| The objectives of the present study, using four dietary corn silage levels, were to evaluate the effect of dietary forage levels on the changes in the nutrients intake, rumination, rumen fermentation, blood metabolites, nutrients digestibility, growth, and global liver gene expression profiles in Holstein heifers.Twenty four half-sib Holstein heifers (Bull ID:11106002; 8 to 10 mo of age and 253±29 kg of BW, respectively, at the beginning of the experiment) were used for a 4 weeks feeding trial following after a pre-experimental period for 4 weeks. Corn silage was used as the sole forage source to reduce the potential interference from forage types and sources. During the pre-experimental period, all the heifers were fed with the adaption diet contained 50% corn silage (DM basis). At the conclusion of the pre-experimental period, heifers were blocked by BW and age in a randomized complete block design in equal numbers and assigned randomly to one of four diets that contained different corn silage levels (20,40,60, and 80%; DM basis, namely the group S20, S40, S60, S80). All diets were provided as TMR and calculated to provide similar intakes of ME to allow for 800 g/d of ADG. In the first part of the study, nutrients intake and rumination were monitored. Results from this part showed that the DMI increased quadratically with the increase in dietary silage levels (.P<0.001). The intake of CP was similar for each group (about 685 g/d). The most obvious difference was observed in the intakes of carbohydrates. The intake of Forage NDF (FNDF) (P=0.016) and NDF (,P=0.013) increased quadratically with increasing dietary silage levels. The intake of NFC decreased quadratically (P<0.001) with increasing dietary silage levels. With the increase in dietary corn silage concentration, daily ruminating time, ruminanting min/kg of DMI and min/kg of OMI of heifers were increased linearly (P<0.001), while the ruminating min/kg NDFI decreased linearly (P<0.001).The blood metabolites and ruminal fermentation were measured in the second part of the study. The rumen contents were collected approximately 3-4 h after the morning feeding by the oral stomach tube method. Ruminal pH, NH3-N, and VFA concentrations were tested. At the last week of both pre-experimental period and experimental period, blood samples were collected from the jugular vein into 10-mL evacuated tubes. All the tubes were centrifuged at 3,000 g at 4 ℃ for 15 min to obtain plasma. Metabolites in plasma were analysed by Automatic Biochemical Analyzer and Radioimmunoassay Analyzer. Results showed that with the increase in dietary corn silage concentration, ruminal pH, mole ratio percentage of acetate, and acetate/propionate ratio increased linearly (-P<0.001), while NH3-N concentration, mole ratio percentage of propionate and butyrate decreased linearly (P<0.001). Increasing dietary corn silage concentration had cubic effects on the plasma glucagon and GHRIH (growth hormone releasing-inhibiting hormone) concentrations (P<0.001), whild the changing trends of T3 was all opposite to that of glucagon and GHRIH (P=0.018). The most obvious change was the activity of ALT, which was increased linearly with the increasing dietary corn silage levels.The nutrients digestibility and growth of heifers was monitored in the third part of the study. Duing the last week of the experimental period, fecal grab samples were collected for 4 consecutive dary from each heifer. The content of AIA, DM, OM, CP, NDF, and ADF was measured, and then the apparent total tract digestibility of these nutrients was calculated by using AIA as an internal standard. During the pre-experimental and experimental periods, body weights were taken on 2 consecutive days weekly. Concurrent with weekly weighing, structural measurements and BCS were recorded for each heifer. Results showed that with the increasing dietary silage levels, the apparent digestibility of DM, OM, and CP decreased linearly (P<0.05), while that of EE, NDF, and ADF increased linearly (P<0.05). The paunch girth and ADG increased linearly with increasing dietary silage levels y (P<0.01). No difference was obsearved in body weight, wither height, body length, heart girth, cannon circumference, hip width, hip height and BCS among treatments.At the last day of both pre-experimental period and experimental period, approximately 2 g of liver biopsy was taken from each heifer. Total RNA was prepared from 100 mg of fragmented frozen liver tissue. Cluster generation and sequencing were performed on the Illumina HiSeq 2500. Paired-end reads of 2×100 bp were produced. A total of 10005 unigene successfully matched the genes in the database, 532 of which were shown to be DEG at a false discovery rate of less than 0.05 and fold change>1.5. There were 81 DEG between S20 and S40,225 DEG between S20 and S60,258 DEG between S20 and S80,112 DEG between S40 and S60,161 DEG between S40 and S80, and 138 DEG between S60 and S80, respectively. Several significant GO categories were enriched (P<0.01), including GO Terms related to oxidation reduction, transferase activity, cholesterol biosynthetic, and carboxylic acid biosynthetic. By using the STEM software package, the expression trend of the 532 DEG was generally divided into 20 clusters according to their dynamic expression patterns among the 4 dietary corn silage levels. Additionally,3 significant enriched profiles were also detected:profile 17 (112 DEG, up-up-up), 19 (73 DEG, up-up-down) and 4 (47 DEG, down-down-down). GO analysis showed that DEG clustered in profile 17 mainly realated to negative regulation of protein kinase activity, carboxylic acid biosynthetic, activation of MAPKK activity, and amine biosynthetic; DEG clustered in profile 19 mainly riched in cholesterol biosynthetic, oxidation reduction, and regulation of cell activation; DEG clustered in profile 4 mainly related to retinoic acid receptor signaling pathway, transaminase activity, and glutathione transferase activity. Results from the present experiment showed that hepatic gene expression profile and biological processes related to oxidation reduction, lipid metabolism and cholesterol biosynthetic were significantly affected by dietary corn silage level.In conlusion, when the ME and CP intake of heifers was similar, increasing dietary corn silage level resulted in greater fiber intake, daily ruminating time, mole ratio percentage of acetate, plasma ALT activity, and apparent digestibility of NDF and ADF. The growth of heifers was unaffected by corn silage level, while the gene expression profile was significantly regulated by dietary corn silage level. |
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