Dietary proline supplementation alters colonic luminal microbiota and bacterial metabolite composition between days 45 and 70 of pregnancy in Huanjiang mini-pigs

Background: Pregnancy is associated with important changes in gut microbiota composition. Dietary factors may affect the diversity, composition, and metabolic activity of the intestinal microbiota. Among amino acids, proline is known to play important roles in protein metabolism and structure, cell differentiation, conceptus growth and development, and gut microbiota re-equilibration in case of dysbiosis.Results: Dietary supplementation with 1% proline decreased(P < 0.05) the amounts of Klebsiella pneumoniae,Peptostreptococcus productus, Pseudomonas, and Veillonella spp. in distal colonic contents than that in the control group. The colonic contents of Butyrivibrio fibrisolvens, Bifidobacterium sp., Clostridium coccoides, Clostridium coccoides-Eubacterium rectale, Clostridium leptum subgroup, Escherichia coli, Faecalibacterium prausnitzii,Fusobacterium prausnitzii, and Prevotella increased(P < 0.05) on d 70 of pregnancy as compared with those on d 45 of pregnancy. The colonic concentrations of acetate, total straight-chain fatty acid, and total short-chain fatty acids(SCFA) in the proline-supplemented group were lower(P < 0.05), and butyrate level(P = 0.06) decreased as compared with the control group. Almost all of the SCFA displayed higher(P < 0.05) concentrations in proximal colonic contents on d 70 of pregnancy than those on d 45 of pregnancy. The concentrations of 1,7-heptyl diamine(P = 0.09) and phenylethylamine(P < 0.05) in proximal colonic contents were higher, while those of spermidine(P = 0.05) and total bioamine(P = 0.06) tended to be lower in the proline-supplemented group than those in the control group. The concentrations of spermidine, spermine, and total bioamine in colonic contents were higher(P < 0.05) on d 70 of pregnancy than those measured on d 45 of pregnancy. In contrast, the concentration of phenylethylamine was lower(P < 0.05) on d 70 than on d 45 of pregnancy.(Continued on next page)(Continued from previous page)Conclusion: These findings indicate that L-proline supplementation modifies both the colonic microbiota composition and the luminal concentrations of several bacterial metabolites. Furthermore, our data show that both the microbiota composition and the concentrations of bacterial metabolites are evolving in the course of pregnancy. These results are discussed in terms of possible implication in terms of luminal environment and consequences for gut physiology and health.

Development of a human rotavirus induced diarrhea model in Chinese mini-pigs

AIM: to establish a new animal model for the research of human rotavirus(HRV) infection, its pathogenesis and immunity and evaluation of potential vaccines.METHODS: 5-d, 30-d and 60-d-old Chinese mini-pigs, Guizhou and bamma, were inoculated with a single oral dose of attenuated strain Wa, G1, G3 of HRV, and PbS(control), respectively, and fecal samples of pigs from 0 to 7 d post infection(DPI) were collected individually. Enzyme linked immunosorbent assay was used to detect HRV antigen in feces. the HRV was tested by real-time PCR(Rt-PCR). the sections of the intestinal tissue were stained with hematoxylin and eosin to observe the morphologic variation by microscopy. Immunofluorescence was used to determine the HRV in intestinal tissue. HRV particles in cells of the ileum were observed by electron micrography.RESULTS: When inoculated with HRV, mini-pigs younger than 30 d developed diarrhea in an agedependent manner and shed HRV antigen of the sameinoculum, as demonstrated by Rt- PCR.Histopathological changes were observed in HRV inoculated mini-pigs including small intestinal cell tumefaction and necrosis. HRV that was distributed in the small intestine was restricted to the top part of the villi on the internal wall of the ileum, which was observed by immunofluorescence and transmission electron microscopy. Virus particles were observed in Golgi like follicles in HRV-infected neonatal minipigs. Guizhou mini-pigs were more sensitive to HRV than bamma with respect to RV antigen shedding and clinical diarrhea.CONCLUSION: these results indicate that we have established a mini-pig model of HRV induced diarrhea. Our findings are useful for the understanding of the pathogenic mechanisms of HRV infection.

Correlation between the Polymorphism of PPARγ-2 gene and the Susceptibility of Type 2 Diabetes Mellitus in Guangxi Bama Mini-pigs

[ Objective] The research aimed to discuss the relationship between the polymorphism of PPARy.2 gene and the susceptibility of type 2 diabetes mellitus （T2DM） in Guangxi Bama mini-pigs. [ Method] 24 Guangxi Bama mini-pigs were fed with high-fat and high-sucrose diet, and partial sequences of exon 2 of PPARy-2 gene were amplified by using PCR method. In addition, the contents of fasting blood glucose and insulin （INS） in Guangxi Bama mini-pigs were determined, and the glucose tolerance test （GTT） was also carried out. [ Result] There was one SNP site （19813A/G） Jn partial sequence of exon 2 of the cloned PPAFly-2 gene, and AA （7 pigs） and AG （17 pigs） genotype were detected. The contents of fasting insulin and 60-min blood glucose in GTT in AG-genotype Guangxi Bama mini-pigs were significantly higher than those of AA genotype （ P 〈0.05）, while the incidence of T2DM in AG-genotype Guangxi Bama mini-pigs （71.4%） was obviously higher than that of AA gen- otype （5.9%）. [ Conclusion] The polymorphism of 19813A/G in exon 2 of PPARy-2 gene was related with the susceptibility of T2DM in Guangxi Bama mini-pigs.

Dietary supplementation with betaine or glycine improves the carcass trait,meat quality and lipid metabolism of finishing mini-pigs

The objective of the study is to evaluate and compare the effects of betaine or glycine on carcass trait,meat quality and lipid metabolism of finishing Huan Jiang mini-pigs.Betaine called trimethylglycine is a methyl derivative of glycine,but few researches were conducted to compare the impact of dietary betaine and glycine on pigs.One hundred and forty-four Huan Jiang mini-pigs(body weight=10.55±0.15 kg;70 d)were randomly divided to 3 treatment groups(basal diet,glycine or betaine).Results indicated that dietary betaine increased the average daily gain(ADG)and final weight(P<0.05).Dietary glycine or betaine markedly reduced average backfat thickness(P<0.05)and heightened lean percentage(P<0.01)compared to the control group.Moreover,in comparison with the control group,betaine significantly improved the redness(a*)and tenderness(shear force)of the longissimus dorsi(LD)muscle(P<0.05),whereas glycine only raised the value of a*of the LD muscle(P<0.05).These results showed that diet supplemented with 0.25%betaine and equimolar amounts of glycine could regulate cascass trait and meat quality of finishing Huan Jiang mini-pigs,and the effect of betaine was superior to that of glycine.

Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

Cloning and Sequence Analysis of Adiponectin Receptor 1 and Receptor 2 cDNA from Guangxi Bama Mini-pig

[ Objective] To clone and analyze the sequence of Adiponectin receptor 1 （ AdipoR1 ） and receptor 2 （AdipoR2） cDNA of Guangxi Bama mini-pig. [Method] The Adiponectin receptors cDNAs were amplified by RT-PCR using skeletal muscle total RNA as template and then ligated into pMD18-T vector after purification. The recombinant pMD18-T vector was transformed into the E. coil DH5α for identification and sequencing. And the results were compared with the cDNA sequence from other species. [Result] The fragments, 1 128 bp and 1 161 bp in size, were amplified by RT-PCR and respectively consistent with the coding sequence of AdipoR1 gene and AdipoR2 gene. The homology analysis showed that the sequences of AdipoR1 gene and AdipoR2 gene were respectively 99.8% and 99.7% homologous to the sequence of domestic pig reported in GenBank with one base and three base missense mutations correspondingly. [ Conclusion] The AdipoR1 gene and AdipoR2. gene were successfully amplified from Guangxi Bama mini-pig, laying the foundation for the further study of the biological function of AdipoR genes and the design of novel drugs with AdipoR as target.

16S rRNA gene sequencing analysis of gut microbiome in a mini-pig diabetes model

Background:Currently,increasing attention is being paid to the important role of intestinal microbiome in diabetes.However,few studies have evaluated the characteristics of gut microbiome in diabetic miniature pigs,despite it being a good model animal for assessing diabetes.Methods:In this study,a mini-pig diabetes model(DM)was established by 9-month high-fat diet(HFD)combined with lowdose streptozotocin,while the animals fed standard chow diet constituted the control group.16S ribosomal RNA(rRNA)gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini-pigs.Results:The results showed that microbial structure in diabetic mini-pigs was altered,reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1,which were positively correlated with diabetes,and decreases in levels of the bac-teria Rikenellaceae,Clostridiales_vadinBB60_group,and Bacteroidales_RF16_group,which were inversely correlated with blood glucose and insulin resistance.Moreover,PICRUSt-predicted pathways related to the glycolysis and Entner-Doudoroff superpathway,enterobactin biosynthesis,and the L-tryptophan biosynthesis were significantly elevated in the DM group.Conclusion:These results reveal the composition and predictive functions of the intestinal microbiome in the mini-pig diabetes model,further verifying the relationship between HFD,gut microbiome,and diabetes,and providing novel insights into the application of the mini-pig diabetes model in gut microbiome research.

Effects of dietary protein/energy ratio on growth performance,carcass trait,meat quality,and plasma metabolites in pigs of different genotypes

Background： The protein/energy ratio is important for the production performance and utilization of available feed resources by animals. Increased protein consumption by mammals leads to elevated feed costs and increased nitrogen release into the environment. This study aimed to evaluate the effects of dietary protein/energy ratio on the growth performance, carcass traits, meat quality, and plasma metabolites of pigs of different genotypes. Methods： Bama mini-pigs and Landrace pigs were randomly assigned to two dietary treatment groups （Chinese conventional diet with low protein/energy ratio or National Research Council diet with high protein/energy ratio; n = 24 per treatment） in a 2 x 2 factorial arrangement. Blood and muscle samples were collected at the end of the nursery, growing, and finishing phases. Results： We observed significant interactions （P 〈 0.05） between breed and diet for total fat percentage, intramuscular fat （IMF） content, protein content in biceps femoris （BF） muscle, and plasma urea nitrogen （UN） concentration in the nursery phase; for average daily gain （ADG）, average daily feed intake （ADFI）, dry matter, IMF content in psoas major （PM） muscle, and plasma total protein and albumin concentrations in the growing phase; and for drip loss and plasma UN concentration in the finishing phase. Breed influenced （P 〈 0.05） growth performance, carcass traits, and meat quality, but not plasma metabolites. Throughout the trial, Landrace pigs showed significantly higher （P 〈 0.0_5） ADG, ADFI, dressing percentage, lean mass rate, and loin-eye area than did Bama mini-pigs, but significantly lower （P 〈 0.0.5） feed/gain ratio, fat percentage, backfat thickness, and IMF content. Dietary protein/energy ratio influenced the pH value, chemical composition of BF and PM muscles, and plasma activities of glutamic-pyruvic transaminase and gamma-glutamyl transpeptidase, and plasma concentration of UN. Conclusions： Compared with Landrace pigs, Bama mini-pigs showed slower growth and lower carcass performance, but had better meat quality. Moreover, unlike Landrace pigs, the dietary protein/energy ratio did not affect the growth performance of Bama mini-pigs. These results suggest that, in swine production, low dietary protein/energy ratio may be useful for reducing feed costs and minimizing the adverse effects of ammonia release into the environment.