Development Of Microecologic Preparation For Disorders Of Energy Metabolism In Dairy Cows And Its Effects

Three strains of dominant propionic acid-producing bacteria were separated from ruminal fulids of healthy Holstein dairy cow identified by their biochemical characteristics and 16S rRNA PCR, and named Selenomonas ruminantium 6 (S6), Megasphaera elsdenii 3 (M3), Veillonella parvula 2 (V2).Efficient propionic acid-producing genetic engineering bacteria were developed by Tn5 transposon tagging with selective mediums containing kanamycin and fluoroacetic acid.PCR of 16S rRNA and Tn5 analyses revealed that the mutants belonged to PTA gene deletion engineering bacteria (named TnS6, TnM3 and TnV2, respectively) and the genetic engineering bacteria were successfully constructed. Culture in vitro showed that TnS6, TnM3 and TnV2, as well as S6, M3 and V2, were all capable of producing VFA using lactic acid as a precursor. In spite of producing propionic acid at a lower level compared to original bacteria, genetic engineering bacteria significantly decreased ratio of C2/C3, owing to remarkablely decline of acetic acid concentration.Co-cultured of genetic engineering bacteria with different strains of yeasts revealed that the ability of propionic acid-producing was best when bacterias were combined with Candida Rugosa, Candida Utilis and Saccharomyces Cerevisiae.Microecologics were developed by mixing genetic engineering bacteria with the three strains of yeasts. A series of studies were conducted to observe the effect of microecologics on dairy energy metabolism both in vitro and in vivo. The in vitro ruminal fermentation revealed that the microecologics promoted utilization of lactic acid and production of VFA. With delayed fermentation time, output of propionic acid raised continuously, and the ratio of C2/C3 decreased gradually. Inoculating with microecologics in dairy rumens showed that the concentration of lactic acid and the ratio of C2/C3 significantly decreased while that of propionic acid increased. Above results initially proved that the fermentation type of microecologics were inclined to production of propionic acid type.Peripartum healthy Holstein Cows (n=6) fed with microecologics developed no adverse reactions during 21 d. It was verified by blood biochemical analysis and metabolic function test, indicating that the microecologics were innocuous, safe and reliable. Healthy cows in perinatal stage and cows with ketosis fed with microecologics both showed a escalating trend of blood sugar over time and a down trend of BHBA concentration, preliminarily indicating that microecologics is effective for prevention and treatment of ketosis in dairy.