Molecular ecology of rumen bacterial populations in steers fed molasses diets

Transcription

1 Molecular ecology of rumen bacterial populations in steers fed molasses diets A thesis presented in fulfilment of the requirements for admission to the degree of Doctor of Philosophy School of Animal Studies The University of Queensland Brisbane, Australia Maria Ximena Tolosa Alvarez 2006

2

3 Declaration The work presented in this thesis is, to the best of my knowledge and belief, original and my own work except as acknowledged in the text. This material has not been submitted, in whole or in part, for a degree at this or any other institution. Experiment 1 and Experiment 2 of this thesis were carried out in collaboration with Tuyen V. Dinh, a PhD student. Tuyen examined the effect of the diets used on nutritional parameters. He carried out analysis for microbial protein synthesis, ammonia concentration in rumen fluid, outflow rate of liquid- and solid-phase of the rumen and kindly shared the data with me. M. Ximena Tolosa September 2006 iii

4

5 Abstract The rumen bacterial community structure and phylogenetic diversity of fluid- and solidassociated bacteria (FAB and SAB, respectively) was investigated in Brahman-cross steers fed several diets based on low-quality pangola grass hay and molasses. Diets used in Experiment 1 (as fed basis) consisted of 100% pangola hay (0M), 75:25 hay/molasses (25M), 50:50 hay/molasses (50M), and 25:75 hay/molasses (75M). Diets used in Experiment 2 (as fed basis) consisted of molasses/hay (HM, 50:50), hay/molasses/urea (MU, 50:48:2), hay/molasses/casein (MCAS, 50:45:5), and hay/molasses/cottonseed meal (MCSM, 30:50:20). In both experiments, four rumen-cannulated steers were fed once daily at 0800 h. Steers were allocated to one of the four diets in a 4x4 Latin square design. Rumen fluid samples were taken during the initial period of molasses introduction (Experiment 1). Following adaptation to the diets, rumen fluid and solid phase samples were taken immediately prior to feeding and 8 h after feeding over two consecutive days (Experiments 1 and 2). Rumen FAB and SAB communities were analysed by amplification of the V2-V3 region of the 16S rdna gene, followed by denaturing gradient gel electrophoresis (DGGE). Bacterial diversity was estimated by applying statistical ecology indices to DGGE data. Intake, digestibility, microbial protein production, fluid and solid fractional outflow rate, ph, NH 3 N and VFA content in the rumen were also measured. Monitoring changes in FAB structure during the period of gradual introduction of molasses to the diets indicated that bacteria responded slowly to a dietary change. Soon after the start of the dietary change (days 1-8), the appearance of transient dominant bands (species) became evident as these bands were not present three weeks after the diet change. Comparison of morning and afternoon DGGE bacterial profiles from steers adapted to the diets fed in Experiments 1 and 2 (after 27 and 20 days on the new diet, respectively) revealed that the structure of the rumen bacterial community was particularly stable over at least two days (days 27 and 28, for Experiment 1, and days 20 and 21 Experiment 2). These results indicate that the sampling time had no influence on the assessment of the bacterial community structure and diversity once the animal had adapted to a diet. The inclusion of different amounts of molasses in the diet resulted in significantly different FAB and SAB population structures, in terms of DGG banding patterns (i.e., differences in position and intensity of bands DGGE in the profile). v

6 The main finding in terms of estimated species diversity in samples of rumen contents from Experiment 1 was that steers fed hay-only (0M) or hay plus low levels of molasses (25M) displayed a more diverse FAB population when compared to steers consuming high levels of molasses (50M and 75M). Therefore, it appears that replacing slowly degradable low-quality fibre for readily available non-structural carbohydrate resulted in decreased FAB species diversity, as determined by diversity indexes calculated from the number and intensity of DGGE bands in each profile. FAB DGGE patterns were significantly different to SAB patterns for the 0M diet but not for the 75M diet. Feeding a diet of pangola hay and molasses (50:50) with or without different nitrogen sources (Experiment 2) had no effect on FAB structure; however, differences in SAB structure were detected between the HM and MCAS. The response of the SAB population to casein addition, a highly degradable true protein source may be associated with a greater supply of branched-chain fatty acids that became available after degradation of branched-chain amino acids. Diversity indices for FAB and SAB samples collected in Experiment 2 showed no diet or steer effect. No differences were found in the structure of FAB and SAB between steers consuming the same diet. Phylogenetic diversity in samples from Experiment 1 was assessed by cloning, sequencing and phylogenetic analysis of excised DGGE bands. Between one and four bands were excised per profile. Dominant FAB and unique SAB bands were reamplified and cloned. A total of 269 partial 16S rdna sequences were retrieved from FAB and SAB DGGE clone libraries. Phylogenetic relationships between unique sequences and their closest culturable rumen bacteria were estimated. The majority of the phylotypes (47-67%) belonged to the Firmicutes phylum and this result was independent of the diet the steer was fed. Phylotypes belonging to Bacteroidetes represented between 33-44% of the total phylotypes. The following phyla were also represented: Fibrobacteria (6%; detected in 25M only), and the proposed OP11 phylogenetic division (3% in both 0M and 25M diets). The majority of the phylotypes identified in this study were unknown species. Some of the phylotypes identified, although unknown, had been reported to be present in the gastrointestinal system of native African herbivores, Norwegian reindeer, and cattle and sheep consuming diverse diets. Of the 90 phylotypes identified, four had sequences identical to those of known culturable bacteria. These were: Butyrivibrio fibrisolvens (detected in 0M, 25M and 75M diets), Clostridium polysaccharolyticum (25M and 75M diets), Ruminococcus albus (0M diet), and Fibrobacter succinogenes vi

7 (25M diet). Phylogenetic analysis demonstrated that dominant bacterial populations in the rumen of steers fed hay and hay plus a small amount of molasses were different from those recovered from the rumen of steers fed high levels of molasses. In particular, phylotypes related to unclassified clostridia appeared to be present only when the proportion of hay in the diets was high; whereas Selenomonas-Quinella type bacteria were more prevalent in the rumen of steers fed high-molasses diets. The presence of Quinella ovalis-like bacteria as dominant organisms in high-molasses diets was confirmed by real time PCR enumeration and microscopy. Q. ovalis-like bacteria tended to increase as the percentage of molasses in the diet increased while the total rumen bacterial populations remained relatively stable. Q. ovalis-like bacteria comprised between 1 and 61% of the total bacteria population (20% average) in 75M. DM intake and digestible organic matter increased quadratically while DM digestibility of the diet, MPS production and the efficiency of MPS increased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen ph, ammonia and VFA concentration in the rumen fluid, urine ph or fluid and solid outflow rate. Significant correlations were found between rumen fluid ph, total and individual volatile fatty acids content, fractional outflow rate of the fluid and solid phases, microbial protein synthesis, Isotricha population and changes in abundance of certain rumen FAB phylotypes. Thus the populations of Prevotella ruminicola-like bacteria were negatively correlated to MPS. It is possible that these bacteria, which were abundant in the rumen of low quality hay-fed steers, had low growth rates. The positive correlation between the populations of Quinella ovalis-like bacteria (dominant in 50M and 75M diets) and the proportion of propionate in the rumen fluid suggested that these organisms were associated with propionate production. The populations of Ruminococcus albus and Fibrobacter succinogenes were linked to the abundance of the ciliate Isotricha. The results from the present study have shown that the level of molasses influenced the structure of rumen bacterial populations. Phylogenetic shifts in dominant bacteria in contrasting diets were also identified. An increase in molasses resulted in significant changes in nutritional parameters, in particular, an increase in the supply of microbial protein, which the phylogenetic study has shown is comprised of different bacterial species and proportions thereof. vii

8

9 Acknowledgements I should first thank the stars because when I decided to do a PhD in ruminant nutrition in Australia I wanted Dennis Poppi as my supervisor. After applying to the only Australian university I had to, I was accepted and got to work with Dennis. I was thrilled then and after four years, still am. I will concurrently thank Debbie Poppi and the Poppi daughters for receiving the students at home regularly and being so friendly with us. Dennis was not only my principal supervisor but also a mentor. He has encouraged me to move out in the research world. I highly value each and every time Dennis introduced me to local and international colleagues in the field. God bless Dennis s connections. I would like to express my sincere gratitude to Athol Klieve, for giving me the opportunity to work on this project knowing I had neither previous experience nor much knowledge in molecular biology. Athol s guidance and enthusiasm during the course of this work was invaluable. I would like to thank Stuart McLennan for his availability to discuss my project at the early stages, his suggestions to improve my literature review and his great sense of humour. This work was made possible by an International Postgraduate Research Scholarship provided by The University of Queensland to which I am sincerely grateful. I am grateful to the Federation of European Microbiological Societies for providing a grant that enabled me to travel to France to attend the Gut Microbiology Conference. I am particularly thankful to Wayne Bryden, Head of the School of Animal Studies, for partly funding my trip to France and for providing an extension to my scholarship. I wish to thank Tuyen Dinh, PhD student, with whom I collaborated in both animal experiments. Les Gardiner, Andrew Gibbon and Jim Kidd were involved in the animal experimentation phase at Mt. Cotton Research Farm. I truly appreciate their expert assistance in animal care and sample collection. Neil McMeniman and the vet students that performed the cannulation surgery on the steers and Chris Edgecumbe for technical assistance during the first animal experiment are also cordially acknowledged. Diane Ouwerkerk, my unofficial supervisor, provided critical support in my day to day lab life. It is a pleasure to acknowledge her guidance, assistance, and patience. I very much appreciate the help provided by Lyle McMillen in real time PCR analyses and for offering constructive advice on drafts of this thesis. I am totally grateful to Tony Swain for making time in his very busy schedule to offer statistical assistance. Many others contributed to this project; I specially thank Jess Morgan for offering me insights into phylogenetics and for answering my questions. I thank Claus Christophersen, a PhD student at the University of Western Australia, for the DGGE gradient mixer gift. I would still be repeating gels if it wasn t for him! I am thankful to Pat Blackball for sharing with me the precious BioNumerics software, to Adam Pytko for facilitating office space and resources for the writing stage of the thesis. Many thanks go to the technical staff at the rumen ecology group, Department of Primary Industries and Fisheries, especially Morgan O Leary for pouring more than she had to and Kerrin Morrisy for proofreading drafts of this thesis. A special thanks goes to Maree Bowen, Fran Cowley, Sounthi Subaaharan, Susana Chaves, Sol Rojas, Jamie Taratoot, Ximenita Trejo, Sue Monk and Jo Wrigley for their friendship during these years and to Chris McCorkindale for his company all those long weekends at the lab. Este trabajo esta dedicado a Fernanda Alvarez, mi madre, soporte invariable de mis proyectos. ix

10

11 Publications and presentations Tolosa, X., Dinh Van, T., Klieve, A.V., Poppi, D.P., McLennan, S.R., Kidd, J.F., Ouwerkerk, D., Maguire, A.J Effect of diet type on rumen microbial populations. Abstract. Presented at MicroNZ 2003: Combined annual scientific meeting of the Australian and New Zealand Societies for Microbiology. 28 September-2 October. Auckland, New Zealand. Tolosa, M.X., Dinh Van, T., Klieve, A.V., Ouwerkerk, D., Poppi, D.P., McLennan, S.R Effect of molasses diets on population profiles of rumen bacteria. Reproduction Nutrition Development Supplement 1 Special Edition June th INRA-RRI Symposium on Gut Microbiology. Concerns and Responses to food Safety, Health and Environmental Issues June. Clermont-Ferrand, France. Tolosa, M.X., Dinh Van, T., Klieve, A.V., Ouwerkerk, D., Poppi, D.P., McLennan, S.R Molecular characterisation of rumen bacterial population in cattle fed molasses diets. Abstract. Animal Production in Australia 25, 328. Australian Society of Animal Production 25 th Biennial Conference. 4-8 July. Melbourne, Australia. xi

12

13 List of Abbreviations μg Microgram μl Microlitre μm Micrometer μm Micromolar 16S rrna 16S ribosomal RNA gene ADF Acid detergent fibre AEC Animal Ethics Committee ANOVA Analysis of variance ATP Adenosine triphosphate BCFA Branched-chain fatty acids bp Base pairs BW Body weight CCA Canonical correlation analysis CrEDTA Chromium-EDTA CP Crude protein CSM Cottonseed meal DGGE Denaturing gradient gel electrophoresis DM Dry matter DMD Dry matter digestibility DMI Dry matter intake DNA Deoxyribonucleic acid DOM Digestible organic matter DOMI Digestible organic matter intake E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EV Explanatory variables emcp Efficiency of microbial protein synthesis FAB Solid-associated bacteria HM 50% molasses-50% pangola hay kb kilobase L Litre LB Luria Bertani media LSD Least significant difference MGBNFQ Minor Grove Binding Non-Fluorescence Quencher min Minute ml Millilitre mm Millimolar MPS Microbial protein synthesis N Nitrogen NCBI National Center for Biotechnology Information NDF Neutral detergent fibre ng Nanogram NJ Neighbour joining method NSC Non-structural carbohydrates NSC:RDP Ratio between non-structural carbohydrate and rumen degradable protein OMADR Organic matter apparently digested in the rumen OMTDR Organic matter truly digested in the rumen PCA Principal component analysis xiii

14 PCO PCR PD pm rdna rrna RDP RNase SAB SC:NSC sdh 2 O sec TBE TE UPGMA V V2-V3 VFA w/v w/w Yb Principal coordinate analysis Polymerase chain reaction Purine derivatives Picomolar Ribosomal deoxyribonucleic acid Ribosomal ribonucleic acid Rumen degradable protein Ribonuclease Fluid-associated bacteria Structural carbohydrates to non-structural carbohydrates ratio Sterile distilled water Second Tris-Borate-EDTA Tris-EDTA Unweighted pair group method with arithmetic mean variable regions of the 16S rrna gene Variable regions 2 and 3 of the 16S rrna gene Volatile fatty acids Weight by volume Weight by weight Ytterbium xiv

15 TABLE OF CONTENTS Chapter 1 Dietary effect on rumen microbial protein synthesis and the rumen bacterial community: A review of the literature Introduction Features of tropical animal production systems Importance of microbial protein for cattle nutrition Nutritional factors that affect the efficiency of microbial protein synthesis Carbohydrates Protein Interaction between carbohydrate and protein Physiological and chemical factors that affect the efficiency of microbial protein synthesis Rumen dilution rate The interaction between diet, emps and rumen microbial ecology Features of high-molasses feeding Overview of rumen microbial ecology Bacteria Protozoa Fungi Bacteriophages Molecular techniques to study rumen ecology Phylogeny determination: From DNA extraction to bacterial identification DNA extraction Polymerase Chain Reaction (PCR) Denaturing Gradient Gel Electrophoresis (DGGE) Analysis of DGGE patterns Correlation between DGGE patterns and environmental variables Phylogenetic tree construction Real time Polymerase Chain Reaction Key issues identified in the literature Thesis proposal...56 Chapter 2 General materials and methods Introduction Animals, ethical approval and experimental site Experimental design and diets Experiment Experiment Collection of rumen fluid and rumen solid phase samples Sampling method Sampling regime...62 xv

16 2.5. Processing of solid-phase samples Bacterial DNA extraction DNA quantitation PCR amplification of 16S rrna gene PCR amplification of V2-V3 region of the 16S rrna gene Agarose gel electrophoresis Denaturing gradient gel electrophoresis (DGGE) DGGE image analysis Calculation of diversity indices and species evenness from DGGE data DGGE band excision and amplification Purification of PCR products Cloning of PCR products Transformation of competent cells Analysis of transformants Plasmid DNA extraction Sequencing Sequence assembly and alignment Phylogenetic analysis Statistical analysis DGGE patterns and diversity estimators Correlation between DGGE patterns and rumen environmental variables...74 Chapter 3 Studies on the stability of the rumen bacterial population Introduction Materials and methods Experiment 1: Animals and diets Experiment 2: Animals and diets Sample collection and processing Denaturing gradient gel electrophoresis image analysis Statistical analysis of DGGE data Results Analysis of DGGE patterns: Experiment Analysis of DGGE patterns: Experiment Bacterial diversity indices Discussion DGGE profiles from fluid-associated bacteria DGGE profiles from solid-associated bacteria...94 xvi

17 3.4.3 Bacterial diversity Conclusion...97 Chapter 4 Effect of molasses-hay diets on molecular profiles of rumen bacteria Introduction Materials and methods Samples used for DGGE analysis Denaturing Gradient Gel Electrophoresis Statistical analysis Results Analysis of DGGE patterns during molasses introduction (Experiment 1) Effect of replacing hay by molasses on FAB and SAB DGGE patterns (Experiment 1) Effect of diet on fluid-associated bacteria Effect of diet on solid-associated bacteria Comparison of fluid- and solid-associated bacteria within two steers Effect of hay/molasses plus different sources of N on FAB and SAB DGGE patterns: Experiment Effect of diet on fluid-associated bacteria Effect of diet on solid-associated bacteria Discussion DGGE patterns during stepwise increase of molasses in the diets Effect of diet on FAB DGGE profiles and diversity Effect of diet on SAB DGGE profiles and diversity Differences between FAB and SAB DGGE profiles and diversity Conclusion Chapter 5 Dominant bacteria present in the rumen of molassesfed steers: Phylogenetic studies Introduction Materials and methods Results Discussion Conclusion xvii

18 Chapter 6 Enumeration of Quinella ovalis-like bacteria in the rumen of steers fed high-molasses diets Introduction Materials and methods Animals, diets and sampling DNA extraction, amplification and sequencing Real time PCR Primer and probe design for total rumen bacterial assay Total rumen bacterial assay conditions Total rumen bacterial assay calibration Primer and probe design for Q. ovalis-like bacteria assay Q. ovalis-like bacterial assay conditions Q. ovalis-like bacterial assay calibration Primer specificity test for Q. ovalis-like bacteria Statistical analysis Results Real time PCR of total rumen bacteria Real time PCR of Q. ovalis-like bacteria Discussion Conclusion Chapter 7 Relationship between rumen environmental parameters and rumen bacterial population structure in molasses-fed steers Introduction Materials and methods Animals and diets Sample collection and processing bacterial molecular studies Sample collection and processing animal nutrition studies Statistical analysis Results Discussion Conclusion Chapter 8 General discussion: Molecular ecology of rumen bacterial populations in steers fed molasses diets FAB structure fluctuates during the adaptation phase to new diets and is stable after 2 to 3 weeks xviii

19 8.2 Changes in structure, diversity and phylogeny of dominant rumen bacteria Dominance of Quinella ovalis-like organisms in the rumen of animals fed high-molasses diets Relationship between the abundance of dominant rumen bacteria and parameters indicative of rumen function Future research Conclusions Appendix I. Steps involved in the study of structure and phylogeny of rumen bacterial communities Appendix II.A Multiple sequence alignment of 16S rrna partial sequences retrieved from the fluid and solid-phase of the rumen of steers fed hay/molasses diets Appendix II.B Pairwise similarity matrix of sequences retrieved from the fluid and solid-phase of the rumen of steers fed hay/molasses diets Appendix III.A Parameters measured in the rumen of steers consuming hay and different proportions of molasses Appendix III.B Protozoal concentration (%) in the rumen of four cannulated steers consuming hay and different proportions of molasses References xix

20 TABLE OF FIGURES Figure 1.1 Effect of microbial growth rate on yield of: cellulolytic bacteria (curve A), mixed rumen bacteria (curve B), and Streptococcus bovis (curve C) from various feed components. Curves A, B and C indicate that microbial growth is influenced by energy used for maintenance (M). (reproduced from Sniffen and Robinson, 1987)....4 Figure 1.2 Effect of addition of increasing levels of urea (level 1 to level 6 equivalent to 10.2 to mg NH 3 -N/L, respectively) on ammonia concentration in fluid ( ), DM disappearance of mature spear grass hay ( ), and microbial protein synthesis (non-ammonia N (NAN), ) (Morrison and Mackie, 1996 adapted from Morrison et al., 1998)...8 Figure 1.3 The effect of non-structural carbohydrate:degradable intake protein ratio (NSC:DIP) on the efficiency of microbial protein synthesis (g microbial N/kg DMD) 54% NSC; 37% NSC; and 25% NSC (Stokes et al., 1991a)...13 Figure 1.4 DGGE profiles of bacteria present in rumen fluid phase (F), associated with (As) or adherent (Ad) to plant particles. Fluid and digesta samples were collected from four sheep fed either a mixture of corn and hay and grass hay only. Reproduced from Larue et al. (2005) Figure 1.5 Phylogenetic placement of 16S rdna sequences of rumen bacteria. Numbers by each node are confidence levels generated from 1000 bootstrap trees (Mackie et al., 1999)...52 Figure 2.1 Diagram of molecular methods used to study the rumen bacterial community structure Figure 3.1 Cluster analysis of 16 FAB DGGE profiles of PCR-amplified V2-V3 region of 16S rdna from four cannulated steers consuming four different diets (Experiment 1). Similarities among samples (profiles) taken in the morning and afternoon on the last two days of experimental period two were calculated Figure 3.2 Cluster analysis of 16 SAB DGGE profiles of PCR-amplified V2-V3 region of 16S rdna from four steers consuming four different diets (Experiment 1). Similarities among samples (profiles) taken in the morning and afternoon on the last two days of experimental period two were calculated Figure 3.3 Principal coordinate analysis plot of FAB DGGE profiles obtained from four steers consuming four different diets ( = 0M, steer 106; = 25M, steer 076; = 50M steer 102; and = 75M, steer 112; Experiment 1). Samples (DGGE profiles) were taken in the morning (unfilled symbols) and afternoon (filled symbols) on two consecutive days from experimental period two, after a three-week adaptation period...84 Figure 3.4 Principal coordinate analysis plot of SAB DGGE profiles obtained from four steers consuming four different diets ( = 0M, steer 106; = 25M, steer 076; = 50M steer 102; and = 75M, steer 112; Experiment 1). Samples were taken in the morning (unfilled symbols) and afternoon (filled symbols) on two consecutive days from experimental period two, after a three-week adaptation period Figure 3.5 Cluster analysis of 16 FAB DGGE profiles of PCR-amplified V2-V3 region of 16S rdna from four cannulated steers consuming four different diets (Experiment 2). Similarities among samples (profiles) taken in the morning and afternoon on the last two days of experimental period one were calculated xx

21 Figure 3.6 Cluster analysis of 16 SAB DGGE profiles of PCR-amplified V2-V3 region of 16S rdna from four steers consuming four different diets (Experiment 2). Similarities among samples (profiles) taken in the morning and afternoon on the last two days of experimental period one were calculated...87 Figure 3.7 Principal coordinate analysis plot of FAB DGGE profiles obtained from four steers consuming four different diets (Ο = HM, steer 106; = MU, steer 112; = MCAS steer 102; and = MCSM, steer 076; Experiment 2). Samples were taken in the morning (unfilled symbols) and afternoon (filled symbols) on two consecutive days in experimental period one, after a two-week adaptation period Figure 3.8 Principal coordinate analysis plot of SAB DGGE profiles obtained from four steers consuming four different diets ( = HM, steer 106; = MU, steer 112; = MCAS steer 102; and = MCSM, steer 076; Experiment 2). Samples were taken in the morning (unfilled symbols) and afternoon (filled symbols) on two consecutive days in experimental period one, after a two-week adaptation period Figure 4.1 Cluster analysis of 14 FAB DGGE patterns obtained from four steers in Experiment 1. Similarities among profiles from all steers during the first eight days of the first period and on the last day of the first period (day 28) were calculated Figure 4.2 Principal coordinate analysis plot of FAB DGGE profiles obtained from four steers during the period of stepwise introduction of molasses to the diets (Experiment 1). Samples (DGGE profiles) were taken on five occasions: day one ( = 0M), day three ( = 25M), day six ( = 50M), day eight ( = 75M), and day 28. The arrows mark samples taken from four steers on the last occasion (day 28). The fours steers are distinguished by colour (black = steer 106, red = steer 112, green = steer 102, and blue = steer 076) Figure 4.3 Cluster analysis of 16 FAB DGGE patterns obtained from four steers in Experiment 1. Similarities among samples (profiles) taken from all steers on the last day of each experimental period (day 28) were calculated Figure 4.4 Principal coordinate analysis plot of FAB DGGE profiles obtained from four steers on day 28 of each period (Experiment 1). Symbols indicate diets ( = 0M, = 25M, = 50M, = 75M) and colours indicate steers (black = steer 106, red = steer 112, green = steer 102, and blue = steer 076) Figure 4.5 Cluster analysis of 16 SAB DGGE patterns obtained from four steers on in Experiment 1. Similarities among samples (profiles) taken from all steers on the last day of each experimental period (day 28) were calculated Figure 4.6 Principal coordinate analysis plot of SAB DGGE profiles obtained from four steers on day 28 of each period (Experiment 1). Symbols indicate diets ( = 0M, Δ = 25M, = 50M, = 75M) and colours indicate steers (black = steer 106, red = steer 112, green = steer 102, and blue = steer 076) Figure 4.7 Cluster analyses of four FAB and their corresponding four SAB DGGE patterns. Dendrogram A shows similarities among samples from steer 112 consuming 75M (5:75 pangola hay/molasses) diet and dendrogram B presents similarities among samples from steer 106 consuming 0M (100% pangola hay). Similarities among samples taken in the morning and afternoon of days 27 and 28 of period two were calculated (Experiment 1) Figure 4.8 Principal coordinate analysis plots of four FAB (unfilled symbols) and their corresponding four SAB DGGE profiles (filled symbols) obtained from steer 112 consuming 75M diet (plot A) and from steer 106 consuming 0M diet xxi

22 (plot B). Samples were taken in the morning and afternoon on day 27 and 28 of period two (Experiment 1). Symbols indicate diets ( = 0M; = 75M) Figure 4.9 Cluster analysis of 16 FAB DGGE patterns obtained from four steers in Experiment 2. Similarities among samples (profiles) taken from all steers on the last day of each experimental period (day 21) were calculated Figure 4.10 Principal coordinate analysis plot of FAB DGGE profiles obtained from four steers on day 21 of each period of Experiment 2. Symbols indicate diets ( = HM, = MU, = MCAS, = MCSM) and colours indicate steers (black = steer 106, red = steer 112, green = steer 102, and blue = steer 076) Figure 4.11 Cluster analysis of 16 SAB DGGE patterns obtained from four steers in Experiment 2. Similarities among samples (profiles) taken from all steers on the last day of each experimental period (day 21) were calculated Figure 4.12 Principal coordinate analysis plot of SAB DGGE profiles obtained from four steers on day 21 of each period (Experiment 2). Symbols indicate diets ( = HM, = MU, = MCAS, = MCSM) and colours indicate steers (black = steer 106, red = steer 112, green = steer 102, and blue = steer 076) Figure 5.1-A Denaturing gradient gel electrophoresis (DGGE) fingerprint patterns of V2-V3 fragments obtained from the rumen fluid of two steers (076 and 112) consuming the following diets: 100% pangola hay (0M), 75:25 pangola hay/molasses, (25M), 50:50 pangola hay/molasses (50M) and 25:75 pangola hay/molasses (75M) Figure 5.1-B Denaturing gradient gel electrophoresis (DGGE) fingerprint patterns of V2-V3 fragments obtained from the rumen fluid of two steers (106 and 102) consuming the following diets: 100% pangola hay (0M), 75:25 pangola hay/molasses, (25M), 50:50 pangola hay/molasses (50M) and 25:75 pangola hay/molasses (75M) Figure 5.1-C Denaturing gradient gel electrophoresis (DGGE) fingerprint patterns of V2-V3 fragments obtained from the solid phase of two steers (076 and 112) consuming the following diets: 100% pangola hay (0M), 75:25 pangola hay/molasses, (25M), 50:50 pangola hay/molasses (50M) and 25:75 pangola hay/molasses (75M) Figure 5.1-D Denaturing gradient gel electrophoresis (DGGE) fingerprint patterns of V2-V3 fragments obtained from the solid phase of two steers (106 and 102) consuming the following diets: 100% pangola hay (0M), 75:25 pangola hay/molasses, (25M), 50:50 pangola hay/molasses (50M) and 25:75 pangola hay/molasses (75M) Figure 5.2 Neighbour joining phylogenetic tree of dominant sequences identified in the rumen of four steers fed molasses/hay diets Figure 6.1 Cell numbers of total rumen bacteria present in the rumen fluid of four steers consuming four diets as follows: 100% pangola hay (0M), 25% molasses plus 75% pangola hay (25M), 50% molasses plus 50% pangola hay (50M) and 75% molasses plus 25% hay (75M) Figure 6.2 Representative Real Time PCR assay for quantification of Q. ovalis-like bacteria Figure 6.3 Standard curve of the data presented in Figure Figure 7.1 CCA plot showing the six most dominant DGGE bands and their relationship to MPS, emps and digestibility of OM, DM and NDF (represented by the arrows) xxii

23 TABLE OF TABLES Table 1.1 Summary of main metabolic activities of typical and predominant rumen bacterial isolates (extracted from Mackie et al., 2002)...30 Table 1.2 Effects of protozoa on rumen characteristics and animal performance (Williams and Coleman, 1997)...33 Table 2.1 Final chemical composition of diets used in Experiment 1 (see text for treatment description)...60 Table 2.2 Dry matter content and final chemical composition of diets used in Experiment 2 (see text for description of diets)...61 Table 3.1 Species richness estimate (total number of bands), Simpson and Shannon diversity indices and species evenness for FAB and their corresponding SAB samples obtained from steers consuming molasses/pangola hay diets, on two consecutive days. Samples were collected on the second period of Experiment Table 3.2 Species richness estimate (total number of bands), Simpson and Shannon diversity indices and species evenness for rumen FAB and their corresponding SAB samples obtained from steers consuming molasses/pangola hay with or without N supplementation, on two consecutive days. Samples were collected on the first period of Experiment Table 4.1 Mean values for species richness estimate (number of bands), Simpson and Shannon diversity indices and species evenness for rumen FAB samples obtained from four steers consuming pangola hay/molasses diets on days 1, 3, 6, 8 and 28 of period one, Experiment Table 4.2 Species richness estimate (number of bands), Simpson and Shannon diversity indices and species evenness values for rumen FAB samples obtained from four steers consuming molasses/pangola hay diets (all periods, Experiment 1) Table 4.3 Predicted values for total number of bands, Simpson and Shannon diversity indices and species evenness for SAB samples obtained after gel and run effects were removed. Samples were collected from four steers consuming molasses/pangola hay diets (all periods, Experiment 1) Table 4.4 Species richness estimate (number of bands), Simpson and Shannon diversity indices and species evenness values for rumen FAB samples and their SAB counterparts obtained from two steers consuming contrasting diets (0M and 75M) on days 27 and 28 of the second period of Experiment Table 4.5 Species richness estimate (total number of bands), Simpson and Shannon diversity indices and species evenness values for rumen FAB samples obtained from four steers consuming pangola hay/molasses with or without N supplementation on day 21 of each period of Experiment Table 4.6 Species richness estimate (total number of bands), Simpson and Shannon diversity indices and species evenness predicted mean values for rumen SAB samples obtained after gel and run effect were removed. Samples were collected from four steers consuming pangola hay/molasses with or without N supplementation on day 21 of each period of Experiment Table 5.1 Closest matches to previously reported sequences in the GenBank public database Table 5.2 Phylotype classification by diet and phase of the rumen Table 6.1 Quantification of Q. ovalis-like bacteria (cells/ml rumen fluid ± s.d.) present in the rumen fluid of steers during the period of molasses introduction to the xxiii

24 diets (Experiment 1) by real time PCR. Samples were taken between days 1 and 8 of period one. See text for diet designation Table 6.2 Quantification of total bacteria (E. coli cell equivalents (ECCE)/ml) and Q. ovalis-like bacteria (cells/ml rumen fluid ± s.d.) present in the rumen fluid of four steers fed molasses plus hay by real time PCR. All samples were taken on day 28 of each period. See text for diet designation Table 7.1 Pearson correlation values between DGGE band intensity and chemical, biological, and physiological parameters measured in the rumen. See text for details on units for each measurement xxiv