Differences in Immune Characteristics and Related Gene Expression in Spleen among Ningxiang, Berkshire Breeds and Their Hybrid Pigs.

To investigate the differential immunology in Ningxiang and Berkshire pigs and their F1 offspring (F1 offspring), physiological and biochemical indicators in the plasma and spleen were analyzed. Then, transcriptomic analysis of the spleen identified 1348, 408, and 207 differentially expressed genes (DEGs) in comparisons of Ningxiang vs. Berkshire, Berkshire vs. F1 offspring, and Ningxiang vs. F1 offspring, respectively. In Ningxiang vs. Berkshire pigs, the gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the DEGs included CD163, MARCO, CXCL14, CCL19, and PPBP, which are associated with immunity. GO and KEGG analyses were also conducted comparing F1 offspring and their parents. The DEGs, including BPIFB1, HAVCR2, CD163, DDX3X, CCR5, and ITGB3, were enriched in immune-related pathways. Protein-protein interaction (PPI) analysis indicated that the EGFR and ITGA2 genes were key hub genes. In conclusion, this study identifies significant immune DEGs in different pig breeds, providing data to support the exploration of breeding strategies for disease resistance in local and crossbred pig populations.

Dietary Eucommia ulmoides leaf extract improves laying performance by altering serum metabolic profiles and gut bacteria in aged laying hens.

The leaves of Eucommia ulmoides are rich in bioactive constituents that have potential gastrointestinal benefits for animals. In aged laying hens, intestinal health issues contribute to a significant decline in egg-laying capacity during intermediate and later stages. It remains unclear whether E. ulmoides leaf extract (ELE) can improve intestinal health and enhance egg production in elderly laying hens, and the underlying mechanisms are yet to be elucidated. Therefore, we conducted a study with 480 laying hens (65 weeks old) randomly allocated into four groups: a control group fed with the basal diet, and three treatment groups supplemented with 500, 1,000, and 2,000 mg/kg of ELE, respectively. The primary active constituents of ELE include flavonoids, polysaccharides, terpenoids, and phenolic acids. Dietary supplementation with ELE at 1,000 mg/kg (ELE1000) significantly improved laying performance and egg quality compared to the other groups. ELE1000 stimulated the maturation of intestinal epithelial cells, increased villus height, and reduced crypt depth. It also influenced the levels of proteins associated with tight junctions (claudin-1 and claudin-2) and intestinal inflammatory factors (IL-6, IL-1ß, and IL-2) in different intestinal sections. Integrative analysis of serum metabolomics and gut microbiota revealed that ELE1000 improved nutrient metabolism by modulating amino acid and ubiquinone biosynthesis and influenced the abundance of intestinal microbiota by enriching pivotal genera such as Bacteroides and Rikenellaceae_RC9_gut_group. We identified 15 metabolites significantly correlated with both gut microbiota and laying performance, e.g., DL-methionine sulfoxide, THJ2201 N-valerate metabolite, tetracarbonic acid, etc. In conclusion, ELE1000 improved laying performance in elderly laying hens by affecting intestinal morphology, barrier function, microbiota, and serum metabolite profiles. These findings suggest that ELE can be a beneficial feed additive for extending the peak producing period in aged laying hens.

Integrative Analysis of Transcriptomic and Lipidomic Profiles Reveals a Differential Subcutaneous Adipose Tissue Mechanism among Ningxiang Pig and Berkshires, and Their Offspring.

Adipose tissue composition contributes greatly to the quality and nutritional value of meat. Transcriptomic and lipidomic techniques were used to investigate the molecular mechanisms of the differences in fat deposition in Ningxiang pigs, Berkshires and F1 offspring. Transcriptomic analysis identified 680, 592, and 380 DEGs in comparisons of Ningxiang pigs vs. Berkshires, Berkshires vs. F1 offspring, and Ningxiang pigs vs. F1 offspring. The lipidomic analysis screened 423, 252, and 50 SCLs in comparisons of Ningxiang pigs vs. Berkshires, Berkshires vs. F1 offspring, and Ningxiang pigs vs. F1 offspring. Lycine, serine, and the threonine metabolism pathway, fatty acid biosynthesis and metabolism-related pathways were significantly enriched in comparisons of Berkshires vs. Ningxiang pigs and Berkshires vs. F1 offspring. The DEGs (PHGDH, LOC110256000) and the SCLs (phosphatidylserines) may have a great impact on the glycine, serine, and the threonine metabolism pathway. Moreover, the DEGs (FASN, ACACA, CBR4, SCD, ELOV6, HACD2, CYP3A46, CYP2B22, GPX1, and GPX3) and the SCLs (palmitoleic acid, linoleic acid, arachidonic acid, and icosadienoic acid) play important roles in the fatty acid biosynthesis and metabolism of fatty acids. Thus, the difference in fat deposition among Ningxiang pig, Berkshires, and F1 offspring may be caused by differences in the expression patterns of key genes in multiple enriched KEGG pathways. This research revealed multiple lipids that are potentially available biological indicators and screened key genes that are potential targets for molecular design breeding. The research also explored the molecular mechanisms of the difference in fat deposition among Ningxiang pig, Berkshires, and F1 pigs, and provided an insight into selection for backfat thickness and the fat composition of adipose tissue for future breeding strategies.

Genome-Wide Association Study and Identification of Candidate Genes for Intramuscular Fat Fatty Acid Composition in Ningxiang Pigs.

Ningxiang pigs exhibit a diverse array of fatty acids, making them an intriguing model for exploring the genetic underpinnings of fatty acid metabolism. We conducted a genome-wide association study using a dataset comprising 50,697 single-nucleotide polymorphisms (SNPs) and samples from over 600 Ningxiang pigs. Our investigation yielded novel candidate genes linked to five saturated fatty acids (SFAs), four monounsaturated fatty acids (MUFAs), and five polyunsaturated fatty acids (PUFAs). Significant associations with SFAs, MUFAs, and PUFAs were found for 37, 21, and 16 SNPs, respectively. Notably, some SNPs have significant PVE, such as ALGA0047587, which can explain 89.85% variation in Arachidic acid (C20:0); H3GA0046208 and DRGA0016063 can explain a total of 76.76% variation in Elaidic Acid (C18:1n-9(t)), and the significant SNP ALGA0031262 of Arachidonic acid (C20:4n-6) can explain 31.76% of the variation. Several significant SNPs were positioned proximally to previously reported genes. In total, we identified 11 candidate genes (hnRNPU, CEPT1, ATP1B1, DPT, DKK1, PRKG1, EXT2, MEF2C, IL17RA, ITGA1 and ALOX5), six candidate genes (ALOX5AP, MEDAG, ISL1, RXRB, CRY1, and CDKAL1), and five candidate genes (NDUFA4L2, SLC16A7, OTUB1, EIF4E and ROBO2) associated with SFAs, MUFAs, and PUFAs, respectively. These findings hold great promise for advancing breeding strategies aimed at optimizing meat quality and enhancing lipid metabolism within the intramuscular fat (IMF) of Ningxiang pigs.

Modulating AHR function offers exciting therapeutic potential in gut immunity and inflammation.

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a classical exogenous synthetic ligand of AHR that has significant immunotoxic effects. Activation of AHR has beneficial effects on intestinal immune responses, but inactivation or overactivation of AHR can lead to intestinal immune dysregulation and even intestinal diseases. Sustained potent activation of AHR by TCDD results in impairment of the intestinal epithelial barrier. However, currently, AHR research has been more focused on elucidating physiologic AHR function than on dioxin toxicity. The appropriate level of AHR activation plays a role in maintaining gut health and protecting against intestinal inflammation. Therefore, AHR offers a crucial target to modulate intestinal immunity and inflammation. Herein, we summarize our current understanding of the relationship between AHR and intestinal immunity, the ways in which AHR affects intestinal immunity and inflammation, the effects of AHR activity on intestinal immunity and inflammation, and the effect of dietary habits on intestinal health through AHR. Finally, we discuss the therapeutic role of AHR in maintaining gut homeostasis and relieving inflammation.

Optimization of HS-SPME-GC-MS for the Determination of Volatile Flavor Compounds in Ningxiang Pork.

This study attempts to explore the suitable conditions for the detection of volatile flavor compounds (VFCs) in Ningxiang pork by headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS). Ningxiang pigs were harvested from a slaughterhouse and a longissimus dorsi sample was collected from each animal. The VFCs of Ningxiang pork can be strongly impacted by the detection conditions (columns, weight of meat samples, heat treatment time, equilibrium conditions, and extraction conditions) that need to be optimized. Our results also provided the optimal test conditions: weighing 5 g of meat samples, grinding for 30 s in a homogenizer, heat treatment at 100 °C for 30 min, equilibration at 70 °C for 30 min, and extraction at 100 °C for 50 min. Furthermore, the feasibility and representativeness of the test method were confirmed based on principal component analysis and a comparison of the three pork VFCs. These findings offer researchers a unified and efficient pretreatment strategy to research pork VFCs.

Predicting the brain age of children with cerebral palsy using a two-dimensional convolutional neural networks prediction model without gray and white matter segmentation.

Background: Abnormal brain development is common in children with cerebral palsy (CP), but there are no recent reports on the actual brain age of children with CP. Objective: Our objective is to use the brain age prediction model to explore the law of brain development in children with CP. Methods: A two-dimensional convolutional neural networks brain age prediction model was designed without segmenting the white and gray matter. Training and testing brain age prediction model using magnetic resonance images of healthy people in a public database. The brain age of children with CP aged 5-27 years old was predicted. Results: The training dataset mean absolute error (MAE) = 1.85, r = 0.99; test dataset MAE = 3.98, r = 0.95. The brain age gap estimation (BrainAGE) of the 5- to 27-year-old patients with CP was generally higher than that of healthy peers (p < 0.0001). The BrainAGE of male patients with CP was higher than that of female patients (p < 0.05). The BrainAGE of patients with bilateral spastic CP was higher than those with unilateral spastic CP (p < 0.05). Conclusion: A two-dimensional convolutional neural networks brain age prediction model allows for brain age prediction using routine hospital T1-weighted head MRI without segmenting the white and gray matter of the brain. At the same time, these findings suggest that brain aging occurs in patients with CP after brain damage. Female patients with CP are more likely to return to their original brain development trajectory than male patients after brain injury. In patients with spastic CP, brain aging is more serious in those with bilateral cerebral hemisphere injury than in those with unilateral cerebral hemisphere injury.

Revealing the Regulatory Mechanism of lncRNA-LMEP on Melanin Deposition Based on High-Throughput Sequencing in Xichuan Chicken Skin.

The therapeutic, medicinal, and nourishing properties of black-bone chickens are highly regarded by consumers in China. However, some birds may have yellow skin (YS) or light skin rather than black skin (BS), which causes economic losses every year. Long noncoding RNAs (lncRNAs) are widely present in living organisms, and they perform various biological functions. Many genes associated with BS pigmentation have been discovered, but the lncRNAs involved and their detailed mechanisms have remained untested. We detected 56 differentially expressed lncRNAs from the RNA-seq of dorsal skin (BS versus YS) and found that TCONS_00054154 plays a vital role in melanogenesis by the combined analysis of lncRNAs and mRNAs. We found that the full length of the TCONS_00054154 sequence was 3093 bp by RACE PCR, and we named it LMEP. Moreover, a subcellular localization analysis identified that LMEP is mainly present in the cytoplasm. After the overexpression and the interference with LMEP, the tyrosinase content significantly increased and decreased, respectively (p < 0.05). In summary, we identified the important lncRNAs of chicken skin pigmentation and initially determined the effect of LMEP on melanin deposition.

Pretreatment with IPA ameliorates colitis in mice: Colon transcriptome and fecal 16S amplicon profiling.

3-Indolepropionic acid (IPA) is a tryptophan metabolite that has anti-inflammatory properties. The present study try to investigate the phylactic effects of IPA on dextran sodium sulfate (DSS)-induced colitis mice. The results showed that IPA pretreatment ameliorated the DSS-induced decrease in growth performance, and intestinal damage and enhanced immunity in mice. RNA-seq analysis of mouse colon samples revealed that the differentially expressed genes (DEGs) were mainly enriched in immune-related pathways. 16S rRNA sequencing showed that IPA pretreatment ameliorated DSS-induced colonic microbiota dysbiosis. Moreover, the expression levels of gut immune genes were positively correlated with the relative abundance of several probiotics, such as Alloprevotella and Catenibacterium. In conclusion, IPA alleviates DSS-induced acute colitis in mice by regulating inflammatory cytokines, balancing the colonic microbiota and modulating the expression of genes related to inflammation, which would also provide a theoretical basis for IPA as a strategy to improve intestinal health.

Melatonin Alleviates Oxidative Stress Induced by H2O2 in Porcine Trophectoderm Cells.

Placental oxidative stress has been implicated as a main risk factor for placental dysfunction. Alleviation of oxidative stress and enhancement of antioxidant capacity of porcine trophectoderm (PTr2) cells are effective means to maintaining normal placental function. The present study was conducted to evaluate the protective effect of melatonin (MT) on H2O2-induced oxidative damage in PTr2 cells. Our data revealed that pretreatment with MT could significantly improve the decrease in cell viability induced by H2O2, and reduce intracellular reactive oxygen species (ROS) levels and the ratio of apoptotic cells. Here, we compared the transcriptomes of untreated versus melatonin-treated PTr2 cells by RNA-seq analysis and found that differentially expressed genes (DEGs) were highly enriched in the Wnt signaling, TGF-beta signaling and mTOR signaling pathways. Moreover, pretreatment with MT upregulated the antioxidant-related genes such as early growth response3 (EGR3), WAP four-disulfide core domain1 (WFDC1), heme oxygenase1 (HMOX1) and vimentin (VIM). These findings reveal that melatonin protects PTr2 cells from H2O2-induced oxidative stress damage.

Effect of Dietary Tryptophan on Growth, Intestinal Microbiota, and Intestinal Gene Expression in an Improved Triploid Crucian Carp.

Tryptophan (Trp) has received increasing attention in the maintenance of intestinal function. In this study, improved triploid crucian carp (ITCC) fed diets containing 6.35 g kg-1 Trp had higher average daily gain (ADG) and improved villus height (VH) and crypt depth (CD) in the intestine compared to the control group. To elucidate the potential mechanisms, we used RNA sequencing (RNA-seq) to investigate changes in the intestinal transcriptome and 16S rRNA gene sequencing to measure the intestinal microbiota in response to 6.35 g kg-1 Trp feeding in ITCC. Dietary Trp altered intestinal gene expression involved in nutrient transport and metabolism. Differentially expressed transcripts (DETs) were highly enriched in key pathways containing protein digestion and absorption and the AMPK signaling pathway. 16S rRNA sequencing showed that 6.35 g kg-1 Trp significantly increased the abundance of the genus Cetobacterium, and the Firmicutes/Bacteroidetes ratio at the phylum level (P < 0.05). In addition, bacterial richness indices (Simpson index) significantly increased (P < 0.05) community evenness in response to 6.35 g kg-1 Trp. In conclusion, appropriate dietary Trp improves the growth performance, and influences the intestinal flora of ITCC. This study might be helpful to guide the supply of dietary exogenous Trp in ITCC breeding.

L-Tryptophan activates the aryl hydrocarbon receptor and induces cell cycle arrest in porcine trophectoderm cells.

During implantation, the proliferation of trophectoderm cells (the outer epithelium of blastocysts) is related to conceptus elongation and placenta formation. Tryptophan (Trp) is a key regulator of embryogenesis and embryonic implantation during pregnancy. We sought to determine whether different concentrations of Trp alters porcine trophectoderm (pTr) cell proliferation. pTr cells were cultured in medium containing 40, 500, or 1000 µM Trp. The cell proliferation rate and the progression of the cells through the cell cycle were determined. To identify differentially expressed genes (DEGs) in the pTr cells, we compared mRNA transcriptomes by RNA-Seq after cell treatment with different concentrations of Trp. Some candidate DEGs were identified by quantitative reverse transcription PCR (qPCR). High L-Trp levels (500 and 1000 µM) inhibited cell proliferation and induced cell cycle arrest. We identified 19 DEGs between the 500 µM L-Trp and 40 µM L-Trp groups and 168 DEGs between the 1000 µM L-Trp and 40 µM L-Trp groups and subsequently used qPCR to validate some genes that were upregulated or downregulated. The functional gene networks in which the DEGs were most enriched included those associated with regulating DNA replication and the cell cycle, and the majority of the DEGs in both of these functional pathways was downregulated. The results showed that the addition of 500 and 1000 µM Trp significantly increased the abundance of proteins in the Aryl Hydrocarbon Receptor (AHR) signaling pathway. Collectively, these results indicate a novel and important role for Trp in mediating the proliferation of porcine placental cells largely via the AHR signaling pathway. Additionally, these findings help to explain the side effects of excessive Trp supplementation on placenta development and embryo growth in mammals.

Associations among Dietary Omega-3 Polyunsaturated Fatty Acids, the Gut Microbiota, and Intestinal Immunity.

Omega-3 polyunsaturated fatty acids (omega-3 PUFAs), which are essential fatty acids that humans should obtain from diet, have potential benefits for human health. In addition to altering the structure and function of cell membranes, omega-3 PUFAs (docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA), and docosapentaenoic acid (DPA)) exert different effects on intestinal immune tolerance and gut microbiota maintenance. Firstly, we review the effect of omega-3 PUFAs on gut microbiota. And the effects of omega-3 PUFAs on intestinal immunity and inflammation were described. Furthermore, the important roles of omega-3 PUFAs in maintaining the balance between gut immunity and the gut microbiota were discussed. Additional factors, such as obesity and diseases (NAFLD, gastrointestinal malignancies or cancer, bacterial and viral infections), which are associated with variability in omega-3 PUFA metabolism, can influence omega-3 PUFAs-microbiome-immune system interactions in the intestinal tract and also play roles in regulating gut immunity. This review identifies several pathways by which the microbiota modulates the gut immune system through omega-3 PUFAs. Omega-3 supplementation can be targeted to specific pathways to prevent and alleviate intestinal diseases, which may help researchers identify innovative diagnostic methods.

Breeding history and candidate genes responsible for black skin of Xichuan black-bone chicken.

BACKGROUND: Domesticated chickens have a wide variety of phenotypes, in contrast with their wild progenitors. Unlike other chicken breeds, Xichuan black-bone chickens have blue-shelled eggs, and black meat, beaks, skin, bones, and legs. The breeding history and the economically important traits of this breed have not yet been explored at the genomic level. We therefore used whole genome resequencing to analyze the breeding history of the Xichuan black-bone chickens and to identify genes responsible for its unique phenotype. RESULTS: Principal component and population structure analysis showed that Xichuan black-bone chicken is in a distinct clade apart from eight other breeds. Linkage disequilibrium analysis showed that the selection intensity of Xichuan black-bone chickens is higher than for other chicken breeds. The estimated time of divergence between the Xichuan black-bone chickens and other breeds is 2.89 ka years ago. Fst analysis identified a selective sweep that contains genes related to melanogenesis. This region is probably associated with the black skin of the Xichuan black-bone chickens and may be the product of long-term artificial selection. A combined analysis of genomic and transcriptomic data suggests that the candidate gene related to the black-bone trait, EDN3, might interact with the upstream ncRNA LOC101747896 to generate black skin color during melanogenesis. CONCLUSIONS: These findings help explain the unique genetic and phenotypic characteristics of Xichuan black-bone chickens, and provide basic research data for studying melanin deposition in animals.

Combined transcriptomics and proteomics forecast analysis for potential genes regulating the Columbian plumage color in chickens.

BACKGROUND: Coloration is one of the most recognizable characteristics in chickens, and clarifying the coloration mechanisms will help us understand feather color formation. "Yufen I" is a commercial egg-laying chicken breed in China that was developed by a three-line cross using lines H, N and D. Columbian plumage is a typical feather character of the "Yufen I" H line. To elucidate the molecular mechanism underlying the pigmentation of Columbian plumage, this study utilizes high-throughput sequencing technology to compare the transcriptome and proteome differences in the follicular tissue of different feathers, including the dorsal neck with black and white striped feather follicles (Group A) and the ventral neck with white feather follicles (Group B) in the "Yufen I" H line. RESULTS: In this study, we identified a total of 21,306 genes and 5,203 proteins in chicken feather follicles. Among these, 209 genes and 382 proteins were differentially expressed in two locations, Group A and Group B, respectively. A total of 8 differentially expressed genes (DEGs) and 9 differentially expressed proteins (DEPs) were found to be involved in the melanogenesis pathway. Additionally, a specifically expressed MED23 gene and a differentially expressed GNAQ protein were involved in melanin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapped 190 DEGs and 322 DEPs to 175 and 242 pathways, respectively, and there were 166 pathways correlated with both DEGs and DEPs. 49 DEPs/DEGs overlapped and were enriched for 12 pathways. Transcriptomic and proteomic analyses revealed that the following pathways were activated: melanogenesis, cardiomyocyte adrenergic, calcium and cGMP-PKG. The expression of DEGs was validated by real-time quantitative polymerase chain reaction (qRT-PCR) that produced results similar to those from RNA-seq. In addition, we found that the expression of the MED23, FZD10, WNT7B and WNT11 genes peaked at approximately 8 weeks in the "Yufen I" H line, which is consistent with the molting cycle. As both groups showed significant differences in terms of the expression of the studied genes, this work opens up avenues for research in the future to assess their exact function in determining plumage color. CONCLUSION: Common DEGs and DEPs were enriched in the melanogenesis pathway. MED23 and GNAQ were also reported to play a crucial role in melanin synthesis. In addition, this study is the first to reveal gene and protein variations in in the "Yufen I" H line during Columbian feather color development and to discover principal genes and proteins that will aid in functional genomics studies in the future. The results of the present study provide a significant conceptual basis for the future breeding schemes with the "Yufen I" H line and provide a basis for research on the mechanisms of feather pigmentation.

Transcriptome Analysis of the Breast Muscle of Xichuan Black-Bone Chickens Under Tyrosine Supplementation Revealed the Mechanism of Tyrosine-Induced Melanin Deposition.

The Xichuan black-bone chicken, which is a rare local chicken species in China, is an important genetic resource of black-bone chickens. Tyrosine can affect melanin production, but the molecular mechanism underlying tyrosine-induced melanin deposition in Xichuan black-bone chickens is poorly understood. Here, the blackness degree and melanin content of the breast muscle of Xichuan black-bone chickens fed a basic diet with five levels of added tyrosine (i.e., 0.2, 0.4, 0.6, 0.8, and 1.0%; these groups were denoted test groups I-V, respectively) were assessed, and the results showed that 0.8% tyrosine was the optimal level of added tyrosine. Moreover, the effects of tyrosine supplementation on the proliferation and tyrosinase content of melanocytes in Xichuan black-bone chickens were evaluated. The results revealed a dose-dependent relationship between tyrosine supplementation and melanocyte proliferation. In addition, 417 differentially expressed genes (DEGs), including 160 upregulated genes and 257 downregulated genes, were identified in a comparative analysis of the transcriptome profiles constructed using the pooled total RNA from breast muscle tissues of the control group and test group IV, respectively (fold change ≥2.0, P < 0.05). These DEGs were mainly involved in melanogenesis, the calcium signaling pathway, the Wnt signaling pathway, the mTOR signaling pathway, and vascular smooth muscle contraction. The pathway analysis of the DEGs identified some key genes associated with pigmentation, such as DCT and EDNRB2. In summary, the melanin content of breast muscle could be markedly enhanced by adding an appropriate amount of tyrosine to the diet of Xichuan black-bone chickens, and the EDNRB2-mediated molecular regulatory network could play a key role in the biological process of tyrosine-induced melanin deposition. These results have deepened the understanding of the molecular regulatory mechanism of melanin deposition in black-bone chickens and provide a basis for the regulation of nutrition and genetic breeding associated with melanin deposition in Xichuan black-bone chickens.