Pathogenesis of subacute ruminal acidosis in sheep

Transcription

1 Pathogenesis of subacute ruminal acidosis in sheep Joshua Patrick Fanning A thesis submitted for fulfilment of the requirements for the degree of Doctorate of Philosophy School of Animal and Veterinary Science University of Adelaide January 2016

2 Table of contents Abstract... 8 Declaration Acknowledgements Introduction and literature review The ruminal environment Ruminal epithelium Morphology Epithelial absorption, metabolism and buffering Ruminal digesta Ruminal contents passage Saliva secretion Acute ruminal acidosis Subacute ruminal acidosis Conclusions Common methods Histology Volatile fatty acid and lactic acid analysis Ruminal ph loggers Prevalence of acidosis in weaner lambs: A field trial using five different diets Introduction Materials and Methods Animal husbandry and diets Statistical analyses of results Dietary effects on ruminal fluid ph Dietary effects on weight change and ruminal volatile fatty acid and lactic acid concentration The relationship between weight change and volatile fatty acid concentration and ruminal fluid ph Pathogenesis of subacute ruminal acidosis in sheep Page 2 of 182

3 3.3 Results Dietary effects on ruminal fluid ph Dietary effects on weight change and ruminal volatile fatty acid and lactic acid concentration The relationship between weight change and volatile fatty acid concentration and ruminal fluid ph Analysis of percentage increase in live weight Analysis of quantitative live weight change adjusted for live weight Discussion Development of experimental diets and experimental design Dietary effects on ruminal fluid ph Dietary effects on weight change, and ruminal fluid volatile fatty acid and lactic acid concentration The effect of diet on live weight The effect of diet on volatile fatty acids and lactic acid concentrations The relationship between weight change and volatile fatty acid concentration and ruminal fluid ph Conclusions The relative contributions of the ruminal epithelium and the ruminal fluid to the development of acidosis of sheep fed high-energy rations: a transfaunation model Introduction Methods Overview of the experimental design Animals, treatments, and experimental design Surgical procedures and transfaunation Samples taken during surgery Placement of the ph logger Processing and analysis of ruminal biopsy samples Glucose challenge and sampling protocols In-vitro incubation of ruminal fluid with glucose or water Pathogenesis of subacute ruminal acidosis in sheep Page 3 of 182

4 4.2.9 Statistical analysis of data Comparison between sheep on high and low energy density diets prior to transfaunation In-vivo glucose challenge post transfaunation Results Feed Intake Rumen ph logger variation Comparison between sheep on high and low energy density diets prior to transfaunation Ruminal epithelium Ruminal fluid In-vivo glucose challenge post transfaunation Qualitative assessment of ruminal fluid ph, volatile fatty acid and lactic acid concentrations Areas under the curve for ruminal fluid ph and ruminal volatile fatty acid and lactate concentrations for the four experimental groups Discussion Comparison between sheep on each diet at transfaunation Ruminal epithelial differences between the sheep fed the low and high energy density diets Ruminal fluid Summary of ruminal adaptation prior to transfaunation In-vivo glucose challenge post transfaunation Experimental Model Qualitative and statistical assessment Conclusions Evaluation of an experimental diet to induce Subacute Ruminal Acidosis (SARA) in lambs Introduction Methods Ruminal surgical fistulation and cannulation Pathogenesis of subacute ruminal acidosis in sheep Page 4 of 182

5 5.1.3 Results Discussion Conclusion The effect of ruminal fluid ph, volume and papillae on ruminal absorption and clearance of volatile fatty acids Introduction Methods Sheep housing, diets and experimental procedures Marker infusion and sampling protocol Chromium analysis Calculation of rumen fluid clearance rate and valeric acid absorption rate Statistical analysis The effect of diet on ruminal papillae The effect of diet on absorption and clearance rates The relationship between the ruminal papillae and absorption Ruminal ph effects on the absorption rate of valeric acid and the ruminal fluid clearance rate in sheep fed a lucerne chaff and rolled barley diet Results Rumen ph logger variation The effect of diet on ruminal papillae The effect of diet on absorption and clearance rates The relationship between the ruminal papillae and absorption Ruminal ph effects on the absorption rate of valeric acid and the ruminal fluid clearance rate in sheep fed a lucerne chaff and rolled barley diet Discussion Suitability of the marker infusion The effect of diet on the absorption and clearance rates The impact of diet on the ruminal papillae The relationship between the ruminal papillae and absorption Pathogenesis of subacute ruminal acidosis in sheep Page 5 of 182

6 6.4.5 The association between ruminal ph and the absorption rate of valeric acid or the ruminal fluid clearance rate in sheep fed a lucerne chaff and rolled barley diet Conclusions General Discussion Normal rumen function and physiology Ruminal acidosis The prevalence of acidosis The effect of the ruminal papillae and ruminal fluid on acidosis An experimental diet to induce subacute ruminal acidosis Absorption and clearance Critical appraisal of current experiments and suggested refinements Ruminal sampling techniques Individual Experiments Prevalence of acidosis in weaner lambs: A field trial using five different diets The relative contributions of the ruminal epithelium and the ruminal fluid to the development of acidosis of sheep fed high-energy rations: a transfaunation model Evaluation of an experimental diet to induce Subacute Ruminal Acidosis (SARA) in lambs The effect of ruminal fluid ph, volume and papillae on ruminal absorption and clearance of volatile fatty acids Ruminal papillae Variation in ruminal papillae Rumen papillae analysis techniques Statistical analyses in relation to interactions Important questions still to be answered Definitions and relevance of subacute and acute ruminal acidosis in the sheep industry Statistical analyses in relation to interactions and required experimental units Conclusion Appendices Pathogenesis of subacute ruminal acidosis in sheep Page 6 of 182

7 8.1 Appendix 1: Prevalence of acidosis in weaner lambs: Ruminal fluid ph measurements Appendix 2: Dry Matter Intake The relative contributions of the ruminal epithelium and the ruminal fluid to the development of acidosis of sheep fed high-energy rations: a transfaunation model The effect of ruminal fluid ph, volume and papillae on ruminal absorption and clearance of volatile fatty acids logger drift Appendix 3: Ruminal fluid ph logger drift The relative contributions of the ruminal epithelium and the ruminal fluid to the development of acidosis of sheep fed high-energy rations: a transfaunation model Comparison between sheep on high and low energy density diets prior to transfaunation logger drift In-vivo glucose challenge post transfaunation logger drift The effect of ruminal fluid ph, volume and papillae on ruminal absorption and clearance of volatile fatty acids logger drift References Pathogenesis of subacute ruminal acidosis in sheep Page 7 of 182

8 Abstract Ruminal acidosis is an economically important condition in ruminants fed diets with a high energy density. Two forms of ruminal acidosis have been described; subacute ruminal acidosis (SARA), when the ruminal fluid in in the ph range and acute ruminal acidosis (ARA), when the ruminal fluid ph is <5.0. Animals with SARA, when compared to normal animals with ruminal fluid ph >5.5, have increased ruminal volatile fatty acid (VFA) concentrations and decreased lactate concentrations. Animals with ARA have increased ruminal lactic acid concentrations, and lower ruminal VFA concentrations, when compared to normal animals. The prevalence of SARA in sheep fed diets with a high energy density is unknown. The pathogenesis of SARA is also poorly understood in cattle and sheep. Mechanisms that regulate the ruminal fluid ph include acid buffering by saliva, which contains high concentrations of bicarbonate, VFA absorption through the ruminal epithelium, VFA metabolism by ruminal microbes and by ruminal fluid clearance/passage from the rumen by passage into the more distal gastrointestinal tract. Subacute ruminal acidosis occurs when the regulatory mechanisms that maintain the ruminal fluid ph >5.5 fail. Their relative contribution of absorption of clearance mechanisms to the pathogenesis of SARA is unknown. Adaptation of ruminal microbes and the ruminal epithelium is thought to be essential in reducing the prevalence and severity of SARA Microbial and ruminal epithelial adaptation in the pathogenesis of SARA are poorly understood. The aims of the thesis were to investigate the occurrence of SARA in sheep on diets with a high energy density and to evaluate the role of dietary adaptation in the pathogenesis of SARA in sheep. The first study examined the prevalence of SARA in sheep fed a range of diets with high energy densities. Between 2 and 16 % of lambs fed a high energy density diet were diagnosed with SARA, but 50% of the lambs were at risk of SARA as defined by a ph range of There was no correlation observed between ruminal fluid ph and lamb growth rate.. The second experiment was designed to investigate the relative impact of two variables: rumen epithelium adaptation and rumen fluid adaptation. No significant differences could be identified within the power of the experiment. The results also highlighted that lactic acid can be present in the rumen with a ruminal fluid ph between ph 5.0 to 6.0 suggesting that the categorisation of VFA and ARA based upon relative VFA and lactate concentrations may be flawed and that a graded continuum exists. Pathogenesis of subacute ruminal acidosis in sheep Page 8 of 182

9 A further study investigated VFA absorption and clearance from the ruminal fluid of sheep fed a high energy density diet formulated to induce SARA. These results showed an increased absorption rate and an increased ruminal clearance rate with a ruminal fluid ph in the ph range compared to >6.0. However, no significant relationship was found between the VFA absorption and clearance rate and a rumen fluid ph in the subacute ruminal acidosis ph range. A significant contribution of dietary adaptation and rumen fluid clearance in sheep on the regulation of VFA concentration and rumen fluid ph in the development of SARA could not be identified within the power of these experiments. Further research is required to identify a more consistent model of SARA to quantify the importance of the condition within the sheep feedlot industry. Pathogenesis of subacute ruminal acidosis in sheep Page 9 of 182

10 Declaration I certify that this work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. In addition, I certify that no part of this work will, in the future, be used in a submission for any other degree or diploma in any university or other tertiary institution without the prior approval of the University of Adelaide and where applicable, any partner institution responsible for the joint-award of this degree. I give consent to this copy of my thesis, when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act I also give permission for the digital version of my thesis to be made available on the web, via the University s digital research repository, the Library catalogue and also through web search engines, unless permission has been granted by the University to restrict access for a period of time. Joshua Fanning Pathogenesis of subacute ruminal acidosis in sheep Page 10 of 182

11 Acknowledgements The production of this thesis would not have been possible without the assistance of the following people. The mentoring provided by my two supervisors was vital in the completion of my experiments and this thesis. Professor Peter Cockcroft has been consistently available, and provided valuable support in experimental procedures, data collection and thesis construction. Professor Phil Hynd has provided significant support in experimental development and procedures. I will be forever grateful for their support. This PhD involved large animal experiments and numerous laboratory methods. Dr Brian Siebert has been instrumental in developing methods for ruminal fluid analysis and assisting with running the gas chromatograph. Dr David Tivey also provided guidance in revamping a gas chromatograph machine when required. I would like to thank Nicole Heberle, Natasha Penno and Rebecca Forder for teaching me the histological methods required for analysis of the ruminal papillae. Surgery days and some days of data collection required significant help due to the numerous tasks required to run animal experiments. The following people provided assistance when required; Sarah Greenslade, Kylie Chenoweth, Michael Wilkes, Ruth Lindsay, Susan Secomb, Kath Brice, Robyn Terry, Kate Lennon, Sharna Coleman, Margie McEwen, Cate Crieghton, Sarah Weaver, Lisa Treatch, Amy Dunstone and Helen Brodie. Data for two of the four experiments within this thesis were collected from experiments that were run by others with assistance from me. Data for the SARA model experiment was collected from an honours experiment run by Amy Dunstone under the guidance of Phil Hynd, Peter Cockcroft and me. The field trial using weaner lambs was run as part of a third year Animal nutrition course, and the students managed the sheep for the experimental period under the guidance of Jim Franklin-McEvoy and Phil Hynd. In both of these trials data used with this experiment was collected by myself with assistance or under my guidance. The analysis of the large amounts of data collected throughout this series of experiments was complicated and beyond my knowledge. Dr David Rutley provided statistical support, spending numerous hours teaching me model design and providing the appropriate knowledge for data analysis. When David was not available Cathy Dodd and Wayne Pitchford provided additional assistance. Pathogenesis of subacute ruminal acidosis in sheep Page 11 of 182

12 Editing a thesis can be tiresome and I would to acknowledge Paul Fanning, Dad, for dedicating time in the final weeks before submission, to complete a final edit for grammar and spelling. A job before the completion of a PhD, particularly in a different field of research can lead to difficulties completing. Support from my current manager Dr Grant Hollaway, and the Pathology team at the Department of Environment or Primary Industries, Horsham, Victoria, has provided invaluable assistance in providing the necessary time and motivation to complete this PhD. Finally I would like to thank the staff and students from the School of Animal and Veterinary Science at the University of Adelaide. A PhD receives support from many sources and the discussions, seminars, lab groups and impromptu conversations has provided assistance that is often underestimated, and thus I wish to acknowledge it. Pathogenesis of subacute ruminal acidosis in sheep Page 12 of 182