What Constitutes a Pony

Aspects of adiposity in ponies Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Philosophy by Alexandra Helena Anne Dugdale May 2011 0 Abstract Aspects of adiposity in ponies. Alexandra H.A. Dugdale Obesity is a growing problem for humans and their horses and ponies, yet emaciated animals still form an important part of the work of equine welfare charities. Non-invasive yet reliable methods of assessing equine body fat are required in order to promote management procedures to improve animal welfare. The overall objective of this work was to investigate the application of a horse-specific body condition scoring system in ponies in order to either validate or revise it, or even replace it with a novel system if necessary. Seasonal differences in appetite, body mass (BM) gain, body condition score (BCS) change and direct (ultrasound) and indirect (morphometry and D2O dilution) measures of body fat were explored in two relatively homogeneous groups of mature Welsh mountain pony mares, studied over summer (June –September 2007) and winter (January-April 2008). The ponies in each group were paired so that, at study outset, two ponies were „thin‟ (BCS, 1-3/9); two were „moderate‟ (BCS, 4-6/9); and two were „obese‟ (BCS, 7-9/9). The greatest appetites (peak 4.6% BM as DMI), increases in body mass (~60kg) and in BCS (~3 points) were recorded for ponies of non-obese outset condition in summer (non-ObS, n=4). For ponies of non-obese outset condition in winter (non-ObW, n=3), appetites peaked at 3.5% BM as DMI, BM increased by a mean of 50kg and BCS increased by ~2 points over the 3 month study period. Appetites for all obese (Ob, n=4) ponies remained almost constant (~2% BM as DMI; peak 2.3% BM as DMI) and minimal changes in BM (n=3) and BCS (n=4) were recorded, regardless of season. All measures of body fat increased for non-Ob ponies (non-ObS>non-ObW). An exponential relationship was determined between body fat content and BCS and for values > 6, BCS was not a useful predictor of actual body fat content. The endogenous circannual mechanisms to encourage winter weight loss were insufficient to prevent the development of obesity in ad libitum fed ponies. The effects of dietary restriction to 1% BM as DMI were studied in a group of 5 overweight or obese mature pony mares (BCS 5.6-8/9). Those measures outlined above were likewise recorded. All ponies remained healthy throughout the 12 week trial. Overall, BM reduced by 1% of outset BM per week. Approximately half the lost BM comprised fat, but fatter animals lost relatively more fat. Despite an average loss of ~30 kg BM, BCS did not change appreciably suggesting that BCS was a relatively poor indicator of early weight/fat loss in obese ponies. The relationships between BCS, direct (ultrasonic) and indirect (morphometric and D2O dilution derived) measures of body fat and actual body fat content determined by both physical dissection and chemical cadaver analysis were explored using 7 donated mature Welsh pony mares (BCS 1.25 to 7/9). Body „fat‟ content (dissected white adipose tissue or chemically-extracted lipid fractions) was the most variable constituent of the cadavers (up to 1/3rd body mass), and was non-linearly related to BCS. From these studies, it was also possible to validate the D2O dilution technique for the measurement of total body water and fat in ponies. Contemporaneously gathered data for BCS and body fat (D2O dilution) from 48 separate observations were explored statistically. A non-linear association between body fat content and BCS was confirmed, with a cut off value of BCS 7/9, above which BCS was less useful for determining body fat content. A novel BCS system was created and is undergoing field trials. 1 Acknowledgements First and foremost, my grateful thanks go to World Horse Welfare for their substantial financial support of, and enthusiastic interest in, all the work presented in this thesis. Roly Owers, Andrew Higgins, Sam Chubbock, Chris Williamson, Tony Fleming and Tony Tyler formed the part of World Horse Welfare‟s team that we got to know well. Furthermore, I am indebted to the Biotechnology and Biological Sciences Research Council for the provision of my stipend through the Doctoral Training Award scheme, which enabled the greatest use of charitable funding for research purposes. I would also like to thank Dr.s Caroline Argo and Patricia Harris, my main supervisors, for the concept and creation of the PhD programme and for their continued enthusiastic support and guidance throughout its development and fruition. Thanks also go to my other supervisors, Prof.s Derek Knottenbelt and Hilary Dobson, for their assistance and advice. Many thanks are due to Gemma Curtis, my partner in crime, who helped me overcome, on numerous occasions, a flooded lab, flooded stables and unserviceable equipment to get the data gathered and analysed more or less on schedule. Keep smiling! Jean Wheeler and Helen Braid assisted in developing our numerous risk assessments and Jean Routly, Hilary Purcell and David Jones helped with FSIGTs and running insulin ELISAs. Jim Khambatta from Mercodia also requires a special thank you for conceding to my negotiation for doing us a great deal on the ELISA kit price! Roy Broadbank from Sartec was an enormous help in keeping the bomb calorimeter working and Peter Taylor and Sue Quinn were fantastic in teaching us how to use the biochemistry analyser and allowing us to run our hundreds of samples. They also helped us set up the NEFA assay, taught us how to stain for Heinz bodies in blood smears and helped us measure fructosamine for a student project. Thanks too go to Anja Kipar, Udo Hetzl and the histopathology team for helping us process, stain and image some of our samples. Ray Smith from Trouw Nutrition and Linda Turtle were instrumental in my learning the basics of proximate analysis. Nigel Jones and team were also a great support for various aspects of the work (laboratory space, land rover use, horse barn, paddocks etc.) – thank you all for your friendly welcome and help. The team at the Rowett Institute in Aberdeen deserves an extra special mention. Eric Milne from the mass spectrometry group was truly inspiring. His patience and expertise in helping me with the deuterium analyses were second to none. Through Eric I also discovered David Brown and Christine Horrocks to whom I‟m extremely grateful for basically letting me take over their whole laboratory to analyse NDFs and nitrogens. Thanks are also due to Sue, Donna, all the Garys and the librarian who looked after me so well; not forgetting Louise Cantlay, Eric‟s new assistant, who has helped analyse all our latest samples. Helen Brough and Holly Oakley deserve special thanks for their stamina during hundreds of hours of pony dissection and even bringing their friends with them to help! I am also indebted to several students who similarly came to join in on Sundays and bank holiday Mondays, persistent offenders being Claire Dixon and 2 Bryony Few. Val Turner‟s team were also amazing for helping with the initial processing. Dissection was facilitated by the awesome and artistic de-boning done by Geoff Partridge. Thank you Geoff - and you must remind me how you sharpen those blades! On the subject of which, W.R. Wright and Sons, Wholesale Butcher Supplies in Liverpool, played an important role in keeping us in sharp band saw blades. So many avenues were explored en route to finding out about body and body- part mincing and the analysis of parts thereafter that there are many people to thank. Peter Molcher was our first contact but, via Eddie Clutton, Uncle Sam and Billy Steele, I found John Corbett at Nottingham, who postponed his retirement to teach us how to use the Wolfking mincer. After retirement, his place was taken by Mick Baker, who also brought the bone-grinder back into action. My many thanks go to Mick and his boss David Edwards, for enabling multiple visits to use their facilities (including their freezers). Thanks too to Claire‟s mum, Vanessa, for letting me stay overnight near to Sutton Bonington. The story was similar as far as proximate analysis was concerned. My many thanks go to Marcus Mitchell from Nottingham and Andrew Mackie and Craig Thomas from Edinburgh who put up with my many questions about freeze-drying, oven-drying and the many different methods of lipid extraction. David Eden of the MLC put me in touch with Kim Matthews, who was a minefield of information, provided me with multiple documents and facilitated an extremely fruitful day sat in the library with a truly amazing librarian. I also thank Kim for putting me in touch with Fran Whittington, Mike Kitcherside and Kathy Hallet at Bristol, who constituted the team that finally processed all our pony samples. Mike delayed his retirement to help and Fran and Kathy gave up precious time from their own work to assist in completing the analyses before the laboratory was moth-balled. My very grateful thanks therefore go to all three for their expert tuition and expertise in getting a mammoth task completed. My thanks are also due to Prof. Jeff Wood for his enthusiastic interest in my work and for his help in finding various manuscripts. Closer to home, my many thanks are due to Rachel James for her tuition in ultrasound imaging and for her valued friendship; and to my anaesthesia colleagues who enabled me to step off the rota for several months to complete the various experiments.

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