Factors that limit maintenance of recombinant rumen bacterium in sheep rumen |
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| Authors: | Yasuo KOBAYASHI Mayu YAMAMOTO |
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| Institution: | Faculty of Bioresources, Mie University, Tsu-shi, Japan |
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| Abstract: | Factors limiting the maintenance of recombinant ruminal bacterium in the rumen were evaluated in vitro , using batch culture prepared from rumen fluid of sheep. Butyrivibrio fibrisolvens expressing a foreign xylanase gene ( B. fibrisolvens NO4) was used as a tested recombinant that was selectable on an erythromycin-containing agar medium. The recombinant tended to reduce its level slowly in the rumen fluid of sheep on a high hay diet, while its initial decrease was more apparent in the rumen fluid of sheep on a high concentrate diet. Incubation with cell-free ruminal fluid revealed a significant decrease of inoculated recombinant, suggesting the presence of antibacterial factors limiting maintenance of the recombinant. In particular, during the first 12 h of incubation this inhibition was more notable in culture prepared from rumen fluid of sheep given the high concentrate diet. Autoclaving the cell-free rumen fluid inactivated the inhibition. Numbers of the recombinant for inoculation did not influence the final level of survived recombinant, that is, the initial depression was larger as more recombinant was inoculated. Subculturing with xylan before inoculation and/or direct addition of xylan to the batch culture did not improve survival of the recombinant. From these results it is suggested that the level of survived recombinant is limited to 102–4/mL of in vitro batch culture with restricted energy supply and that initial depression of the recombinant is mainly caused by the heat-sensitive antibacterial factors not associating with microbial cells in the rumen. |
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| Keywords: | limiting factors maintenance recombinant rumen bacterium rumen bacteria survival |
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