Kennedy P M, Hazlewood G P, Milligan L P
Br J Nutr. 1984 Sep;52(2):403-17. doi: 10.1079/bjn19840106.
Four sheep, each fitted with cannulas in the rumen and proximal duodenum, were given two diets (1390 g dry matter (DM)/d) consisting of lucerne (Medicago sativa) pellets (24.2 g nitrogen/kg DM) plus pelleted reed canary grass (Phalaris arundinacea; 14.1 g N/kg DM) or chopped hay (11.8 g N/kg DM) at intervals of 2 h. Flow of duodenal digesta measured by reference to the markers 51Cr-EDTA and 103Ru-phenanthroline indicated a net gain of 5.8-7.5 g non-ammonia-N (NAN) between mouth and duodenum. The proportion of microbial N in duodenal digesta N was estimated using 15N and 35S incorporation into bacteria and digesta. Two methods of analysis for 35S content, the Bird & Fountain (1970; B&F method) and the Mathers & Miller (1980; M&M method), were used. (15NH4)2SO4 and Na2(35)SO4 were infused into the rumen for 3.5 d before and 4.0 d during sampling. A bacterial fraction was prepared from the fluid phases of sampled duodenal digesta and rumen contents by differential centrifugation. In addition, samples of ground canary grass and of lucerne were incubated in nylon bags in the rumen for 3-48 h during the infusion. Each of the 35S analytical methods yielded similar values of 35S content of isolated rumen or duodenal bacteria, but there was more (P less than 0.05) incorporation of 15N into rumen than into duodenal bacteria. Relative to values obtained using the M&M method and 15N incorporation, the B&F method for S analysis yielded higher (P less than 0.05) estimates of microbial content of duodenal digesta from sheep given chopped reed canary grass. 35S activity associated with washed nylon-bag residues increased rapidly with time-period of incubation and was substantially greater (P less than 0.05) when analysed by the B&F method compared with the M&M method. The 35S content (/g DM) of adherent bacteria removed from nylon-bag residues by homogenization in a second experiment varied from 0.65 to 1.88 that of free-living bacteria isolated from rumen fluid by differential centrifugation. The difference in 35S content in digesta and nylon-bag residues as measured using the two analytical methods was considered in relation to 35S-labelled extracellular material postulated to be produced by bacteria adherent to plant residues. Estimates of disappearance of dietary N from nylon bags after correction for microbial contamination indicated a disparity with estimates based on in vivo information.
选用4只绵羊，每只在瘤胃和十二指肠近端安装套管，每隔2小时给予两种日粮（1390克干物质（DM）/天），日粮分别由苜蓿（紫花苜蓿）颗粒（24.2克氮/千克DM）加颗粒状虉草（虉草；14.1克氮/千克DM）或切碎的干草（11.8克氮/千克DM）组成。通过参考标记物51Cr - EDTA和103Ru - 菲咯啉测定十二指肠食糜流量，结果表明口腔至十二指肠间非氨态氮（NAN）净增加5.8 - 7.5克。利用15N和35S掺入细菌和食糜中的方法，估算十二指肠食糜氮中微生物氮的比例。采用两种分析35S含量的方法，即伯德和方丹（1970年；B&F法）以及马瑟斯和米勒（1980年；M&M法）。在采样前3.5天和采样期间4.0天，将(15NH4)2SO4和Na2(35)SO4注入瘤胃。通过差速离心从采样的十二指肠食糜和瘤胃内容物的液相中制备细菌组分。此外，在注入期间，将磨碎的虉草和苜蓿样品装在尼龙袋中在瘤胃中培养3 - 48小时。两种35S分析方法得到的瘤胃或十二指肠分离细菌的35S含量值相似，但15N掺入瘤胃细菌的量比掺入十二指肠细菌的量更多（P<0.05）。相对于使用M&M法和15N掺入法得到的值，用B&F法进行硫分析时，对饲喂切碎虉草绵羊的十二指肠食糜微生物含量的估计值更高（P<0.05）。与洗涤过的尼龙袋残渣相关的35S活性随培养时间迅速增加，与M&M法相比，用B&F法分析时显著更高（P<0.05）。在第二个实验中，通过匀浆从尼龙袋残渣中去除的附着细菌的35S含量（/克DM）是通过差速离心从瘤胃液中分离的自由生活细菌的0.65至1.88倍。考虑到两种分析方法测定的食糜和尼龙袋残渣中35S含量的差异与假定由附着在植物残渣上的细菌产生的35S标记细胞外物质有关。校正微生物污染后尼龙袋中日粮氮消失量的估计值与基于体内信息的估计值存在差异。
