A cell transcriptomic profile p ovides insights into adipocytes of porcine mammary gland across development

Background Studying the composition and developmental mechanisms in mammary gland is crucial for healthy growth of newborns. The mammary gland is inherently heterogeneous, and its physiological function dependents on the gene expression of multiple cell types. Most studies focused on epithelial cells, disregarding the role of neighboring adipocytes.Results Here, we constructed the largest transcriptomic dataset of porcine mammary gland cells thus far. The dataset captured 126,829 high-quality nuclei from physiological mammary glands across five developmental stages(d 90 of gestation, G90;d 0 after lactation, L0;d 20 after lactation, L20;2 d post natural involution, PI2;7 d post natural involution, PI7). Seven cell types were identified, including epithelial cells, adipocytes, endothelial cells, fibroblasts cells, immune cells, myoepithelial cells and precursor cells. Our data indicate that mammary glands at different developmental stages have distinct phenotypic and transcriptional signatures. During late gestation(G90), the differentiation and proliferation of adipocytes were inhibited. Meanwhile, partly epithelial cells were completely differentiated. Pseudo-time analysis showed that epithelial cells undergo three stages to achieve lactation, including cellular differentiation, hormone sensing, and metabolic activation. During lactation(L0 and L20), adipocytes area accounts for less than 0.5% of mammary glands. To maintain their own survival, the adipocyte exhibited a poorly differentiated state and a proliferative capacity. Epithelial cells initiate lactation upon hormonal stimulation. After fulfilling lactation mission, their undergo physiological death under high intensity lactation. Interestingly, the physiological dead cells seem to be actively cleared by immune cells via CCL21-ACKR4 pathway. This biological process may be an important mechanism for maintaining homeostasis of the mammary gland. During natural involution(PI2 and PI7), epithelial cell populations dedifferentiate into mesenchymal stem cells to maintain the lactation potential of mammary glands for the next lactation cycle.Conclusion The molecular mechanisms of dedifferentiation, proliferation and redifferentiation of adipocytes and epithelial cells were revealed from late pregnancy to natural involution. This cell transcriptomic profile constitutes an essential reference for future studies in the development and remodeling of the mammary gland at different stages.

Genetic Parameters and Genome-Wide Association Studies for Body Size Traits of Shuxuan Cattle in China

In domestic cattle,the body size traits have important implications in terms of breed characteristics and production performance.Shuxuan cattle is a dual-purpose breed mainly raised in Sichuan province,China,for which we have known less about the genetic parameters and underlying candidate genes in relation to the body size traits.In this study,we obtained the genome-wide single nucleotide polymorphisms(SNPs)using the Illumina Bovine BeadChip in 275 Shuxuan cattle.These SNPs were first used for estimating genetic parameters for the withers height(WH)and diagonal body length(BL).Using the bivariate animal model,the estimates(±standard error)of heritabilities were 0.71±0.22 and 0.49±0.29 for BL,and their genetic correlation was 0.64±0.37.Second,the genome-wide association study(GWAS)was performed.However,these did not result into genome-wide significant SNPs for both WH and BL traits.According to a less stringent suggestive significance,some positional candidate genes were found,and some of them(such as FAM110B,TAS1R2,PAX3,and FHIT)were previously reported in literature to be associated with body size traits in cattle.In conclusion,we estimated the genetic parameters in Shuxuan cattle using genomic information for the first time,which are required for implementing the genomic selection programs in the future.

Analyses of Chicken Tenderness Traits Based on Transcriptome Sequencing

The calpain system is ubiquitous in cells, mainly comprising calpains and calpain inhibitors, and is a widespread calcium-dependent cysteine protease in organisms that is involved in many cellular processes such as muscle degradation in vivo and affects the tenderness of meat after animal slaughter. The study found 128 DEGs that probably regulated tenderness traits were selected from 16 significantly enriched GO terms by transcriptome sequencing analysis, and found that the developmental changes in the expression levels of the CAPN1 gene in the pectoral and leg muscles were significantly positively correlated ( P <0.05) with the cumulative growth values of live weight and comb weight. The developmental changes in the expression levels of the CAST gene in the pectoral and leg muscles were not significantly correlated with the cumulative growth values of live weight and comb weight. Our results helped demonstrate the potential molecular mechanisms of tenderness in chickens and provide valuable information for chicken breeding.

miRNA sequencing analysis of healthy and atretic follicles of chickens revealed that miR-30a-5p inhibits granulosa cell death via targeting Beclin1

Background:The egg production performance of chickens is affected by many factors,including genetics,nutrition and environmental conditions.These factors all play a role in egg production by affecting the development of follicles.MicroRNAs(miRNAs)are important non-coding RNAs that regulate biological processes by targeting genes or other non-coding RNAs after transcription.In the animal reproduction process,miRNA is known to affect the development and atresia of follicles by regulating apoptosis and autophagy of granulosa cells(GCs).Results:In this study,we identified potential miRNAs in the atretic follicles of broody chickens and unatretic follicles of healthy chickens.We identified gga-miR-30a-5p in 50 differentially expressed miRNAs and found that gga-miR-30a-5p played a regulatory role in the development of chicken follicles.The function of miR-30a-5p was explored through the transfection test of miR-30a-5p inhibitor and miR-30a-5p mimics.In the study,we used qPCR,western blot and flow cytometry to detect granulosa cell apoptosis,autophagy and steroid hormone synthesis.Confocal microscopy and transmission electron microscopy are used for the observation of autophagolysosomes.The levels of estradiol(E2),progesterone(P4),malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.The results showed that miR-30a-5p showed a negative effect on autophagy and apoptosis of granulosa cells,and also contributed in steroid hormones and reactive oxygen species(ROS)production.In addition,the results obtained from the biosynthesis and dual luciferase experiments showed that Beclin1 was the target gene of miR-30a-5p.The rescue experiment conducted further confirmed that Beclin1 belongs to the miR-30a-5p regulatory pathway.Conclusions:In summary,after deep miRNA sequencing on healthy and atretic follicles,the results indicated that miR-30a-5p inhibits granulosa cell death by inhibiting Beclin1.

Research Progress on Effects of Stocking Density on Performance, Health and Welfare of Duck

Stocking density is an important factor for animal production performance.It appears particularly urgent and important to research systematically on stocking density for the dual restriction of environmental protection and animal welfare.This review dealt with the influence of stocking density on ducks'productivity and health through production performance,animal behavior and animal welfare,and analyzed the possible mechanism of high stocking density reducing production performance,resulting in abnormal behavior and stress,causing welfare and health problems.We consider that it might be associated with heat stress,more spatial competition(to gather the food,drinking water,sports area,etc.),as well as the quality deterioration of litter and house air caused by high stocking density.Finally,we put forward some suggestions on the study of stocking density's effects on ducks,and proposed the future work that need to be studied and problems that need to be solved imminently.

Growth and Development Analysis of Kongshan Cattle

[Objectives]In order to better understand the performance of Kongshan cattle,their growth and development was analyzed.[Methods]The body weights of 106 kongshan cattle at different month ages were measured,and the growth curves and body measurement indices of Kongshan cattle after one year old were analyzed by SPPS18.0.[Results]Kongshan cattle have good body shape.Compared with 1985,the performance of body measurements and weight was improved significantly;and the body measurements and body weight of Kongshan cattle increased significantly after 24 months old,indicating that Kongshan cattle still have good fattening performance in adulthood.[Conclusions]This study analyzed the growth and development laws of Kongshan cattle and the status quo of the population,and will provide some help for the selection and utilization of Kongshan cattle breed more intuitively.

Establishment and Analysis of Growth Curve of Muchuan Black Chicken

To find out the growth and development regularity,the growth curves of Muchuan black chicken at the age of 0-21 weeks were established by Logistic,Bertalanffy and Gompertz models in this study.The results showed that all the three models can well fit the growth curve of Muchuan black chicken with a degree above 0.988,while the Gompertz model and the Bertalanffy model was the best for hens and cocks,respectively.Finally,the weight of hens and cocks were analyzed according to their corresponding high fitting models.Results showed that the inflexion age of Muchuan black chicken was 6.8-6.9 weeks old with an inflexion weight of 692.98 g in hens and 805.57 g in cocks.In conclusion,it was feasible to establish the growth curve of Muchuan black chicken with Logistic,Bertalanffy and Gompertz model.

Differentiation of Expression Profiles of SREBF1 and SREBF2 Gene in Chicken

Sterol regulatory element-binding factor-1 and-2(SREBF1 and SREBF2)are important transcription factors involved in the regulating lipid homeostasis.Based on the essential role of SREBF1 and SREBF2,we measured the mRNA expression levels of the two genes in six various tissues at different growth points.Our results showed that the SREBF1 and SREBF2 were expressed in all six tissues examined in Erlang mountainous chicken(SD02)at 42 d,and were expressed abundantly in the uropygial gland and liver,with relatively lowest levels of expression in the abdominal fat,sebum cutaneum and leg muscle.The expression ratio of SREBF1 and SREBF2 in breast muscle,leg muscle,sebum cutaneum and uropygial gland exhibited a"decline-rise"trend.However,in liver,the expression ratio of these two genes exhibited a"decline-rise-decline"trend.Meanwhile,the expression level of SREBF1 gene of all tissues was lower than that of SREBF2 except for uropygial gland.The findings will provide important references for further function investigation of the two genes involved in fat deposition in chickens.

Zinc lactate alleviates oxidative stress by modulating crosstalk between constitutive androstane receptor signaling pathway and gut microbiota profile in weaned piglets

This study aimed to determine the regulatory mechanism of dietary zinc lactate(ZL)supplementation on intestinal oxidative stress damage in a paraquat(PQ)-induced piglet model.Twenty-eight piglets(mean body weight 9.51±0.23 kg)weaned at 28 d of age were randomly divided into control,ZL,PQ,and ZL+PQ groups(n=7 in each group).The ZL-supplemented diet had little effect on growth performance under normal physiological conditions.However,under PQ challenge,ZL supplementation significantly improved average daily gain(P<0.05)and reduced the frequency of diarrhea.ZL improved intestinal morphology and ultrastructure by significantly increasing the expression level of the jejunal tight junction protein,zonula occludens-1(ZO-1)(P<0.05),and intestinal zinc transport and absorption in PQ-induced piglets,which reduced intestinal permeability.ZL supplementation also enhanced the expression of antioxidant and antiinflammatory factor-related genes and decreased inflammatory cytokine expression and secretion in PQinduced piglets.Furthermore,ZL treatment significantly inhibited the activation of constitutive androstane receptor(CAR)signaling(P<0.01)in PQ-induced piglets and altered the structure of the gut microbiota,especially by significantly increasing the abundance of beneficial gut microbes,including UCG_002,Ruminococcus,Rikenellaceae_RC9_gut_group,Christensenellaceae_R_7_group,Treponema,unclassified_Christensenellaceae,and unclassified_Erysipelotrichaceae(P<0.05).These data reveal that pre-administration of ZL to piglets can suppress intestinal oxidative stress by improving antioxidant and anti-inflammatory capacity and regulating the crosstalk between CAR signaling and gut microbiota.

Transcriptomic analysis elucidates the enhanced skeletal muscle mass, reduced fat accumulation, and metabolically benign liver in human follistatin-344 transgenic pigs

Follistatin(FST) is an important regulator of skeletal muscle growth and adipose deposition through its ability to bind to several members of the transforming growth factor-β(TGF-β) superfamily, and thus may be a good candidate for future animal breeding programs. However, the molecular mechanisms underlying the phenotypic changes have yet to be clarified in pig. We generated transgenic(TG) pigs that express human FST specifically in skeletal muscle tissues and characterized the phenotypic changes compared with the same tissues in wild-type pigs. The TG pigs showed increased skeletal muscle growth, decreased adipose deposition, and improved metabolism status(P<0.05). Transcriptome analysis detected important roles of the PIK3–AKT signaling pathway, calcium-mediated signaling pathway, and amino acid metabolism pathway in FST-induced skeletal muscle hypertrophy, and depot-specific oxidative metabolism changes in psoas major muscle. Furthermore, the lipid metabolism-related process was changed in adipose tissue in the TG pigs. Gene set enrichment analysis revealed that genes related to lipid synthesis, lipid catabolism, and lipid storage were down-regulated(P<0.01) in the TG pigs for subcutaneous fat, whereas genes related to lipid catabolism were significantly up-regulated(P<0.05) in the TG pigs for retroperitoneal fat compared with their expression levels in wild-type pigs. In liver, genes related to the TGF-β signaling pathway were over-represented in the TG pigs, which is consistent with the inhibitory role of FST in regulating TGF-β signaling. Together, these results provide new insights into the molecular mechanisms underlying the phenotypic changes in pig.

A Monophyletic Status of Axis Genus in Subfamily Cervinae Supported by the Complete Mitochondrial Genome of Chinese Hog Deer(Axis porcinus)

Hog deer(Axis porcinus)is a small mammal and listed in the International Union for Conservation of Nature.However,phylogenetic position of hog deer within Axis genus has remained controversial.In the present study,we first assembled complete mitochondrial genome of Chinese hog deer reared in Chengdu Zoo,Sichuan,by the second-generation sequencing technology.This newly assembled mitochondrial genome of hog deer is 16376 bp in length and consists of 13 protein-encoding genes,23 transfer RNA genes and 2 ribosomal RNA genes.Phylogenetic analyses based on complete mitochondrial genome and cytochrome b gene sequences revealed that hog deer is closely clustered together and placed with sister taxon of spotted deer(A.Axis),which therefore supported monophyletic statue of Axis genus.Furthermore,considerable genetic differentiation,up to 139 mutations of complete mitochondrial genome was revealed between geographical populations of hog deer in France and Southeast Asia.However,only six variable sites(nucleotide diversity of 0.00007)and four haplotypes(haplotype diversity of 0.533)were totally detected among ten newly sequenced Chinese hog deer.The results provide a better understanding on the phylogeny of hog deer.

Study on PRRSV Receptor Genes Differential Expression in Lung Tissues in Different Breeds of Pigs after Infecting with HP-PRRSV

[ Objective] In order to study the susceptibility molecular mechanism of highly pathogenic porcine reproductive and respiratory syndrome virus （ HP- PRRSV） JXA1 isolate on Tibetan pig, Zangmei pig and Yorkshire pig. [ Method ] In the study, real-time quantitative RT-PCR method was established to compare and analyze the differential expression of five porcine reproductive and respiratory syndrome virus （PRRSV） receptor genes （HSPG2, SIGLEC1, CD163, VIM and NMMHC-H A） in lung tissues in Tibetan pig, Zangmei pig and Yorkshire pig before the challenge and at the 4th ,7th and 14th days after the challenge with JXAI isolate. [ Results ] HSPG2 expression in Tibetan pig lung tissues increased significantly at the 4th and 14th days after the challenge with JXAI （ P 〈 0.05 ）, while decreased significantly at the 7th day after the challenge （P 〈 0.05 ）, HSPG2 expression in Zangmei pig lung tissues increased significantly at the 14th day after the challenge （P〈0.05）. SIGLECl expression in Tibetan pig lung tissues increased significantly at the 4th and 14th days after the infection（P 〈 0.05 ）, while SIGLEC 1 expression in Yorkshire pig decreased significantly at the 4th, 7th and 14th days after the challenge （P 〈0. 05 ）. CD163 expression in lung tissues of Tibetan pig and Zangmei pig both increased significantly at the 14th day after the challenge （P 〈 0.05 ）, while CD163 expression in lung tissues of Yorkshire pig decreased significantly at the 7th and 14th days after the challenge （ P 〈 0. 05 ）. VIM expression in lung tissues of Tibetan pig increased significantly at the 7th day after the challenge （ P 〈 0. 05 ）, while which of Yorkshire pig at the 7th day after the challenge decreased significantly （ P 〈 0. 05 ）. NMMHC-II A expression in lung tissues of Zangmei pig increased significantly at the 4th day after the challenge （ P 〈 0. 05 ）, and which of Yorkshire pig increased significantly at the 4th and 14th days after the challenge （P 〈 0. 05 ）. [ Conclusion] SIGLEC1 and VIM genes might be the potential key genes affecting the susceptibility of JXA1 isolate on Tibetan pig, Zangrnei pig and Yorkshire pig. Key words JXA1 isolate; Tibetan pig; Zangmei pig; Yorkshire pig; Porcine reproductive and respiratory syndrome virus receptor genes; Differential expression

Production Performance of Different Generations of Muchuan Blackbone Chicken

In order to formulate feasible conservation measures and develop and utilize Muchuan black-bone chicken rationally,two generations of Muchuan black-bone chicken were bred,and their production performance and slaughter traits( at the age of 300 d) were compared. The results showed that the reproductive performance,slaughter performance and body size of the Muchuan black-bone chicken of Generation 1( G_1)were all improved compared with those of the Generation 0( G_0)( P <0. 05). At the age of 300 d,the live weight,carcass weight,and leg muscle weight of the G_1 were significantly higher than those of the G_0( P <0. 05); and the chest width of the cocks of the G_1 was significantly higher than that of the G_0( P <0. 05). It suggests that after rigorous selection and utilization of modern poultry breeding techniques,the reproductive performance,slaughter performance and survival rate of Muchuan black-bone chicken can be improved significantly.

Diarrhea induced by insufficient fat absorption in weaned piglets:Causes and nutrition regulation

Fat is one of the three macronutrients and a significant energy source for piglets.It plays a positive role in maintaining intestinal health and improving production performance.During the weaning period,physiological,stress and diet-related factors influence the absorption of fat in piglets,leading to damage to the intestinal barrier,diarrhea and even death.Signaling pathways,such as fatty acid translocase(CD36),pregnane X receptor(PXR),and AMP-dependent protein kinase(AMPK),are responsible for regulating intestinal fat uptake and maintaining intestinal barrier function.Therefore,this review mainly elaborates on the reasons for diarrhea induced by insufficient fat absorption and related signaling pathways in weaned-piglets,with an emphasis on the intestinal fat absorption disorder.Moreover,we focus on introducing nutritional strategies that can promote intestinal fat absorption in piglets with insufficient fat absorption-related diarrhea,such as lipase,amino acids,and probiotics.

Dietary iron regulates intestinal goblet cell function and alleviates Salmonella typhimurium invasion in mice

Iron is an important micronutrient that plays a vital role in host defenses and bacterial pathogenicity. As iron treatments increase the risk of infection by stimulating the growth and virulence of bacterial pathogens, their roles in anti-infection immunity have frequently been underestimated. To estimate whether adequate dietary iron intake would help defend against pathogenic bacterial infection, mice were fed iron-deficient(2 mg kg-1feed), iron-sufficient(35 mg kg-1feed), or iron-enriched diet(350 mg kg-1feed) for 12 weeks, followed by oral infection with Salmonella typhimurium. Our results revealed that dietary iron intake improved mucus layer function and decelerated the invasion of the pathogenic bacteria, Salmonella typhimurium. Positive correlations between serum iron and the number of goblet cells and mucin2 were found in response to total iron intake in mice.Unabsorbed iron in the intestinal tract affected the gut microbiota composition, and the abundance of Bacteroidales, family Muribaculaceae, was positively correlated with their mucin2 expression. However, the results from antibiotic-treated mice showed that the dietary iron-regulated mucin layer function was not microbial-dependent. Furthermore, in vitro studies revealed that ferric citrate directly induced mucin2 expression and promoted the proliferation of goblet cells in both ileal and colonic organoids. Thus, dietary iron intake improves serum iron levels, regulates goblet cell regeneration and mucin layer function, and plays a positive role in the prevention of pathogenic bacteria.

Crosstalk between trace elements and T-cell immunity during early-life health in pigs

With gradual ban on the use of antibiotics, the deficiency and excessive use of trace elements in intestinal health is gaining attention. In mammals, trace elements are essential for the development of the immune system, specifically T-cell proliferation,and differentiation. However, there remain significant gaps in our understanding of the effects of certain trace elements on T-cell immune phenotypes and functions in pigs. In this review, we summarize the specificity, development, subpopulations, and responses to pathogens of porcine T cells and the effects of functional trace elements(e.g., iron, copper, zinc, and selenium) on intestinal T-cell immunity during early-life health in pigs. Furthermore, we discuss the current trends of research on the crosstalk mechanisms between trace elements and T-cell immunity. The present review expands our knowledge of the association between trace elements and T-cell immunity and provides an opportunity to utilize the metabolism of trace elements as a target to treat various diseases.

Small RNA Sequencing Reveals Differentially Expressed piRNAs Related to Hypoxia in Tibetan Chickens

The relevance of genetic mechanism to the phenotype of hypoxic adaptation remains elusive.Tibetan chickens typically used to investigate the mechanism for the adaptation of hypoxia and the recognition of hypoxia-related piRNA remains an open issue.The purpose of this study was to illustrate whether the piRNAs were related to hypoxic adaptation.First of all,the differentially expressed piRNAs(DEpiRNAs)were identified through RNA sequencing between the Tibetan chickens and Daheng broilers.Subsequently,the target genes of DEpiRNAs were predicted and annotated by software.The network was constructed by Cytoscape.In our study,a total of 277 DEpiRNAs(33 down-regulated,244 up-regulated)were identified in the Tibetan chickens compared with the Daheng broilers.All of the 277 DEpiRNAs predicted 36658 targeted genes.Gene Ontology(GO)analysis showed that the target genes were significantly enriched in the biological process correlated with proliferation and apoptosis of cells,including cell cycle,mitochondrial outer membrane permeabilization,and positive regulation of stress-activated mitogen-activated protein kinase cascade.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis implicated that the DEpiRNAs were mainly involved in immune and metabolism,including natural killer cell-mediated cytotoxicity,toll-like receptor signaling pathway and fatty acid metabolism.Furthermore,a predicted network with four piRNAs acted on 11 pathways via interacting with 22 target genes,in which piR-gga-1368839 regulated metabolic pathways by acting on DHCR24.In conclusion,we determined the DEpiRNAs in the Tibetan chickens and found that these piRNAs were associated with metabolism,which may be favorable for researching the biological adaptation to hypoxic stress.

Glutamate and aspartate alleviate testicular/epididymal oxidative stress by supporting antioxidant enzymes and immune defense systems in boars

Several potential oxidative agents have damaging effects on mammalian reproductive systems.This study was conducted to investigate the effects of glutamate(Glu)and aspartate(Asp)supplementation on antioxidant enzymes and immune defense systems in the outer scrotum of boars injected with H2O2.A total of 24 healthy boars were randomly divided into 4 treatment groups:control(basal diet,saline-treated),H2O2(basal diet,H2O2-challenged outer scrotum(1 m L kg^–1 BW)),Glu(basal diet+2%Glu,H2O2-challenged),and Asp(basal diet+2%Asp,H2O2-challenged).Our results showed that both Glu and Asp supplementation improved testicular morphology and decreased the genital index in the H2O2-treated boars.Glu and Asp administration increased the antioxidant enzyme activities and affected the testicular inflammatory cytokine secretion but had no effect on sex hormone levels.Furthermore,the m RNA expression of CAT,Cu Zn SOD,and GPx4 was altered in the testes and epididymis of boars treated with Asp and Glu.Glu and Asp supplementation also modulated the expression of TGF-β1,IL-10,TNF-α,IL-6 and IL-1βin the testis and epididymis.These results indicate that dietary Glu and Asp supplementation might enhance antioxidant capacity and regulate the secretion and expression of inflammatory cytokines to protect the testes and epididymis of boars against oxidative stress.

Dietary polyphenols in lipid metabolism: A role of gut microbiome

Polyphenols are a class of non-essential phytonutrients,which are abundant in fruits and vegetables.Dietary polyphenols or foods rich in polyphenols are widely recommended for metabolic health.Indeed,polyphenols(i.e.,catechins,resveratrol,and curcumin)are increasingly recognized as a regulator of lipid metabolism in host.The mechanisms,at least in part,may be highly associated with gut microbiome.This review mainly discussed the beneficial effects of dietary polyphenols on lipid metabolism.The potential mechanisms of gut microbiome are focused on the effect of dietary polyphenols on gut microbiota compositions and how gut microbiota affect polyphenol metabolism.Together,dietary polyphenols may be a useful nutritional strategy for manipulation of lipid metabolism or obesity care.

An emerging role of vitamin D_(3) in amino acid absorption in different intestinal segments of on-growing grass carp(Ctenopharyngodon idella)

Vitamin D_(3)(VD_(3)),an essential nutrient for animals,has been demonstrated to stimulate the uptake of certain amino acids.However,the role of VD_(3) in the intestine,the primary site for digestion and absorption of nutrients,remains poorly characterized.Here,the grass carp(Ctenopharyngodon idella)was studied to assess the influence of different doses of VD_(3)(15.2,364.3,782.5,1,167.9,1,573.8,and 1,980.1 IU/kg)on growth performance,intestinal morphology,digestive absorption,amino acid transport,and potential signaling molecule levels in a feeding experiment.As a result,dietary VD_(3) improved growth performance,intestinal structure,and digestive and brush border enzyme activities.Additionally,most intestinal free amino acids and their transporters were upregulated after VD_(3) intake,except for Ala,Lys,Asp,Leu,solute carrier(SLC)7A7,SLC1A5,and SLC1A3 mRNA in different segments,Leu and SLC6A14 mRNA in the proximal intestine,and SLC7A5 mRNA in the mid and distal intestine.In the crucial target of rapamycin(TOR)signal pathway of amino acid transport,the gene and protein expression of TOR,S6 kinase 1,and activating transcription factor 4 were elevated,whereas 4E-binding protein 1 was decreased,further suggesting an advanced amino acid absorption capacity in the fish due to VD_(3) supplementation.Based on percentage weight gain,feed efficiency,and trypsin activity,the VD_(3) requirements of on-growing grass carp were estimated to be 968.33,1,005.00,and 1,166.67 IU/kg,respectively.Our findings provide novel recommendations for VD_(3) supplementation to promote digestion and absorption capacities of fish,contributing to the overall productivity of aquaculture.