATP [35]. In most eukaryotes, lower abundance of these ribosomal proteins in the CS oxidative phosphorylation in the mitochondria involving group suggests depressed efficiency of protein translation, a series of ATPases is the prime metabolic pathway to contributing to lower milk protein in these cows [31]. generate energy [36]. In the study, we observed that the For protein processing, proteins and peptides must be expression of several ATPase components (including transported into the endoplasmic reticulum (ER) or ATPsynGL, ATP6V0A1, ATP5H and ATP6V0D1) and Golgi, where they acquire modifications that allow them one mitochondrial enzyme NADH dehydrogenase (ubi- to be biologically active [43]. Specific three-dimensional quinone) 1 subunit C1 (NDUFC1) involved in oxidative conformations acquired through folding of newly trans- phosphorylation were lower in CS-fed cows. The lower lated polypeptides and/or refolding of unfolded proteins levels of these enzymes may potentially contribute to a are essential for protein function formation and main- lower ATP production in the mammary tissue of these tenance [44]. The protein DNAJB1 is a heat shock protein animals. For high-yield dairy cows, particularly during (HSP) [an ER molecular chaperone that can protect other negative energy balance, ketone bodies (included acetoa- proteins against occurrence of incorrect folding, but may cetate, β-hydroxy-butyrate, and acetone) are produced in also stimulate ER-associated degradation (ERAD)] [45]. the liver but utilized in other tissues of the body (includ- As an ER molecular chaperone, the protein DNAJB11 is ing the mammary gland) as an energy source [37]. essential to prevent the processes such as protein aggrega- Acetyl-CoA acetyltransferase (ACAT1) plays a major role tion, allowing protein folding and assembly to proceed in ketone body synthesis, transferring one acetyl group correctly [46]. In this study, expression of DNAJB1 and to another acetyl-CoA (the crucial substrate for ketone DNAJB11 was higher at the mRNA and protein levels in body generation) [38]. Furthermore, the rate-limiting the CS-fed cows, suggesting that these cows may have enzyme in ketone body synthesis is HMGCS1, which higher degree of protein non-aggregation and protein deg- promotes β-hydroxy-butyrate production [39]. The in- radation in the mammary gland. The protein heat shock creased expression of both ACAT1 and HMGCS1 in 105 kDa/110 kDa protein 1 (HSPH1), another HSP, can be CS-fed cows may reflect an increased level of mammary induced by several kinds of environmental stress [47]and ketone body production in cows consuming CS, which is associated with mammary tumor tissues [48]. In the results in an increased level of ketone body utilization as current study, the highly levels of HSPH1 in the CS-fed energy for the mammary gland of these cows, in line cows may suggest that the CS diet induces ER-stress in with the high abundance of β-hydroxybutyric acid and the mammary gland. The increased amount of protein acetoacetyl-CoA in blood of CS-fed cows versus AH-fed degradation in the mammary gland is in accordance with cows [7]. the reduced milk protein and lactation performance in the Amino acids are fundamental proteogenic substrates CS group [6]. for milk synthesis in dairy cows, most of which derive The proteasome is a large, 26S-multicatalytic protease from dietary proteins. After digestion and absorption in that degrades poly-ubiquitinated proteins to small pep- the small intestine, amino acids are taken up from the tides [49]. The proteasome is composed of two blood by the mammary gland via amino acid trans- sub-complexes: a core catalytic 20S particle and a regula- porters. The protein SLC7A8 is a system L amino acid tory 19S particle [50]. Expression of two 19S regulatory transporter that functions by a Na -independent and particles [proteasome 26S subunit, ATPase 6 (PSMC6) electroneutral transport mechanism for neutral amino and proteasome 26S subunit, ATPase 2 (PSMC2)], the acids [40]. In this study, mammary expression of two 20S core particles [proteasome subunit alpha 3 Dai et al. BMC Genomics (2018) 19:403 Page 16 of 18 (PSMA3) and proteasome subunit alpha 2 (PSMA2)] and cows. Taken together, expression changes of multiple proteasome 26S subunit, non-ATPase 14 (PSMD14) was genes involved in fatty acid β-oxidation and fatty acid higher in the mammary gland of cows fed the CS-based transport indicate a more active fatty acid metabolism diet, supporting enhanced protein degradation in these to supply energy in the CS-fed cows compared to cows. In addition, many intracellular proteins become co- AH-fed cows. This observation is consistent with our valently modified with ubiquitin (UB) or ubiquitin-like previous finding that the CS-fed cows had a lower proteins (UBLs) [51]. Then, ubiquitin-conjugating enzymes supply of acetate which is the major energy source in (E1s) transfers the activated modifier to a family of E2 ruminants [6]. ubiquitin-conjugating enzymes, leading to their degradation Finally, six collagen proteins and five genes encoding [52]. In this study, expression of ubiquitin-conjugating en- collagen proteins were lower in the mammary gland of zyme 2B (UBE2B), ubiquitin-conjugating enzyme E2H CS-fed cows. Collagens are the main structural proteins (UBE2H) and target recognizing subunit kelch-like family in extracellular matrix (ECM), and are important member 9 (KLHL9) was higher in CS-fed cows, further indi- regulators of the differentiated phenotype of mammary cating active protein degradation in the mammary glands of epithelial cells in culture [31, 32]. Lower levels of these these animals. Taken together, the higher abundance of the proteins may indicate a different mammary gland proteasome-related and ubiquitin-dependent proteins is morphology in these animals. Specifically, a decrease in consistent with a higher degree of protein degradation in the levels of the protein COL4A2 was found to be the mammary cells of CS-fed cows. Both the possibly in- associated with a loss of basement membrane integrity, creased protein degradation and reduced protein synthesis accompanied by a dramatic alteration of alveolar morph- may result in lower milk protein yield in the CS group. ology with decreased size and shrunken lumen contain- Peroxisomes are essential organelles that play a key ing little β-casein [32]. Mammary structural changes role in lipid homeostasis. The protein SCP2 acts in per- may be another factor contributing to the reduction of oxisome cholesterol transport through the cytoplasm, milk yield in the CS-fed cows compared with cows fed and loss of SCP2 can result in defects in fatty acid AH [31]. β-oxidation [53]. Higher SCP2 measured at mRNA andprotein levels in cows fedCS-baseddiet sug- gested an enhanced level of fatty acid β-oxidation in Conclusions themammarygland. Theprotein isocitrate dehydro- By integrating transcriptomic and proteomic data, this genase [NADP] 1 (IDH1) is an enzyme that catalyzes study suggested four major possible mechanisms con- oxidative decarboxylation of isocitrate, producing tributing to the lower milk production in dairy cows fed alpha-ketoglutarate (α-ketoglutarate) and CO .Cyto- CS-diet compared to AH-diet: (i) reduced mammary solic IDH1 (EC 1.1.1.41) and mitochondrial IDH2 (EC growth/development through lower expression of 1.1.1.42) catalyze the same reaction outside the con- COL4A2, MAPK3, IKBKB, and LGALS3, (ii) less oxida- text of the citric acid cycle and use NADP as a co- tive phosphorylation through lower expression of ATP- factor instead of NAD [54]. Cytosolic IDH1 plays a synGL, ATP6VOA1, ATP5H, ATP6VOD1, and NDUFC1 complementary role in reductive glutamine metabol- with enhanced lipid uptake and fatty acid beta-oxidation ism, possibly through its oxidative function in an to supply energy through higher expression of SLC27A6, IDH2/IDH1 shuttle that transfers high energy elec- FABP4, SOD2, ACADM, ACAT1, IDH1, SCP2, ECHDC1, trons in the form of NADPH from mitochondria to and HMGCS1, (iii) less AA/protein transport and me- cytosol, especially under hypoxia [55]. In this study, tabolism through lower expression of SLC38A2, SLC7A8, the higher IDH1 and the lower levels of IDH2 in RAB5a, and VPS18, and less protein translation through CS-fed cows may indicate that the complementary lower expression of RPS6, RPS12, RPS16, RPS19, RPS20, IDH1-dependent carboxylation pathway is enhanced and RPS27, and (iv) more proteasome- and in CS-fed cows, enabling higher citrate production for ubiquitin-mediated protein degradation through higher ex- energy supply. Acyl-CoA dehydrogenases (ACADs) pression of the protease components PSMC2, PSMC6, are a class of enzymes that function to catalyze the PSMD14, PSMA2, PSMA3, and ubiquitin-conjugating en- initial step in each cycle of fatty acid β-oxidation in zymes UBE2B, UBE2H, KLHL9, HSPH1, DNAJA1, and the mitochondria of cells [56]. Higher expression of CACYBP, and protein disassembly-associated enzymes acyl-CoA dehydrogenase (ACADM), combined with SEC63, DNAJC3, DNAJB1, DNAJB11, and DNAJC12.These the higher levels of long fatty acid transporter mechanisms involved in milk production of dairy cows fed (SLC27A6), in CS-fed cows may indicate a higher CS-forage-based diet can direct future work to further transport activity of long fatty acid from the plasma understand how the mammary gland adapts to low nutrient into the mammary cells, thus supporting a higher availability and, ultimately, to change feeding strategies so level of fatty acid oxidation for energy supply in these that cows can utilize low-quality forage more efficiently. Dai et al. BMC Genomics (2018) 19:403 Page 17 of 18 Additional files Acknowledgements The authors gratefully thank the personnel of Hangjiang Dairy Farm (Hangzhou, China) for their assistance in the feeding and care of the animals. Additional file 1: Table S1. Primers used in real-time RT-PCR. (XLSX 11 kb) Additional file 2: Table S2. Summary of the transcriptome data in the Funding mammary gland of dairy cows fed corn stover (CS) or alfalfa hay (AH). This research was supported by grants from the National Key Research and Table S3. Summary of the proteome data in the mammary gland of Development Program of China (2016YFD0500503) and the National Natural dairy cows fed corn stover (CS) or alfalfa hay (AH)). (ZIP 13 kb) Science Foundations of China (31672447, 31372336). The funders had no role Additional file 3: Table S4. Differentially expressed genes in the in study design, data collection, analysis and interpretation, or preparation of mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH). The the manuscript. cutoff is set at 1.5-fold change and p < 0.05. Table S5. Differentially expressed proteins in the mammary gland of cows fed corn stover (CS) Availability of data and materials vs. alfalfa hay (AH). The cutoff is set at 1.2-fold change and p < 0.05. All data in this study are included in the article and its additional files. Table S6. Differentially expressed genes found in both transcriptomic and proteomic analyses in the mammary gland of cows fed corn stover Authors’ contributions (CS) vs. alfalfa hay (AH). (ZIP 164 kb) DWT and LHY conceived of the study and designed the project. DWT Additional file 4: Table S7. A list of common differentially expressed performed the lab work, analyzed data and wrote the manuscript. WQJ genes and differentially expressed proteins in the mammary gland of helped the qRT-PCR experiment and the KEGG pathway analysis. LHY, ZFQ cows fed corn stover (CS) vs. alfalfa hay (AH). (XLSX 17 kb) and LJX revised the manuscript. All the authors have read and approved the final version of the manuscript. Additional file 5: Table S8. The full name and abbreviation of proteins listed in Figs. 6 and 7. (XLSX 14 kb) Ethics approval and consent to participate Additional file 6: Table S9. The full name and abbreviation of proteins The procedures of this study were approved by the Animal Care and Use in Fig. 8. (XLSX 16 kb) Committee of Zhejiang University (Hangzhou, China) and were in accordance with the university’s guidelines for animal research. Abbreviations Competing interests AARS2: Probable alanyl-tRNA synthetase; ACADM: Acyl-CoA dehydrogenase, The authors declare that they have no competing interests. C-4 to C-12 straight chain; ACAT1: Acetyl-CoA acetyltransferase; ACN: Acetonitrile; ACSS1: Acetyl-coenzyme A synthetase 1; AGC: Automatic Publisher’sNote gain control; AH: Alfalfa hay based diet; ATP5H: ATP synthase subunit d; Springer Nature remains neutral with regard to jurisdictional claims in ATP6V0A1: Isoform 1 of V-type proton ATPase 116 kDa subunit a isoform 1; published maps and institutional affiliations. ATP6V0D1: V-type proton ATPase subunit d1; ATPsynGL: ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G-like; CACYBP: Calcyclin Author details binding protein; CHAPS: 3-[(3-Cholamidopropy) dimethylammonio] propane- Institute of Dairy Science, College of Animal Sciences, Zhejiang University, sulfonate; COL4A2: Collagen, type IV, alpha 2; CS: Corn stover based diet; Hang Zhou 310058, People’s Republic of China. Laboratory of Lactation and DBT: Dihydrolipoamide branched chain transacylase E2; Metabolic Physiology, Department of Animal and Veterinary Sciences, DMGDH: Dimethylglycine dehydrogenase; DNAJA1: DnaJ (Hsp40) homolog, University of Vermont, Burlington, VT 05405, USA. subfamily A, member 1; DNAJB1: DnaJ (Hsp40) homolog, subfamily B, member 1; DNAJB11: DnaJ homolog subfamily B member 11; DNAJC12: DnaJ Received: 6 July 2017 Accepted: 21 May 2018 homolog subfamily C member 12; DNAJC3: DnaJ homolog subfamily C member 3; ECHDC1: Enoyl CoA hydratase domain containing 1; FA: Formic acid; FABP4: Fatty acid binding protein 4; FDR: False discovery rate; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HK1: Hexokinase 1; References HMGCS1: Hydroxymethylglutaryl-CoA synthase; HSPH: Heat shock 105 kDa/ 1. Kendall C, Leonardi C, Hoffman P, Combs D. 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Understanding the regulatory mechanisms of milk production using integrative transcriptomic and proteomic analyses: improving inefficient utilization of crop by-products as forage in dairy industry

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Life Sciences; Life Sciences, general; Microarrays; Proteomics; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics and Genomics
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Abstract

Background: Bovine milk is an important nutrient source for humans. Forage plays a vital role in dairy husbandry via affecting milk quality and quantity. However, the differences in mammary metabolism of dairy cows fed different forages remain elucidated. In this study, we utilized transcriptomic RNA-seq and iTRAQ proteomic techniques to investigate and integrate the differences of molecular pathways and biological processes in the mammary tissues collected from 12 lactating cows fed corn stover (CS, low-quality, n = 6) and alfalfa hay (AH, high-quality, n =6). Results: A total of 1631 differentially expressed genes (DEGs; 1046 up-regulated and 585 down-regulated) and 346 differentially expressed proteins (DEPs; 138 increased and 208 decreased) were detected in the mammary glands between the CS- and AH-fed animals. Expression patterns of 33 DEPs (18 increased and 15 decreased) were consistent with the expression of their mRNAs. Compared with the mammary gland of AH-fed cows, the marked expression changes found in the mammary gland of CS group were for genes involved in reduced mammary growth/development (COL4A2, MAPK3, IKBKB, LGALS3), less oxidative phosphorylation (ATPsynGL, ATP6VOA1, ATP5H, ATP6VOD1, NDUFC1), enhanced lipid uptake/metabolism (SLC27A6, FABP4, SOD2,ACADM,ACAT1, IDH1,SCP2,ECHDC1),more active fatty acid beta-oxidation (HMGCS1),less amino acid/ protein transport (SLC38A2, SLC7A8, RAB5a, VPS18), reduced protein translation (RPS6, RPS12, RPS16, RPS19, RPS20, RPS27), more proteasome- (PSMC2, PSMC6, PSMD14, PSMA2, PSMA3) and ubiquitin-mediated protein degradation (UBE2B, UBE2H, KLHL9, HSPH1, DNAJA1 and CACYBP), and more protein disassembly-related enzymes (SEC63, DNAJC3, DNAJB1, DNAJB11 and DNAJC12). Conclusion: Our results indicate that the lower milk production in the CS-fed dairy cows compared with the AH-fed cows was associated with a network of mammary gene expression changes, importantly, the prime factors include decreased energy metabolism, attenuated protein synthesis, enhanced protein degradation, and the lower mammary cell growth. The present study provides insights into the effects of the varying quality of forages on mammary metabolisms, which can help the improvement of strategies in feeding dairy cows with CS-based diet. Keywords: Dairy cow, Forage source, Mammary gland, Milk protein production, Proteomics, Transcriptomics * Correspondence: hyliu@zju.edu.cn Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hang Zhou 310058, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dai et al. BMC Genomics (2018) 19:403 Page 2 of 18 Background proteome (expressed protein set from genome) [9]. To Forage accounts for 50% or more of the diet of dairy date, iTRAQ-based quantitative proteomics has exten- cows [1]. Therefore, the quality of forage has a large ef- sively improved protein identification coverage, thus fect on bovine milk production in the dairy industry. In providing more comprehensive linking of proteins to China, alfalfa hay (AH) is widely used as a high-quality their metabolic functions. Recently, integrating tran- forage, but approximately 230 million kilogram of AH scriptomic and quantitative proteomic analyses have has to be imported from other countries annually be- been widely used to promote a better understanding of cause of a shortage [2], substantially increasing milk pro- the molecular mechanisms driving biological process in duction cost. Furthermore, the gap between supply and cells and tissues [11–15]. Here our objective was to elu- demand for high-quality forage is increasing by approxi- cidate a more complete understanding of molecular mately 10% annually [3]. However, China produces ap- mechanisms underlying mammary gland adaptation to proximately 100 million tons of corn stover (CS; a main the alternative nutrient supplies provided by two rations crop byproduct produced in North China) each year, with different forage sources (AH and CS). We per- and most of this crop byproduct is disposed or burned formed transcriptomic and proteomic analyses of the [4]. The use of CS in dairy feed has been limited due to its mammary gland tissue samples collected from 12 lactat- low nutritional value. From an environmental prospective ing cows fed either AH) or CS). and to reduce the dependence on imported AH, it is stra- tegically important for China to explore how to efficiently Methods utilize these low quality forages in dairy production. Experiment design and sample collection Therefore, it is our goal to investigate the regulatory All animal care and procedures in this study were ap- mechanism through which various forages exert their ef- proved by the Animal Care Committee, Zhejiang Uni- fects on dairy production, which will help us to develop versity (Hangzhou, P. R. China) and were in accordance new methods to improve the efficiency of utilizing with the Zhejiang University’s guidelines for animal re- low-quality forages (such as CS) in dairy feeding. search. Twelve multiparous Chinese Holstein dairy cows Compared with AH, CS has lower protein content were randomly assigned into two blocks, among which (crude protein, rumen degradable protein, and rumen six cows with the milk yield of 30.3 ± 5.1 kg/d (mean ± ungradable protein) and non-fiber carbohydrates [5]. In SD) and the body weight of 604 ± 37.4 kg were fed CS as recent studies from our group, cows fed AH-based diets forage, and the other six cows with the milk yield of had better production performance in milk yield, milk 29.2 ± 4.4 kg/d and the body weight of 608 ± 41.4 kg protein content, and milk efficiency (milk yield/dry mat- were fed AH as forage. The two blocks of cows were fed ter intake) compared to cows fed CS-based diets [6]. To 55% concentrate (dry matter, DM), 15% corn silage, and explore the mechanisms underlying the low production either 30% CS (CS group; n = 6) or 23% AH and 7% performance of cows fed a CS diet, we performed the Chinese wild rye hay (AH group; n = 6). The dietary following studies. First, we found that the dairy cows fed compositions and nutritional values of both diets were an AH-based diet had enhanced metabolisms of several reported previously [6]. The experiment was conducted amino acids, including phenylalanine, serine, threonine, over a 14-wk period, with the first 2 wk. for dietary tyrosine, arginine and proline, in the mammary gland adaptation, in Hangjiang Dairy Farm (Hangzhou, China). compared to cows consuming CS-based diets [7]. Sec- The cows were slaughtered at the end of experiment, ond, we demonstrated that low feed and nitrogen effi- and mammary tissue samples were collected from each ciency may play a vital role in contributing to low milk cow, placed in sterile tubes, immediately snap-frozen in protein production in the mammary glands of cows con- liquid N , and subsequently stored at − 80 °Cfor extrac- suming low-quality forage of CS and rice straw [8]. tion of RNA and protein. However, the regulatory mechanisms by which the different-quality forages affect bovine mammary metabo- RNA preparation, cDNA synthesis and RNA sequencing lisms and the subsequent milk production remained RNA was extracted from mammary tissue with Trizol re- largely unknown. agent (Invitrogen, Carlsbad, CA, USA) and purified with Sustained developments in the nucleotide sequencing Qiagen RNeasy kit (Qiagen, Valencia, CA, USA). RNA technology, especially RNA-sequencing, has resulted in quality was examined with the Agilent 2100 Bioanalyzer an explosive growth in the number and quality of tran- (Agilent Technologies, Palo Alto, CA), and RNA quantity scriptome sequenced for various tissues in both prokary- was measured with a NanoDrop (NanoDrop Technologies, otes and eukaryotes [9, 10] and their potential changes Inc. Wilmington, DE, USA). The RNA integrity number under different conditions. Also, a certain of dynamicity (RIN) [16] was more than 7.0. Approximately 5 μgof high by post-transcriptional and translational regulations of quality RNA was used for cDNA library construction ac- gene expression can be estimated by measuring its cording to the Illumina RNA ligation-based method [17]. Dai et al. BMC Genomics (2018) 19:403 Page 3 of 18 2.5 μg RNA from the mammary gland of two cows in each 7500 real-time PCR system (Applied Biosystems, Foster group were combined (three biological replicates per City,CA, USA).Each20 μL reaction included 50 ng of re- group; Fig. 1), and RNA sequencing was performed using a verse transcription product, 40 nM of each forward and re- HISeq 2000 sequencing system (Illumina, San Diego, CA, verse primer [Additional file 1:Table S1,designedby USA) from LC Sciences (Houston, TX, USA). Reads were Primer 5 software (Premier Biosoft International, Palo Alto, processed with Bowtie version 0.12.7 (http://bowtie-bio.- CA, USA)], and SYBR Premix Taq (Takara). The PCR pro- sourceforge.net), aligned in Tophat version 1.3.2 (http:// gram was one cycle of 95 °C for 30 s plus 40 cycles of amp- ccb.jhu.edu/software/tophat/index.shtml), and then mapped lification at 95 °C for 5 s and 58 °C for 30 s, followed by an to bovine reference genome UMD3.1 (ftp://ftp.ensem- additional 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C bl.org/pub/release-79/fasta/bos_taurus/dna/)[18]. Gene to generate melt curves. The relative gene expression −ΔΔCt abundance was estimated as reads per kilobase of exon values were calculated by the 2 method [19]. The model per million mapped reads (RPKM) with Cufflinks gene expression levels were normalized against the internal version 1.2.1 as previously described [17, 18]. Genes with a control genes β-actin and GAPDH. cut-off of 1.5-fold expression changes and p-value less than 0.05 between CS and AH groups were defined as differen- Protein preparation and digestion tially expressed. Protein preparation and digestion were performed as in the previous studies [20, 21]. Briefly, 500 mg mammary The qRT-PCR analysis tissue was ground to a fine powder in liquid N , lysed Total RNA extracted from the mammary gland was reverse with the lysis buffer A (7 M Urea, 2 M Thiourea, 4% RT transcribed for cDNA synthesis using a PrimeScript Re- CHAPS (3-[(3-Cholamidopropy) dimethylammonio] agent Kit with gDNA Eraser (Takara, Tokyo, Ostu, Japan) propane-sulfonate), 40 mM Tris-HCl, pH 8.5), and re- following the manufacturer’s instructions. The qRT-PCR duced with 10 mM DTT at 56 °C for 1 h, followed by al- was performed in triplicate using the Applied Biosystems kylation with 55 mM IAM (Iodoacetamide) in a Fig. 1 An overview of the transcriptomic and proteomic experiment. Schematic diagram of workflow of the RNA-seq transcriptomic and iTRAQ-based proteomic experiments. Six cows were fed either corn stover (CS) or alfalfa hay (AH) as forage for 14 weeks. RNA from two cows and protein samples from three cows within each group were pooled for transcriptomic (3 replicates/group) and proteomic (2 replicates/group) analyses. For transcriptomic assay, the pooled RNA was sequenced on the Illumina platform, and subsequently the reads were aligned and mapped to the Bos taurus genome. For the proteomic assay, the extracted proteins were digested with trypsin, and the peptides are labeled with different iTRAQ reagents, which contain reporter groups of different masses (114, 115, 116, 117), balance groups of different masses (191, 190, 189, 188), and a reactive group (R). The labeled peptides are then mixed equivalently and fractionated by strong cation exchange (SCX) chromatography. Fractions were separated by liquid chromatography (LC) and analyzed by two-step mass spectrometry (MS) Dai et al. BMC Genomics (2018) 19:403 Page 4 of 18 darkroom for 1 h. The reduced and alkylated protein selected mass range of 350–2000 mass/charge (m/z). mixtures were precipitated by adding 4 × volume of The electrospray voltage applied was 1.6 kV. For MS chilled acetone and incubating at − 20 °C overnight. scans, the m/z scan range was 350 to 2000 Da. For After centrifugation at 4 °C and 30,000×g, the pellet was MS/MS scans, the m/z scan range was 100–1800. dissolved in 0.5 M TEAB (Triethylamine borane; Ap- MS/MS data was acquired using the top 15 most plied Biosystems, Milan, Italy) and sonicated on ice. abundant precursor ions with the ion count more After centrifugation at 30,000×g at 4 °C again, an aliquot than 20,000 in the MS scan. These were selected with of the supernatant was assigned for determination of an isolation window of 2 m/z and were fragmented protein concentration by the Bradford method [22]. The via high energy collisional dissociation under normal- proteins in the supernatant were kept at − 80 °C for fur- ized collision energies of 30 eV. For the MS scan, the ther analysis. resolving power was set to 70,000 at m/z 200, the maximal ion injection time was 10 ms and, dynamic The iTRAQ labeling and strong cationic exchange (SCX) exclusion of the selected precursor ions was 15 s. fractionation Automatic gain control (AGC) was used to optimize Total protein (150 μg obtained by mixing 50 μg protein the spectra generated from the orbitrap, and the AGC from the mammary glands of three cows in each group, target value was 3,000,000 for full MS and 100,000 two biological replicates per group; Fig. 1) was digested for MS , respectively. For the MS/MS scans, the re- with Trypsin Gold (Promega, Madison, WI, USA) at 37 ° solving power was set to 17,500 at m/z 200; max- C for 16 h with the ratio of protein: trypsin = 30: 1. After imum ion injection times for MS/MS scans were at digestion, peptides were dried by vacuum centrifugation. 60 ms; and the underfill ratio was defined as 0.1%. Peptides were then reconstituted in 0.5 M TEAB and processed following the manufacturer’sprotocolfor Protein identification and quantification 4-plex iTRAQ reagent (Applied Biosystems) [21]. The The raw files were first merged and transformed to an protein samples from the CS and AH groups were labeled MGF file with Proteome Discoverer ver. 1.2 (Thermo with iTRAQ reagents 114, 115, 116 and 117. Strong cat- Fisher Scientific, San Jose, CA, USA) and were then ionic exchange chromatography was performed with an probed on the Mascot search engine (ver. 2.3.02; Matrix LC-20AB HPLC pump system (Shimadzu, Kyoto, Japan). Science, London, UK) of the Uniprot database of bovine The procedures for SCX fractionation including the elu- (Bos tauru) with 31,661 entries. For protein identifica- tion were essentially the same as in the study of Meng et tion, the parameters were set the same as in the study by al. [20]. Finally, the eluted peptides were pooled into 20 Yang et al. [23] with some minor changes: carbamido- fractions, desalted with a Strata X C18 column (Phenom- methyl (C), iTRAQ 4-plex (N-term), and iTRAQ 4-plex enex, Torrance, CA, USA), and vacuum-dried. (K) were defined as fixed modifications; Gln→pyro-Glu (N-term Q), oxidation (M), deamidated (NQ) were the Liquid chromatography–tandem mass spectrometry (LC/MS) possible variable modifications. The decoy database pat- analysis tern was considered as the reverse of the target database. The sample fractions described above were further The method of peptide identification by false discovery separated and identified on an LC-20 AD nano-HPLC rate (FDR) was performed as Sheng et al. described [24]. system (Shimadzu) loaded with Q-Extractive mass Relative quantification of the identified proteins was spectrometer (Thermo Fisher Scientific, San Jose, CA, performed with the Proteome Discoverer software de- USA). Buffer C consisted of 2% acetonitrile (ACN) scribed above and calculated by the weighted and and 0.1% formic acid (FA) in Milli-Q water; buffer D normalized ratios of uniquely identified peptides that consisted of 98% ACN and 0.1% FA. After resuspen- belong to the specific individual protein. The integra- sion with buffer C, 10 μL sample supernatant was tion window tolerance of the peak was set to 20 ppm. loaded by the auto-sampler onto a C18 trap column Statistics analysis was conducted using Fisher’stest. (2 cm × 100 μm, 5 μm) and then separated on the Proteins with a cutoff of 1.2-fold change between CS reverse-phase analytical C18 column (100 mm × and AH samples and p < 0.05 were determined as sig- 75 μm, 3 μm). The samples were loaded at 8 μL/min nificantly differentially expressed proteins. for 4 min, then a 44 min gradient was run at 300 nL/min starting from 2 to 35% buffer D, followed by 2 min linear gradient to 80% buffer D, and then Bioinformatics analysis maintenance at 80% buffer D for 4 min, and finally Functional annotations were performed using Blast2GO return to 5% buffer D in 1 min. program against the non-redundant protein database (NR; Peptide analysis was performed with a Q-Exactive NCBI). Metabolic pathway analysis was conducted using mass spectrometer in a positive ion mode with a R software (R version 3.2.3) according to the Kyoto Dai et al. BMC Genomics (2018) 19:403 Page 5 of 18 Encyclopedia of Genes and Genomes (KEGG) pathway through one-way ANOVA in SAS (SAS 9.0). The statis- database (http://www.genome.jp/kegg/). The pathway en- tical significance was declared at p ≤ 0.05. richment statistics were performed by Fisher’s exact test with a p-value ≤ 0.05 considered as significant. Results Overview of transcriptomic and quantitative proteomic analyses Western blot analysis Figure 1 shows the workflow of our integrative RNA-seq Approximately 40 μg protein per sample was separated transcriptomic and iTRAQ-based proteomic experi- on 12% SDS (sodium dodecyl sulfate) polyacrylamide ments. In the transcriptomic analysis, 78,138,798 and gels. Proteins were transferred onto 0.45 μm PVDF 90,522,588 raw/clean reads were detected in the CS and membranes (IPVH00010; Millipore, Boston, Massachu- AH groups, respectively (Additional file 2: Table S2), setts, USA) and blocked with blocking buffer (Beyotime, and in proteomic analysis, 62,367 unique spectra were Jiangsu, China). The membranes were incubated with strictly matched to 24,606 unique peptides and further primary antibodies to SCP2 (non-specific lipid-transfer mapped to 3744 unique proteins (Additional file 2: Table protein 2, ab140126; Abcam, Cambridge, MA, USA), S3). With a cutoff of 1.5-fold change and a p-value < IDH2 (isocitrate dehydrogenase 2; ab131263, Abcam), 0.05, a total of 1631 differentially expressed genes SLC7A8 [(solute carrier family 7 (amino acid transporter, (DEGs) were identified between CS and AH groups in L system), member 8, ab75610, Abcam)], COL4A2 (col- transcriptomic analysis, among which 1046 DEGs were lagen, type IV, alpha 2, sc-70,243, Santa Cruz biotechnol- up-regulated and 585 were down-regulated (Fig. 2 and ogy; Cambridge, MA, USA), and β-actin (Beyotime). Additional file 3: Table S4). Using a threshold of 1.2-fold After washing with TBST [tris-buffered saline containing change and p < 0.05, 346 differentially expressed proteins 0.02% (v/v) Tween-20] three times, the membranes were (DEPs; 138 up-regulated and 208 down-regulated) were incubated with goat anti-rabbit IgG or goat anti-mouse detected between CS and AH groups in proteomic ana- IgG secondary antibodies conjugated with horseradish lysis (Fig. 2 and Additional file 3: Table S5). By compar- peroxidase (Beyotime), incubated with ECL (electroche- ing the RNA-seq data with the proteomic data (Fig. 2 miluminescence) Western Blotting Substrate Kits (Beyo- and Additional file 3: Table S6), 40 genes displayed dif- time), and finally visualized with a Kodak Image Station ferential expression at both mRNA and protein levels, of 2000MM (Kodak Molecular Imaging Systems, New which 18 and 15 genes were consistently up- or Haven, USA). The relative intensities of bands were cal- down-regulated, respectively, whereas the remaining 7 culated with ImagePro Plus 6.0 software (Media Cyber- genes had inconsistent expression in mRNA and protein netics, Washington, MD, USA) using β-actin as the levels, which may result from post-translational reference protein. modifications. Statistical analysis The gene ontology (GO) analysis of DEGs and DEPs The data on relative mRNA expression by qRT-PCR and The GO analysis of all the DEGs and DEPs in cows fed protein expression by Western blot were analyzed CS versus AH is shown in Fig. 3. In the cellular Fig. 2 The venn diagram of the differentially expressed genes (DEGs) and proteins (DEPs) in the mammary gland of cows fed either corn stover (CS) or alfalfa hay (AH). The cut-off of differential expression of mRNA is set at 1.5-fold change and p < 0.05, whereas the cutoff of differential expression of protein is set at 1.2-fold and p < 0.05 Dai et al. BMC Genomics (2018) 19:403 Page 6 of 18 Fig. 3 Gene ontology (GO) categories assigned to the differentially expressed genes (DEGs, inner cycle) and proteins (DEPs, outer cycle) in the mammary gland of cows fed either corn stover (CS) or alfalfa hay (AH). The differentially expressed genes were classified into cellular component, biological process, and molecular function by WEGO (Web Gene Ontology Annotation Plot) according to the GO terms component category, most of the DEGs and DEPs were predominant functions of the DEGs and DEPs, and a mainly assigned to cell (25.8% genes and 24% proteins), relatively low proportion of DEGs and DEPs were associ- cell part (25.8% genes and 24% proteins), organelle ated with various biological activities such as molecular (18.9% genes and 19.2% proteins) and organelle part transducer (3.3% genes and 3.2% proteins), transcription (9.8% genes and 10.2% proteins). Notably, a small num- regulator (2.9% genes and 1.4% proteins), transporter ber of the DEGs and DEPs were located in the extracel- (2.2% genes and 7.8% proteins), structural molecule lular region (3.9% genes and 5.4% proteins), (1.6% genes and 6.0% proteins) and electron carrier membrane-enclosed lumen (3.9% genes and 3.0% pro- (0.4% genes and 1.1% proteins). In general, the DEGs teins) and macromolecular complexes (6.5% genes and and DEPs displayed similar GO annotation patterns. 6.9% proteins). In the biological process category, a large We also performed further functional analysis of the number of DEGs and DEPs were involved in cellular up- and down-regulated genes and proteins using the process (16.9% genes and 17.2% proteins), metabolic UniProt knowledgebase and GO database. The increased process (13.0% genes and 12.6% proteins) and biological genes (Fig. 4) in the CS group were significantly and regulation (10.9% genes and 9.0% proteins). Noticeably, abundantly enriched for genes involved in the positive some DEGs and DEPs were assigned to response to regulation of apoptosis, unfolded protein binding, nega- stimulus (4.8% genes and 7.3% proteins), immune system tive regulation of protein ubiquitination and response to process (2.3% genes and 2.4% proteins), cellular compo- stress. In addition, we found that some genes with in- nent biogenesis (2.3% genes and 2.3% proteins) and cell creased abundance were enriched in the negative regula- growth (0.8% genes and 0.9% proteins). For the molecu- tions of translation, DNA binding, NF-κB transcription lar function category, the GO terms including binding factor activity, phosphorylation, cell cycle and in (43.1% genes and 41.7% proteins), enzyme regulator ac- proteasome-mediated/ubiquitin-dependent protein cata- tivity (29.1% genes and 5.1% proteins) and catalytic ac- bolic processes. Notably, nine genes with up-regulated tivity (16.6% genes and 30.6% proteins) were the expression were enriched in the GO term GDP binding. Dai et al. BMC Genomics (2018) 19:403 Page 7 of 18 Fig. 4 Functional characterization of the increased or decreased transcripts in the mammary gland of cows fed alfalfa hay (AH) vs. corn stover (CS) by gene ontology analysis. DEG indicates differentially expressed genes. The x-axis shows the functional categories of the increased or decreased genes, the left y-axis shows the value of –Log (p-value) and the right y-axis shows the number of increased/decreased genes In contrast, the decreased genes (Fig. 4) were principally pathway. Notably, four decreased genes were significantly enriched in cell adhesion, positive regulation of cell pro- enriched in the GO term “cellular response to amino acid liferation, binding activities of actin/receptor/carbohy- stimulus”. Furthermore, the GO term “ATPase activity” drate, structural molecule activity and protein binding was significantly enriched in six down-regulated genes. (295 decreased genes enriched). Additionally, some The up-regulated proteins (Fig. 5) were primarily down-regulated genes were related to the positive regu- enriched in negative regulation of multicellular organis- lation of several transcriptional factors (Stat3 phosphor- mal and developmental processes. In addition, we ob- ylation and NF-κB signaling) and cell-growth associated served a small proportion of increased proteins enriched processes (cell proliferation, multicellular organism in the GO terms related to lipid translocation including growth, and cell growth). Importantly, a small number of lipid transport and long-chain fatty acid binding, and in decreased genes were associated with the cytoskeleton/ GO terms involved in cation homeostasis and ion chan- extracellular matrix and cytokine-mediated signaling nel inhibitor activity. Specifically, eight up-regulated Dai et al. BMC Genomics (2018) 19:403 Page 8 of 18 Fig. 5 Functional characterization of the increased or decreased proteins in the mammary gland of cows fed alfalfa hay (AH) vs. corn stover (CS) by gene ontology analysis. DEP indicates differentially expressed proteins. The x-axis shows the functional categories of increased or decreased proteins, the left y-axis shows the value of –Log (p-value) and the right y-axis shows the number of increased/decreased proteins proteins were enriched in enzyme inhibitor activity, biosynthetic process and three decreased proteins were which may suggest a general reduction of the related to ATPase activity. enzyme-mediated metabolism. The GO annotation ana- lysis of the down-regulated proteins is shown in Fig. 5. KEGG pathway analysis of DEGs and DEPs Notably, the most abundant GO term was extracellular Table 1 shows the KEGG pathway enrichment analysis matrix organization enriched in ten down-regulated pro- of DEGs. In the analysis, a total of 7 decreased DEGs teins. In addition, the body defense-related GO terms, were significantly enriched in the pathway of protein di- such as response to external stimulus, cellular response gestion and absorption (p = 0.0269). In contrast, 10 in- to amino acid stimulus, and defense response, were sig- creased DEGs were significantly enriched in protein nificantly enriched with some decreased proteins. Some processing in endoplasmic reticulum (p = 0.045). Add- proteins of down-regulation were involved in tissue de- itionally, we found that 4, 11, and 7 increased genes were velopment, organ morphogenesis, biological adhesion significantly enriched in protein degradation-related pro- and response to transforming growth factor beta. Im- cesses ubiquitin mediated proteolysis, spliceosome and portantly, 16 down-regulated proteins were involved in proteasome, respectively. In particular, the significant substrate-specific transporter activity. Furthermore, six pathways (p ≤ 0.05) enriched by some decreased DEGs decreased proteins were enriched in glycosyl-compound were related to protein synthesis (such as ribosome Dai et al. BMC Genomics (2018) 19:403 Page 9 of 18 Table 1 The KEGG pathway enrichment by up−/down-regulated genes in the mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH) KEGG ID Pathway Name P-value of No. of ID of Increased Genes Gene Symbol of No. of ID of Decreased Genes Gene Symbol of Fisher’ Increased Increased Genes Decreased Decreased Genes exact Test Genes Genes ko04974 Protein digestion 0.0269 0 7 XLOC_022008;XLOC_001311; SLC38A2; SLC7A8; and absorption XLOC_000568;XLOC_027248; BT.23508; COL12A1; XLOC_000569;XLOC_017584; COL6A2; COL17A1; XLOC_020771 COL6A3 ko04141 Protein processing 0.0450 10 XLOC_000859;XLOC_003872;XLOC_006584; HSPH1;DNAJB1;DNAJA1; 0 in endoplasmic XLOC_009297;XLOC_020319;XLOC_022100; DDIT3;CRYAB;SAR1B;BT.59327; reticulum XLOC_024455;XLOC_025681;XLOC_026233; MAN1A2;SEC63;DNAJB11 XLOC_027001 ko04120 Ubiquitin mediated 0.0277 4 XLOC_024865;XLOC_018298;XLOC_021649; UBE2B;HERC4;UBE2H; 1 XLOC_025357 KEAP1 proteolysis XLOC_025981 BT.19212 ko0970 Aminoacyl-tRNA 0.0312 1 XLOC_025065 RARS biosynthesis ko03010 Ribosome 0.0201 1 XLOC_028013 RPS23 ko03008 Ribosome biogenesis 0.0219 2 XLOC_001726; XLOC_010292 FCF1;UTP6 3 XLOC_025000;XLOC_023340; TCOF1;RRP7A;NOL6 in eukaryotes XLOC_026685 ko03040 Spliceosome 0.0388 11 XLOC_000309;XLOC_004289;XLOC_007964; SNRPB2;TRA2B;SMNDC1;TRA2A; 1 XLOC_012196 SFRS4 XLOC_012182;XLOC_012566;XLOC_017604; BCAS2;SF3B1;PHF5A;BT.59135; XLOC_019531;XLOC_021421;XLOC_023325; SLU7;BT.91058;BT.91058;PLRG1 XLOC_025841; XLOC_028252 ko03050 Proteasome 0.0142 7 XLOC_001624;XLOC_001843;XLOC_003577; BT.22570;PSMD14;PSMA3; XLOC_004136;XLOC_012473;XLOC_020934; BT.56882;PSMA2;POMP;PSMC2 XLOC_021072 ko04142 Lysosome 0.0394 0 5 XLOC_001832;XLOC_006153; BT.35140; LAPTM5; XLOC_010442;XLOC_012190; ARSB; CD68; CTSH XLOC_013979 ko04150 mTOR signaling 0.0300 1 XLOC_018127 DDIT4 1 XLOC_013054; RICTOR pathway ko04350 TGF-beta signaling 0.0190 2 XLOC_003540;XLOC_018178 ID2;BT.48514 3 XLOC_004021;XLOC_004591; FST;ID1;TFDP1 pathway XLOC_013257 ko04115 p53 signaling 0.0247 5 XLOC_007111;XLOC_012771;XLOC_025054; GADD45G;SESN1;BT.36413; 2 XLOC_021046;XLOC_018555 IGFBP3;BT.33239 pathway XLOC_026769;XLOC_026996 SESN2;CCNG1 ko04310 Wnt signaling 0.0458 1 XLOC_007394 CACYBP 1 XLOC_007966 SFRP2 pathway ko03050 Proteasome 0.0142 7 XLOC_001624;XLOC_001843;XLOC_003577; BT.22570;PSMD14;PSMA3; XLOC_004136;XLOC_012473;XLOC_020934; BT.56882;PSMA2;POMP;PSMC2 XLOC_021072 Dai et al. BMC Genomics (2018) 19:403 Page 10 of 18 biogenesis in eukaryotes, aminoacyl-tRNA biosynthesis VPS18 protein (VPS18), and the uncharacterized protein and lysosome). Also, a small number of DEGs were ORAI1] were involved in the processes of “protein significantly enriched in the pathways of mTOR sig- localization” and “transport”. Intriguingly, two naling pathway, p53 signaling pathway, and two up-regulated genes DHFR and KRT15 at mRNA and cell-growth-associated signaling pathways (the protein levels were related to one carbon metabolism TGF-beta signaling and Wnt signaling). and staphylococcus aureus infection, respectively. Add- The KEGG pathway enrichment analysis of the DEPs itionally, the 3 collagens (COL1A1, COL1A2 and COL4A2) is shown in Table 2, and a total of 29 KEGG pathways and mitogen-activated protein kinase 3 (MAPK3) were significantly enriched. Importantly, 7 decreased down-regulated at both mRNA and protein levels were in- DEPs were significantly enriched (p = 0.0101) in the volved in the protein synthesis-related pathways of “pro- “protein digestion and absorption” pathway, while 7 in- tein digestion and absorption” and “ECM-receptor creased DEPs were significantly enriched (p = 0.037) in interaction”. the “protein processing in endoplasmic and reticulum” pathway. Several DEPs were involved in pathways associ- Verification of DEGs by qRT-PCR and DEPs by western ated with energy metabolism― glycolysis/gluconeogene- blot analysis sis (p = 0.0302), citrate cycle (p = 0.0399), pentose Nineteen genes involved in energy metabolism, transcrip- phosphate pathway (p = 0.0352), and PI3K-Akt signaling tion/translation, protein processing/transport, protein (p = 0.0375). Additionally, the KEGG pathways enriched degradation, amino acid metabolism/ transport, fatty acid by a small number of DEPs were related to the protein oxidation, and mammary gland growth and development synthesis/processing (including ribosome, p = 0.0069; were selected for qRT-PCR analysis (Additional file 5: aminoacyl-tRNA biosynthesis, p = 0.0236; mTOR signal- Table S8). Among these genes (Fig. 6), expression levels of ing pathway, p = 0.035; lysosome, p = 0.0350; protein 15 genes were significantly altered between CS- and AH- processing in endoplasmic reticulum, p = 0.0370; and groups, and the abundance of 11 of the 15 gene abun- spliceosome, p = 0.0232) and the metabolisms of several dance were consistent with expression patterns measured amino acids. Noticeably, we also found that a certain of by RNA-seq. Four proteins, including IDH2, SLC7A8, proteins were significantly enriched in two SCP2,and COL4A2 were selected for Western blot ana- cell-growth-signaling pathways (Wnt and TGF-beta sig- lysis (Fig. 7 and Additional file 5: Table S8). Western blot naling pathways; p = 0.0375 and 0.0272, respectively). analysis showed that the protein levels of IDH2, SLC7A8, and COL4A2 in the mammary glands of cows fed the Functional analysis of the common DEGs and DEPs CS-based diet were lower than those cows fed the AH The 40 common expressed genes at mRNA and protein diet, whereas the level of SCP2 was higher. All the immu- levels were further analyzed by the GO and KEGG path- noblot results were consistent with the findings from way (Additional file 4: Table S7). Importantly, 3 in- proteomic analysis. creased genes at mRNA and protein levels (DnaJ homolog subfamily B members—DNAJB11, DNAJB1, Relationship between the DEGs/DEPs and mammary and DNAJC12) were related to protein unfolding and in- metabolism volved in the pathway of “protein processing in endo- A comprehensive view of the molecular mechanisms plasmic reticulum”. Also, two common genes of underlying milk production was summarized based on up-regulation (dihydrolipoamide branched chain transa- the proteomic and transcriptomic data collected (Fig. 8 cylase, DBT and hydroxymethylglutaryl-CoA synthase, and Additional file 6: Table S9). The regulatory subsec- HMGCS1) were involved in the process of “valine, leu- tions proposed include: energy metabolism, amino acid cine and isoleucine degradation”. Noticeably, the de- (AA) /fatty acid metabolism, protein degradation, pro- creased genes IDH2 and ATPase 2 (ATP2B4) at mRNA tein synthesis, protein processing, AA/protein transport, and protein levels were involved in TCA cycle and ATP and cell growth and development. Each of these regula- synthesis, respectively; in contrast, 4 common genes of tory functions is involved in the regulation of mammary up-regulated expression [HMGCS1, SCP2, DBT and metabolism, and the collective analysis shows how the isopentenyl-diphosphate delta-isomerase 1 (IDI1))] were mammary gland adapts to the low nutrient availability of all associated with fatty-acyl-CoA metabolic process. In the CS ration compared with the higher nutrient avail- particular, the commonly decreased gene dimethylgly- ability on the AH ration. Despite that the gene- and cine dehydrogenase (DMGDH) was associated with “gly- protein-level responses are not always consistent, the cine, serine and threonine metabolism”. In addition, relatively higher degree of agreement between analyses several commonly expressed genes of down-regulation of DEGs and DEPs at the functional level suggests that [such as erythrocyte membrane protein (EPB41L3), transcriptomic analysis of the mammary gland might be odorant-binding protein (MGC151921), DMGDH, sufficient to characterize tissue functional responses to Dai et al. BMC Genomics (2018) 19:403 Page 11 of 18 Table 2 The KEGG pathway enrichment by up−/down-regulated proteins in the mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH) KEGG ID Pathway Name P-value of No. of ID of Increased Proteins Gene Symbol of Increased Proteins No. of ID of Decreased Proteins Gene Symbol of Fisher’ exact Increased Decreased Decreased Proteins Test Transcripts Proteins ko03040 Spliceosome 0.0232 4 IPI00687479;IPI00715218; SNRNP40;LSM3; SNRPB;PRPF8 6 IPI00690232;IPI00699558; MAGOHB;SNRPD3; IPI00702381; IPI00687395 IPI00687560;IPI00687015; PCBP1;SNRPD2;SF3B4;BUD31 IPI00717302; IPI00688521 ko04141 Protein 0.0370 7 IPI00702891;IPI00699038; ERP29; TXNDC5; DNAJC3;DNAJB11; 1 IPI00692963 SEC23 processing in IPI00693007; IPI00699107; CALR; SSR2; DNAJB1 endoplasmic IPI00691963; IPI00696616; reticulum IPI00688461 ko04974 Protein digestion 0.0101 0 7 IPI00707857;IPI00708244; COL4A2; COL3A1; and absorption IPI00711933;IPI00712524; COL5A2; COL1A1; IPI00731432;IPI00826022; COL1A2;COL11A1; IPI00905045 COL18A1 ko04142 Lysosome 0.0314 2 IPI00711862; IPI00706203 NPC2; HEXB 4 IPI00697314;IPI00699372; CTSC; ATP6V0A1; IPI00717554;IPI00716195 NAGLU; ATP6V0D1 ko03010 Ribosome 0.0069 0 6 IPI00695732;IPI00699146; RPS2; RPS16; RPS20; IPI00707431;IPI00713536; RPS19; RPS12; RPS27 IPI00714445;IPI00715091 ko00260 Glycine, serine 0.0090 1 IPI00698589 PGAM1 3 IPI00698059;IPI00707303; SARDH; DMGDH; MAOA and threonine IPI00715285 metabolism ko04150 mTOR signaling 0.0350 1 IPI00700182 EIF4B 2 IPI00903663; IPI00732002 IKBKB; MAPK3 pathway ko03008 Ribosome 0.0374 2 IPI00705941;IPI00708018 REXO2; RAN 1 IPI00852474 NAT10 biogenesis in eukaryotes ko00280 Valine, leucine 0.0468 3 IPI00711918;IPI00717256; DBT; ACAT1; HMGCS1 and isoleucine IPI00968674 degradation ko00380 Tryptophan 0.0381 1 IPI00711918 ACAT1 1 IPI00698059 MAOA metabolism ko00330 Arginine and 0.0370 0 2 IPI00698059;IPI00838420 P4HA2; MAOA proline metabolism ko00970 Aminoacyl-tRNA 0.0236 0 2 IPI00689365;IPI00703906 TARS2; AARS2 biosynthesis ko00360 Phenylalanine 0.0473 0 1 IPI00698059 MAOA metabolism ko00340 Histidine 0.0461 0 1 IPI00698059 MAOA metabolism ko00350 Tyrosine 0.0365 0 1 IPI00698059 MAOA metabolism ko00270 0.0432 1 IPI00694739 APIP Dai et al. BMC Genomics (2018) 19:403 Page 12 of 18 Table 2 The KEGG pathway enrichment by up−/down-regulated proteins in the mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH) (Continued) KEGG ID Pathway Name P-value of No. of ID of Increased Proteins Gene Symbol of Increased Proteins No. of ID of Decreased Proteins Gene Symbol of Fisher’ exact Increased Decreased Decreased Proteins Test Transcripts Proteins Cysteine and methionine metabolism ko00010 Glycolysis / 0.0302 3 IPI00696912; IPI00698589; ACSS1; PGAM1; GALM 2 IPI00687211;IPI00715799 HK1; GAPDHS Gluconeogenesis IPI00712164 ko00190 Oxidative 0.0287 0 5 IPI00697768;IPI00699372; ATPsynGL;ATP6V0A1; phosphorylation IPI00712252;IPI00716163; ATP5H;NDUFC1; IPI00716195 ATP6V0D1 ko00020 Citrate cycle 0.0399 2 IPI00702781;IPI00708438 IDH1; SUCLG1 1 IPI00714468 IDH2 (TCA cycle) ko00640 Propanoate 0.0423 3 IPI00696912;IPI00708438; ACSS1; ACAT1; SUCLG1 metabolism IPI00711918 ko00030 Pentose phosphate 0.0352 2 IPI00728589;IPI00904104 TKT; RBKS pathway ko04146 Peroxisome 0.0169 4 IPI00686601;IPI00702781; SOD2; SCP2; IDH1; ECH1 1 IPI00714468 IDH2 IPI00704382;IPI00714468 ko03320 PPAR signaling 0.0263 4 IPI00686601;IPI00699355; SCP2; FABP4; APOA1; PPARD pathway IPI00715548;IPI00839653 ko04975 Fat digestion 0.0370 2 IPI00695965;IPI00715548 APOA4; APOA1 1 IPI00710056 APOB and absorption ko04540 Gap junction 0.0352 0 2 IPI00695917;IPI00732002 MAPK3; GNAS ko04210 Apoptosis 0.0261 1 IPI00704835 DFFA 2 IPI00709124; IPI00903663 ENDOG; IKBKB ko04151 PI3K-Akt signaling 0.0301 1 IPI00700182 EIF4B 9 IPI00697595;IPI00707857; COL4A2; IKBKB; pathway IPI00708244;IPI00712524; COL3A1; COL5A2; IPI00731432;IPI00732002; COL1A1; COL1A2; IPI00826022;IPI00903663; COL11A1; MAPK3; ITGA1 IPI00905045 ko04310 Wnt signaling 0.0375 1 IPI00708311 CACYBP 1 IPI00699355 PPARD pathway ko04350 TGF-beta signaling 0.0272 0 1 IPI00732002 MAPK3 pathway Dai et al. BMC Genomics (2018) 19:403 Page 13 of 18 Fig. 6 Real time PCR analysis of mRNA expression changes of genes involved in mammary metabolism of cows fed corn stover (CS) and alfalfa hay (AH). Relative mRNA expression levels were normalized by the levels of β-actin. Error bars represent the standard deviation. ** and * indicate that the difference in gene expression between CS and AH groups reached p < 0.01, and 0.01 < p < 0.05, respectively altered states but might not always be reflective of shifts milk production in the mammary gland. In this study, in specific proteins. we detected 1046 up-regulated and 585 down-regulated genes by RNA-seq transcriptomics as well as 138 in- Discussion creased and 208 decreased proteins by iTRAQ proteo- Roughage is one of the most important components in mics in the mammary gland of dairy cows fed CS- ruminant diets, the quality of which has a large impact versus AH-based diets. There were only 33 genes with on milk performance in dairy cows [25]. Our previous consistent expression patterns measured by both tran- study showed that compared to cows fed AH as the for- scriptomic and proteomic analyses, suggesting that the age source, cows fed CS had lower yields of milk (kg/d; post-transcriptional regulation may play an important 23.5 vs. 19.4), milk fat (kg/d; 0.98 vs. 0.82), milk protein role in gene expression. The relatively limited overlap of (kg/d; 0.77 vs. 0.62) and lactose (kg/d; 1.15 vs. 0.94) (P < proteins and mRNAs was also seen in other studies in 0.01) [6]. In addition, as a major crop byproduct, a large human [14], mouse [11], fish [26] and bacteria [27]. quantity of corn stover is burned by humans, and this The GO term annotation can help to characterize inefficient utilization of crop residues subsequently pro- physiological and functional changes associated with the duces heavy environmental pollution. Therefore, it is ur- changes in mRNA and protein expression in cells and gent for us to determine strategies to improve the tissues [28]. In up-regulated genes in CS-fed cows versus utilization of low-quality forage (CS) in the dairy indus- AH-fed cows, the GO term “ubiquitin-dependent/pro- try and reduce damage to the environment caused by teasome-mediated protein catabolic process” was signifi- burning CS. However, little is known about the effects of cantly enriched, which may indicate enhanced mammary roughage consumption on the molecular mechanisms in protein catabolism in cows fed CS. Enrichment in the Fig. 7 Western blot analysis of expression of IDH2, SLC7A8, SCP2, and COL4A2 proteins in the mammary gland of cows fed corn stover (CS) and alfalfa hay (AH). β-Actin was used as a sample loading control. ** and * indicate p < 0.01 and 0.01 < p < 0.05, respectively. IDH2: Isocitrate dehydrogenase 2; SLC7A8: also referred as LAT2, L type amino acid transporter 2; SCP2: sterol carrier protein 2; COL4A2: collagen type IV alpha 2 Dai et al. BMC Genomics (2018) 19:403 Page 14 of 18 Fig. 8 An overview of possible biological changes that might contribute to low milk production in cows fed corn stover-based diet vs. cows fed alfalfa hay-based diet. The color coding for the individual genes is as follows: black letters and pink background represents the up-regulated genes, yellow letters and pink background represents increased proteins, black letters and green background represents the down-regulated genes, and blue letters and green background represents decreased proteins. The half blue/half black letters with green background represents genes that were both down-regulated at mRNA and protein levels. The half black/half yellow letters with pink background represent the genes that were up-regulated at both protein and gene levels. The full name of each protein is listed in Additional file 6: Table S9 GO terms of negative regulation of translation, NF-κB associated with positive regulation of Stat3 phosphoryl- transcription factor activity, and DNA binding activity ation and NF-κB signaling pathway in CS-fed cows was suggested inhibited protein synthesis in the mammary consistent with the reduced protein synthesis in these gland, consistent with the reduced milk protein yield in animals. Meanwhile, the lower expression levels of the CS-fed cows [6]. In addition, several proteins that were proteins involved in biological adhesion, extracellular up-regulated in CS-fed cows were enriched in the GO matrix organization, tissue development, and organ mor- term “enzyme inhibitor activity”, which is also consistent phogenesis were consistent with possible reduced mam- with an overall reduced mammary metabolism in these mary cell growth and mammary tissue development in cows. Furthermore, some increased proteins were in- CS-fed cows. Furthermore, the proteins that were lower volved in positive regulation of apoptosis and negative in abundance were enriched in the GO term “response regulations of multicellular organismal processes and de- to transforming growth factor beta”, indicating that velopmental processes, in line with the reduced cell TGF-β may be a major signaling molecule in the regula- growth in the CS group as shown in our previous micro- tion of mammary cell growth in cows fed CS. Collect- RNAome analysis [8]. Interestingly, the fact that the GO ively, GO analysis indicated that the reduced cell growth terms of lipid transport and long-fatty acid binding were and metabolism, attenuated protein synthesis, and en- enriched in the up-regulated proteins in CS-fed cows sup- hanced protein degradation may play major roles leading ported the idea that the mammary gland of these cows to the low milk production in CS-fed dairy cows. may take up more fatty acids from the blood for mam- The KEGG pathway analysis has also been widely used mary metabolism as these cows had a lower acetate supply for systematic understanding of the gene functions in to the mammary gland than AH-fed cows [6]. cells or organisms from large-scale molecular data sets Consistently, the genes that were expressed lower in [29, 30]. In this study, the up-regulated genes were CS-fed cows were enriched for the GO terms of cell ad- enriched in ubiquitin mediated proteolysis, and the hesion, positive regulation of cell proliferation, and genes with lower abundance were enriched in ribosome multicellular organism growth, which also indicated the (the main machinery for protein synthesis), suggesting attenuated cell growth in the mammary gland of CS-fed that these pathways may play an integrative role in low- animals. In addition, down-regulation of genes ering milk protein production in the mammary gland of Dai et al. BMC Genomics (2018) 19:403 Page 15 of 18 CS-fed cows [6]. Additionally, some down-regulated SLC7A8 genes was 0.38- fold lower in CS-fed cows com- proteins from CS-fed cows were enriched in the mTOR pared to the AH-fed cows, indicating a reduced system signaling pathway and metabolisms of several amino L amino acid transporter activity for neutral amino acids acids, including glycine, serine, threonine, arginine, pro- (including isoleucine, leucine, methionine, phenylalanine, line, phenylalanine, histidine, and tyrosine, which is also threonine, alanine and serine) in the mammary gland of consistent with the lower milk protein in the CS group CS-fed cows [41]. This finding was in consistent with [6, 8]. In particular, three down-regulated proteins in our previous observations of amino acid uptake in these CS-fed cows were associated with valine, leucine and cows [6, 31]. isoleucine degradation, indicating a reduction of Ribosomes consist of a small 40S and a large 60S sub- branched-amino acids production in milk [31]. More- units, and ribosomal proteins are required for different over, the pathways enriched among differentially stages of ribosome biogenesis and/or for distinct steps of expressed proteins included the Wnt and TGF-β signal- the translation process [42]. All six ribosomal protein sub- ing pathways. These pathways are known to play roles in units (RPS2, RPS12, RPS16, RPS19, RPS20 and RPS27) regulating mammary growth and differentiation [32–34]. expressed at lower levels in CS-fed cows were essential Milk production is a highly energy-dependent process components of the small ribosomal subunit (40S). The and requires sufficient ATP [35]. In most eukaryotes, lower abundance of these ribosomal proteins in the CS oxidative phosphorylation in the mitochondria involving group suggests depressed efficiency of protein translation, a series of ATPases is the prime metabolic pathway to contributing to lower milk protein in these cows [31]. generate energy [36]. In the study, we observed that the For protein processing, proteins and peptides must be expression of several ATPase components (including transported into the endoplasmic reticulum (ER) or ATPsynGL, ATP6V0A1, ATP5H and ATP6V0D1) and Golgi, where they acquire modifications that allow them one mitochondrial enzyme NADH dehydrogenase (ubi- to be biologically active [43]. Specific three-dimensional quinone) 1 subunit C1 (NDUFC1) involved in oxidative conformations acquired through folding of newly trans- phosphorylation were lower in CS-fed cows. The lower lated polypeptides and/or refolding of unfolded proteins levels of these enzymes may potentially contribute to a are essential for protein function formation and main- lower ATP production in the mammary tissue of these tenance [44]. The protein DNAJB1 is a heat shock protein animals. For high-yield dairy cows, particularly during (HSP) [an ER molecular chaperone that can protect other negative energy balance, ketone bodies (included acetoa- proteins against occurrence of incorrect folding, but may cetate, β-hydroxy-butyrate, and acetone) are produced in also stimulate ER-associated degradation (ERAD)] [45]. the liver but utilized in other tissues of the body (includ- As an ER molecular chaperone, the protein DNAJB11 is ing the mammary gland) as an energy source [37]. essential to prevent the processes such as protein aggrega- Acetyl-CoA acetyltransferase (ACAT1) plays a major role tion, allowing protein folding and assembly to proceed in ketone body synthesis, transferring one acetyl group correctly [46]. In this study, expression of DNAJB1 and to another acetyl-CoA (the crucial substrate for ketone DNAJB11 was higher at the mRNA and protein levels in body generation) [38]. Furthermore, the rate-limiting the CS-fed cows, suggesting that these cows may have enzyme in ketone body synthesis is HMGCS1, which higher degree of protein non-aggregation and protein deg- promotes β-hydroxy-butyrate production [39]. The in- radation in the mammary gland. The protein heat shock creased expression of both ACAT1 and HMGCS1 in 105 kDa/110 kDa protein 1 (HSPH1), another HSP, can be CS-fed cows may reflect an increased level of mammary induced by several kinds of environmental stress [47]and ketone body production in cows consuming CS, which is associated with mammary tumor tissues [48]. In the results in an increased level of ketone body utilization as current study, the highly levels of HSPH1 in the CS-fed energy for the mammary gland of these cows, in line cows may suggest that the CS diet induces ER-stress in with the high abundance of β-hydroxybutyric acid and the mammary gland. The increased amount of protein acetoacetyl-CoA in blood of CS-fed cows versus AH-fed degradation in the mammary gland is in accordance with cows [7]. the reduced milk protein and lactation performance in the Amino acids are fundamental proteogenic substrates CS group [6]. for milk synthesis in dairy cows, most of which derive The proteasome is a large, 26S-multicatalytic protease from dietary proteins. After digestion and absorption in that degrades poly-ubiquitinated proteins to small pep- the small intestine, amino acids are taken up from the tides [49]. The proteasome is composed of two blood by the mammary gland via amino acid trans- sub-complexes: a core catalytic 20S particle and a regula- porters. The protein SLC7A8 is a system L amino acid tory 19S particle [50]. Expression of two 19S regulatory transporter that functions by a Na -independent and particles [proteasome 26S subunit, ATPase 6 (PSMC6) electroneutral transport mechanism for neutral amino and proteasome 26S subunit, ATPase 2 (PSMC2)], the acids [40]. In this study, mammary expression of two 20S core particles [proteasome subunit alpha 3 Dai et al. BMC Genomics (2018) 19:403 Page 16 of 18 (PSMA3) and proteasome subunit alpha 2 (PSMA2)] and cows. Taken together, expression changes of multiple proteasome 26S subunit, non-ATPase 14 (PSMD14) was genes involved in fatty acid β-oxidation and fatty acid higher in the mammary gland of cows fed the CS-based transport indicate a more active fatty acid metabolism diet, supporting enhanced protein degradation in these to supply energy in the CS-fed cows compared to cows. In addition, many intracellular proteins become co- AH-fed cows. This observation is consistent with our valently modified with ubiquitin (UB) or ubiquitin-like previous finding that the CS-fed cows had a lower proteins (UBLs) [51]. Then, ubiquitin-conjugating enzymes supply of acetate which is the major energy source in (E1s) transfers the activated modifier to a family of E2 ruminants [6]. ubiquitin-conjugating enzymes, leading to their degradation Finally, six collagen proteins and five genes encoding [52]. In this study, expression of ubiquitin-conjugating en- collagen proteins were lower in the mammary gland of zyme 2B (UBE2B), ubiquitin-conjugating enzyme E2H CS-fed cows. Collagens are the main structural proteins (UBE2H) and target recognizing subunit kelch-like family in extracellular matrix (ECM), and are important member 9 (KLHL9) was higher in CS-fed cows, further indi- regulators of the differentiated phenotype of mammary cating active protein degradation in the mammary glands of epithelial cells in culture [31, 32]. Lower levels of these these animals. Taken together, the higher abundance of the proteins may indicate a different mammary gland proteasome-related and ubiquitin-dependent proteins is morphology in these animals. Specifically, a decrease in consistent with a higher degree of protein degradation in the levels of the protein COL4A2 was found to be the mammary cells of CS-fed cows. Both the possibly in- associated with a loss of basement membrane integrity, creased protein degradation and reduced protein synthesis accompanied by a dramatic alteration of alveolar morph- may result in lower milk protein yield in the CS group. ology with decreased size and shrunken lumen contain- Peroxisomes are essential organelles that play a key ing little β-casein [32]. Mammary structural changes role in lipid homeostasis. The protein SCP2 acts in per- may be another factor contributing to the reduction of oxisome cholesterol transport through the cytoplasm, milk yield in the CS-fed cows compared with cows fed and loss of SCP2 can result in defects in fatty acid AH [31]. β-oxidation [53]. Higher SCP2 measured at mRNA andprotein levels in cows fedCS-baseddiet sug- gested an enhanced level of fatty acid β-oxidation in Conclusions themammarygland. Theprotein isocitrate dehydro- By integrating transcriptomic and proteomic data, this genase [NADP] 1 (IDH1) is an enzyme that catalyzes study suggested four major possible mechanisms con- oxidative decarboxylation of isocitrate, producing tributing to the lower milk production in dairy cows fed alpha-ketoglutarate (α-ketoglutarate) and CO .Cyto- CS-diet compared to AH-diet: (i) reduced mammary solic IDH1 (EC 1.1.1.41) and mitochondrial IDH2 (EC growth/development through lower expression of 1.1.1.42) catalyze the same reaction outside the con- COL4A2, MAPK3, IKBKB, and LGALS3, (ii) less oxida- text of the citric acid cycle and use NADP as a co- tive phosphorylation through lower expression of ATP- factor instead of NAD [54]. Cytosolic IDH1 plays a synGL, ATP6VOA1, ATP5H, ATP6VOD1, and NDUFC1 complementary role in reductive glutamine metabol- with enhanced lipid uptake and fatty acid beta-oxidation ism, possibly through its oxidative function in an to supply energy through higher expression of SLC27A6, IDH2/IDH1 shuttle that transfers high energy elec- FABP4, SOD2, ACADM, ACAT1, IDH1, SCP2, ECHDC1, trons in the form of NADPH from mitochondria to and HMGCS1, (iii) less AA/protein transport and me- cytosol, especially under hypoxia [55]. In this study, tabolism through lower expression of SLC38A2, SLC7A8, the higher IDH1 and the lower levels of IDH2 in RAB5a, and VPS18, and less protein translation through CS-fed cows may indicate that the complementary lower expression of RPS6, RPS12, RPS16, RPS19, RPS20, IDH1-dependent carboxylation pathway is enhanced and RPS27, and (iv) more proteasome- and in CS-fed cows, enabling higher citrate production for ubiquitin-mediated protein degradation through higher ex- energy supply. Acyl-CoA dehydrogenases (ACADs) pression of the protease components PSMC2, PSMC6, are a class of enzymes that function to catalyze the PSMD14, PSMA2, PSMA3, and ubiquitin-conjugating en- initial step in each cycle of fatty acid β-oxidation in zymes UBE2B, UBE2H, KLHL9, HSPH1, DNAJA1, and the mitochondria of cells [56]. Higher expression of CACYBP, and protein disassembly-associated enzymes acyl-CoA dehydrogenase (ACADM), combined with SEC63, DNAJC3, DNAJB1, DNAJB11, and DNAJC12.These the higher levels of long fatty acid transporter mechanisms involved in milk production of dairy cows fed (SLC27A6), in CS-fed cows may indicate a higher CS-forage-based diet can direct future work to further transport activity of long fatty acid from the plasma understand how the mammary gland adapts to low nutrient into the mammary cells, thus supporting a higher availability and, ultimately, to change feeding strategies so level of fatty acid oxidation for energy supply in these that cows can utilize low-quality forage more efficiently. Dai et al. BMC Genomics (2018) 19:403 Page 17 of 18 Additional files Acknowledgements The authors gratefully thank the personnel of Hangjiang Dairy Farm (Hangzhou, China) for their assistance in the feeding and care of the animals. Additional file 1: Table S1. Primers used in real-time RT-PCR. (XLSX 11 kb) Additional file 2: Table S2. Summary of the transcriptome data in the Funding mammary gland of dairy cows fed corn stover (CS) or alfalfa hay (AH). This research was supported by grants from the National Key Research and Table S3. Summary of the proteome data in the mammary gland of Development Program of China (2016YFD0500503) and the National Natural dairy cows fed corn stover (CS) or alfalfa hay (AH)). (ZIP 13 kb) Science Foundations of China (31672447, 31372336). The funders had no role Additional file 3: Table S4. Differentially expressed genes in the in study design, data collection, analysis and interpretation, or preparation of mammary gland of cows fed corn stover (CS) vs. alfalfa hay (AH). The the manuscript. cutoff is set at 1.5-fold change and p < 0.05. Table S5. Differentially expressed proteins in the mammary gland of cows fed corn stover (CS) Availability of data and materials vs. alfalfa hay (AH). The cutoff is set at 1.2-fold change and p < 0.05. All data in this study are included in the article and its additional files. Table S6. Differentially expressed genes found in both transcriptomic and proteomic analyses in the mammary gland of cows fed corn stover Authors’ contributions (CS) vs. alfalfa hay (AH). (ZIP 164 kb) DWT and LHY conceived of the study and designed the project. DWT Additional file 4: Table S7. A list of common differentially expressed performed the lab work, analyzed data and wrote the manuscript. WQJ genes and differentially expressed proteins in the mammary gland of helped the qRT-PCR experiment and the KEGG pathway analysis. LHY, ZFQ cows fed corn stover (CS) vs. alfalfa hay (AH). (XLSX 17 kb) and LJX revised the manuscript. All the authors have read and approved the final version of the manuscript. Additional file 5: Table S8. The full name and abbreviation of proteins listed in Figs. 6 and 7. (XLSX 14 kb) Ethics approval and consent to participate Additional file 6: Table S9. The full name and abbreviation of proteins The procedures of this study were approved by the Animal Care and Use in Fig. 8. (XLSX 16 kb) Committee of Zhejiang University (Hangzhou, China) and were in accordance with the university’s guidelines for animal research. Abbreviations Competing interests AARS2: Probable alanyl-tRNA synthetase; ACADM: Acyl-CoA dehydrogenase, The authors declare that they have no competing interests. C-4 to C-12 straight chain; ACAT1: Acetyl-CoA acetyltransferase; ACN: Acetonitrile; ACSS1: Acetyl-coenzyme A synthetase 1; AGC: Automatic Publisher’sNote gain control; AH: Alfalfa hay based diet; ATP5H: ATP synthase subunit d; Springer Nature remains neutral with regard to jurisdictional claims in ATP6V0A1: Isoform 1 of V-type proton ATPase 116 kDa subunit a isoform 1; published maps and institutional affiliations. ATP6V0D1: V-type proton ATPase subunit d1; ATPsynGL: ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G-like; CACYBP: Calcyclin Author details binding protein; CHAPS: 3-[(3-Cholamidopropy) dimethylammonio] propane- Institute of Dairy Science, College of Animal Sciences, Zhejiang University, sulfonate; COL4A2: Collagen, type IV, alpha 2; CS: Corn stover based diet; Hang Zhou 310058, People’s Republic of China. Laboratory of Lactation and DBT: Dihydrolipoamide branched chain transacylase E2; Metabolic Physiology, Department of Animal and Veterinary Sciences, DMGDH: Dimethylglycine dehydrogenase; DNAJA1: DnaJ (Hsp40) homolog, University of Vermont, Burlington, VT 05405, USA. subfamily A, member 1; DNAJB1: DnaJ (Hsp40) homolog, subfamily B, member 1; DNAJB11: DnaJ homolog subfamily B member 11; DNAJC12: DnaJ Received: 6 July 2017 Accepted: 21 May 2018 homolog subfamily C member 12; DNAJC3: DnaJ homolog subfamily C member 3; ECHDC1: Enoyl CoA hydratase domain containing 1; FA: Formic acid; FABP4: Fatty acid binding protein 4; FDR: False discovery rate; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HK1: Hexokinase 1; References HMGCS1: Hydroxymethylglutaryl-CoA synthase; HSPH: Heat shock 105 kDa/ 1. Kendall C, Leonardi C, Hoffman P, Combs D. Intake and milk production of 110 kDa protein 1; HSPH1: Heat shock 105 kDa/110 kDa protein 1; cows fed diets that differed in dietary neutral detergent fiber and neutral IDH1: Isocitrate dehydrogenase [NADP]; IDH2: Isocitrate dehydrogenase detergent fiber digestibility. J Dairy Sci. 2009;92(1):313–23. [NADP]; IKBKB: Inhibitor of kappa light polypeptide gene enhancer in B-cells, 2. Wang JQ. Five key indicators of leading the direction of China dairy kinase beta; KLHL9: Kelch-like family member 9; LGALS3: Galectin-3-binding industry. Feed and Husbandry. 2011;4:003. protein; MAOA: Amine oxidase [flavin-containing] A; MAP K3: Mitogen- 3. Li XL, Wan LQ. Research progress on Medicago sativa silage technology [J]. activated protein kinase 3; NDUFC1: NADH dehydrogenase [ubiquinone] 1 Acta Pratac Sci. 2005;2:001. subunit C1; PDHX: Pyruvate dehydrogenase complex, component X; 4. Pang Y, Liu Y, Li X, Wang K, Yuan H. 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BMC GenomicsSpringer Journals

Published: May 29, 2018

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