Effect of dietary fiber and starch level and barley processing on relative variation of microbial population in gastrointestinal tract of Horse by using Real-time PCR

Understanding and description of degradation mechanism in digestive ecosystem of horse especially posterior part of gastrointestinal tract (GIT) is of great importance in proper nourishment. Despite its importance in host nutritional status, fermentation in posterior part of GIT is not completely understood and studies on contribution of posterior microbial population in nitrogen and energy requirements were rarely investigated. The literature is rather unanimous on ecology and microbial diversity in GIT of horses that based on microbial culture. Most of the time, the microbial culture is timeconsuming and difficult to implement and may include some part of microbial diversity. However, modern molecular methods like real-time PCR are independent tool of microbial culture which specifies varieties of bacterial species and total bacterial count with high accuracy and sensitivity. The aim of current study was to investigate the effect of dietary fiber and starch level and processing type on relative changes of microbial population in GIT.
In order to do this experiment, eight 6-12 years old Turkmen male horse with body weight of 270-300 kg were used. The rations were placed in two meals in the morning and evening and water was freely available to them. Two base rations containing high fiber and high starch were used for horses. Treatments included: 1) high fiber diet with steam flake barley (A), 2) high fiber diet with pelleted barley (B), 3) high starch diet with steam flake barley (C) and 4) high starch diet with pelleted barley (D). The experiment was conducted in a changeover design with four treatments and four periods, each period including 14 days of adaptation to the new diet and 7 days of sampling. Samples from feces of horses were collected on the last day of each period and stored in a freezer at -80 °C until the microbial population was determined. DNA extraction was done by phenol-chloroform method. In order to clone the genes, after extraction of their sequence from the NCBI database for each gene, a pair of primers (lead and follower primers) was designed using the AllelID software. Finally, the relative quantity of bacteria and protozoa was determined using a polymerase chain reaction method in real time.
Protozoa population in the diet containing high starch and flaked barley was higher than other diets and had a significant difference. The high starch diet containing pelleted barley and the high fiber diet containing flaked barley were 54 and 106 times higher than the high fiber diet and pelleted barley, respectively. The relative populations of Streptococcus bovis showed a significant increase in the high starch diets and flaked barley compared to other diets. In this experiment, the high fiber diet, with flaked barley increased the Ruminococcus flavefaciens 1400000 times relative to the high starch and flaked barley diet. The difference between high-fiber diet with pelleted barley and high-starch diet with flaked barley was also significant, and the concentration of Ruminococcus flavefaciens in a highfiber diet with pelleted barley was 6000 times of high starch diet and flaked barley. The highest concentrations of Fibrobacter succinogen observed in the high fiber with pelleted barley diet, that were 33800 times compared to high starch diets and flaked barley. In the next stage, there were high dietary fiber and pelleted barley, high-starch diet and flaked barley that were 1690 and 1470 times higher than high starch diet and flaked barley, but these two diets did not differ significantly in terms of concentration of Fibrobacter succinogen.
The results of this study showed that the use of diet high in starch and flaked barley increases the relative population of Streptococcus bovis and protozoan populations.