| The brooding period is a critical period for intestinal development and microbial colonization of layers.Chick diarrhea and nutritional malabsorption caused by the problem of intestinal development often occur in production,causing irreversible damage to later growth and production.In addition,the intestine is colonized by a large variety of microbiota,which is important for the early intestinal development of chicks.In this paper,the effects of in ovo feeding?IOF?of L-Arginine?L-Arg?on the intestinal development and microflora of layers during the embryonic and early posthatch stage were studied by combining 16 S r RNA high-throughput sequencing and metabolomics technology,and the role of early microbial colonization in the intestinal development was discussed,so as to provide a new idea for early nutrition regulate the intestinal development of poultry.Experiment 1.Effects of in ovo feeding of L-Arg on the early intestinal development of pullets.The treatments included the non-injected control,saline-injected control,and saline containing 2,6,or 10 mg/egg L-Arg groups.The results showed that significant differences in hatchability and hatching weight among groups were not observed in response to IOF of L-Arg?P > 0.05?,but IOF of L-Arg increased the body weight of pullets on d 21 and 42?P < 0.05?.Compared with the control group,the crypt depth of duodenum in the Arg10 group was reduced and the greater villus height/crypt depth of jejunum was observed on d 3?P < 0.05?.And the increased villus height of duodenum,villus height/crypt depth and epithelial cell proliferation index of jejunum were also found in the Arg10 group on d 14?P < 0.05?.However,there was no significant effect on the intestinal morphology of pullets was found on d 42?P > 0.05?.Compared with NC group,serum triglyceride content of pullets were significantly decreased on d 3 and 14 by IOF of L-Arg?P < 0.05?,and serum total cholesterol and low density liptein cholesterol contents were significant increased?P < 0.05?.Compared with SC group,serum high density liptein cholesterol and glucose contents of pullets were significantly increased on d 14 by IOF of L-Arg?P < 0.05?.This experiment suggested that the early intestinal development of pullets were accelerated by IOF of 10 mg/egg L-Arg,and the structure of intestinal villus and growth of pullets were also promoted;According to the F/G of pullets from d 0 to d 21,the appropriate injection dose is 7 mg L-Arg/egg.Experiment 2.Effects of in ovo feeding of L-Arg on intestinal microflora and metabolism of pullets.Experiment design was the same as experiment 1.The results showed that compared with the control group,the microbial ? diversity was significantly increased in Arg6 and Arg10 groups on d 3.Principal coordinate analysis?PCoA?showed that the intestinal microbiota of pullets in each groups were significantly different on d 3.As the age increased,the differences between the groups gradually narrowed.Compared with the control group,we found that the IOF groups increased the relative abundance of Firmicutes and decreased the relative abundance of Proteobacteria?P < 0.05?.We also found that the colonization time of cecal microbiota for Lactobacillus,norankfRuminococcaceae and ClostridialesvadinBB60group in Arg groups were earlier than that in the NC group.In addition,bacteria that significantly positively relating to the development of intestinal morphology were mostly short chain fatty acid-producing bacteria,while some potentially pathogenic bacteria were significantly negatively related to the intestinal morphology by the method of Spearman correlation analysis?P < 0.05?.The metabolomics indicated that the IOF L-Arg groups were significantly different from the control group in galactose metabolism,primary bile acid metabolism,and lipid metabolism.This experiment revealed that IOF of L-Arg can accelerate intestinal development and succession of cecal microflora and colonization of probiotics of pullets in the early stage,and the galactose metabolism,primary bile acid metabolism,and lipid metabolism of host were changed,which making more energy and nutrients used for early intestinal development of pullets.Experiment 3.Effects of in ovo feeding of L-Arg on intestinal development and microflora in embryonic stage.According to the type of breeding eggs?SPF breeding eggs and common breeding eggs?and IOF?no injection,L-Arg injection?,a two-factor test design was used.Each type of breeding egg was treated with no injection?control?and L-Arg injection,and was divided into 4 groups.The amniotic cavity of embryos were injected with 7 mg/egg L-Arg on d 17.5 of incubation.The results showed that the microbial DNA copy number in the common breeding eggs group?COM?was significantly higher than that in the SPF breeding eggs group?SPF??P < 0.05?.Before injection,there was no significant difference in embryos weight,yolk sac-free embryo weight,yolk sac index and intestinal index between SPF group and COM group at the age of embryos?E?17.Compared with no injection groups,intestinal index of embryos,villus height and villus width were all increased by the IOF of L-Arg at E 19 and E 21.IOF of L-Arg significantly increased intestinal index,villus height and villus width at E 19 and E 21?P < 0.05?.However,no significant difference in intestinal index and intestinal morphology of COM and SPF groups were found?P > 0.05?.In addition,compared with the COM group,IOF of L-Arg significantly increased the relative expression of mTOR and 4E-BP1 mRNA in the embryonic jejunum of SPFA and COMA groups?P < 0.05?.Compared with the COM group,significant increased the intestinal microbial ? diversity were found in the IOF of L-Arg groups at E 21?SPFA and COMA??P < 0.05?,but no significant difference in intestinal microbial ? diversity was found among them at E 19?P > 0.05?.PCoA analysis showed that there was no significant difference in the microbiota at E 19,but the microbial composition of the SPFA and the COMA groups were more similar,and significant difference was found at E 21.The relative abundance of Firmicutes in the intestine of the SPFA and COMA groups were significantly higher than that in the COM group?P < 0.05?,and the relative abundance of Proteobacteria and Acidobacteria in the intestine showed a downward trend?P < 0.10?.At the genus level,the relative abundances of Blautia,Bifidobacterium,Streptococcus,Agathobacter,Ruminococcus1,Chryseobacterium,and Holdemanella were significantly increased in the intestine of the SPFA and COMA groups?P < 0.05?.The microbial function prediction results showed that two carbohydrate metabolism pathways?starch and sucrose metabolism,fructose and mannose metabolism?and three lipid metabolism pathways?primary bile acid synthesis,secondary bile acid synthesis and steroid synthesis?,1 amino acid metabolism pathway?cyanoamino acid metabolism?,1 digestive system pathway?carbohydrate digestion and absorption?,and 1 membrane transport pathway?phosphotransferase system?in environmental information processing were significantly enriched in the SPFA and COMA groups?P < 0.05?.In addition,IOF of L-Arg significantly increased the contents of formic and butyric acids in the microbial metabolites of SPFA and COMA groups?P < 0.05?.This experiment revealed that intestinal development and the succession of microflora in embryos were accelerated by IOF of L-Arg,and the contents of metabolites short chain fatty acids were increased,providing more energy for intestinal development.In addition,the number of bacteria carried by the breeding eggs didn't affect effects of L-Arg on intestinal development and microflora maturation of embryos.In summary,the intestinal development and succession of microflora can be promoted by IOF of LArg,and the growth of pullets was promoted;The early colonization of probiotics can also be accelerated by IOF of L-Arg,the contents of metabolites short chain fatty acids were increased,and the carbohydrate,lipid,bile acid,and membrane transport metabolism were regulated to provide more energy for intestinal development of pullets. |
PDF Full Text Request