Effects of a wide range of dietary forage-toconcentrate ratios on nutrient utilization and hepatic transcriptional profiles in limitfed Holstein heifers

- However, the specific metabolic changes in the liver under conditions of limit-feeding remain unclear and require further study.
- Moreover, the ruminal fermentation profiles, growth characteristics, and levels of metabolites in the liver and plasma of the heifers were monitored..
- Nine DEGs were enriched in the related pathways, namely HMGCS1 , HMGCR , MSMO1 , MVK , MVD , IDI1 , FDPS , LSS , and DHCR7.
- Full list of author information is available at the end of the article.
- 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.
- With rapid growth in the human population, there is increasing demand for livestock products.
- However, details of the associated physiological and metabolic mechanisms and changes remain to be fur- ther characterized..
- Coordin- ation of the flux and inter-conversion of nutrients and metabolites in the liver might thus be altered by changes in the diet.
- With sensitive, unbiased detection of all expressed genes, this method has been successfully used in the identifica- tion of potential transcriptional mechanisms and compo- nents in bovine species subjected to different phenotypic and physiological changes.
- Therefore, RNA-Seq based transcriptomic profiling was used in the present study to identify the effects and underlying mechanism of four F:C diets on hepatic gene expression profiles in heifers with an equal intake of metabolizable energy (ME).
- The animal procedures were approved by the Ethical Committee of the College of Animal Science and Technology of China Agricultural University..
- The animals were privately owned by the Beijing Sanyuan Lvhe Dairy Group and permissions for the animals to be used were obtained prior to initiation of the study.
- At the end of the feeding period (day 28), rumen contents (100 mL/heifer) were collected using an oral stomach tube approximately 4 h after morning feeding, according to previously reported procedures [10].
- Blood samples were collected about 6 h after morning feeding from the jugular vein into 5-mL lithium heparin vacuum tubes (Hebei Xinle Medical, Shijiazhuang, China) on day 28 of the experimental period.
- A stab incision was made through the skin in the right 11th intercostals space at the level of the greater trochanter, through which a biopsy needle was inserted into the liver and around 500–1000 mg liver tissue was collected.
- The TC and TG con- centrations in the liver were analyzed using a commercially available enzymatic kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)..
- A transcriptome library was con- structed for sequencing, according to the protocols of the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530S) and NEBNext Multiplex Oligos for Illumina (NEB, E7500S).
- 10 nt, and low-quality reads (more than half of the reads with a phred base quality score of <.
- An index of the reference genome was built using Bowtie v1.1.2, and paired-end clean reads for each indi- vidual were aligned to the bovine reference genome (UMD3.1) using TopHat v2.1.0, and then assembled using the Cufflinks package [14, 25].
- The differentially expressed genes (DEGs) were detected by Cuffdiff, which is included in the Cufflinks package.
- the number of fragments were normalized by the transcript’s length and total yield of the fragments to ensure accurate quanti- fication of the gene’s expression [25].
- The quantitative real-time PCR (qRT-PCR) protocol To validate the repeatability and reproducibility of gene expression data obtained by RNA sequencing in the Holstein heifers, quantitative real-time PCR was carried.
- Data for ruminal fermentation parameters, growth charac- teristics, liver and plasma metabolites, and qRT-PCR were analyzed in a randomized complete block design using the PROC MIXED procedure of the SAS software (SAS Insti- tute Inc., Cary, NC, USA).
- 0.01) in the S60 group, which had the lowest value among all treatments.
- 0.01) with increasing levels of forage in the diets (Table 1).
- By the end of the experiment, no significant differ- ences were found in BW among the treatments (Table 2).
- 0.001) re- sponse with increasing dietary forage levels.
- Sequencing and characterization of the bovine liver transcriptome.
- Among these were uniquely aligned reads that were used for further analysis, to verify the reliability of the results..
- When selected gene expression levels between qRT- PCR and RNA-Seq platforms were compared, a strong average correlation (r = 0.88) was observed, confirming the high reproducibility of the data.
- For all five genes, the fold changes among treatments in qRT-PCR were consistent with those in the RNA-Seq data (Fig.
- Table 1 Effects of dietary forage levels on ruminal fermentation in Holstein heifers.
- NH 3 -N, mg/dL 8.27 a 3.93 b 2.22 b 2.40 b 0.583 <0.01 <0.01 <0.01 0.78.
- Acetate 53.85 c 58.04 bc 62.48 ab 65.97 a 1.135 <0.01 < .
- Butyrate 16.91 a 12.42 b 11.05 b 9.69 b 0.688 <0.01 < .
- A:P g 2.16 b 2.53 ab 2.89 ab 3.18 a 0.115 <0.01 < .
- f SEM standard error of the mean.
- Table 2 Effects of dietary forage levels on growth characteristics.
- d SEM standard error of the mean.
- A total of 10,005 unigenes were detected in the bovine liver.
- The details of the DEGs, including gene IDs, symbols, descriptions, and statistical informa- tion are shown in Additional file 2: Table S4.
- The heat- map of the DEGs was generated by hierarchical cluster analysis of gene expression traits (Fig.
- enriched (P ≤ 0.05) in the KEGG analysis, most of which were related to lipid, amino acid, and carbohydrate meta- bolic pathways (Fig.
- Expression pattern and functional analysis of DEGs involved in the significant gene expression profiles.
- The list of DEGs and their expression levels in the three profiles are shown in Additional file 1: Table S5..
- Consistent with previous studies [8, 12], all groups had a similar intake of ME and CP, indicating that the limit- feeding model was successfully established in the present study.
- As the amount of feed offered was determined by the ME of the diets, the DMI was increased with increasing dietary forage levels (Additional file 1: Table S2).
- The net release of acetate and propionate in the rumen accounted for about 70% and 55% of their portal net releases, respectively [34].
- The greater percentage of propionate combined with the similar levels of total volatile fatty acids (TVFAs) in the rumen indicate that an increased supply.
- Conse- quently, the ADG and FE were greater in the low-forage groups (Table 2), which is consistent with the findings of previous studies .
- Nevertheless, it should be noted that the experimental period might not have been long enough to produce reliable estimates of FE, and the general equation used to predict gut fill might not have been suitable for the specific situations of the present.
- After digestion, the chemical constituents of the feed are further metabolized and subsequently transported to the liver [14].
- In the present study, some key genes associated with lipid metabolism, particularly cholesterol and steroid metabolism, were significantly altered in the liver when subjected to differ- ent F:C diets..
- Cholesterol performs a number of essential functions in the body.
- It is critically important that the cells of the body have a steady, appropriate supply of cholesterol.
- The lower left-hand corner contains the P -value of the number of assigned genes compared with the expected value.
- Table 6 Summary of the KEGG analysis of significant differentially expressed genes clustered in STEM.
- nine key genes, namely: HMGCS1, HMGCR, MSMO1, MVK, MVD, IDI1, FDPS, LSS, and DHCR7 that encode the enzymes involved in these reactions were found to be significantly differentially expressed in the present study (Fig.
- Key enzymes of the cholesterol biosynthetic pathway that are also associated with rate-controlling steps have been identified as HMGCR, HMGCS1, and IDI .
- Moreover, terpenoid backbone biosyn- thesis (KEGG map00900) shared some steps with choles- terol biosynthesis, and was one of the precursor steps for steroid biosynthesis (KEGG map00100) in the KEGG pathway analysis.
- This explains the simultaneous enrich- ment of these two pathways in the present study [46]..
- All of the aforementioned genes were enriched in pro- file 19 and showed a quadratic increase with increasing dietary forage levels, with the S60 group yielding the high- est values (Fig.
- This finding was con- firmed by the TC levels in the liver (Table 3).
- As one of the main precursors of cholesterol, about 60%–80% of acetate is absorbed in the rumen, and protein-mediated transport pathways play a major role in its absorption [47].
- The reduced cholesterol synthesis observed in the S80 group could be due to the limited absorption capacity of acetate transporter proteins when excessive acetate is produced (Table 1).
- the activity of protein-mediated transport pathways of acetate absorption in the rumen were warranted..
- In the liver, cholesterol either becomes involved in bile synthesis, or is secreted in VLDL that is delivered to the systemic circulation [48].
- The lower concentrations of VLDL-C observed in the S60 group could be attributed to the fact that they were processed in intermediate- density lipoproteins and then metabolized to HDL..
- The genes HSPB1, ATF4, GADD45B, GADD45G, and FGF21 were differentially expressed and enriched in the MAPK signaling pathway in profile 17.
- By contrast, in the present study, both the MAPK signaling pathway and steroid biosyn- thesis were stimulated with increasing dietary forage levels, indicating that the MAPK signaling pathway was executing its role in regulating cholesterol homeostasis..
- In this study, GADD45B, GADD45G, and CDKN1A were also significantly differentially expressed and enriched in the p53 signaling pathways of profile 17 (Table 4).
- The GADD45 genes are important for intra- cellular communication in the immune system, and were found to be upregulated in the liver of cattle that had higher serum concentrations of cholesterol, or lower residual feed intake, as well as lower levels of cellular growth and proliferation, and lipid metabolism [18, 50]..
- The p53 signaling pathway and MAPK signaling path- way shared the same significant DEGs (GADD45B and GADD45G) in the present study.
- Thus, it is reasonable to infer that the changes observed in the p53 signaling pathway might be related to cholesterol biosynthesis..
- However, this inference should be made with caution, as only three genes were enriched in the p53 signaling pathway in the present study..
- Therefore, it is reasonable to infer that the increased cholesterol synthesis from acetate observed in the present study, might be one of the main reasons for the reduced efficiency of energy utilization observed in high-forage-fed heifers..
- The FGF21 protein has been identified as a novel hormonal factor produced by the liver that is involved in the regulation of metabolic homeostasis and energy balance (particularly the processes of glucose and lipid metabolism) in cattle [56, 57].
- In the present study, FGF21 mRNA expression increased with increasing dietary for- age levels, and this could have occurred as a consequence of energy reassignment.
- 3) were all significantly enriched in the liver and might have been involved in the regulation of feed efficiency..
- Alanine and glutamine are the most glucogenic, and account for 40%–60% of the glucose formed from amino acids in ruminants [62, 63].
- This finding is in line with the changes observed in alanine, aspartate and glutamate metabolism and glutathione metabolic pathways in the liver.
- [17] reported that CYP11A1, UGT2a1, SULTE1, and CYP7A1 were altered in the steroid hormone bio- synthesis pathway when cows were subjected to severe negative energy balance.
- In the present study, these four genes were all found to be altered in the same pathway (Additional file 2: Table S4).
- ability to catalyze the rate-limiting step of the conversion of cholesterol to bile acids [17].
- The MAPK signaling pathway, which may have an import- ant role in the regulation of steroid metabolism, also showed a linear increase with increasing dietary forage levels.
- Therefore, increased hepatic lipid metabolism might be responsible for the lower energy utilization efficiency ob- served in the heifers fed high-forage diets.
- This study might be useful in the future identification of individual heifers within the same dietary group that are high-efficiency phe- notypes, using the upregulation of cholesterol metabolism as a proxy.
- In conclusion, the results of the present study provide an insight into the biology of energy utilization in heifers, and they have the potential to promote a favorable strategy to improve feed efficiency in ruminants..
- SEM: Standard error of the means.
- Animal procedures were approved by the Ethical Committee of the College of Animal Science and Technology of China Agricultural University..
- Effects of energy and protein allowances in the diets of prepubertal heifers on growth and milk production.
- Energy metabolism in the digestive tract and liver of cattle: influence of physiological state and nutrition.
- Cholesterol synthesis in the lactating cow: induced expression of candidate genes.
- Production and absorption of volatile fatty acids in the rumen..
- Bicarbonate- dependent and bicarbonate-independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep.
- Role of the gut in lipid homeostasis.
- Changes in the expression of hepatic genes involved in cholesterol homeostasis in dairy cows in the transition period and at different stages of lactation.
- Expression of fibroblast growth factor 21 in the liver of dairy cows in the transition period and during lactation.
- Growth hormone stimulates transcription of the fibroblast growth factor 21 gene in the liver through the signal transducer and activator of transcription 5..
- Effect of dietary supplementation with glutamic-acid or glutamine on the splanchnic and muscle metabolism of Glucogenic amino-acids in the rat

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