High-throughput Sequencing Analysis Of Normal Intestinal Flora In Small Tail Han Sheep And The Development Of Sheep Source Probiotics

The animals intestinal flora have the function of promoting nutrition and metabolism, improving immunity and protection, maintaining the balance of intestinal flora, improving the intestinal environment, and promoting animals’ growth. However, when animals suffer the stress of cold, heat, shock, long-distance transport, bacteria and viruses, the intestinal flora may be disorder, and then the animals may develop the diarrhea. Besides, the immune function and growth index of animals will decrease. This may bring huge economic losses to the cultivation industry. The small tail Han sheep is one of the local high-quality and dual-purpose sheep breeds. They are widely reared because of their some excellent features such as, big body, precocious puberty, crude feed tolerance, fast growth, high reproduction rate, disease resistance, genetic stability, adaptability and so on. However, with the expansion of their breeding scales, the gastrointestinal diseases are becoming more prominent, which lead to a serious abuse of antibiotics. While antibiotics can treat some gastrointestinal diseases, but also can break the balance of intestinal flora, this resulting in the intestinal flora disturbance. Micro-ecological preparation as an alternative to antibiotics has many advantages, such as no residue, no drug resistance, non-toxic side effects, significant effects, not polluting the environment, etc. It can not only prevent and cure diseases, improving feed conversion rate and animals performance, but also can improve their health. But there is no suitable micro-ecological preparation for sheep now, and we have not enough knowledge for the gut microflora of sheep. We currently use the traditional molecular biology techniques, such as real-time PCR and DGGE to study the intestinal microflora. These methods are not only time consuming but also a heavy workload, and can only detect a certain abundance of microbes.It is also not comprehensive enough for studying the complex intestinal microflora with these methods. However, we can sequence all the gut microbial DNA sequences by Illumina high-throughput sequencing technology, with a comprehensive analysis of the diversity and abundance of intestinal microflora, in order to satisfy our understanding of the intestinal microflora. Therefore, we use the Illumina Mi Seq high-throughput sequencing technology to analyze the intestinal flora composition and diversity of small tail Han sheep, and results of the analysis as a basis for screening the gut bacteria of small tail Han sheep. Finally we develop the micro-ecological preparation with strains screened, and the effects of the micro-ecological preparation have been tested.We used the Illumina Mi Seq high-throughput sequencing technology to analyze the flora composition, α diversity, β diversity and sample clustering of the jejunum, cecum and rectum in small tail Han sheep. The results showed that the top five predominant populations in the cecum and rectum were Bacteroides, Ruminococcus, Lactobacillus, Incertae_Sedis and Flavonifractor at the genus level. While in the jejunum, the top five predominant populations were Lactobacillus, Chloroplast, Incertae_Sedis, Acetitomaculum and Ruminococcus. We can saw that there were no significant difference between cecum and rectum about the flora composition of different individuals. However, it had significant difference between the jejunum of different small tail Han sheep individuals. Besides, we could guess that from the duodenum to the rectum of small tail Han sheep, the abundance of Lactobacillus was decreasing, and the abundance of Bacteroides was growing. The bacterial abundance and diversity in cecum and rectum were greater than jejunum, and there were no significant difference between cecum and rectum. Besides, the bacterial communities in cecum and rectum were similar in evolution, while they were different from jejunum.According to the results of high-throughput sequencing, we used the traditional biological methods to isolate and identify the bacteria in small tail Han sheep gut, and the isolated strains were screened by the test of growth performance, resistance to acid and bile, and inhibiting the growth of harmful bacteria. Then, three strains were screened by the tests, they were M1(Lactobacillus casei), M2(Lactobacillus plantarum) and L1(Bacillus subtilis).We developed the micro-ecological preparation with the three strains and tested its safety by feeding the mice. The result showed that the micro-ecological preparation we developed was safe.The micro-ecological preparation we developed was mixed in drinking water for rabbits of low, medium and high dose group respectively, and then the growth performance and immune function of rabbits were detected. The results showed that the micro-ecological preparation could improve the average daily gain and lower feed conversion rate in rabbits, and also could improve the serum antibody titer of RHDV, serum IFN-γ and Lysozyme concentration. Among them, the medium and high dose group had the best effects(P < 0.05), while the difference between the medium and high dose group was not significant(P > 0.05). Besides, we fed sheep of diarrhea with the micro-ecological preparation we developed, comparing with the diarrhea control group and healthy control group. After five days of conditioning treatment, all the sheep in diarrhea test group returned to normal, which indicated that the micro-ecological preparation could treat sheep with diarrhea. We can conclude that the micro-ecological preparation we developed can be used in practical production.