Evaluation of rumen methanogen diversity in cattle fed diets containing dry corn distillers grains and condensed tannins using PCR-DGGE and qRT-PCR analyses

Abstract

Condensed tannins (CT) and corn distillers grains from ethanol production have the potential to reduce enteric CH4 production from ruminants because of their anti-microbial properties and high lipid contents, respectively. However, their effects on the community of rumen methanogens in cattle rumens have not been investigated. Our objective was to evaluate effects of dry corn distillers grain with solubles (DDG) and CT from Acacia mearnsii added to the diet of cattle on rumen methanogens using PCR denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time (qRT)-PCR. Eight ruminally cannulated beef heifers were used in a replicated 4×4 Latin square design with four 35d periods. Diets were (g DDG dry matter [DM]/kg DM): control (Ctrl; 0 DDG), 200 DDG, 400 DDG and 400 DDG plus 25g CT (400 DDG+CT). Rumen digesta samples were collected before feeding on d 25 and 28 of each period from 4 sites in the rumen. Total DNA extracted from rumen digesta samples (i.e., 0.5ml liquid+0.5g solid pooled by heifer for each period) was used to generate amplicons of partial 16S rRNA genes. The PCR products obtained were subjected to PCR-DGGE. Detectable methanogenic PCR-DGGE profiles from periods 1 and 2 clustered while periods 3 and 4 formed a second cluster. A total of 25 distinct PCR-DGGE bands were identified, of which 16 bands were successfully cloned and sequenced. The sequences of 11 bands had 97–100% similarity with Methanobrevibacter sp., and this was supported by clustering of 10 of these bands with this species upon phylogenetic analysis of the sequences. The remaining bands clustered with Methanosphaera stadmanae. Although no effect of diet on total methanogenic PCR-DGGE profiles occurred, the proportion of bands 4, 5, 6, 7, 9 and 11 increased and bands 16 and 18 decreased with increased DDG in the diet. Bands 13 and 19 were in all diets except 400 DDG+CT. Quantitative RT-PCR analysis revealed that the copy numbers of total methanogenic 16S rRNA gene did not differ among diets. Findings from PCR-DGGE and qRT-PCR analysis suggest that inclusion of DDG or a mixture of DDG and CT altered the diversity of rumen methanogens without affecting total methanogen associated 16S rRNA copy numbers thereby indicating that copy numbers of particular species of methanogens may be required to explain the altered diversity of rumen methanogens.This article is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.