عنوان مقاله [English]
Introduction: Rumen microbial manipulation has been of interest to ruminant nutritionists in order to achieve improvement in the profitability and health of lactating cows. Concerns regarding the use of antibiotics and other growth stimulants in animal feed industry have caused attention to find other alternative agents to replace antibiotics. For these reasons, in ruminant, microbial cultures have been used to replace antibiotics to enhance milk production in dairy cattle and improve feed efficiency and daily gain in beef cattle. Rumen bacteria, that have ability to ferment carbohydrate, are primarily responsible for causing lactic acidosis in ruminant. Propionibacteria are natural inhabitants of the rumen that comprise 1.4 % of the ruminal microflora and produce propionic and acetic acid in the rumen. Therefore, propionibacteria have been used as a direct-fed microbial to prevent the risk of acidosis in feedlot cattle. Last studies have reported that combinations of propionic bacteria and lactobacilli resulted in increased average daily gain and improved feed efficiency in feedlot cattle. The purpose of this study were preparation of live bacterial culture in laboratory and investigate the effect of adding live bacterial culture on digestibility and ruminal fermentation parameters on in vitro condition.
Materials and Methods: Three in vitro experiments designed in order to determine the effects of bacteria strains supplementation on dry matter and organic matter digestibility, pH, VFA and ammonia nitrogen concentration. Bacterial pure strains were Propioni bacterium freudenreichii and Enterococcus faecium. Stock cultures of freeze-dried strains were individually inoculated into 5 mL Brain Heart infusion (BHI) and sodium lactate broth (SLB) respectively and incubated at 37 °C for 24 h under anaerobic conditions. By pour plating serial 10 fold dilutions (in sterile ringer’s solution) on demand, Rogosa, sharp agar and SLB agar plates were incubated anaerobically at 37 °C for 48h. 108 cfu /ml of culture were produced after 24 and 36 h of culturing. Rumen fluid was collected 3 h after morning feeding from three ruminally fistulated sheep with mean body weight of 45+2.5 kg. Buffer was prepared as proposed by Goering and Van Soest (1970). In an anaerobic condition, 50 ml of buffered rumen fluid (ratio of buffer to rumen fluid was 1:1), was dispensed into a 100 ml serum bottle containing 0.5 g DM of the experimental diet (four replicate) for each experimental incubation time (2, 4, 6, 12 and 24 h). The experimental treatments were: CON) control (basal diet without any additive), P) basal diet inoculated with 108 cfu Propionibacteria feriderinrichii, E) basal diet inoculated with 108 cfu Enterococcus faecium and P+E) basal diet inoculated with 108 cfu Propionibacteria fredrinrichii + Enterococcus faecium. Rumen fluid was collected before morning feeding from three ruminally fistulated sheep. In all experiments a diet based on concentrate were used with the ratio 90: 10 of concentrate to forage. In the second experiment 1 mL sucrose (10% w/w) injected to the diet and in third experiment pH adjusted to 5.5 at the beginning of experiment. After 2, 4, 6, 12 and 24 h of the incubation, the bottles were respectively transferred to an ice bath to stop fermentation and then opened to measure medium pH using a pH meter (Metrhom pH meter, Model 691). Then, each bottle content was filtered (42 μm pore size) and a 5 ml sample of each filtrate bottle was taken and acidified with 5 ml of 0.2 N HCl and frozen at -20°C. for analyzing VFA by gas chromatography, 4 mL of each filtrate bottle were stabilized with 4 mL meta-phosphoric acid Liquid effluent was collected in flasks containing a solution of H2SO4 to maintain pH values below 2, and samples were taken for volatile fatty acids The filtrated residual was oven dried (60 °C for 48 h) and used to calculate in vitro dry matter and organic matter disappearances.
Results and Discussion: DM and OM digestibility for all treatments increased during incubation. Control treatment had lowest DM digestibility. Changes in OM digestibility was in same manner with DM digestibility in all treatments and control had lowest OM digestibility significantly. In comparison with initial pH, treatments control, Propionibacterium fredrinrichii, enterococcus faecium and mixed of Propionibacterium fredrinrichii + Enterococcus faecium had decreased 0.91, 0.73, 0.76 and 0.58 unit, respectively. NH3-N concentration after 2h after incubation was low in all treatments and increased during incubation, but for control it was low in compare with other treatments during incubation. Adding bacterial culture had significantly effect on VFA concentration. There was significantly difference among treatments on acetate: propionate ratio and treatments 2 and 4 had lowest ratio.
Conclusion: Generally, DM and OM digestibility and VFA concentration can be affected positively and significantly by live bacterial cultures additives. These changes would be associated with the stabilization of rumen pH and provide favorable conditions for microorganisms’activity in the rumen.