Effects Of Age And Dietary Forage-to-concentrate Ratios On Rumen Microbial Flora Of The Shaanbei White-cashmere Goat

Rumen microorganisms have a symbiotic relationship with ruminant animals, a principal function of the microbiome is the conversion of plant materials into digestible compounds that can be used by the animal host. This function is of tremendous importance as it allows the conversion of solar energy stored in plant fibers into food products, such as milk and meat. Optimizing rumen microbial flora has an important role in maintaining host health, improving performance, reducing environmental pollution, and ensuring food and animal product safety. Additionally, rumen contains a large number of micro-organisms and functional genomics resources. Therefore, studies of ruminal microbes represent a key area of nutrition research in ruminants. Lactobacilli are considered to have beneficial effects on human and animal health, which is one of the important advantages in the gastro-intestinal tract of humans and animals. Lactobacilli not only can play the normal functioning of the metabolic activity in the body, modulate immune system function, enhance metabolic capacity of animals, lower blood pressure and treat a variety of animal diseases, but also has played a very important role in improving animal performance and regulating animal gastrointestinal tract microflora balance. Additionally, they are generally regarded as safe by the FDA, which makes them attractive as vehicles for oral vaccination. It is therefore necessary to know the distribution and movement of lactobacilli in the gastro-intestinal tract, as well as in which tract the strain play its role. Thus, this study aims to characterize the colonization process by ruminal microorganisms in goats from 80 to 110-day-old of age using high-throughput next-generation sequencing; Studying effects of dietary forage-to-concentrate ratios on rumen microbial flora of the Shaanbei white-cashmere goat, this aims to provide a theoretical basis for the Shaanbei white-cashmere goat production and nutritional regulation; we constructed the recombinant Lactobacillus plantarum using GFP as the reporter protein. Then the Shaanbei white-cashmere goats were fed with the transformants using GFP as a visible marker for tracking this strain introduced into the gastrointestinal tract and observing its colonization capability, this is designed to provide a theoretical basis for the lactobacilli as foreign gene and the expression system of Lactobacilli from ruminant. The results of our experiment were as follow: 1. Rumen bacterial diversity of 80 to 110-day-old goats using 16 S r RNA sequencingThe age of the goats showed a statistically significant effect on rumen microbial flora of the Shaanbei white-cashmere goat. At the phylum level, within the 80-, 90- and 100-day-old groups, Firmicutes and Synergistete were the dominant phyla; in the 110-day-old group, Bacteroidetes and Firmicutes were the dominant phyla. With age, the relative abundances of Bacteroidetes and Proteobacteria significantly increased; however, the relative abundance of Firmicutes and Synergistetes significantly declined(P<0.05). At the genus level, in the 80-day-old group, the BS11_gut_group was predominant; in the 90-day-old group, the Roseburia was predominant; in the 100- and 110-day-old group, the Prevotella was predominant. With age, the relative abundances of Prevotella, RC9_gut_group and p2534-18B5_gut_group significantly significantly increased; however, the relative abundance of Ruminococcus and Selenomonas significantly declined(P<0.05). We also noticed that there were many unclassified and uncultured bacteria in the samples from the different age groups, which significantly declined with age(P<0.05).2. Effects of dietary forage-to-concentrate ratios on rumen flora of the Shaanbei white-cashmere goat.The forage-to-concentrate ratios had significant effect on rumen bacterial composition of the Shaanbei white-cashmere goat. At the phylum level, in different dietary groups, Bacteroidetes and Firmicutes were the dominant phyla. With the increase in the proportion of dietary concentrate, the relative abundances of Actinobacteria and Proteobacteria significantly significantly increased; however, the relative abundance of Bacteroidetes significantly declined(P<0.05). At the genus level, in different dietary groups, Prevotella was predominant. With the increase in the proportion of dietary concentrate, the relative abundances of Prevotella, Selenomonas and Treponema significantly significantly increased; however, the relative abundance of Oscillospira and Succiniclasticum significantly declined(P<0.05). These cellulose-degrading bacteria in this study were present at a low overall relative abundance(<1% of the total microbial community). With the increase in the proportion of dietary concentrate, the relative abundances of Ruminococcus and Butyrivibrio significantly declined(P<0.05).The forage-to-concentrate ratios had significant effect on rumen fungal composition of the Shaanbei white-cashmere goat. At the phylum level, in different dietary groups, Ascomycota was predominant. With the increase in the proportion of dietary concentrate, the relative abundances of Ascomycota, Basidiomycota and Zygomycota significantly increased; however, the relative abundance of Chytridiomycota significantly declined(P<0.05). At the genus level, in C30 dietary group, Neocallimastix was predominant; in C50 and C70 dietary groups, Candida was predominant. With the increase in the proportion of dietary concentrate, the relative abundances of Alternaria, Aspergillus, Neocallimastix, Orpinomyces, Piromyces and Stachybotrys significantly increased; however, the relative abundance of Candida, Penicillium and Trichosporon significantly declined(P<0.05).3. Use of green fluorescent protein to monitor Lactobacillus plantarum in the gastrointestinal tract of goatWe isolated 10 Lactobacilli from the rumen of Shaanbei white-cashmere goat using screen MRS medium. They then identified by 16 Sr DNA anylysis. The results are: 6 strain are Lactobacillus plantarum, 2 strain are lactobacillus curvatus, 1 strain is lactobacillus casei, 1 strain is lactobacillus spp.We constructed a constitutive expression vector p LEM415-gfp-p32 for Lactobacilli p LEM415-gfp-p32 using GFP as the reporter protein. Then, it was electroporated the recombinant into Lactobacillus plantarum which was isolated from Shanbei White cashmere goats. Green fluorescent protein(GFP) was expressed successfully in Lactobacillus plantarum. After post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, concentrations of bacteria were decreased at first, which reached minimum at 42 h post-oral administration, and then increased. However, it was decreased constantly in the duodenum.In a word, this work found that the age of the goats showed a statistically significant effect on rumen microbial flora of the Shaanbei white-cashmere goat; the forage-to-concentrate ratios had significant effect on rumen microbial flora of the Shaanbei white-cashmere goat; Lactobacillus plantarum could colonize in the rumen but not in the duodenum.