Yumao Li

Effects of dietary supplementation of quercetin on performance, egg quality, cecal microflora populations, and antioxidant status in laying hens

Plant polyphenols, especially flavonoids, are of great interest due to their wide range of biological activities. Quercetin, a ubiquitous flavonoid, is known to have antioxidant and antibacterial effects. In this study, we investigated the effect of quercetin on performance, egg quality, cecal microflora populations, and antioxidant status in laying hens. Two hundred forty 28-wk-old Hessian laying hens, with an average laying rate of lay 85% at the start, were randomly allotted to 4 treatments and fed 1 of 4 diets (negative control, 0.2, 0.4, and 0.6 g of quercetin/kg of diet) for 8 wk. Layer performance responses, egg quality parameters, cecal microflora populations and antioxidant status were measured at the end of the experiment. Results showed that feed conversion decreased as the quercetin level increased. Laying rate had a quadratic correlation with the level of quercetin (P = 0.056) and was maximized by the supplementation level of 0.2 g/kg of diet. However, no significant quercetin effect was observed on egg quality. Regression analysis showed that the population of total aerobes and coliforms decreased and the population of Bifidobacteria increased as the level of quercetin increased. Regression analysis also showed the activities of Cu-Zn-superoxide dismutase increased as the level of quercetin increased (P < 0.05). Results of the study suggest that the appropriate level of supplementation is 0.367 to 0.369 g of quercetin/kg of feed based on the improvement of laying rate (with 88.55 as maximum value) and feed conversion (with 2.0725 as minimum value). Our observations provided further evidence that dietary supplementation of quercetin improved performance by modulation of intestinal environment and liver superoxide dismutase content in laying hens. Quercetin has the potential as functional feed additive in animal production.

Transdifferentiation of fibroblasts into adipocyte-like cells by chicken adipogenic transcription factors

Adipocyte differentiation is a complex process that is regulated mainly by a cascade of transcription factors. Among these, CCAAT/enhancer binding protein (C/EBP)alpha, peroxisome proliferator-activated receptor (PPAR)gamma and sterol regulatory element-binding protein-1 (SREBP-1) have been identified as key regulators of adipocyte differentiation. In mammals, ectopic expression of adipogenic transcription factors C/EBPalpha, PPARgamma and SREBP-1 can induce transdifferentiation of a variety of different cell types into adipocyte-like cells. However, in birds, whether C/EBPalpha, PPARgamma and SREBP-1 can induce transdifferentiation is unknown. The current study was designed to investigate whether chicken embryo fibroblasts (CEFs) can be induced to transdifferentiate into adipocyte-like cells by the ectopic expression of chicken C/EBPalpha, PPARgamma and SREBP-1 via retrovirus-mediated gene transfer. The results showed that any one of these three adipogenic transcription factors was sufficient to trigger the adipogenic program in CEFs, as demonstrated by accumulation of cytoplasmic lipid droplets and expression of the adipocyte marker gene (adipocyte fatty acid binding protein, A-FABP). This suggests that C/EBPalpha, PPARgamma and SREBP-1 play a crucial role in chicken adipogenesis.

Expression profiling of preadipocyte microRNAs by deep sequencing on chicken lines divergently selected for abdominal fatness.

Through posttranscriptional gene regulation, microRNA (miRNA) is linked to a wide variety of biological processes, including adipogenesis and lipid metabolism. Although miRNAs in mammalian adipogenesis have been worked on extensively, their study in chicken adipogenesis is still very limited. To find miRNAs potentially important for chicken preadipocyte development, we compared the preadipocyte miRNA expression profiles in two broiler lines divergently selected for abdominal fat content, by sequencing two small RNA libraries constructed for primary preadipocytes isolated from abdominal adipose tissues. After bioinformatics analyses, from chicken miRNAs deposited in miRBase 20.0, we identified 225 miRNAs to be expressed in preadipocytes, 185 in the lean line and 200 in the fat line (derived from 208 and 203 miRNA precursors, respectively), which corresponds to 114 miRNA families. The let-7 family miRNAs were the most abundant. Furthermore, we validated the sequencing results of 15 known miRNAs by qRT-PCR, and confirmed that the expression levels of most miRNAs correlated well with those of Solexa sequencing. A total of 33 miRNAs was significantly differentially expressed between the two chicken lines (P<0.05). Gene ontology analysis revealed that they could target genes enriched in the regulation of gene transcription and chromatin function, response to insulin stimulation, and IGF-1 signaling pathways, which could have important roles in preadipocyte development. Therefore, a valuable information and resource of miRNAs on chicken adipogenesis were provided in this study. Future functional investigations on these miRNAs could help explore related genes and molecular networks fundamental to preadipocyte development.

Comparison of serum biochemical parameters between two broiler chicken lines divergently selected for abdominal fat content

In humans, obesity is associated with increased or decreased levels of serum biochemical indicators. However, the relationship is not as well understood in chickens. Due to long-term intense selection for fast growth rate, modern broilers have the problem of excessive fat deposition, exhibiting biochemical or metabolic changes. In the current study, the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) were used to identify differences in serum biochemical parameters between the 2 lines. A total of 18 serum biochemical indicators were investigated in the 16th, 17th, and 18th generation populations of NEAUHLF, and the genetic parameters of these serum biochemical indicators were estimated. After analyzing the data from these 3 generations together, the results showed that the levels of 16 of the tested serum biochemical parameters were significantly different between the lean and fat birds. In the fat birds, serum concentrations of high-density lipoprotein cholesterol (HDL-C), HDL-C:low-density lipoprotein cholesterol (LDL-C), total bile acid, total protein, albumin, globulin, aspartate transaminase (AST):alanine transaminase (ALT), γ-glutamyl transpeptidase (GGT), uric acid, and creatinine were very significantly higher (P < 0.01), whereas LDL-C, albumin:globulin, glucose, AST, ALT, and free fatty acids concentrations in serum were very significantly lower than those in the lean birds (P < 0.01). Of these 16 serum biochemical parameters, 5 (LDL-C, HDL-C:LDL-C, total bile acid, albumin, and albumin:globulin) had high heritabilities (0.58 ≤ h2 ≤ 0.89), 6 (HDL-C, total protein, globulin, AST:ALT, GGT, and creatinine) had moderate heritabilities (0.29 ≤ h2 ≤ 0.48), and the remaining 5 had low heritabilities (h2 < 0.20). Serum HDL-C, HDL-C:LDL-C, and glucose had higher positive genetic correlation coefficients (rg) with abdominal fat traits (0.30 ≤ rg ≤ 0.80), whereas serum globulin, AST, and uric acid showed higher negative genetic correlations with abdominal fat traits (–0.62 ≤ rg ≤ –0.30). The remaining 10 serum biochemical parameters had lower genetic correlations with abdominal fat traits (–0.30 < rg < 0.30). In conclusion, we identified serum HDL-C and HDL-C:LDL-C levels as potential biomarkers for selection of lean birds. These findings will also be useful in future studies for investigating obesity and lipid metabolism in humans as well as in other animal species.

Genetic epistasis analysis of 10 peroxisome proliferator-activated receptor γ-correlated genes in broiler lines divergently selected for abdominal fat content

Chicken peroxisome proliferator-activated receptor γ (PPARγ), which is highly expressed in adipose tissues, is a key factor in fat accumulation in the abdominal fat pad. In this study, association and pairwise epistasis analyses were performed for all the polymorphisms detected in PPARγ and for 9 genes from PPARγ-correlated lipid metabolic pathways for abdominal fat weight (AFW) in 10th-generation populations of Northeast Agricultural University broiler lines divergently selected for abdominal fat content. Epistatic networks were then reconstructed with the identified epistatic effects. Single-marker association analyses showed that 5 of the 20 screened polymorphisms were significantly associated with AFW (P < 0.05), and CCAAT/enhancer-binding protein α (C/EBPα) c.552G>A was 1 of the 5 significant loci. Pairwise interaction analyses showed that 15 pairs of polymorphisms reached a significance level of P < 2.64 × 10(-4) (adjusted by Bonferroni correction) in the lean line, 41 pairs reached significance in the fat line, and 7 pairs reached significance in both lines. Interestingly, many other loci interacted with C/EBPα c.552G>A in both lines. In epistatic network analyses, C/EBPα c.552G>A seemed to behave as a hub for the epistatic network in both lines. All these results revealed that the genetic architecture of C/EBPα c.552G>A for AFW seemed to be an apparent individual main-effect QTL but that it could be dissected into a genetic epistatic network. Our results suggest that C/EBPα c.552G>A might be the most important locus contributing to phenotypic variation in AFW among all the polymorphisms detected in this study.

Differential expression of six genes and correlation with fatness traits in a unique broiler population

Previous results from genome wide association studies (GWASs) in chickens divergently selected for abdominal fat content of Northeast Agricultural University (NEAUHLF) showed that many single nucleotide polymorphism (SNP) variants were associated with abdominal fat content. Of them, six top significant SNPs at the genome level were located within SRD5A3, SGCZ, DLC1, GBE1, GALNT9 and DNAJB6 genes. Here, expression levels of these six candidate genes were investigated in abdominal fat and liver tissue between fat and lean broilers from the 14th generation population of NEAUHLF. The results showed that expression levels of SRD5A3, SGCZ and DNAJB6 in the abdominal fat and SRD5A3, DLC1, GALNT9, DNAJB6 and GBE1 in the liver tissue differed significantly between the fat and lean birds, and were correlated with abdominal fat traits. The findings will provide important references for further function investigation of the six candidate genes involved in abdominal fat deposition in chickens.

Differential expression of six chicken genes associated with fatness traits in a divergently selected broiler population.

A genome-wide association study has shown a number of chicken (Gallus gallus) single nucleotide polymorphism (SNP) markers to be significantly associated with abdominal fat content in Northeast Agricultural University (NEAU) broiler lines selected divergently for abdominal fat content (NEAUHLF). The six significant SNPs are located in the kinase insert domain receptor (KDR), tumor suppressor candidate 3 (TUSC3), phosphoribosyl pyrophosphate amidotransferase (PPAT), exocyst complex component 1 (EXOC1), v-myb myeloblastosis viral oncogene homolog (avian)-like 2 (MYBL2) and KIAA1211 (undefined) genes. In this study, the expression levels of these genes were investigated in both abdominal fat and liver tissues using 32 14th generation chickens from the NEAUHLF. The levels of expression of KDR in abdominal fat and KDR and TUSC3 in liver differed significantly between the two lines. The expression level of KDR in the abdominal fat was significantly correlated with the abdominal fat weight (AFW) and abdominal fat percentage (AFP). The expression levels of KDR, TUSC3 and PPAT in liver were significantly correlated with AFW and AFP, indicating that the six genes, especially KDR and TUSC3, could be associated with fat traits in domestic chickens. This study could provide insight into the mechanisms underlying the formation of abdominal fat in chickens.

Production of transgenic broilers by non-viral vectors via optimizing egg windowing and screening transgenic roosters

&NA; The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)‐transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid‐liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP‐positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence‐activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene‐positive roosters can improve the production of germline‐transmitted transgenic birds.

Genetic parameters for the prediction of abdominal fat traits using blood biochemical indicators in broilers

ABSTRACT 1. Excessive deposition of body fat, especially abdominal fat, is detrimental in chickens and the prevention of excessive fat accumulation is an important problem. The aim of this study was to identify blood biochemical indicators that could be used as criteria to select lean Yellow-feathered chicken lines. 2. Levels of blood biochemical indicators in the fed and fasted states and the abdominal fat traits were measured in 332 Guangxi Yellow chickens. In the fed state, the genetic correlations (rg) of triglycerides and very low density lipoprotein levels were positive for the abdominal fat traits (0.47 ≤ rg ≤ 0.67), whereas total cholesterol, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) showed higher negative correlations with abdominal fat traits (–0.59 ≤ rg ≤ −0.33). Heritabilities of these blood biochemical parameters were high, varying from 0.26 to 0.60. 3. In the fasted state, HDL-C:LDL-C level was positively correlated with abdominal fat traits (0.35 ≤ rg ≤ 0.38), but triglycerides, total cholesterol, HDL-C, LDL-C, total protein, albumin, aspartate transaminase, uric acid and creatinine levels were negatively correlated with abdominal fat traits (–0.79 ≤ rg ≤ −0.35). The heritabilities of these 10 blood biochemical parameters were high (0.22 ≤ h2 ≤ 0.59). 4. In the fed state, optimal multiple regression models were constructed to predict abdominal fat traits by using triglycerides and LDL-C. In the fasted state, triglycerides, total cholesterol, HDL-C, LDL-C, total protein, albumin and uric acid could be used to predict abdominal fat content. 5. It was concluded that these models in both nutritional states could be used to predict abdominal fat content in Guangxi Yellow broiler chickens.

Structural Characterization and Association of Ovine Dickkopf-1 Gene with Wool Production and Quality Traits in Chinese Merino

Dickkopf-1 (DKK1) is an inhibitor of canonical Wnt signaling pathway and regulates hair follicle morphogenesis and cycling. To investigate the potential involvement of DKK1 in wool production and quality traits, we characterized the genomic structure of ovine DKK1, performed polymorphism detection and association analysis of ovine DKK1 with wool production and quality traits in Chinese Merino. Our results showed that ovine DKK1 consists of four exons and three introns, which encodes a protein of 262 amino acids. The coding sequence of ovine DKK1 and its deduced amino acid sequence were highly conserved in mammals. Eleven single nucleotide polymorphisms (SNPs) were identified within the ovine DKK1 genomic region. Gene-wide association analysis showed that SNP5 was significantly associated with mean fiber diameter (MFD) in the B (selected for long wool fiber and high-quality wool), PW (selected for high reproductive capacity, high clean wool yield and high-quality wool) and U (selected for long wool fiber with good uniformity, high wool yield and lower fiber diameter) strains (p < 4.55 × 10−3 = 0.05/11). Single Nucleotide Polymorphisms wide association analysis showed that SNP8 was significantly associated with MFD in A strain and fleece weight in A (selected for large body size), PM (selected for large body size, high reproductive capacity and high meat yield) and SF (selected for mean fiber diameter less than 18 μm and wool fiber length between 5 and 9 cm) strains (p < 0.05), SNP9 was significantly associated with curvature in B and U strains (p < 0.05) and SNP10 was significantly associated with coefficient of variation of fiber diameter in A, PW and PM strains and standard deviation of fiber diameter in A and PM strains (p < 0.05). The haplotypes derived from these 11 identified SNPs were significantly associated with MFD (p < 0.05). In conclusion, our results suggest that DKK1 may be a major gene controlling wool production and quality traits, also the identified SNPs (SNPs5, 8, 9 and 10) might be used as potential molecular markers for improving sheep wool production and quality in sheep breeding.