Responses in ileal and cecal bacteria to low and high amylose/amylopectin ratio diets in growing pigs.

Dietary starch that escapes digestion in the small intestine may serve as a carbon source for bacterial fermentation in the distal intestine. This study aimed to compare the bacterial community in the ileal and cecal digesta of growing pigs fed diets with low (0.14, LR pigs) and high (0.43, HR pigs) amylose/amylopectin ratio. Pyrosequencing based on MiSeq 2000 platform showed that in ileum digesta, Bacteroidetes of LR pigs was markedly higher than that in HR pigs (P < 0.05). Megasphaera and Prevotella were the two most predominant genera in LR pigs, and Prevotella was significantly higher in LR pigs than in HR pigs (P < 0.05). Prevotella was predominant in cecal samples from both LR and HR pigs, although no significant differences were found between the two groups. In the ileum, Megasphaera elsdenii and Mitsuokella multacida were significantly (P < 0.01) higher in LR pigs along with an increase of acetate and butyrate concentrations. Halomonas pacifica, Escherichia fergusonii, and Actinobacillus minor which belong to class Gammaproteobacteria were significantly lower (P < 0.01) in HR pigs with a significant increase (P < 0.01) of Lactobacillus acetotolerans-like bacteria. Therefore, the changed bacterial community may lead to a transformation of microbial function, such as the alteration of fermentation mode which is showed on the change of microbial metabolites like the concentration of short-chain fatty acids (SCFAs), to a response to the switch of dietary composition, and in turn, to help host absorb and utilize nutrients efficiently. The increase of dietary amylose induced the reduction of conditioned pathogens which may probably be due to the increase of some probiotics such as Lactobacillus, thus reducing the risk of intestinal disease.

Effects of different starch sources on Bacillus spp. in intestinal tract and expression of intestinal development related genes of weanling piglets.

The study was conducted to evaluate the effects of different starch sources on Bacillus spp. in intestinal tract and expression of intestinal development related genes of weanling piglets. Twenty-eight PIC male piglets were divided into four homogeneous groups according to initial body weight (similar birth and parity, weaned at 21 ± 1.5 days). Diets for the four treatments consisted of corn starch, wheat starch, tapioca starch and pea starch with the determined ratio for amylose to amylopectin of 0.21, 0.24, 0.12 and 0.52 respectively. Real-time quantitative polymerase chain reaction was applied to: (1) detect genomic DNA of Bacillus and to quantify the number of Bacillus in the intestinal tract chyme of piglets with the primers and probe which designed based on the 16S rRNA sequences of maximum species of Bacillus on GenBank; (2) measure the mRNA level of glucagon-like peptide 2 (GLP-2), insulin-like growth factors 1 (IGF-1) and epidermal growth factor (EGF) in duodenum, jejunum and ileum. Results showed that the number of Baciilus and the percentage based on all bacteria in the whole intestinal content of piglets fed pea starch was highest in all groups (P < 0.05). There was no significant differance on copy numbers for all bacteria and Bacillus in the whole intestinal tract of piglets between the corn starch group and wheat starch group (P > 0.05). In addition, the expression level of GLP-2, IGF-1 mRNA in jejunum and ileum of pea starch treatment (the high amylose/amylopectin ratio) were increased while the tapioca starch decreased their mRNA level significantly compared to other three treatments (P < 0.05). There was no significant difference for the mRNA level of EGF in each group. The present study revealed that high amylose/amylopectin ratio of starches significantly enhanced the numbers of Bacillus in all segments of intestine and the mRNA level of intestinal development related genes.

Effect of maternal folic acid supplementation on hepatic one-carbon unit associated gene expressions in newborn piglets.

Intrauterine growth retardation (IUGR) induces alterations to hepatic gene expressions which might program poor postnatal growth and health status. Maternal folic acid supplementation was administered in gilt diets to test whether hepatic mRNA expressions of some important genes induced by IUGR could be rescued by folic acid supplementation. Thirty-two Yorkshire gilts were allotted to two treatment groups of control (C folic acid 1.3 mg/kg) or folic acid supplementation (FS folic acid 30 mg/kg) after mating, to study the effects of maternal folic acid supplementation on the mRNA expression of methionine adenosyltransferase (MAT), cystathionine-ß-synthase (CBS), methylenetetrahydrofolate reductase (MTHFR), DNA methyltransferase1 (DNMT1), peroxisomal proliferator-activated receptor (PPARγ), glucocorticoid receptor (GR), obesity receptor (ob-R) and Acyl-CoA oxidase (AOX) in the liver of IUGR and NBW piglets. Blood and liver samples were collected for determinations of serum folic acid and gene expressions. The total number of born piglets, number of piglets born alive, average birth weight and 21 days average weight were not affected by dietary treatment (P>0.05), and serum folic acid concentration of piglets was greater in FS than C groups (P<0.05). Real-time PCR indicated that gene expression of MAT1A, MAT2A and DNMT1 were lower in IUGR piglets but could be elevated by maternal folic acid supplementation. Transcript expression levels of PPARγ, GR and AOX were higher in IUGR piglets, but were decreased to the level of normal piglets by maternal folic acid supplementation. Our results suggested that maternal folic acid supplementation be an effective way to rescue the gene expressions negatively induced by IUGR.

Design, expression and characterization of recombinant hybrid peptide Attacin-Thanatin in Escherichia coli.

Antimicrobial peptides will be attractive and potential candidates as peptide drugs because of their efficient action against microbes and low toxicity to mammal cells. To improve their antibacterial activity, some modifications needs to be made. In this research, the hybrid peptide gene Attacin-Thanatin with 642 bp in length with preferred codons of E. coli was generated using the technology of Gene splicing by overlap extension. The gene was inserted in-frame into E. coli expression plasmid pET-32a (+) and induced to express in E. coli Rosetta. The recombinant protein was partial purified and its biological activity was determined. Analysis of the E. coli Rosetta induced with IPTG revealed that the molecular weight of fusion protein was approximately 41.8 kDa, which perfectly matched the mass calculated from the amino acid sequence. Biological activity detection showed that this peptide effectively inhibited the growth of the test bacteria including E. coli DH5α, E. coli BL21 (DE3), Salmonella choleraesuis and Staphylococcus aureus. Among these bacteria, the Gram-negative E. coli was the most sensitive. Furthermore, there was minor hemolysis activity for porcine red blood cells. So, the results indicated that the hybrid peptide Attacin-Thanatin could be served as a promising candidate for the chemical antibiotics.

Molecular cloning, expression in Escherichia coli of Attacin A gene from Drosophila and detection of biological activity.

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS-PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS-PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G- bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.

Upregulation of amino acid transporter expression induced by L-leucine availability in L6 myotubes is associated with ATF4 signaling through mTORC1-dependent mechanism.

OBJECTIVE: Essential amino acids, especially l-leucine, initiate the signaling of the mammalian target of rapamycin complex-1 (mTORC1) and protein synthesis in skeletal muscle. Current information on the relation between amino acid transporter mechanisms and mTORC1 signaling is sparse. The objectives of this study were to determine whether an increase in leucine availability upregulates the gene transcription and translation of amino acid transporters and other amino acid members in an mTORC1-dependent pathway that control amino acid use (general control non-repressed-2 and activating transcription factor-4) and to measure the factors related to protein synthesis and proteolysis. METHODS: L6 skeletal muscle cells that had been treated with l-leucine (0.105 g/L) were incubated for 30 min to stimulate the transcription of L-type amino acid transporter-1, CD98, and sodium-coupled neutral amino acid transporter-2 and increase activating transcription factor-4 protein, which is dependent on the mTORC1 signaling pathway. RESULTS: A rapid, high level of p70 S6 kinase-1 phosphorylation was detected but was suppressed by rapamycin (P < 0.05). The addition of leucine decreased the atrogin-1 transcription abundance in an insulin-involved manner (P < 0.05), which could not be completely blocked by rapamycin (P = 0.055). CONCLUSIONS: Our findings indicate that the mTOR is a component of the nutrient signaling pathway, which regulates system A and L amino acid transporters, the initiation factors involved in mRNA translation, and is downstream of forkhead box-O in L6 myotubes.

Effects of active immunization against myostatin on carcass quality and expression of the myostatin gene in pigs.

The study was conducted to investigate the effects of active immunization against myostatin on the titer of myostatin antibody, carcass evaluation, activity of creatine kinase and the expression of the myostatin gene in pigs. Eighteen pigs were allotted into three groups (six pigs per group), and pigs in treatment 1, 2 and 3 were immunized with physiological saline, 1 mg or 4 mg myostatin per pig, respectively. Six pigs were killed by electrical stunning followed by exsanguination at BW of 100 kg. The results indicated that the titer of myostatin antibody was increased in treated groups compared to the control group on day 42 (P < 0.01) and d 84 (P < 0.01). The carcass lean percentage was significantly increased in the treatment groups compared to the control group (P < 0.01), and intramuscular fat was significantly decreased in the 4 mg group compared to the control group (P < 0.05). The muscle creatine kinase activity of pigs treated with 1 mg and 4 mg myostatin was lower than the control group. The immunization of myostatin significantly decreased the myostatin gene expression levels in muscle. It was concluded that optimal active immunization against myostatin could increase the content of myostatin antibody, suppress the activity of creatine kinase and the expression of myostatin gene, and therefore improve the carcass lean percentage for pigs.