Transcriptome analysis reveals mechanism underlying the differential intestinal functionality of laying hens in the late phase and peak phase of production

- functionality of laying hens in the late phase and peak phase of production.
- Background: The compromised performance of laying hens in the late phase of production relative to the peak production was thought to be associated with the impairment of intestinal functionality, which plays essential roles in contributing to their overall health and production performance.
- In the present study, RNA sequencing was used to investigate differences in the expression profile of intestinal functionality-related genes and associated pathways between laying hens in the late phase and peak phase of production..
- Results: A total of 104 upregulated genes with 190 downregulated genes were identified in the ileum (the distal small intestine) of laying hens in the late phase of production compared to those at peak production.
- These upregulated genes were found to be enriched in little KEGG pathway, however, the downregulated genes were enriched in the pathways of PPAR signaling pathway, oxidative phosphorylation and glutathione metabolism..
- Similarly, there were lower activities of total superoxide dismutase, glutathione S-transferase and Na + /K + -ATPase, and reductions of total antioxidant capacity and ATP level, along with an elevation in malondialdehyde content in the ileum of laying hens in the late phase of production as compared with those at peak production..
- Conclusions: The intestine of laying hens in the late phase of production were predominantly characterized by a disorder of lipid metabolism, concurrent with impairments of energy production and antioxidant property.
- This study uncovers the mechanism underlying differences between the intestinal functionality of laying hens in the late phase and peak phase of production, thereby providing potential targets for the genetic control or dietary modulation of intestinal hypofunction of laying hens in the late phase of production..
- Layer industry is one of the key components contribut- ing to sustainable food sources in the world.
- accounts for a large part of the whole cycle of layer production, during which laying hens are known to be characterized by the de- clined production performance and poor egg quality as compared with those at peak production, resulting in a restricted economic benefit of layer production [1, 2]..
- One crucial reason for the compromises of production performance and egg quality of laying hens in the late phase of production could be the corresponding impair- ment of intestinal functional state [3, 4].
- Since there was a deterioration of intestinal functioning such as absorption and barrier dysfunction, immune and defense defects in older animals as compared with young animals [10, 11], the laying hens in the late and peak phase of production were speculated to display distinct differences in terms of intestinal functioning.
- This could be supported by the findings that aged laying hens had a destructed intestinal structure and an increased susceptibility of gut mucosal system to lose its integrity, as well as being more vulner- able to intestinal inflammatory responses relative to the young counterparts [12, 13]..
- It seems that the intestinal hypofunction of laying hens in the late phase of production after having undergone the intensive metabolism at peak production is associated with the aging-related down-regulations of the expression of certain functional molecules in the intestine [14, 15], as supported by the finding that the age-related decline in the absorption of nutrients (carbohydrates, lipids and amino acids) was linked to the reduced abundances of their transporters in the intestine of rats [16, 17], besides, aging-induced disorder of energy generation in the intes- tine was responsible by the mitochondrial respiratory chain deficiency, being mediated by the reduced expres- sion of cytochrome c oxidase and succinate dehydrogen- ase [18].
- To date, comprehensive knowledge on the age- related discrepancies of intestinal functions between laying hens at different production stages is poorly understood..
- And far less is known regarding the differences between the intestinal functions of laying hens in the late phase and peak phase of production at the molecular level..
- In this study, the RNA next-generation sequencing was employed to re- veal intestinal differences in transcriptome profiles of laying hens at different laying periods, aiming to identify the important genes and critical pathways associated.
- with the underlying mechanism for differences between the complex intestinal functionality of laying hens in the late phase and peak phase of production, thereby provid- ing potential targets for improving the performance of laying hens in the late phase of production..
- Biochemical indices of the layer intestine.
- 0.10) of the activities of ALP and Ca 2+ /Mg 2.
- ATPase in the layer intestine of LP group relative to PP group (Table 2)..
- There was an obvious difference in gene expression profile of the layer intestine between groups, as revealed by the principal component analysis plot (Additional file 1).
- A total of 294 DGEs were identified in the intestine between groups, including 104 upregulated and 190 downregulated genes in LP group relative to PP group (Fig.
- Volcano plot visualized the difference in the expression profile of intestinal genes in these two groups (Fig.
- 2), suggesting that the RNA sequencing reliably identified differentially expressed mRNAs in the ileal transcriptome..
- To obtain valuable information for functional predic- tion of DEGs, searches were made on standard uni- genes in the COG and GO databases.
- The most abundant terms annotated to the DEGs in the category of biological progress were cellular process, single-organism process, and metabolic process.
- 0.10) in the pathway of SNARE interactions in ves- icular transport (Table 4).
- 0.05) in the pathways of peroxisome proliferator-activated receptor (PPAR) signaling.
- 0.10) in the pathways of drug metabolism-cytochrome P450 (RF = 13.1), metab- olism of xenobiotics by cytochrome P450 (RF = 12.4), and glycine, serine and threonine metabolism (RF = 11.8)..
- In the PPAR signaling pathway, fatty acid-binding protein 1 (FABP1|FC = 0.38), FABP2 (FC = 0.49), FABP3 (FC = 0.41), FABP5 (FC = 0.69), FABP6 (FC = 0.58), lipoprotein lip- ase (LPL|FC = 0.56), apolipoprotein A1 (APOA1|FC = 0.56), sterol carrier protein 2 (SCP2|FC = 0.75) and perilipin-1 (PLIN1|FC = 0.59) were lower expressed in LP group rela- tive to PP group (Table 6).
- The downregulated genes in LP group that implicated in the pathway of glutathione metabolism were glutathione S-transferase (GST) omega-1 (GSTO1|FC = 0.7 3), GST mu 2 (GSTM2|FC = 0.59), GST alpha 3 (GS TA3|FC = 0.69) and ornithine decarboxylase 1 (ODC1|FC = 0.68).
- Remarkably, the downregulated expression of GSTO1, Table 1 Comparison of intestinal antioxidant status 1 of laying hens between groups 2 (n = 8).
- 2 PP laying hens in the peak phase of production, LP laying hens in the late phase of production.
- Table 2 Comparison of intestinal enzyme 1 activities of laying hens between groups 2 (n = 8) ALP.
- GSTM2 and GSTA3 in LP group also mediated the decreas- ing trend of the pathways of drug metabolism-cytochrome P450 and metabolism of xenobiotics by cytochrome P450..
- metabolism, energy production and oxidation resistance Since pathway analysis revealed that DEGs were predom- inantly enriched in the pathways of PPAR signaling pathway, oxidative phosphorylation and glutathione me- tabolism, the DEGs were subjected to deep-level GO clus- tering analysis in relation to lipid metabolism, energy generation and oxidation resistance, in order to better understand the network that responsible for the difference between groups.
- 0.05) of the clusters of transport, regulation of intestinal cholesterol absorption, phospholipid efflux, posi- tive regulation of cholesterol esterification, reverse choles- terol transport, ATP synthesis coupled proton transport, hydrogen peroxide catabolic process, and removal of superoxide radicals within the category of biological process in LP group as compared to PP group.
- PPAR signaling pathway is a key regulator of metabolism of the intestine [22], which together with the liver are considered as important sites for lipid metabolism [7].
- In the present study, the lipid metabolism-related genes such as FABP1, FABP2, FABP3, FABP5, FABP6, LPL and APOA1 that mapped to PPAR signaling pathway were downregulated in LP group relative to PP group..
- LP, laying hens in the late phase of production.
- PP, laying hens in the peak phase of production.
- Specifically, ileal-type FABP that located in the distal small intestine is regarded as the cytosolic re- ceptor for bile acids, although it has a low binding affin- ity for fatty acids [26].
- Therefore, the reduced expression of FABP6 with the resultant downregulations of GO clusters of transport and transporter activity might sug- gest a compromised reabsorption of luminal bile acids into enterocytes [26], resulting in a disordered regulation of lipid metabolism of the laying hens in LP group.
- In this study, although no difference in the expression of PPARs was observed.
- 2 Validation of the differentially expressed genes (DEGs) by RT-PCR (n = 8).
- a Comparison (fold change) of the RNA-Seq data of LP group relative to PP group.
- 1 PP laying hens in the peak phase of production, LP laying hens in the late phase of production.
- APOA1, an essential structural and functional component of chylomicron, can be synthesized in the intestine [7].
- Accordingly, the downregulations of APOA1 and LPL in LP group probably caused an inefficient utilization of dietary lipids that serve as a momentous energy source for animals, presumptively favoring the compromised performance of laying hens.
- Besides participating in the.
- Indeed, the current study showed that the downregulated expression of APOA1 and APOA4 induced reductions of cholesterol metabolism-related GO clusters such as regulation of in- testinal cholesterol absorption, cholesterol transporter activity, very-low density lipoprotein particle, positive regulation of cholesterol esterification and reverse chol- esterol transport, indicating perturbations of cholesterol absorption, transport and excretion of laying hens in LP group.
- Thus, the lower GO clus- ters of phosphatidylcholine-sterol O-acyltransferase acti- vator activity and phospholipid efflux in LP group may exacerbate the impaired efflux of cholesterol, triggering cholesterol accumulation inside the body of laying hens in LP group..
- This might thus deteriorate the deficiency of oxidative phos- phorylation of laying hens in LP group..
- This is accomplished by the respira- tory chain in the inner mitochondrial membrane [38], comprising five complexes including complex I (NADH-CoQ dehydrogenase), complex II (succinate- CoQ dehydrogenase), complex III (reduced CoQ- cytochrome c reductase), complex IV (cytochrome C oxidase) and complex V (ATP synthase) (Additional file 4).
- Consequently, mitochondrial dysfunction could re- strict nutrient absorption and metabolism, therefore favoring the declined performance of laying hens.
- Similarly, it was reported that aging induced reduced expression of the subunits of respiratory chain complexes (III, IV and V) in the brain of mice [42], as well as the sub- units of all the respiratory chain complexes in rat heart [43].
- K + -ATPase, a major ion pump in basolateral membrane of enterocytes and drives the co-absorption of sodium with selected nutrients [44], confirming a disturbance of intestinal mitochondria to supply en- ergy for laying hens in LP group.
- This could inevitably obstruct various metabolic processes with energy ex- penditure such as active transport of nutrients, pre- sumably conducing to the impaired performance of laying hens in the late phase of production..
- GSTs are broadly spread in various cell compartments inside the body, among which GST A, M, P, K and Z can reside in the mitochondria [45].
- Specifically, GSTA3 is found to exist in the mitochondria and capable to clear various peroxidation products [45], while GSTM2 protects against mitochondrial dysfunction by acting on V-type proton ATPase [49].
- Similarly, the expression of GSTs in the visceral or- gans (liver and lung) of rats was reported to be de- creased due to aging [52].
- Since mitochondria in the intestinal tissue is highly sensitive to oxidative stress that can lead to an inactivation of respiratory chain enzymes [54], the depressed oxida- tion resistance of the intestine presumably induced an inefficiency of energy production of laying hens in LP group [41].
- However, there were enzyme systems such as GSTs cap- able of biotransformation of xenobiotics in the intestine, which consequently influenced the overall bioavailability of these chemicals [56].
- This study demonstrated that there were disturbances of lipid metabolism, energy production and oxidation re- sistance of the intestine of laying hens in the late phase of production as compared to those at peak production..
- LP group mediated by the downregulation of PPAR sig- naling pathway together with the GSTs-mediated down- regulation of glutathione metabolism may aggravate the dysfunction of oxidative phosphorylation, conducing to the compromised energy generation in the intestine of laying hens in the late phase of production.
- The results described herein provide insights into the mechanism for differences between the intestinal functionality of lay- ing hens in the late phase and peak phase of production, which may serve as a resource for future studies on the genetic control or dietary regulation of intestinal hypo- function of laying hens in the late phase of production..
- A total of 96 Hy-Line Brown laying hens in the peak phase of pro- duction (35-wk-old, PP group) and 96 Hy-Line Brown lay- ing hens in the late phase of production (60-wk-old, LP group) were separately allocated into 8 replicates with 12 birds per replicate cage in a randomized block design..
- Three weeks before the beginning of the experiment, all the.
- laying hens were kept in one new room to acclimate the en- vironment and received the same diet.
- 2 of the experiment, one bird was randomly se- lected from each replicate cage.
- The remainder laying hens were raised continuously until elimination (about 70-wk old).
- Approximately 0.1 g of frozen mucosa sample from the ileum of laying hens from each replicate cage (n = 8) was ho- mogenized with 1:10 (w/v) cold buffer (pH 7.4) containing 10 mM Tris-HCl, 0.1 mM EDTA-Na 2 and 0.85% (w/v) NaCl.
- 4 Summarization of the mechanism underlying the differential intestinal functionality of laying hens in the late phase and peak phase of production.
- The sequencing results have been submitted to the Sequence Read Archive of the NCBI (ac- cession number: SRR9650692)..
- Additional file 1: Principal component analysis (PCA) plot of gene expression profile of the layer intestine between groups..
- Additional file 2: Clusters of Orthologous Genes (COG) classification of differentially expressed genes of the layer intestine between groups..
- Additional file 7: Composition of the basal diet..
- LP: laying hens in the late phase of production.
- PP: laying hens in the peak phase of production.
- The authors thank Yaoming Cui, Jianmin Zhou and Fengdong Zhang (Chinese Academy of Agricultural Sciences, Beijing, China) for their assistance in the process of animals feeding and sample collection.
- Apart from provid- ing funds, they were not involved in the study design, data collection, ana- lysis, interpretation, or manuscript writing..
- Datasets supporting the results of this article are also included in the Additional files 1-8.
- The RNA-seq data sets are available in the Sequence Read Archive of the NCBI (accession number: SRR9650692)..
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