The association between rumen microbial protein production with rumen microbial community structure.

Abstract Beef production in tropical northern Australia is generally limited by poor feed quality during the critical dry season. The digestibility of this poor quality feed is directly related to rumen microbial activity and community composition, and despite this important relationship, a review of available literature highlighted our limited understanding of this. Supplementation with nitrogen (N) is a practical strategy to improve the liveweight (LW) gain of cattle grazing low quality pastures, however, the effect of various forms of N supplementation on the composition of the rumen microbes is unknown. N supplementation will increase microbial crude protein (MCP) production but the efficiency of MCP production (EMCP) is difficult to increase. Supplementation with spirulina algae increased EMCP and therefore provided a working model to compare the rumen function and community structure of steers with low and high EMCP values when fed tropical pastures. This study was initiated to develop a better understanding of rumen ecology the factors that govern microbial community structure and growth to increase the EMCP and associated animal performance in tropical production systems. The aims of this study were firstly to identify the changes in microbial species of the rumen when various forms of N were supplemented, and secondly to examine the relationship between the microbial community structure, EMCP and LW gain, as it relates to tropical pastures and supplement type. Experiment 1 investigated the use of the N supplements, urea and spirulina algae, to enhance the EMCP of steers fed a basal diet of Mitchell Grass (Astrebla spp.). Data from a parallel study on rumen function showed a significant improvement in the EMCP with spirulina algae supplementation. In this experiment denaturing gradient gel electrophoresis (DGGE) was applied to compare the effects of treatments on rumen bacterial community structure. Phylogenetic sequence analysis was performed on cloned DGGE bands which represented bacterial species common across all treatments (core bacteria) and supplement specific bacteria. Experiment 2 investigated the effect of N supplements (urea, spirulina algae and cottonseed meal (CSM)) on LW gain of steers fed a basal diet of Speargrass (Heteropogon contortus). LW data from a parallel component of the experiment demonstrated significant LW gains under N supplementation. The 454 pyrosequencing technique was applied providing a comprehensive examination of microbial diversity, community structure, and identification of the core bacteria and bacteria specific to supplement treatments. The core bacteria, which were represented as common bands across all treatments, were identified in Experiment 1. The 454 pyrosequencing (Experiment 2) identified and validated a stable core group of bacterial operational taxonomic units (OTUs) present in all steers. The core group of bacteria could not be characterized to a species level because there are no cultured representatives in the available databases. Supplement specific bacteria were identified in Experiment 1 and 2 using the different molecular techniques however bacterial species again could not be characterized. In both experiments there was animal to animal variation in microbial community structure that was not fully attributable to N supplement treatments. Each steer appeared to have its own unique bacterial profile. In Experiment 1, due to the incomplete Latin square design, it was shown that the profile for each animal was stable when changes in supplement were imposed across the three runs. In Experiment 2, there was considerable animal to animal variation in microbes at both the phyla and the genera level. Across both experiments, a greater bacterial diversity was found in rumen samples from animals fed diets supplemented with the medium (130-170 gRDP/kg DOM in Experiment 1 and 0.14-0.16 g N/kgLW/day in Experiment 2) levels of protein supplement. The bacterial diversity did not correlate with EMCP values in Experiment 1, however, it is likely that any effects were confounded since with high spirulina algae supplementation, rumen retention time was significantly reduced, which is likely to affect microbial populations. In Experiment 2 the higher bacterial diversity was strongly associated with greater LW gains. In an attempt to provide a better understanding of the diversity changes, rumen bacteria community profiles were compared in both experiments. Comparing DGGE profiles in Experiment 1, there were significant changes only at the high and low planes of N with high and low EMCP steers clustering separately, suggesting a relationship between bacterial community profile and EMCP at the extremities of the N supplementation range. The use of 454 pyrosequencing in Experiment 2 provided more clarity of the changes in the OTUs with supplementation by means of OTU networks. These networks showed two distinct clusters that related to urea and protein supplementation treatments, however many OTUs were common across all treatments. It may be concluded that supplementation of protein increases the diversity of rumen bacteria as well as improves the growth conditions for the entire bacterial community, therefore providing more nutrients to the host animal leading to improved EMCP and increased LW gains