Effects of dietary vitamin C, vitamin E, and alpha-lipoic acid supplementation on the antioxidant defense system and immune-related gene expression in broilers exposed to oxidative stress by dexamethasone.

Vitamin C, vitamin E, and alpha-lipoic acid (ALA) are potent nutritional antioxidants, which are important for enhancing immunity. This study compared the effects of supplementation with vitamin C, vitamin E, or ALA on the antioxidant defense system and the expression of immune-related genes under oxidative stress induced by dexamethasone (DEX) in broilers. In total, 240 one-day-old female Recessive White Rock chickens were assigned randomly to either a basal diet (control group) or basal diet supplemented with vitamin C (200 mg/kg diet), vitamin E (100 mg/kg), or ALA (500 mg/kg) for 28 d starting from hatching. At 21 d of age, birds fed the ALA-supplemented diet had the highest plasma total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) enzyme activities, and the lowest plasma malondialdehyde (MDA) activity, as well as the lowest mRNA gene expression levels of interferon gamma (IFN-γ) and lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF). At 23 d of age, the broilers in the 3 treatment groups were injected in the thigh muscle with DEX for 3 alternating days. In addition, the control group was divided into 2 equal groups, in which one was injected with saline and the other was injected with DEX. At 28 d of age, the DEX-ALA group (P < 0.05) had the highest activity levels for T-AOC, T-SOD, and GSH-PX in the plasma and liver (P < 0.05), and the greatest reduction in the MDA level. Dietary ALA significantly decreased the mRNA expression levels of the interleukin 1 ß (IL-1ß), IL-6, IFN-γ, and LITAF genes compared with the other groups during oxidative stress by DEX. In conclusion, this study suggests that in broilers, ALA is more effective for normalizing the oxidative stress induced by DEX than vitamin C or vitamin E.

In ovo injection of ascorbic acid modulates antioxidant defense system and immune gene expression in newly hatched local Chinese yellow broiler chicks.

The present study was conducted to investigate the effects of in ovo injection of ascorbic acid on antioxidant capacity and immune-related gene expression in the newly hatched local Chinese yellow broiler chicks. Fertile Chinese yellow broiler eggs (n = 90) were assigned to three equal groups. The first group was a non-injected control group. The second group was another control group where the eggs were injected with saline in the air sac after 18 days of incubation. The third group was injected with 3 mg/egg AA in the air sac after 18 days of incubation. In ovo injection of 3 mg/egg AA significantly (P < 0.0001) increased plasma glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and significantly reduced plasma malondialdehyde (MDA) at 1 d old. Moreover, in ovo injection of 3 mg/egg AA significantly increased mRNA expression of glutathione peroxidase (GSH-PX) and superoxide Dismutase (SOD) in the chick's spleen.Additionally, the mRNA level of interleukin 1 ß (IL-1ß), interleukin 6 (IL-6), and tumour necrosis factor α (TNF-α) in the spleen were significantly decreased (P < 0.0001), which indicates an improvement in chick's immunity. In conclusion, our data suggest that in ovo injection of AA at 3 mg/egg enhance antioxidant defense system and immune system for newly hatched chicks.

Polymorphisms in the 5'-flanking regions of the GH, PRL, and Pit-1 genes with Muscovy duck egg production.

Somatotropic axis-related genes contribute to reproduction of ducks. Five SNP in the 5'-flanking regions of the growth hormone (GH), prolactin (PRL), and pituitary-specific transcription factor (Pit-1) genes were identified and genotyped in a female population of Muscovy ducks. Association analysis of these SNP with Muscovy duck egg production traits was performed. Results showed that SNP C-515G of GH was significantly associated with egg number in ducks at age 59 wk (E59W; P = 0.0009) and egg number in ducks at age 300 d (E300D; P = 0.0022). Single nucleotide polymorphism C-441T of GH was significantly associated with E59W (P = 0.0014). Significant associations of SNP T-884C and T-335C of PRL with the age at first egg (A1D), E59W, and E300D were detected in this population (P < 0.0001). It was concluded that these 4 SNP might be useful markers to use with the aim of increasing Muscovy duck E59W. On the basis of genetic parameter estimation, the heritability of A1D, E300D, E59W, and molting time were 0.43 ± 0.04, 0.45 ± 0.04, 0.36 ± 0.04, and 0.04 ± 0.03, respectively. Strong positive genetic correlation was noted between E59W and E300D (correlation coefficient = 0.80), whereas a negative association was noted between E59W and A1D (correlation coefficient = -0.80). Therefore, the selection for improved A1D should also increase E59W.

Expression of variant transcripts of the potassium channel tetramerization domain-containing 15 (KCTD15) gene and their association with fatness traits in chickens.

The aim of this study was to characterize the structure, expression, and biological functions of potassium channel tetramerization domain containing 15 (KCTD15) in chickens. We compared the KCTD15 expression level in samples of hypothalamic, adipose, and liver tissue of Xinghua chickens that were maintained on different dietary status. An association analysis of KCTD15 gene variant transcripts with fatness traits in a F2 resource population of chickens was performed. Three KCTD15 transcripts were identified in which the complete transcript was predominantly expressed in adipose tissue and the hypothalamus. The chicken KCTD15 gene was regulated by both feeding and fasting and consumption of a high-fat diet. The expression level of KCTD15 gene was markedly decreased in hypothalamus and liver of fasted and refed chickens (P < 0.05) and significantly downregulated in adipose tissue by the high-fat diet (P < 0.05). Three single-nucleotide polymorphisms of the KCTD15 gene were significantly associated with a number of fatness traits in chicken (P < 0.05). These results suggest that KCTD15 have a potential role regulation of obesity and fat metabolism in chickens.

Identification and characterization of RFRP gene in pigs and its association with reproductive traits.

RFamide-related peptide (RFRP) gene is a crucial gene of the hypothalamo-pituitary-gonadal axis and plays an important role in reproduction regulation. This study aimed to clone and characterize the pig RFRP gene. We obtained a 645-bp cDNA of pig RFRP gene comprising a 546-bp open reading frame, which encoded a peptide of 188 amino acids. The pig RFRP coding sequences have the identities of 81, 68.8, and 76.1% with their counterparts in humans, mice, and rats, respectively. Real-time polymerase chain reaction showed that the pig RFRP gene was expressed predominantly in the stomach and testis of males, and large intestine and uterus of females. It was also found to be abundantly expressed in the cerebrum and liver tissues of both male and female pigs. The pig RFRP gene is 4.6 kb long and contains 3 exons and 2 introns. We further identified a total of 25 single nucleotide polymorphisms and 2 insertion/deletion (indel) polymorphisms across the whole RFRP gene, and 9 of them were in the exons. Marker trait association analysis in Yorkshire and Landrace populations showed that g.45859759C>T was significantly associated with the total number born of second parity (TBA2; P < 0.05). In conclusion, the pig RFRP gene was cloned and characterized in this study, and its polymorphism g.45859759C>T showed significant associations with reproductive traits.

Characterization of chicken natural resistance-associated macrophage protein encoding genes (Nramp1 and Nramp2) and association with salmonellosis resistance.

Natural resistance-associated macrophage protein 1 and 2 encoding genes (Nramp1 and Nramp2) are related to many diseases. We cloned the cDNA of chicken Nramp1 and Nramp2 genes, characterized their expression and polymorphisms, and investigated the association of some SNPs with resistance to salmonellosis. The Nramp1 cDNA was 1746 bp long and the Nramp2 cDNA was 1938 bp long. These cDNAs are similar to previously reported cDNAs, varying by two and one amino acids, respectively. The chicken Nramp1 gene expressed predominantly in liver, thymus and spleen in both females and males. The Nramp2 gene expressed in almost all tissues, but predominantly in breast muscle, leg muscle, cerebrum, cerebellum, lung, kidney, and heart in both females and males. We identified 45 SNPs and 2 indels in the chicken Nramp1 gene; three of 13 SNPs in the exons were missense mutations (Arg223Gln, Ala273Glu and Arg497Gln). Association analysis indicated that A24101991G is significantly associated with chicken salmonellosis resistance. These results will be useful for functional investigation of chicken Nramp1 and Nramp2 genes.

Molecular cloning, expression and variation analyses of the dopamine D2 receptor gene in pig breeds in China.

The dopamine D2 receptor (DRD2) is a crucial mediator for normal physiological processes. We cloned the pig DRD2 gene, investigated its distribution in tissues and identified polymorphisms by RT-PCR, quantitative real-time PCR and direct sequencing. Two Yorkshire pigs from Guangdong Academy of Agricultural Sciences (Guangzhou, China) were selected to clone the gene and investigate its expression; 16 individuals from four pig breeds (Yorkshire, Landrace, small-ear spotted, and Xinchang) were used to scan the variations. The two transcripts (DRD2L and DRD2S), obtained through insertion or deletion of exon 5 and part of 3'UTR, were found to encode 444- and 415-amino acid proteins, respectively. The 574-bp indel in 3'UTR comprises five miRNA targeting sites, based on bioinformatics predictions. The pig DRD2 gene expresses predominantly in the pituitary gland, and then in oviducts and the hypothalamus. Both DRD2L and DRD2S mRNA were detected in cerebrum, cerebellum, hypothalamus, pituitary gland, back muscle, oviduct, uterus, and testis tissues; DRD2L was more abundant than DRD2S. The DRD2 gene is located on chromosome 9 and contains seven exons. Sixty-one different sequences were identified in this gene; among seven in the coding region, only one altered the encoded amino acid. These findings will help us understand the functions of the DRD2 gene in pigs.

Polymorphisms associated with egg number at 300 days of age in chickens.

We looked for variations that could be associated with chicken egg number at 300 days of age (EN300) in seven genes of the hypothalamic-pituitary-gonadal axis, including gonadotrophin-releasing hormone-I (GnRH-I), GnRH receptor (GnRHR), neuropeptide Y (NPY), dopamine D2 receptor (DRD2), vasoactive intestinal polypeptide (VIP), VIP receptor-1 (VIPR-1), prolactin (PRL), and the QTL region between 87 and 105 cM of the Z chromosome. Ten mutations in the seven genes were chosen to do marker-trait association analyses in a population comprising 1310 chickens, which were obtained from a company located in Guangdong Province of China. The C1704887T of VIPR-1 was found to have a highly significant association with EN300. The T5841629C of DRD2 and the C1715301T of VIPR-1 were significantly associated with EN300. A highly significant association was also found between the C1704887T-C1715301T haplotypes of VIPR-1 and EN300. H1H3 had the highest EN300. Four PCR-RFLP variations in the candidate QTL region were selected to investigate their genetic effects on EN300. The haplotypes of T32742468C-G32742603A in this region showed a highly significant association with EN300. Bioinformatics analyses showed that both T32742468C and G32742603A were located in intron 1 of the SH3-domain GRB2-like 2 (SH3GL2) gene. We conclude that five SNPs, including C1704887T and C1715301T of VIPR-1, T5841629C of DRD2, and T32742468C and G32742603A of SH3GL2, would be useful as markers for breeding to increase chicken EN300.

Associations of ATGL gene polymorphisms with chicken growth and fat traits.

Adipose triglyceride lipase (ATGL) catalyses the initial step in triglyceride hydrolysis, so the ATGL gene is a candidate for growth and fat traits in chickens. Nine reported single-nucleotide polymorphisms (SNPs) located in 3 exons of the chicken ATGL gene were chosen for genotyping an F2 population. Only 5 SNPs were confirmed for polymorphisms and used for association analyses. The results show that c.531G>A (p.E177Syn) was not associated with any growth and fat traits (P > 0.05), but c.782G>A (p.S261N) was associated with body weight (BW) on days 14, 21, 35, 63, 70, 77, cingulated fat width and abdominal fat pad weight (P < 0.05), and significantly associated with BW on days 42, 49, and 56 (P < 0.01). Significant associations of c.903C>T (p.F301Syn) with BW on days 49 and 77 days and crude protein content of breast muscle (P < 0.05), and c.1164G>A (p.K388Syn) with BW on day 7 (P < 0.05) were also detected. Additionally, c.1069T>C (p.L357Syn) was associated with breast muscle colour (P < 0.05), and significantly associated with crude fat (ether extract) content of breast muscle (P < 0.01). Thus the missense SNP of c.782G>A (p.S261N) was significantly associated with the largest number of chicken growth and fat traits in this study.

The dopamine D2 receptor gene polymorphisms associated with chicken broodiness.

Chicken broodiness is a polygenic trait controlled by autosomal genes. Prolactin gene is a candidate of great interest in molecular studies of broodiness. However, another candidate dopamine D2 receptor (DRD2) gene has not been studied extensively. The objective of this study was to analyze the genetic effects of the DRD2 gene on chicken broodiness through linkage disequilibrium analyses, tag SNP selection, genetic diversity observation, 2-tailed test, and association analyses. In this study, we assayed 27 variations of this gene in 456 individuals from 6 chicken populations to observe linkage disequilibrium pattern, the tag SNP, and genetic diversity. Among the 6 populations, Taihe Silkies exhibited no characteristic between the square of the correlation coefficient of gene frequencies (r(2)) and physical distance. The other populations including Red Jungle Fowls, Xinghua chickens, Ningdu Sanhuang chickens (NDH), Baier Huang chickens, and Leghorn layers exhibited conspicuous characteristic of decreasing r(2) value over physical distance. Linkage disequilibrium decayed more rapidly in Red Jungle Fowls, Xinghua, and NDH than in Baier Huang and Leghorn layers. Allelic frequencies and genotype distributions in the 5 populations showed that A-38600G, I-38463D, T-32751C, A-16105G, A-6543G, C-6539T, and A+2794G were possibly associated with broodiness. Besides the above 7 sites, another 2 sites that might be associated with broodiness were screened by 2-tailed test. All 9 sites were used for association analyses with broodiness in 644 NDH chickens. A significant association (P < 0.05) was found between A-16105G and broody frequency (%), and the T+619C in intron 1 was significantly associated with duration of broodiness (P < 0.05). These findings suggested that the DRD2 gene should be included in future genetic studies of chicken broodiness and 2 SNP of A-16105G and T+619C might be markers for breeding against broodiness.

Association of single nucleotide polymorphisms of the insulin gene with chicken early growth and fat deposition.

Growth rate, body composition, and fat deposition are important traits in chickens. Insulin plays important roles in hepatic cells, muscle cells, and adipose tissue cells. The purpose of the present study was to analyze association of the insulin (INS) gene with chicken growth and body composition traits. Using a F2 design resource population constructed with the crossing of Chinese native Xinghua chickens and White Recessive Rock chickens, the association of 4 single nucleotide polymorphisms (SNP; A+428G, C+1549T, T+3737C, and A+3971G) of INS gene with 13 growth and body composition traits was studied. The T+3737C genotypes were significantly associated with small intestine length (P = 0.0002), and the A+3971G genotypes were significantly associated with early growth (hatch weight and BW at 28 d of age) (P < 0.0001), breast angle (P = 0.0002), and small intestine length (P < 0.0001). None of the 4 SNP was significantly associated with abdominal fat pad weight (P > 0.05). The haplotypes based on the 4 SNP were also significantly associated with early growth (hatch weight and BW at 28 d of age; P < 0.0001) and breast angle (P < 0.0001) but not with small intestine length (P = 0.0505). These results suggested that variation of the insulin gene was significantly associated with chicken early growth but not with fat deposition. In addition, the data from the present study supported the inference that both the one-SNP-at-a-time and the haplotype-based approaches have their own advantages and disadvantages when association analysis of one SNP and haplotypes with chicken complex traits was conducted.

Single nucleotide polymorphisms of the chicken insulin-like factor binding protein 2 gene associated with chicken growth and carcass traits.

Five single nucleotide polymorphisms (SNP) of the chicken insulin-like factor binding protein 2 (IGFBP2) gene were selected to genotype a F2 designed population with restriction fragment length polymorphisms and single stranded-conformation polymorphisms. The associations of the SNP and their haplotypes with chicken growth and carcass traits were analyzed. Results showed that the difference induced by the haplotypes derived from the 5 SNP was more significant than that by the single SNP in the genotype-phenotype association analysis. The haplotypes were associated with BW at hatch and at 21, 28, 42, 49, 56, and 90 d of age, as well as eviscerated weight with giblets (EWG), eviscerated weight (EW), and weights of heart, liver, and gizzard (HLGW) (P < or = 0.01). The haplotypes were also related to BW at 7, 14, and 35 d of age, breast depth, carcass weight, and breast muscle weight (P < or = 0.05). Significant and suggestive dominant effects of H1H5 diplotype were detected for BW at 7, 14, 21, 28, and 90 d of age, as well as breast depth, carcass weight, eviscerated weight with giblets, eviscerated weight, breast muscle weight, leg muscle weight, and weights of heart, liver, and gizzard. It was concluded that H1H5 was the most advantageous diplotype, and H4H10 was the negative diplotype for growth and carcass traits.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis.

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P> 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.