Krüppel-like factor KLF9 inhibits chicken intramuscular preadipocyte differentiation.

1. Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects tenderness, juiciness and flavour of meat. Krüppel-like transcriptional factors (KLFs) are important regulators of adipocyte differentiation. However, little is known about the KLF9 gene associated with poultry IMF deposition, especially intramuscular adipocyte differentiation.2. Previous work has shown that chicken KLF9 was differentially expressed during adipogenesis of intramuscular preadipocytes differentiation. In this study, the function of KLF9 in chicken intramuscular preadipocytes differentiation was investigated.3. In the chicken preadipocyte differentiation model, KLF9 expression showed a major increase with adipogenic induction. Overexpression of KLF9 down-regulated the expression of the adipogenic marker gene AP2, and impaired triglyceride accumulation. Knockdown of KLF9 in chicken intramuscular preadipocytes increased the expression of PPARG, CEBPA and AP2. In addition, it was proposed that KLF9 may regulate adipogenesis via lncRNAs NONGGAT002209.2, NONGGAT003346.2, NONGGAT000436.2 and NONGGAT006302.2 in chicken.4. The data supported a novel role of KLF9 in regulating chicken intramuscular preadipocyte differentiation. Such findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

[Comparison of different massive parallel sequencing platforms for mutation profiling in formalin-fixed and paraffin-embedded samples].

Objective: To compare the performance of Miseq and Ion Torrent PGM platforms and library construction method for next-generation sequencing (NGS) technology for formalin-fixed and paraffin-embedded (FFPE) samples. Methods: A total of 204 FFPE cancer samples including 100 non-small cell lung cancers at the First Affiliated Hospital of Zhejiang University, and 104 colorectal cancers at West China Hospital of Sichuan University were retrospectively selected from January 2013 to December 2016. By using the same samples, DNA was extracted, and the same amount of DNA was used for library construction with the same kit, and sequenced on Miseq and Ion Torrent PGM respectively, after passing the quality control. Any discordant mutations between two platforms were validated by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR) method and Sanger sequencing. Results: A total of 204 FFPE samples were included and 197 samples were successfully analyzed by both platforms. The number of reads generated by the samples on Miseq platform sequencing was higher than PGM platform (median 391 634 vs. 298 030, P<0.01). Alignment with human reference genome showed that the mapping rate of Miseq platform was higher than PGM platform (median 100.0% vs. 99.7%, P<0.01). The median sequence depth of samples on Miseq was higher than PGM platform (median 853× vs. 698×, P<0.01). A total of 236 mutations were detected by two platforms, of which 221 were detected on both platforms, with a 93.6% concordance. Miseq platform detected 11 mutations not detected on PGM platform, while PGM platform detected 4 more mutations not detected on Miseq platform. With validation by ARMS-PCR and Sanger sequencing, Miseq platform was more reliable for low-frequency mutations. The main reasons for the discordant mutations between two platforms were that mutation frequency on undetected platform was lower than mutation reporting range (5%) and FFPE samples were stored for a long time. Conclusions: Compared with PGM, Miseq platform shows higher sequencing quality in terms of the number of reads, alignment results and coverage depth, and the test results are more reliable. In clinical practice, the appropriate platform should be chosen based on sample size and actual throughput requirements to aid in the molecular characterization of tumors.

[Feasibility of amplicon-based targeted next-generation sequencing of colorectal cancer in endoscopic biopsies].

Objective: To investigate whether small endoscopic biopsies of colorectal cancer were sufficient for quality and accurate mutational analysis by amplicon-based next-generation sequencing (NGS). Methods: By using an amplicon-based targeted NGS panel for mutational detection on Illumina Miseq platform, a total of 109 formalin-fixed and paraffin-embedded (FFPE) endoscopic biopsies of colorectal cancer were retrospectively selected, based on specific histopathologic criteria, from January 2012 to June 2016 at West China Hospital of Sichuan University and Peking University Third Hospital. Twelve of these biopsies had corresponding FFPE surgical resection specimens. Quality control parameters of NGS testing were analyzed and NGS results were confirmed by other methods. Mutation calls of the 12 paired endoscopic biopsies and surgical resections were compared. Results: Of the endoscopic biopsy specimens, 97.2% (106/109) had sufficient DNA and qualified sequencing library. NGS generated excellent sequencing data, with a median of 848× for median read depth and 95.7% for uniformity. The success rate of NGS was 95.4% (104/109). Conventional methods confirmed the results of NGS for KRAS and BRAF, and the concordance rate was 100.0%. The clinically actionable mutations detected in the 12 paired endoscopic biopsies and surgical resections were concordant. Conclusion: FFPE endoscopic biopsies of colorectal cancer is suitable for targeted NGS, providing quality sequencing data and accurate mutational information to guide targeted therapy.

Polymorphisms of Flanking Region of the ASB15 Gene and Their Associations with Performance Traits in Chicken.

Research on the identity of genes and their relationship with traits of economic importance in chickens could assist in the selection of poultry. In this study, an F2 resource population of Gushi chickens crossed with Anka broilers was used to detect single-nucleotide polymorphisms (SNPs) in the flanking region of the ASB15 gene by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). One SNP of -1271 C>T in 5' flanking region of the chicken ASB15 gene and two SNPs of the 10618 A>G and 10716 G>A in 3' flanking region were identified. Furthermore, the 10618 A>G and 10716 G>A in 3' flanking region were in complete linkage. Association analysis results showed that -1271 C>T was not associated with performance traits, while the 10618 A>G and 10716 G>A were significantly associated with BW2, 4, 6, 8, 10, 12, SL12, CD8, CW4, 8, 12, BSL4, 8, 12, and SEW, EW, WW, BMW, LW, CW, SFT. Our results suggest that the ASB15 gene profoundly affects chicken performance traits.

Assessment of correlation between pre-miRNA-1757 polymorphism and chicken performance traits.

Single nucleotide polymorphism in microRNAs (miRNA) may influence their target gene selection and regulation efficiency, leading to animal phenotypic variation. The aim of this study was to evaluate the possible effect of single nucleotide polymorphisms in the miRNA-1757 gene precursor region (pre-mir-1757) on economic-related traits in chicken. Genotyping was performed using Sequenom MassArray® iPLEX GOLD System. Association analysis was performed using SPSS19.0. The data showed that the G/C polymorphism was significantly correlated with semi-evisceration weight, evisceration weight, carcass weight, body weight at 10 weeks of age, shank length at 4 weeks of age, pectoral angle at 8 weeks of age, and body slanting length and pelvis breadth at 12 weeks of age (P < 0.05), and led to the alteration of the RNA secondary structure of pre-mir-1757. Our results provide useful information for further annotation studies of miRNA function.

Identification of single nucleotide polymorphisms in the ASB15 gene and their associations with chicken growth and carcass traits.

ASB15 is a member of the ankyrin repeat and suppressor of cytokine signaling box family, and is predominantly expressed in skeletal muscle. In the present study, an F2 resource population of Gushi chickens crossed with Anka broilers was used to investigate the genetic effects of the chicken ASB15 gene. Two single nucleotide polymorphisms (SNPs) (rs315759231 A>G and rs312619270 T>C) were identified in exon 7 of the ASB15 gene using forced chain reaction-restriction fragment length polymorphism and DNA sequencing. One was a missense SNP (rs315759231 A>G) and the other was a synonymous SNP (rs312619270 T>C). The rs315759231 A>G polymorphism was significantly associated with body weight at birth, 12-week body slanting length, semi-evisceration weight, evisceration weight, leg muscle weight, and carcass weight (P < 0.05). The rs312619270 T>C polymorphism was significantly associated with body weight at birth, 4, 8, and 12-week body weight, 8-week shank length, 12-week breast bone length, 8 and 12-week body slanting length, breast muscle weight, and carcass weight (P < 0.05). Our results suggest that the ASB15 gene profoundly affects chicken growth and carcass traits.

Influence of dietary nicotinic acid supplementation on lipid metabolism and related gene expression in two distinct broiler breeds of female chickens.

This study aimed to evaluate the influence of supplemental dietary nicotinic acid (NA) on lipid metabolism and hepatic expression of related genes in female chickens of two distinct broiler strains [Arbor Acres (AA) and Beijing-You (BJY)]. The treatments were arranged in a 2 × 4 factorial in a completely randomized design. Day-old females (n = 384) were allocated to four treatments with six cages per treatment and fed diets (basal contained approximately 25 mg NA/kg) supplemented with 0, 30, 60 and 120 mg NA/kg. A sample of 72 birds from each breed was slaughtered and sampled at their different market times (8 week for AA and 16 week for BJY). Arbor Acres broilers had thickness of subcutaneous fat plus the skin (SFS), and plasma concentration of low-density lipoprotein cholesterol (LDLC) and lower percentage of abdominal fat (PAF), plasma concentrations of TG, NEFA and adiponectin than the BJY line. The hepatic transcription of apolipoprotein A-I (ApoA-I), apolipoproteinB (ApoB), and adiponectin was significantly higher in AA broilers than in BJY broilers. In both breeds, BW, PAF, SFS, NEFA and TG were increased with increasing supplementation from 0 to 60 mg NA/kg, but then decreased slightly with 120 mg added NA/kg. With increasing supplementation, hepatic expression and plasma concentrations of adiponectin decreased from 0 to 60 mg added NA/kg and then increased with 120 mg added NA/kg. The expression of ApoA-I and ApoB mRNA showed linear response to dietary supplementation with NA. These findings indicate that: (i) supplementation of NA influenced the lipid metabolism and related gene expression; (ii) when supplemented with 120 mg NA/kg, some pharmacologic actions on lipid metabolism appeared; and (iii) changes in BW and fat deposition appeared to be associated with hepatic expression of adiponectin.

Deposition rule of yolk cholesterol in two different breeds of laying hens.

The objective of this study was to investigate the deposition rule of yolk cholesterol in Lushi Green-shelled and Silky Fowl layers. A total of 90 layers of each breed were selected at an age of 15 to 51 weeks. Productive performance was recorded on a weekly basis, whereas yolk cholesterol was determined at 4-week intervals from 21 to 51 weeks of age. The average yolk cholesterol content of Silky Fowl layers during the laying period was higher than that of Lushi Green-shelled layers (58.16 and 49.67%, P > 0.05). Yolk cholesterol content decreased at 21 to 31 weeks of the laying period, whereas a non-significant increasing trend was observed during 31 to 51 weeks of laying period. In conclusion, yolk cholesterol content is not only dependent on the age of hen but also the breed of layers.

Identification of the transcript isoforms and expression characteristics for chicken Lpin1.

Lpin1 was a gene with important effects on controlling lipid/energy metabolism in humans and mice. However, little was known about chicken Lpin1 gene. In the present study, two transcript isoforms of chicken Lpin1 were identified. Lpin1-α was predicted encoding one 902 amino acid protein, whereas Lpin1-δ was predicted encoding one 918 amino acid protein with an insertion of 48-bp fragment from intron 12 of chicken Lpin1-α, and a conservative element was found to be located in intron 12 of chicken Lpin1-α genomic sequence. Ten variants were identified from chicken Lpin1-α coding sequence, and two missense mutations were predicted to affect the protein function of Lpin1. Reverse transcription PCR (RT-PCR) analysis revealed that chicken total Lpin1, Lpin1-α and Lpin1-δ were expressed in all analyzed tissues, and presented clear tissue expression differences. Real-time quantitative RT-PCR revealed that 30% energy restriction significantly elevated the total Lpin1 mRNA expression level in hepatic (P < 0.01) and adipose (P < 0.01) tissues of birds. Chicken total Lpin1 gene mRNA expression level presented a significantly inverse correlation with some traits including abdominal fat rate (P < 0.01), serum high-density lipoprotein (P < 0.05) and total cholesterol (P < 0.05), which would make a foundation for the further study on chicken Lpin1 gene function.

Effects of diet-induced differences in growth rate on metabolic, histological, and meat-quality properties of 2 muscles in male chickens of 2 distinct broiler breeds.

This paper, the second from a comprehensive study, describes the effects of varying growth rate by feeding at different planes of nutrition with a constant ME:CP ratio on muscle characteristics and meat quality in 2 distinct broiler breeds of male chickens (Arbor Acres, a commercial line; and Beijing-You, a Chinese nonimproved line). Experimental diets, differing on average by 2% CP, were formulated with high-, medium-, or low-nutrient densities for 3 growing phases. Male hatchlings (216 of each breed) were randomly assigned to 6 pens of 12 birds in each treatment. Altered histological characteristics of muscle fibers, early postmortem muscle metabolism, and meat quality were investigated in the pectoralis major and biceps femoris. At their market age, Arbor Acres broilers had significantly higher concentrations of plasma protein and lipid metabolites, ratios of white to red and intermediate fibers, pH, L* and b* values, and lower concentrations of plasma glucose metabolites, muscle-fiber diameter, muscle contents of energy stores, a* value, drip loss, and shear force than the values found for the Beijing-You chickens (P < 0.01). Higher nutrient density increased the size of the muscle fibers, decreased glycogen reserve, and reduced the rate and extent of acidification in the Arbor Acres chickens, while accelerating transformation of red and intermediate-to-white fibers, enhancing energy stores, and hastening the decrease in pH postmortem in the Beijing-You chickens (P < 0.05). In each breed, most meat quality variables (e.g., shear force, drip loss, and color) were consistent with the histological and biochemical changes caused by the feeding strategy. Together, dietary nutrient density can influence meat quality as a result of altered histological and initial energy and metabolic characteristics of the muscle. Many of the responses to diet are breed and tissue dependent in broiler chickens.

Effect of dietary supplemental nicotinic acid on growth performance, carcass characteristics and meat quality in three genotypes of chicken.

The effects of dietary supplemental nicotinic acid (NA) on growth performance, carcass characteristics and meat quality were investigated in three genotypes of chicken. Fast-growing AA (Arbor Acres) broilers were compared with two genotypes of a slow-growing local breed, Beijing-You, that had undergone selection for and against intramuscular fat content respectively (BJY+IMF and BJY-IMF). The treatments were arranged 3×4 factorial completely randomized design. Day-old females (n=624) were allocated to four treatments with six replicates per treatment and fed diets (basal contained ~25 mg NA/kg) supplemented with 0, 30, 60 and 120 mg NA/kg. A sample of 72 birds from each genotype was slaughtered at market time (8 weeks of age for AA and 16 weeks of age for BJY). The breast muscles of AA broilers were darker, had less redness and yellowness, lower drip loss and higher shear force as compared to the BJY genotypes (p<0.01). The highest drip loss and the lowest shear force among the three genotypes were apparent in BJY+IMF (p<0.01). Increasing supplementation from 0 to 60 mg NA/kg tended to increase average daily gain (ADG), average daily feed intake, width of intermuscular fat band, thickness of subcutaneous fat (including skin) and percentage of abdominal fat but, for most variables, values decreased slightly with 120 mg NA/kg. Increasing supplementation to 60 mg NA/kg decreased (quadratic, p<0.001) drip loss, but it increased at 120 mg NA/kg. The present results indicate that (i) the AA broilers fed corn-soybean meal based-diets require approximately 60 mg NA/kg to maximize ADG and meat product yield and decrease the drip loss of breast muscle; (ii) the addition of 30 mg NA/kg meets the requirement of BJY genotypes; and (iii) there seems to be no beneficial effect of NA supplementation on chicken meat quality except for limiting the drip loss.

The effect of a mutation in the 3-UTR region of the HMGCR gene on cholesterol in Beijing-you chickens.

The 3-hydroxyl-3-methylglutaryl Coenzyme A reductase (HMGCR) gene was examined for polymorphisms in Beijing-you chickens. A "T" base insert was detected at nucleotide 2749 of the 3-UTR region of the HMGCR gene and was used as the basis for distinguishing a B allele, distinct from the A. Serum and muscle contents of total cholesterol. LDL-cholesterol in serum was significantly lower in AB birds and lowest in BB birds. Real-time PCR showed that the same trends across genotypes occurred in an abundance of HMGCR transcripts in liver, but there was no difference in contents of HMGCR mRNA in breast or thigh muscles. Hepatic expression and serum LDL-cholesterol were meaningfully correlated (partial, with total serum cholesterol held constant, r = 0.923). In muscle, similar genotypic differences were found for the abundance of the LDL receptor (LDLR) transcript. Cholesterol content in breast muscle related to LDLR expression (partial correlation with serum LDL-cholesterol held constant, r = 0.719); the equivalent partial correlation in thigh muscle was not significant. The results indicated that the B allele significantly reduces hepatic abundance of HMGCR transcripts, probably accounting for genotypic differences in serum cholesterol. In muscle, the cholesterol content appeared to reflect differences in LDLR expression with apparent mechanistic differences between breast and thigh.

Live performance, carcass composition, and blood metabolite responses to dietary nutrient density in two distinct broiler breeds of male chickens.

A study was conducted to evaluate the effects of varying nutrient density with constant ME:CP ratio on growing performance, carcass characteristics, and blood responses in 2 distinct broiler breeds of male chickens (Arbor Acres, a commercial line, and Beijing-You, a Chinese nonimproved line). Experimental diets were formulated with high, medium, or low nutrient densities for 3 growing phases. Starter diets (1 to 21 d) contained 23, 21, and 19% CP with 3,059, 2,793, and 2,527 kcal/kg of ME; grower diets (22 to 35 d) contained 21, 19, and 17% CP with 3,150, 2,850, and 2,550 kcal/kg of ME; and finisher diets (36 to 42 d for Arbor Acres and 36 to 91 d for Beijing-You) had 19, 17, and 15% CP with 3,230, 2,890, and 2,550 kcal/kg of ME. Male hatchlings (216 of each breed) were randomly assigned to 6 replicates of 12 birds in each treatment. Arbor Acres broilers had better (P < 0.001) BW gain, feed conversion ratio (FCR), and carcass yield, but had greater (P < 0.001) abdominal and carcass fat deposition. In both breeds, the higher nutrient density increased (P < 0.05) BW gain, protein efficiency ratio, and energy efficiency ratio while decreasing (P < 0.05) feed intake and FCR. The breed differences were increased for FCR, protein efficiency ratio, and energy efficiency ratio in the starter period and decreased for carcass chemical composition, respectively, by higher nutrient density. These findings indicate that 1) genetic improvement has a significant effect on broiler responses to dietary nutrient density, 2) performance differences between breeds are lessened with diets of low nutrient density, 3) carcass quality differences are less when birds were fed diets of high nutrient density, 4) carcass composition is hardly modified by nutrient density and both breeds exhibit similar metabolite responses to dietary concentrations, and 5) optimal diets are deduced for these breeds for the 3 growing phases.