A 314-bp SINE insertion in the ZNF2 promoter region may act as a repressor related to regulation of fat deposition in pigs

Retrotransposons,a type of DNA fragment that can mobilize itself on genome,can generate genetic variations and develop for molecular markers based on the insertion polymorphism.Zinc finger proteins(ZNFs)are among the most abundant proteins in eukaryotic animals,and their functions are extraordinarily diverse and particularly important in gene regulation.In the current study,bioinformatic prediction was performed to screen for retrotransposon insertion polymorphisms(RIPs)in six ZNF genes(ZNF2,ZNF3,ZNF7,ZNF8,ZNF10 and ZNF12).Six RIPs in these ZNFs,including one short interspersed nuclear element(SINE)RIP in intron 1 and one long interspersed nuclear element 1(L1)RIP in intron 3 of ZNF2,one SINE RIP in 5′flanking region and one SINE RIP in intron 2 of ZNF3,one SINE RIP in 3′UTR of ZNF7 and one L1 RIP in intron 2 of ZNF12,were discovered and their presence was confirmed by PCR.The impact of the SINE RIP in the first intron of ZNF2,which is close to the core promoter of ZNF2,on the gene activity was investigated by dual-luciferase assay in three cell lines.Our results showed that the SINE insertion in the intron 1 of ZNF2 repressed the core promoter activity extremely significantly(P<0.01)in cervical cancer cells and porcine primary embryonic fibroblasts(HeLa and PEF),thus SINE may act as a repressor.This SINE RIP also significantly(P<0.05)affected the corrected back fat thickness in Yorkshire pigs.The corrected back fat thickness of individuals with SINE insertion in the first intron of ZNF2 was significantly(P<0.05)higher than that of individuals without SINE insertion.In summary,our data suggested that RIPs play important roles in the genetic variations of these ZNF genes and SINE RIP in the intron 1 of ZNF2 may provide a useful molecular marker for the screening of fat deposition in the pig breeding.

Identification and characterization of genes that control fat deposition in chickens

Background： Fat deposits in chickens contribute significantly to meat quality attributes such as juiciness, flavor, taste and other organoleptic properties. The quantity of fat deposited increases faster and earlier in the fast- growing chickens than in slow-growing chickens. In this study, Affymetrix Genechip~ Chicken Genome Arrays 32773 transcripts were used to compare gene expression profiles in liver and hypothalamus tissues of fast-growing and slow-growing chicken at 8 wk of age. Real-time RT-PCR was used to validate the differential expression of genes selected from the microarray analysis. The mRNA expression of the genes was further examined in fat tissues. The association of single nucleotide polymorphisms of four lipid-related genes with fat traits was examined in a F2 resource population. Results： Four hundred genes in the liver tissues and 220 genes hypothalamus tissues, respectively, were identified to be differentially expressed in fast-growing chickens and slow-growing chickens. Expression levels of genes for lipid metabolism （SULTIB1, ACSBG2, PNPLA3, LPL, AOAH） carbohydrate metabolism （MGAT4B, XYLB, GBE1, PGM1, HKDCl）cholesttrol biosynthesis （FDPS, LSS, HMGCR, NSDHL, DHCR24, IDI1, MEI） HSD17B7 and other reaction or pro- cesses （CYPIA4, CYP1A1, AKR1BI, CYP4V2, DDO） were higher in the fast-growing White Recessive Rock chickens than in the slow-growing Xinghua chickens. On the other hand, expression levels of genes associated with multicellular organism development, immune response, DNA integration, melanin biosynthetic process, muscle organ develop- ment and oxidation-reduction （FRZB, DMD, FUT8, CYP2C45, DHRSX, and CYP2C18） and with glycol-metabolism （GCNT2, ELOVL d, and FASN）, were higher in the XH chickens than in the fast-growing chickens, fiT-PCR validated high expression levels of nine out of 12 genes in fat tissues. The G1257069A and T1247123C of the ACSBG2 gene were significantly associated with abdominal fat weight. The G4928024A of the FASN gene were significantly associ- ated with fat bandwidth, and abdominal fat percentage. The C4930169T of the FASN gene was associated with ab- dominal fat weight while the A59539099G of the ELOVL 6 was significantly associated with subcutaneous fat. The A8378815G of the DDT was associated with fat band width. Conclusion： The differences in fat deposition were reflected with differential gene expressions in fast and slow growing chickens.

Identification of key genes affecting porcine fat deposition based on coexpression network analysis of weighted genes

Background:Fat deposition is an important economic consideration in pig production.The amount of fat deposition in pigs seriously affects production efficiency,quality,and reproductive performance,while also affecting consumers’choice of pork.Weighted gene co-expression network analysis(WGCNA)is effective in pig genetic studies.Therefore,this study aimed to identify modules that co-express genes associated with fat deposition in pigs(Songliao black and Landrace breeds)with extreme levels of backfat(high and low)and to identify the core genes in each of these modules.Results:We used RNA sequences generated in different pig tissues to construct a gene expression matrix consisting of 12,862 genes from 36 samples.Eleven co-expression modules were identified using WGCNA and the number of genes in these modules ranged from 39 to 3,363.Four co-expression modules were significantly correlated with backfat thickness.A total of 16 genes(RAD9A,IGF2R,SCAP,TCAP,SMYD1,PFKM,DGAT1,GPS2,IGF1,MAPK8,FABP,FABP5,LEPR,UCP3,APOF,and FASN)were associated with fat deposition.Conclusions:RAD9A,TCAP,SMYD1,PFKM,GPS2,and APOF were the key genes in the four modules based on the degree of gene connectivity.Combining these results with those from differential gene analysis,SMYD1 and PFKM were proposed as strong candidate genes for body size traits.This study explored the key genes that regulate porcine fat deposition and lays the foundation for further research into the molecular regulatory mechanisms underlying porcine fat deposition.

Dietary methionine deficiency stunts growth and increases fat deposition via suppression of fatty acids transportation and hepatic catabolism in Pekin ducks

Background:Although methionine(Met),the first-limiting dietary amino acid,has crucial roles in growth and regulation of lipid metabolism in ducks,mechanisms underlying are not well understood.Therefore,the objective was to use dietary Met deficiency to investigate the involvement of Met in lipid metabolism and fat accumulation of Pekin ducks.Methods:A total of 150 male Pekin ducks(15-d-old,558.5±4.4 g)were allocated into 5 groups(6 replicates with 5 birds each)and fed corn and soybean meal-based diets containing 0.28%,0.35%,0.43%,0.50%,and 0.58%Met,respectively,for 4 weeks.Met-deficient(Met-D,0.28%Met)and Met-adequate(Met-A,0.43%Met)groups were selected for subsequent molecular studies.Serum,liver,and abdominal fat samples were collected to assess the genes and proteins involved in lipid metabolism of Pekin ducks and hepatocytes were cultured in vivo for verification.Results:Dietary Met deficiency caused growth depression and excess fat deposition that were ameliorated by feeding diets with adequate Met.Serum triglyceride and non-esterified fatty acid concentrations increased(P<0.05),whereas serum concentrations of total cholesterol,low density lipoprotein cholesterol,total protein,and albumin decreased(P<0.05)in Met-D ducks compared to those in Met-A ducks.Based on hepatic proteomics analyses,dietary Met deficiency suppressed expression of key proteins related to fatty acid transport,fatty acid oxidation,tricarboxylic acid cycle,glycolysis/gluconeogenesis,ketogenesis,and electron transport chain;selected key proteins had similar expression patterns verified by qRT-PCR and Western blotting,which indicated these processes were likely impaired.In vitro verification with hepatocyte models confirmed albumin expression was diminished by Met deficiency.Additionally,in abdominal fat,dietary Met deficiency increased adipocyte diameter and area(P<0.05),and down-regulated(P<0.05)of lipolytic genes and proteins,suggesting Met deficiency may suppress lipolysis in adipocyte.Conclusion:Taken together,these data demonstrated that dietary Met deficiency in Pekin ducks resulted in stunted growth and excess fat deposition,which may be related to suppression of fatty acids transportation and hepatic catabolism.

Effects of Maternal Dietary Energy Restriction on Fat Deposition of Offspring

The study was carried out to investigate the effects of maternal dietary energy restriction on growth performance, serum indices and fat deposition of offspring. A total of 400 female Arbor Acres （AA） broiler breeders were studied. These female birds involved three experimental treatments and a control group with normal dietary energy diets （ND, 11.7 MJ of ME. kgt during the laying）. In treatments 2, 3 and 4, the energies of diets were 20%, 30% and 50% （LD20, LD30 and LD50） lower than those of the control, respectively. The study commenced at the beginning of the laying period when the total egg production reached 5% of the flock. All the broiler offspring were fed the same diets. The results showed that in low energy diets, offspring showed decreased 1-day-old weight, but 49-day-old weight was higher in LD20 diet （P〈0.05）. For offspring during days 1-49, the average daily gain （ADG） in LD20 group and the feed conversion ratio in LD50 group were improved as compared with those of the control （P〈0.05）. Compared with the control, abdominal fat percentage increased in 49-day-old offspring from LD30 diet （P〈0.05）; the fat content of breast muscle in offspring increased in broilers fed low energy diets （P〈0.05）. In 28-day-old offspring from breeders given LD20 and LD50 diets, liver fat percentages were higher compared with ND （P〈0.05）. The subcutaneous fat thickness in 28-day-old offspring from LD50 group and 49-day-old offspring from LD30 group was higher （P〈0.05）. On day 49, the serum cholesterol （CHO） of offspring from breeders fed LD20 diet and serum high-density lipoprotein （HDL） of offspring from breeders fed LD50 diet reduced compared with those of the control （P〈0.05）. In addition, a higher triiodothyronine （T3） content in serum was found in offspring from broiler breeders given LD20 and LD30 diets （P〈0.05）. Serum thyroxine （T4） in offspring significantly decreased with the decrease of diet energy （P〈0.05）. In conclusion, to a certain extent, dietary energy restriction in breeders could improve growth performance and promote lipid metabolism of offspring.

Plant-based meat analogues aggravated lipid accumulation by regulating lipid metabolism homeostasis in mice

To determine the effects of plant-based meat analogues on the metabolic health and the possible mechanisms,mice were fed with a real pork diet(AP),a real beef diet(AB),a plant-based pork analogue diet(PP)and plant-based beef analogue diet(PB)for 68 days.Compared with real meat,the plant-based meat analogues increased food and energy intake,body weight,white fat and liver weight and caused adipocyte hypertrophy,hepatic lipid droplet accumulation,and inflammatory responses in mice.Metabolomics revealed that plantbased meat analogues altered the composition of serum metabolites,which regulated lipid metabolism homeostasis.The PB diet upregulated gene expression related to lipid synthesis,lipolysis and adipocyte differentiation while the PP diet upregulated expression of lipolysis-related genes but downregulated expression of adipocyte differentiation-related genes in white adipose tissue.Meanwhile,both PP and PB diets upregulated lipid influx-and synthesis-related genes but downregulated lipid oxidation-related genes in liver.The specific metabolite biomarkers may affect fat accumulation mainly by direct lipid metabolism pathways or indirect amino acid metabolism,protein digestion and absorption,bile secretion,aminoacyl-tRNA biosynthesis,neuroactive ligand-receptor interaction and ABC transporters pathways.These findings provide a new insight into understanding the differences in nutritional functions of meat and plant-based meat analogues.

Molecular cloning and ontogenesis expression of fatty acid transport protein-1 in yellow-feathered broilers

Fatty acid transport protein-1 （FATP-1） is one of the important transporter proteins involved in fatty acid transmembrane transport and fat deposition. To study the relationship between FATP-1 mRNA expression and fat deposition, chicken （Gallus gallus） FATP-1 sequence was first cloned by rapid amplification of cDNA ends （RACE）. Tissue samples of chest muscle, leg muscle, subcutaneous fat, and abdominal fat were collected from six male and six female broilers each, at 22 days, 29 days, and 42 days, respectively. The tissue specificity and ontogenesis expression pattern of the FATP-1 mRNA of yellow-feathered broilers was studied by real-time reverse transcription polymerase chain reaction （RT-PCR）, and the fat deposition laws in different tissues were also compared. A 2,488 bp cDNA sequence of chicken FATP-1 was cloned by RACE （GenBank accession no. DQ352834）, including 547 bp 3＇ end untranslated region （URT） and 1,941 bp open reading frame （ORF）. Chicken FATP-1 encoded 646 amino acid residues, which shared 83.9% and 83.0% identity with those of human and rat, respectively. The results of quantitative PCR demonstrated a constant FATP-1 mRNA expression level in the chest muscle and subcutaneous fat of both male and female broilers at three stages, whereas the expression level of the FATP-1 mRNA in the leg muscle at 42 days was significantly higher than that at 22 days or 29 days. In the abdominal fat of male broilers, the gene expression significantly increased with age, whereas the female broilers showed a dramatic downregulation of FATP-1 expression in abdominal fat at 42 days. This suggested a typical tissue-and gender-specific expression pattern of chicken FATP-1, mediating the specific process of fatty acid transport or utilization in muscle and adipose tissues.

The regulation of IMF deposition in pectoralis major of fast-and slow-growing chickens at hatching

Background： The lipid from egg yolk is largely consumed in supplying the energy for embryonic growth until hatching. The remaining lipid in the yolk sac is transported into the hatchling＇s tissues. The gene expression profiles of fast-and slow-growing chickens, Arbor Acres（AA） and Beijing-You（BJY）, were determined to identify global differentially expressed genes and enriched pathways related to lipid metabolism in the pectoralis major at hatching.Results： Between these two breeds, the absolute and weight-specific amounts of total yolk energy（TYE） and intramuscular fat（IMF） content in pectoralis major of fast-growing chickens were significantly higher（P 〈 0.01,P 〈 0.01, P 〈 0.05, respectively） than those of the slow-growing breed. IMF content and u-TYE were significantly related（r = 0.9047, P 〈 0.01）. Microarray analysis revealed that gene transcripts related to lipogenesis, including PPARG, RBP7, LPL, FABP4, THRSP, ACACA, ACSS1, DGAT2, and GK, were significantly more abundant in breast muscle of fast-growing chickens than in slow-growing chickens. Conversely, the abundance of transcripts of genes involved in fatty acid degradation and glycometabolism, including ACAT1, ACOX2, ACOX3, CPT1 A, CPT2, DAK, APOO, FUT9, GCNT1,and B4 GALT3, was significantly lower in fast-growing chickens. The results further indicated that the PPAR signaling pathway was directly involved in fat deposition in pectoralis major, and other upstream pathways（Hedgehog, TGFbeta, and cytokine–cytokine receptor interaction signaling pathways） play roles in its regulation of the expression of related genes.Conclusions： Additional energy from the yolk sac is transported and deposited as IMF in the pectoralis major of chickens at hatching. Genes and pathways related to lipid metabolism（such as PPAR, Hedgehog, TGF-beta, and cytokine–cytokine receptor interaction signaling pathways） promote the deposition of IMF in the pectoralis major of fast-growing chickens compared with those that grow more slowly. These findings provide new insights into the molecular mechanisms underlying lipid metabolism and deposition in hatchling chickens.

CircDOCK7 facilitates the proliferation and adipogenic differentiation of chicken abdominal preadipocytes through the gga‑miR‑301b‑3p/ACSL1 axis

Background Abdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes,which is a well-orchestrated multistep process involving many regulatory molecules.Circular RNAs(circRNAs)have emergingly been implicated in mammalian adipogenesis.However,circRNA-mediated regulation in chicken adipogenesis remains unclear.Our previous circRNA sequencing data identified a differentially expressed novel circRNA,8:27,886,180|27,889,657,during the adipogenic differentiation of chicken abdominal preadipocytes.This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes,and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.Results Our results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19–22 of the dedicator of cytokinesis 7(DOCK7)gene,abbreviated as circDOCK7.CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life.CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens,and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Functionally,the gain-and loss-of-function experiments showed that circDOCK7 promoted proliferation,G0/G1-to S-phase progression,and glucose uptake capacity of chicken abdominal preadipocytes,in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes.Mechanistically,a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p,which was identified as an inhibitor of chicken abdominal adipogenesis.Moreover,the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p.Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator.Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Conclusions CircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene,thus augmenting adipogenesis in chickens.These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.

Intravenous-Accelerated Saline Particles to Unblock Partially Clogged Blood Vessels Using a Microcontroller

This research assesses the speed of saline fluid in vein vessels using venipuncture medical kit as well as DC submersive pumps that are being controlled by a microcontroller. The microcontroller is monitored and governed using a software IDE interface installed on a powerful laptop. Saline solution is being pumped through a medical syringe at variable speeds up to a maximum of 18.39 cm/second to the vein. The novel technique in this research is the usage of two pumps called Pump 1 and Pump 2. Pump 1 is used to physically model the flow of “blood” in human vein and the second pump (Pump 2) is used to generate the accelerated saline particles that are used to break the yellow grease that is placed on the inside of the vein’s wall. A tiny brush is briefly dipped into yellow grease, and then it is used to place one layer (one turn) of yellow grease on the inside of the vein’s wall, and then this procedure is repeated to place consecutive layers of yellow grease onto the inside of the wall of the vein vessel using a tiny brush. It was found that accelerated saline particles can in fact destroy fats that are built up inside the veins’ walls.

Evaluation of dynamic effects of dietary medium-chain monoglycerides on performance,intestinal development and gut microbiota of broilers in large-scale production

Medium-chain monoglycerides(MG)have been reported to affect the productive performance,gut microbiota and health of broiler chickens reared in ideal experimental conditions at home and abroad.However,the effects of MG on performance,intestinal development and gut microbiota of chickens in large-scale farms during different feed stages remain unknown.The present study was conducted on a modern farm with a total of 12,000 yellow feathered broiler chicks that were randomly allotted to 2groups(1000 chicks/replicate,6 replicates/group)for a 70-day trial.The control group(CON group)received a basal diet,and the treated group(MG group)was fed a basal diet containing 300 mg/kg mixed MG.The results revealed that dietary MG significantly(P<0.05)increased the body weight and average feed intake,but notably reduced the feed conversion and mortality of chickens in large-scale production during the starter phase.The villus height of the duodenum in the MG group at 1,2 and 7 wk of age increased notably,and the villus height to crypt depth ratio at 1,2,5 and 10 wk of age was improved.Dietary MG decreased the serum insulin content of chickens at 5,7 and 10 wk of age,and decreased the serum lipopolysaccharide at 3 and 7 wk of age.The triglyceride level of chickens at 3,5 and 10 wk of age and the low-density lipoprotein cholesterol level of chickens at 7 and 10 wk of age in the MG group decreased notably,while the high-density lipoprotein cholesterol increased significantly.Moreover,MG supplementation selectively increased the relative abundance of genus Bacteroides(family Bacteroidaceae)and Lachnospiraceae_NK4A136_group,but decreased the content of genus Rikenellaceae_RC9_gut_group,Collinsella and family Barnesiellaceae in the cecum of chickens at 3,7 and 10 wk of age.Conclusively,these findings showed that dietary MG notably enhanced chicken performance,health and feed nutrient utilization at early ages by regulating gut microbiota,intestinal development and serum biochemical indices.