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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-08-31.

Permanent Link: http://ufdc.ufl.edu/UFE0025086/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-08-31.
Physical Description: Book
Language: english
Creator: Chen, Chingai
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Chingai Chen.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Hill, Richard C.
Electronic Access: INACCESSIBLE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025086:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025086/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-08-31.
Physical Description: Book
Language: english
Creator: Chen, Chingai
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Chingai Chen.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Hill, Richard C.
Electronic Access: INACCESSIBLE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025086:00001


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1 ENERGY REQUIREMENTS OF I NDOOR ADULT PET CATS By CHING-AI CHEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Ching-Ai Chen

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3 To my Dad Fu-Li Chen

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4 ACKNOWLEDGMENTS I am indebted to many people who have encouraged and supported me during my masters studies. I thank Waltham for supporting the project financially, and all the cat owners for their effort and consistency. I especially acknowle dge Dr. Richard C. Hill, chairperson of my committee, for his dedication, inspiration and excel lent guidance. Special thanks are due to Dr. Karen Scott for many discussions an d technical support. I also appr eciate the expe rtise that was invested for this research by each of my commi ttee members, Dr. Elliott Jacobson and Dr Lori K. Warren. Finally, I express thanks to my pa rents. This study would have been impossible without their encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................... .............11 CHAP TER 1 ENERGY REQUIREMENTS OF I NDOOR ADULT PET CATS ........................................ 13 Introduction .................................................................................................................. ...........13 Methods and Materials ...........................................................................................................16 Sample Selection .............................................................................................................17 Study Design ...................................................................................................................19 Data Manipulation ...........................................................................................................22 Food and energy intake ............................................................................................22 Body weight, body condition score, a nd m orphometric measurements .................. 23 Seasonal comparison in food and energy intake ......................................................23 Statistical Analysis ................................................................................................... 24 Results .....................................................................................................................................24 Part I Energy Requirements of Pet Cats in W inter .......................................................... 24 Animals .................................................................................................................... 24 Body weight, body condition score a nd m orphometric measurements....................25 Diets ......................................................................................................................... 25 Food intake and metabolizable energy ..................................................................... 26 Factors of interest .....................................................................................................27 Correlation between ME intake and m easured factors ............................................. 28 Part II Seasonal Changes in ME intake of Pet Cats ......................................................... 28 Discussion .................................................................................................................... ...........31 Part I Energy Requirements of Pet Cats in W inter .......................................................... 31 Part II Seasonal Changes in ME Intake of Pet Cats ........................................................ 36 2 VALIDATION OF THE DOUBLE-LABLED WAT ER METHOD BY COMPARING AGAINST FOOD INTAKE MEASUREMENTS ................................................................. 67 Introduction .................................................................................................................. ...........67 Methods and Materials ...........................................................................................................69 Experimental Design ....................................................................................................... 69 Data Calculation ..............................................................................................................70 Body Composition ........................................................................................................... 72 Statistical Analysis .......................................................................................................... 73 Results .....................................................................................................................................73

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6 Animals ....................................................................................................................... .....73 Body Weight, Body Condition Score and Morphometric Measurements ....................... 74 Energy intake (EI) and Energy Expenditure (EE) ........................................................... 74 Body Composition Comparison Using Two Methods .................................................... 75 Elimination of the Isotopes Administered (18O and 2H) ..................................................75 Discussion .................................................................................................................... ...........75 APPENDIX A QUESTIONNAIRE FOR STUDY OF ENERGY REQUIREMENTS OF PET CATS ........88 B ACTIVITY MONITOR QUESTIONNAIRE .........................................................................89 C ATTACHMENT FOOD CONSUMPTI ON AND ROOM TEMPERATURE ...................... 90 LIST OF REFERENCES ...............................................................................................................91 BIOGRAPHICAL SKETCH .........................................................................................................97

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7 LIST OF TABLES Table page 1-1 Characteristics of the pet cats enrolled in the food intake study. ....................................... 50 1-2 Body weight, body condition score, and % body fat at week 0, week 2 and week 4 in winter food intake study..................................................................................................... 51 1-3 Diets fed to the 31 cats enrolled in the winter food intake study. ...................................... 52 1-4 Proportion of the metabolizable energy from different types of food fed to each cat ....... 54 1-5 Average of the guaranteed analyses fo r all dry foods, wet f oods, cat treats, and human foods in the food intake study. ............................................................................... 55 1-6 The effect of including the energy as sociated with changes in body weight on estimated daily metabolizable energy. ............................................................................... 56 1-7 Daily metabolizable energy intake of the 31 cats in the winter food intake study expressed with different exponents. ................................................................................... 57 1-8 Daily metabolizable energy intake of adult cats maintaining body weight. ......................58 1-9 Percentage of time cats spent undertak ing activity of different intensities. ......................59 1-10 The relationship of metabo lizable energy intake with f actors that may affect energy requirements. ................................................................................................................. .....60 1-11 Seasonal comparison of diets and their metabolizable energy density .............................. 61 1-12 Comparison of the metabolizable energy in take relative to body weight and fat-free mass between winter and summer .....................................................................................63 1-13 Seasonal comparison of the average ambient temperature within households. ................. 64 1-14 Comparison of body weight, body condition score, fat free mass, percentage of body fat and morphometric measuremen ts between winter and summer ................................... 65 1-15 Seasonal comparison of thyr oid hormone concentrations .................................................66 2-1 Characteristics of the pet cats enrolled in th e double-labeled wate r validation study ....... 85 2-2 The slopes and intercepts of the natural logarithm of the enrich ment of the stable isotopes of hydrogen and oxygen in water over the first and second week and over both weeks after in jection of double-labeled water. ..........................................................86 2-3 Sources for the equations used for calculating carbon dioxide production and energy expenditure ................................................................................................................... ......87

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8 2-4 Comparison of published methodologies that have used the double-labeled water to m easure energy expenditure in cats ................................................................................... 87

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9 LIST OF FIGURES Figure page 1-1 Devices used in the food intake study.. .............................................................................. 39 1-2 Morphometric measurement perfor med in the food intake study.. .................................... 39 1-3 Flow sheet showing the recruitment of participants for the food intake study. ................. 40 1-4 Correlation between body condition score and percent body fat of the 31 cats in the food intake study.. ........................................................................................................... ...41 1-5 Regression of metabolizable energy inta ke against body weight of the 31 cats in the food intake study. ............................................................................................................ ...42 1-6 Regression of metabolizable energy intake against fat free mass of the 31 cats in the food intake study. ............................................................................................................ ...43 1-7 Regression of metabolizable energy intake against body weight of the 31 pet cats in the food intake study.. ........................................................................................................44 1-8 Average winter ambient temperatures of the households of 30 cats during the food intake study. ................................................................................................................. ......45 1-9 Histogram showing the average activity counts per minute of 30 cats over a 7-day period in winter. ............................................................................................................. ....46 1-10 The total activity counts for each minute for the most ac tive, moderately active, and least active cat on a Sunday. .............................................................................................. 47 1-11 Seasonal difference in metabolizable energy intake of the 14 cats that participated during both the winter and summer of 2008. .....................................................................48 1-12 Regression of the difference in meta bolizable energy intake against average body condition score and average percentage of body fat from the two seasons ..................... 49 2-1 Flow sheet showing the r ecruitment of participants in the double-labeled water study. ... 79 2-2 Comparison between the metabolizable en ergy intake estimated from food intake and energy expenditure calculated using the double-labeled water method ...................... 80 2-3 Regression of energy intake against energy expenditure. .................................................. 81 2-4 Regression of energy intake against en ergy expenditure after taking account of the change in body weight. ......................................................................................................82 2-5 Correlation of the fat free mass derived using two methods. ............................................ 83

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10 2-6 Correlation of the percentage body fat derived by two m ethod. ........................................ 84

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ENERGY REQUIREMENTS OF I NDOOR ADULT PET CATS By Ching-Ai Chen August 2009 Chair: Richard C. Hill Major: Veterinary Medical Sciences Studies of laboratory cats have suggested that the average daily meta bolizable energy (ME) intake of lean cat s is 100 kcalkg BW-0.67day-1 and that of overweight cats is 130 kcalkg BW0.4day-1 (1). The first objective of this study was to ev aluate how ME intake of pet cats in singlecat households varies with body weight (BW), body condition score (BCS), indoor room temperature, and activity. Measuring ME intake is impractical in multi-cat households, so the second objective was to compare energy expenditu re (EE) measured using the double-labeled water (DLW) method against ME inta ke in single-cat households to evaluate the utility of using the DLW method in multi-cat households in the future. Food intake was measured in 31 neutered pet cats (exclusively i ndoor, 2-10 year-old, both sexes, single-cat households in Gainesville, Flor ida) that maintained BW for 4 weeks during winter. BW and BCS were measured every 2 w eeks. Relative activity was measured with an accelerometer-based activity monitor for 7 days. The ME density of food was calculated from the proximate analysis of representative sa mples. Saline containing DLW was injected subcutaneously in another 10 ca ts, and the elimination rate of the isotopes was measured and used to estimate EE over 2 weeks during which ME intake was also measured.

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12 Cats consumed on average 76 kcalkg BW-0.568day-1. Cats were divided into two equal groups of lean or overweight cats with a BCS of < or 6, respectively (9 point scale). Lean and overweight cats consumed 38 kcalkg BW-1.067day-1 and 87 kcalkg BW-0.494day-1, respectively, which were 34% and 20% lower than the NRC r ecommendations. ME intake declined slightly with age (p=0.0006) but there was no evidence of an effect of indoor room temperature or activity. ME intake was a mean 13% lower (p=0. 004) still when measured again in 14 of these cats in summer. The difference between ME intake and EE determined by DLW was small in eight cats but large in two cat s, suggesting that the DLW me thod needs further evaluation. This study suggests that neut ered indoor pet cats should be fed less than the NRC recommendation. The validity of the DLW method remains questi onable until more cats have been studied.

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13 CHAPTER 1 ENERGY REQUIREMENTS OF I NDOOR ADULT PET CATS Introduction Obesity has been associated with an increase d risk of disease. Overweight cats are at greater risk of developing insulin-independent di abetes mellitus, in which tissues become less sensitive to insulin, as well as joint, oral, skin and urinary tract disease, and neoplasia (2-5). Excess food intake that results in a positive en ergy balance will cause obesity. Exactly how much to feed pet cats to ensure that cats maintain their body weight (BW) is currently unknown and understanding what factors aff ect energy requirements is cr ucial to prevent obesity. Currently, the National Research Council (N RC) recommends that the average daily metabolizable energy (ME) requiremen t of lean cats is 100 kcalkg BW-0.67day-1 and that of overweight cats is 130 kcalkg BW-0.4day-1(1). The NRC recommendation is based on nine studies conducted using indirect calorimetry (6-14), 10 studies conducted measuring food intake (15-24) and five studies conduc ted using the double-labeled wa ter (DLW) method (25-29). The sex of these cats and the conditions under which they are maintain ed are often not described but most studies have used laboratory cats that are often intact, housed in individual cages or group housed, and maintained in accommodation with a c ontrolled ambient temperature. By contrast, pet cats are now mostly neutered, may roam throughout a house and room temperature may or may not be controlled. The ME intake in free liv ing pet cats was reported in only one abstract and the study details have not b een published (22). All of these studies have also shown a wide variation in daily ME re quirements (20-100 kcalkg BW-1day-1) among cats (1). Th ese variations may be due to the differences among the populati ons studied with respect to the factors that affect energy requirements such as body weight (BW), body condition score (BCS), age, sex,

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14 neuter status, activity, and indoor temperature. Unfortunately, most studies have not reported all the factors that might affect energy requireme nts making comparisons am ong studies difficult. Energy requirements increase with BW but th e principle components of BW, body fat (BF) and fat free mass (FFM), are not equally active meta bolically. The FFM is considered to be more metabolically active and responsible for most of the energy required at rest: the basal metabolic rate (BMR) (30). The fat mass is relatively metabo lically inert so changes in this component will have little impact on basal metabolic rate and, consequently, the ener gy requirement of the individual. Although the BCS ranki ng system is a simple and quick method of estimating the body composition of an animal, ther e are currently no studies eval uating the effect of BCS on energy requirements in cats. Energy is required by homeothermic animals, su ch as dogs and cats, to regulate their body temperature. Energy is required to increase heat production at lo w ambient temperatures and to cool the body at high ambient temperatures. Anim als will actively increase their metabolism rate at any temperature below the lower critical temper ature or above the upper critical temperature. The range of ambient temperatures at which no rmal temperatures are maintained without any increase in metabolic rate is called the thermone utral zone. Previous studies of laboratory cats have suggested that the lower critic al temperature of adult cats (30-35oC) is higher than in dogs (20-25oC) suggesting that cats kept at room temperature are using energy to maintain their body temperature (1). Nevertheless, there are currentl y no studies evaluating the effect of ambient temperature in pet cats. Activity is also thought to affect energy require ments. Most previous studies of cat activity have relied on direct observati on, did not evaluate activity of pet cats, and did not measure ME intake concurrently (31-35). These studies concl uded that cats spend up to 50% of their time

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15 sleeping and resting with food depr ived cats sleeping less because of the need to hunt. There was less variation in the type and amount of activ ity among cats compared to dogs so activity may not affect energy requirements in cats as much as it does in dogs. Of the two studies that compared activity to energy requirements, one evaluated activity subjectively in laboratory cats (14-16.5 month of age) (21), the second measured changes in energy expend iture associated with electronically-detected movement of laboratory cats (median 3 year s of age) confined within a box (21, 36). Thus, there is a n eed to measure the activity of free-living pet cats using a more quantitative method, such as an accelerometer, while also measuring energy intake. Accelerometers provide a continuous, objective, re liable, quantitative measure of the movement of multiple free-living subjects in various lo cations for days to weeks without observer intervention (37). Accelerometers contain a piezo-electric sensor that generates a voltage when the device undergoes a change in acceleration thereby recording the magnitude of physical movements, such as grooming, scratching, wa lking, running, or jumping. One study used accelerometers to evaluate activity in laboratory cats but did not m easure ME intake concurrently (38). Age and neuter status may also affect ener gy requirements. Some studies have shown an effect of aging on energy requi rements in cats (23), wherea s others have not (20, 24, 39). Gonadectomy appears to induce changes in metabolic rate and feeding pattern with the result that neutered cats gain significantly more body fat and body weight than do sexually intact cats (40). Nevertheless, all these studies were performed using laboratory cats and there were no comparable studies in pet cats. Diet-induced thermogenesis represents the en ergy required during the ingestion, digestion, absorption, and assimilation of f ood. Diet-induced thermogenesis may vary with the frequency of

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16 feeding and the relative composition of protein, fat and carbohydrate in the diet. Ingestion of protein, in particular uses more energy than do i ngestion of fat and carbohydrate. Since cats have a high requirement for dietary protein and they often eat many small meals throughout the day, energy required for diet-induced thermogenesis may be higher in cats than in other species. Nevertheless, there have been no studies evaluating how protein in the diet or diet frequency might affect energy requirements in cats (1) Thyroid hormones also play an important role in regulating energy expenditure. Thyroidectomy causes a drop in the normal metabo lism rate by as much as 40 percent, and oversecretion causes a rise which may reach 125 percent above normal (6). Hyperthyroidism is common in pet cats so it is important to select cats with normal thyroid status for metabolic studies. Nevertheless, thyroid hor mones also modulate non-shivering thermogenesis in response to low ambient temperature so thyroid horm one concentrations may change as ambient temperature changes from winter to summer and may affect energy requirements. It is important, therefore, to evalua te how thyroid hormones change with season. Thus, factors affecting the energy requirement s of cats have not been well documented or controlled in past studies and differences in th ese factors among studies could explain the wide variation in energy requirements that have been reported previous ly. The objectives of this study were to determine (a) food and energy intake of pet cats in the home environment, (b) how energy intake is affected by factors such as BW, BCS, sex, activity, indoor te mperature, hair coat density, and (c) whether energy intake and the factors that affect energy requirements differ between summer and winter. Methods and Materials Metabo lizable energy intake of pet cats was measured during the winter (January and February) of 2008 and 2009 and the summer (Sep tember and October) of 2008. This study was

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17 approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida (IACUC study #200701018) and by the Clinical Research Review Committee from the College of Veterinary Medicine. All cat owners agreed to participate by signing an informed consent form. Sample Selection The participation of faculty, students, and sta ff of the College of Veterinary Medicine and of the College of Public Health and Health Professions, University of Florida (UF) (and their friends and relatives) who own si ngle pet cats was solicited by word of mouth and by email. Respondents to this initial enquiry were then asked to complete a questionnaire (APPENDIX A) to determine whether their pet cat conformed to the inclusion criteria, i.e., that each cat should be the only cat in the household, kept exclusively indoors, adult (2 -10 years old) and physically healthy. Cats that live with dogs were enrolled pr ovided that the owner could guarantee that their pets would be eating separately. To ascertain whether cats were healthy, a phys ical examination was performed and a blood sample was obtained from the jugul ar or saphenous vein to meas ure complete blood cell counts (CBC), blood chemistry and thyroid hormone concentrations. Blood samples were collected in EDTA coated vacutainers (BD Vacutainer EDTA Tubes, Becton, Dickinson and Company, Franklin Lakes, NJ) for CBC analysis and in vacuta iner tubes containing sp ray-coated silica and polymer gel (BD Vacutainer SST Tubes) for chemistry and thyroid tests. Blood samples for thyroid hormones were centrifuged, and the serum tr ansferred into cryovials and kept at -20 C until shipment for analysis. The CBC included measurement of the packedcell volume, total white blood cell count and red blood cell count, hemoglobin concentr ation, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular he moglobin concentration, cellular hemoglobin

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18 concentration mean, red cell dist ribution width, hemoglobin distri bution width, platelet counts, icterus index, plasma protein, and fibrinogen. Blood chemistry concentrations measured included alkaline phosphatase, alanine aminotransferase, as partate aminotransferase, total bilirubin, total protein, albumin, globulin, phosphor us, creatinine, blood urea nitr ogen, glucose, cholesterol, magnesium, sodium, potassium, chloride, total CO2, and anion gap. The CBC and chemistry tests were performed at a diagnostic la boratory (the Clinical Pathology Laboratory at University of Florida Veterinary Medical Ce nter, Gainesville, FL). Blood cells were counted using an automatic cell counter (Advia 120, Siemens Cor poration, New York, NY), whereas chemistries were measured using an automatic analyz er (Hitachi 912, Boehringer Mannheim, Mannheim, Germany). Concentrations of total thyroxine (TT4 ), total triiodothyronine (TT3), free thyroxine (fT4), free triiodothyronine (fT3 ), and thyroid stimulating hormone (TSH) were measured by radioimmunoassay at another di agnostic laboratory (the Diagnos tic Center for Population and Animal Health, DCPAH, at Michigan State University, East Lansing, MI) using commercially available kits for TT4 (Gamma Coat M Total T4), fT4 (Gamma Coat Free T4:Two-Step), fT3 (Clinical Assays Gamma Coat Free T3 125I radioimmunoassay, all from DiaSorin, Inc., Stillwater, MN), TSH (Coat-A-C ount Canine TSH, Siemens Medi cal Solutions Diagnostics, Los Angeles, CA) and an in-house kit for TT3 (Michi gan State University DCPAH, Lansing, MI). A second blood sample with or without a urin e sample was obtained from any cat with blood values slightly outside the normal range to determine whether these abnormalities were persistent. Any cats with illness, physical a bnormality other than obesity, or persistently abnormal blood values were excluded from the study. Cat owners who participated during the winter of 2008 were encouraged to participate again in the summer to determine whether there was a seasonal effect on energy intake.

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19 Study Design Owners were asked to weigh all food consum ed during a 4 week study period using a food scale (Escali Model P115CH, Minneapolis, USA) provided by the laborat ory (Figure 1-1A). Owners were asked not to change their feeding routine during this period of time. Food intake was documented daily by weighing the amount of food offered and the amount left after feeding. The time when food was weighed was also record ed. A representative sample (approximately 50 grams) of each food that each cat consumed (including treats and human f ood offered to the cat) was collected and stored at -20 C until shipment for analysis. Pr oximate analysis was performed by a commercial laboratory (Dairy One Forage Testing Laboratory, Cornell University, Ithaca, NY) to determine percentage as fed of mois ture, dry matter (% DM), crude protein (% CP), crude fiber (% CFi), crude fat (% CF) and ash c ontent using Association of Official Analytical Chemists (AOAC) methods. Aliquots of feed were dried (at 60C), ground, and then analyzed for crude protein (using a Leco FP-528 Combusti on analyzer, Leco Corporation, St. Joseph, MI; AOAC 990.03), crude fat (by acid hydrolysis AOAC 954.02), crude fiber (AOAC 962.09) and ash (AOAC 942.05) content. Owners were asked to measure room temperature daily using digital thermometers (Model THT312, Oregon Scientific, Cannon Beach, OR) provided by the laboratory (Figure 1-1A). Owners were instruct ed to position the temperature probe at the location and height where the cat spent most of its time and to record maximum and minimum temperatures daily. Daily midpoint temperature was then calculated by averaging the maximum and the minimum temperature each day. The food scales maximum weight capacity was 5000 grams and sensitivity was 1 gram. These scales were calibrated us ing standard metal weights (rangi ng from 1-1000 g) to assure accuracy before the study. The accuracy of the scales was tested only to 1000 g because cat meals were not expected to exceed more than 1000g. Thermometers were calibrated using

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20 Checktemp 1 (HANNA instruments Inc., Woonsocket, RI) to assure accuracy before the study. Moreover, thermometers were placed on a laborato ry bench and temperature readings of three settings (current temperature, maximum and minimum temperatur e) were recorded for two to three consecutive days to assure that all th ermometers reproduced consistent readings. This study was conducted with the help of one senior biological scientist (KCS) and one veterinary student (ST). The body weight (BW) a nd morphometrics of each cat were measured and body condition score (BCS) was assessed immediately before and after 2 and 4 weeks of the study period. All measurements at week 2 we re performed during home visits to avoid interference with the cats feeding routine. Measurements at w eek 4 were performed either at the Veterinary Medical Center or during home visits. The durati on of the study period was extended to 6 weeks if a cats BW changed more than 5% during the first 4 weeks. If the study period was extended to 6 weeks, only data from the last 4 w eeks when cats maintained stable BW were used for analysis. Body weight was measured us ing a digital scale (Tanita Model 1583, Wholesale Point, Inc., Willowbrook, IL). Body condition score was assessed using a 9-poi nt scale (41) by two investigators (CC and ST) duri ng the winter of 2008 and by one investigator (CC) during the winter of 2009. Body condition was assessed mor phometrically by measuring the circumference of the chest at the level of the cranial 9th rib (CIRC), and the distance between the patella and calcaneal tuberosity (LIMB) in centimeters using a tape meas ure (Ohaus Corporation, Pine Brood, NJ) (Figure 1-2 A&B). Hair coat length was assessed subj ectively by one investigator (KCS) immediately before the study as short or long and also measured objectively (subject to owner agreement) by weighing hair clippe d from a small area (approximately 4 cm2) of the left flank using clippers with a #40 blade. The area clipped was measured (in centimeters) using a

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21 tape measure. Coat density (mg/cm2) was determined by dividing the weight of hair coat (in mg) by the area of the shaved patch (cm2). An accelerometer-based physical activity monitor (Actical TM, Mini Mitter Co., Inc., Bend, OR, dimensions 28x27x10 mm, weight 17 grams) was used to quantify physical activity objectively for 10-14 days during the 4-week study period (Figure 1-1B). For cats that were not acclimated to wearing a collar, a safety collar was distributed to allow cats to become acclimated before an activity monitor was assigned. Only 13 accelerometers were available for use in 2008. During the winter of 2008, cats were randomized into two groups of 13 cats and one group of 2 cats using computer generated random numbers (Microsoft Offi ce Excel 2003, Microsoft Corp., Seattle, WA). The accelerometers we re then allocated to cats of each group in turn for periods of at least 10 days. During the winter of 2009, there were enough accelerometers for all the cats to wear an accelerometer at the same time. The accelerometers were programmed to record the total number of ac tivity counts every minute. After the recording period, data were transferred from the accelerometer to a computer using a standard program (ActiReader, Mini M itter Co., Inc., Bend, OR). Mean activity counts per minute (AC/min) for each cat was determined by averaging the total ac tivity counts during 7 contiguous days. Each owner was asked to comple te a questionnaire concerning the number of people living in the household, the usual time that those people woke up in the morning and retired to sleep at night both during the week an d at the weekend, the numb er of dogs living with the cat and how long the cat was usually alone during the day. The same procedures were followed during th e summer of 2008 with the exception that hair coat was clipped from the opposite flank. A sample of each food fe d during the summer was analyzed. Blood count and chemistry measurements were not repeated, but a blood sample was

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22 obtained to measure any change in thyroid hormone concentrati ons. Also, owners were asked whether living conditions had chan ged from winter to summer. Data Manipulation Food and energy intake Only the days for whic h food intake was documented were used to calculate daily food intake. Daily food intake (g/d ay) was calculated by dividing the total grams of each food consumed by the number of days on which th e owner documented food intake. The number of days on which owners documented food intake is re ported as a perc entage of the number of days between the BW measurements at the start and end of the period of st udy. Metabolizable energy (ME) density (kcal/g as fed) was calculated fro m the proximate analysis of the food using the following equation recomme nded by the NRC(1): ME density = (87.9-0.88 x CFi x100/DM) x (5.7 x CP+9.4 x CF+4.1 x (CFi+NFE))/10000-(0.77 x CP)/100, (1-1) where DM is dry matter, CFi is crude fiber, CP is crude protein, CF is crude fat, and NFE is nitrogen-free extract obtai ned by difference, all as percentages as fed. An estimate of the average daily ME intake (kcal/day) for each cat was then derived by multiplying the daily food intake of each food fed by the ME density of that food. The total daily ME intake was obtained by summing the average ME intake for all foods, incl uding treats, that each cat consumed. The total daily ME intake was also adjusted for any BW gain or loss observed during the 4-week study period, assuming 7 kcal of energy was associated w ith each gram of tissue loss or gain (42). The ME intake for each cat was then compared with the ME requirement predicted using the NRC formula for lean or overweight cats de pending on whether the BCS was < or 6. The BCS cutoff of 6 was chosen to divide cats into two groups of approximately equal numbers. The difference between the two values was reported as a percentage of the predicted value.

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23 Body weight, body condition score, and morphometric measurements Mean BW for each cat was obtained by averaging the three measurements recorded immediately before and after 2 and 4 weeks of the study. The BCS assessed by the two investigators (CC and ST) was averaged to obtain a mean score at each tim e point for each cat. Mean BCS for each cat was then obtained by aver aging the mean scores at the three different time points (before and after weeks 2 and 4 of th e study period). The percentage body fat (%BF) was calculated using the following equation, whic h has been termed the Feline Body Mass Index (FBMITM) (43): %BF = [(CIRC/0.7062)-LIMB]/0.9156-LIMB (1-2) where CIRC represents the circumference of the chest at the level of the cranial 9th rib, and LIMB represents the distance between the patella and calcaneal tuberosity, both in centimeters. Fat free mass (FFM, kg) was then calculated as follows: FFM (kg) = BW (kg) x (100-%BF) (1-3) Mean %BF and mean FFM for each cat were obt ained by averaging the three measurements before and after weeks 2 and 4 of the study. Seasonal comparison in food and energy intake The percentage difference in ME intake ( ME %) between seasons was determined in every cat as follows: ME% = [(MEw-MEs)/MEw] x 100 (1-4) where MEw was the ME intake measured in winter, and MEs was the ME intake measured in summer. To allow comparison with other studies, the change in ME intake was calculated with MEw and MEs expressed as total ME intake in kca l/day and also with the total intake divided by BW and by FFM.

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24 Statistical Analysis All procedures were perf ormed with a comput er statistics progra m (SAS for Windows 9.2, SAS Institute Inc., Cary, NC). Daily ME intake was regressed against mean BW and mean FFM using non-linear regression (44). Factors such as age, BCS, FFM indoor temperature, average activity count and hair coat density were log tran sformed and added sequentia lly to evaluate their effect on the model (45). Mean BCS was regre ssed against mean %BF using linear regression (45). Correlations between factors were assessed visually by c onstructing paired plots. Body weight, BCS, CIRC, LIMB and %BF were all test ed for consistency throughout the study period using the glimmix function (46). The glimmix func tion is a fast flexible procedure capable of running linear models (fixed effects), generalize d linear models (fixed effects), linear mixed models (fixed and random effects) as well as ge neralized linear mixed m odels (fixed and random effects). (46) Differences from summer to winter were assessed using a paired t-test (45). The change in total ME intake was regressed ag ainst the change in midpoint temperature ( T %) between winter and summer with BCS, %BF and hair coat de nsity averaged from the two seasons as factors in the model (45). A probability of error <0.05 was considered significant and a probability of error <0.1 was considered to suggest a trend towards significance. Results are reported as means +/one standard deviation unless otherwise stated. Results Part I Energy Requirements of Pet Cats in Winter Animals Thirty eight cats were recrui ted during the winters of 2008 and 2009. Seven of the cats (6 fe male, 1 male) were subsequently removed from the study because of failure to conform to the inclusion criteria. Reasons for exclusion include: slight but persistently increased liver enzyme concentrations (n=4); persistently increased creatinine concentration when the urine was not

PAGE 25

25 concentrated (n=1); uncooperative during handli ng (n=1) and discovered to be an outdoor cat (n=1) (Figure 1-3). Of the 31 cats that comp leted the study, nine were males and 22 were females, all neutered. Five were pure breed cats (one Bengal, one Burmese, one Himalayan, one Russian blue, and one Turkish Angora) and 26 we re mixed breed domestic cats. Thirteen cats lived with dogs but the owners guaranteed that their animals were eating separately either by feeding each animal in a separate room or by f eeding the cat on a raised surface that dogs were unable to reach. On average, cats had access to a living area of 1035 square feet (range 5002400). Body weight changed less than 5% during the first four weeks of the study in all but two cats. In these two cats, the study was extended to a total of six weeks and food intake was evaluated only during the last four of the six weeks of study, during which BW was stable. Body weight, body condition sco r e and morphometric measurements Mean BW and mean BCS were 5.11 kg and 6.1, respectively for the 31 cats (Table 1-1), and there was no evidence of a change in either mean BW or mean BCS over four weeks (Table 1-2). Mean %BF was 29% of BW and increased slightly at the end of the four week study period (Table 1-1). BCS and % BF were positively correlated (p<0.001), with a coefficient of determination (R2) of 82% (Figure 1-4). The correla tion between BCS and %BF was best described by the equation: %BF=1.6 + 4.6 x BCS (1-5) The investigators (CC & ST in 2008 and CC in 2009) scored body condition consistently over the four weeks of study. The length of LI MB was measured consistently but CIRC measurements increased over time (Table 1-2). Diets Food intake was docum ented by owners on a mean of 85% (median 84%; range 67-100%) of days. Dry food provided 99% (range 89-100%) of the ME consumed by cats fed mostly dry

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26 diets (n=29); wet food provided 98% of the ME consumed by two cats fed mostly canned food (Table 1-3 & 1-4). Seventeen cats were fed a single dry diet, without a dditional wet food, treats, or human food. Ten cats were given treats and/or human food other than their main diet (dry and/or wet). Treats provided 1% (range 0.1-3.7%) of the ME in 10 cats whereas human food provided 0.06% (range 0.02-0.1%) of the ME in two cats (Table 1-4). Only three cats were given treats on a routine basis. Table 1-5 shows the averages of the guaranteed analyses for dry foods, wet foods, treats, and human foods. Food intake and metabolizable energy There was no evidence that taking th e energy from BW change into consideration affected the energy requirement (Table 1-6). The mean da ily ME intake (in kcal ME) without taking changes in BW into account (ME1) was 38 kcalkg BW-1 and 54 kcalkg FFM-1 and was best described by the equations (Figure 1-5 and 1-6): ME1 = 76 x BW 0.568 (1-6) ME1 = 71 x FFM 0.78 (1-7) where BW and FFM are in kg (Table 1-6). The m ean daily ME utilizati on (in kcal ME) when change in body weight was taken into account (ME2) was 38 kcalkg BW-1 and 53 kcalkg FFM1. The coefficient of variati on (CV) of daily ME intake relative to BW or FFM was 19% whichever exponent was used (Table 1-7). Cats were divided into an overweight and a lean group using a BCS cutoff of 6 that was chosen to divide the cats into two groups of ap proximately equal numbers (15 lean cats, 16 fat cats). Lean and fat cats consumed 38 kcalkg BW-1.067 and 87 kcalkg BW-0.494 daily, respectively (Table 1-8 & Figure 1-7). These values were 34 % and 22% lower, respectively, than the NRC recommendation (Table 1-8).

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27 Factors of interest Indoor temperature: T he effect of ambient temperat ure was evaluated in only 30 cats because one of the cat owners failed to record the daily ambient temperature correctly (Figure 18). Mean maximum, midpoint, and minimum temperatures were 75.0+/-3.3 (range 67.6-82.1), 70.9+/-3.4 (range 57.0-76.5), 66.8+/-4.6 F (range 46.2-71.6), respectively. The corresponding m ean values in degree Celsius were 23.8, 21.6 and 19.3 for maximum, midpoint and minimum temperature, respectively. Hair coat thickness: Eleven of the 31 cats were evalua ted subjectively as long-haired and 20 as short-haired. Hair coat de nsity was objectively a ssessed in 28 cats in wh ich owners allowed their cats coat to be cl ipped. Mean hair coat de nsity was 35.0+/-9.0 mg/ cm2 (Table 1-1). There was no evidence of a difference (p=0 .5) in mean hair coat density between long-haired cats (36.6 +/9.0 mg/ cm2, n=10), and short-haired cats (34.1 +/9.5 mg/ cm2, n=18). Activity level: Activity data was not co llected successfully over seven contiguous days in one cat. The mean AC/min for the other 30 cats was 64 (Table 1-1). Of the 30 cats evaluated, there was a skewed distribution in the activity levels with the major ity of the cats being relatively inactive and only a few cats being relatively active (Figure 1-9). To provide a degree of scale for the activity measured by the accele rometers, activity counts registered by the activity monitors were compared for one hour with the activity documented by direct observation in five of the cats. For example, 2 activity coun ts were registered during a minute of sl eeping, 367 activity counts were registered during a minute of walking and layi ng down, 717 activity counts were registered during 30 seconds of grooming plus 30 seconds of laying down, and 1304 activity counts were registered during 11 seconds of walking up stairs plus 7 seconds of running up and down stairs plus 42 seconds of sitting, gr ooming, standing and walking. Based on these observations, activity was divided in to four categories: 1) <100 AC /min representing cats at rest,

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28 2) 100-499 AC/min representing low activity, 3) 500-999 AC/min repr esenting moderate activity, 4) >1000 AC/min representing high activit y. Cats on average spent 85 % of time resting and spent only <1% of time doing high intensity of activities (Table 1-9). Examination of the minute by minute record of activity showed that both active and inactive cats indulged in short episodes of more intense activity at irregular intervals throu ghout both day and night (Figure 110). Correlation between ME intake and measured factors The ME intake was affected by BW %BF, FFM BCS and age (p<0.05). ME intake declined slightly as age increased, but ME intake incr eased slightly as BW, BCS, %BF, FFM increased (Table 1-10).There was a non-significant trend towa rds a decline in ME intake as hair coat density increased (p=0.06) (Tab le 1-10). There was no evidence of an effect of indoor room temperature and activity on ME intake (p>0.05) (Table 1-10). Body weight, BCS, %BF and FFM were all correlated to one another. Part II Seasonal Changes in ME intake of Pet Cats Fourteen owners who had participated during the winter of 2008 agreed to m easure food intake during the summer of the same year (Fi gure 1-3). Most (85%) of these owners claimed that nothing had changed regardi ng the living environment. Two cat owners moved to a different apartment but, in each case, the new apartment was approximately the same size as the old one. Of the 14 cats enrolled in the summer study, 7 ca ts were living with dogs; 9 were female and 5 were male. Five of 14 cats were fed a new diet and one cat had its diet cha nged partially (Table 1-11). There were also slight differences in the proximate analyses of food samples from winter to summer when cats were fed the same food du ring both seasons. When the foods fed to all 14 cats were compared between winter and summer, the change in ME density was <10% in 12 cats and <20% in two cats (Table 1-11) and there was no evidence of a difference in the protein

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29 content (p=0.5). Of the six cats that had their diet changed, the change in protein content between seasons was <10% in five cats a nd 25% in one cat (from 32% of CP as fed in winter to 40% of CP as fed in summer). Of the 14 cats that participated in both seasons, the mean da ily ME intake (in kcal ME) was 38 kcalkg BW-1 in winter and 32 kcalkg BW-1 in summer and was best described by the equations (Figure 1-11): MEw = 97 x BW 0.445 (1-8) MEs = 53 x BW 0.698 (1-9) where MEw represents ME intake in winter an d MEs represents ME intake in summer. The average ME% of the 14 cats was 13%, 15% and 17% hi gher in winter than in summer when calculated using either total in take, intake/BW, and intake/FFM values, respectively (p<0.05) (Table 1-12 & Figure 1-7). Mean maximum, midpoint, and minimum household temperatures were on average 5%, 7% and 9% lower respectively in winter than in summer (p<0.0001) (Table 1-13). The ME% declined slightly as BCS and %BF increased and is best described by the equations (Figure 1-12 A & B): ME (%) = 63.197.7699 x BCS (1-10) ME (%) = 58.2541.4861 x %BF (1-11) There was no evidence of an effect of hair coat density on the ME. Mean maximum, midpoint, and minimum household temperatures were on average 5%, 7% and 9% lower, respectively, in winter than in summer (p<0.0001) (Tab le 1-13). The average midpoint temperature was higher in summer than winter in all bu t two households. The midpoint temperature decreased from summer to winter in one household and did not change in another. For the 12 households where temp erature decreased from summer to winter, daily ME intake

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30 decreased on average 1.8 kcalkg BW-1 for each decrease in ambient temperature by one degree Celsius, When ME relative to the change in midpoint temperature ( T %) was regressed against BCS, %BF and hair coat de nsity in these 12 cats, there was no evidence of an effect of any of these factors. The BW did not change more than 5% in an y of these cats duri ng the four weeks of summer food intake measurement. Nevertheless, there was a non-signifi cant trend towards the mean BW in winter being 2% lower than that in summer (p=0.08) (Table 1-14). There was no evidence of a change in BCS between the two seasons (p=0.8) (Table 1-14). During the summer, there was no evidence of a difference in BCS assessment between the two investigators (CC & ST) (p=0.3) or over time (p=0.8) and there was no interaction between time and investigator. Consistency of the BCS assessment by the two investigators was maintained throughout the summer (p>0.05). There was no evidence of a diffe rence in BCS assessments between the two investigators (CC & ST) (p=0.3) or over time fo r each investigator (p=0.8), and there was no interaction between time and investigator. The es timate of %BF was, on average, 6% higher in winter than in summer (p=0.01), which co rresponded to a 5% decrease in average FFM (p=0.005) (Table 1-14). There was no differen ce in CIRC measurements between the two seasons, whereas LIMB measurements were 6% shorter in winter than in summer (p<0.0001) (Table 1-14). Differences were found in bot h CIRC and LIMB between weeks 2 and 4 of the summer study. Mean concentrations of TT4, TT3, fT4, fT3 were on average 18%, 15%, 52% and 41% higher respectively in winter than in summer (p<0.005), whereas mean TSH concentration was 49% lower in winter than in summer (p< 0.002; Table 1-15). There was no evidence of a difference in activity (p=0.6) or hair coat density (p= 0.7) between the two seasons.

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31 Discussion Part I Energy Requirements of Pet Cats in Winter The ME intake for neutered cats reported in th e current study was on average 38 kcal kg BW-1day-1, which is comparable to the ME intake re ported recently for lean and obese neutered laboratory cats of similar age and weight (47) and ME intake reported for older neutered laboratory cats (48). These values for the ME in take of cats are much lower, however, than the ME intake (50-80 kcal kg BW-1day-1) reported in other feeding studies summarized by the NRC (15, 16, 18, 20, 23, 24, 49). The discrepancy may be attr ibuted to the fact th at previous studies have not distinguished among cats of different ages, BW, gender, reproductive status and the methodology used for ME calculation. The cats in our study were neutered. This ma y explain partly why our cats required only 2/3 of the energy recommended by the NRC (1). Neut ered cats appear to require less energy than sexually intact cats to maintain BW (21, 40). Gonadectomy removes estradiol or testosterone which, in turn, affects voluntary food intake in cats (50). Sexually intact cats appear to selfregulate their food intake, whereas neutered cats te nd to eat almost all of the food available to them (21). Administering estrad iol has been reported to reduce food intake in both male and female neutered cats (50). Neuter ed cats also gained more BW co mpared to sexually intact cats and this weight gain was comprised mostly of fat mass (29, 40, 51, 52). Nowadays, pet cats are usually neutered so reducing energy intake in neutered cats should help to prevent obesity. Body composition was found to affect the relati onship between ME intake and BW in our cats. In the current study, eight of the cats had a %BF <25%, 14 of the cats had a %BF between 26-30% and nine of the cats had a %BF >31%. Using a 9 point scale, a BCS of 5 is considered to be an ideal body shape. The equation obtained in the current study suggests that a BCS of 5 corresponds to a %BF of approximately 25%. This is comparable to two previous studies where

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32 the percentage of body fat in normal weight cat s was reported to be 15-20% of BW and 24% of BW by bioelectrical impedance and dual energy X-ray absorptiometry (DEXA), respectively (53, 54). Thus, one third of our cats might be considered overweight, which may explain why cats in our study were on average consuming le ss than has been reported in the past. The regression slope of ME intake against body weight was steeper for cats with a BCS <6 than for more overweight cats. For cats with a BCS<6, ME intake was roughly proportional to BW, whereas energy intake did not increase gr eatly with body weight in overweight cats. Although there were only four overw eight cats with a BCS >7, it is cl ear that in this as in other studies, obese cats had a di sporportional impact on the exponent (19, 24, 26, 49). A regression of ME intake against FFM to minimize the effect of fat mass gave an exponent of 0.78, which is similar to the exponent suggested by Kleiber for the relationship between basal metabolic rate and BW (0.75) in a range of species. Ne vertheless, individual variability in ME intake relative to FFM was still high with a coefficient of variation of 20% even when the effect of fat mass had been eliminated. Thus, other fa ctors, such as age, activity or indoor temperature, must also affect energy intake. Younger cats required slightly more energy than older cats in the current study, however, reports of the effect of age on ME intake have been inconsistent. Anantharaman-Barr et al. conducted a digestibility study with young (1 y/o), middle-aged (3-5y/ o) and old (>10 y/o) cats and found that mean food intake ap peared to increase wi th increasing age (55) Burger concluded from digestibility trials involving cats from 1-11 years of age and found that there was no evidence of an age effect on the DE requirement ( 39). Taylor et al. observe d a slightly decreasing but not significant trend in daily ME intake with cats aged up to 10 year s old (20). In contrast, Laflamme and Ballam found based on data from 113 cats ranging in age from 2 to 17 years old

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33 that ME intake declines as cats age up to 11 ye ars of age and then incr eases in cats from 12-17 years old (23). In those cats that were younger than 11 years old, ME intake decreased approximately 3% per year. Scarlett et al. suggested that this discrepancy in findings may be due to changes in BW and BCS affecting energy re quirement (56). The higher values of energy requirements reported in previous studies have sometimes been obtained from cats under the age of that they were probably still growing. It is important, therefore, to document age when measuring energy requirements in future studies. In the current study, indoor temperature did not appear to affect ME intake. The lower critical temperature in cats has been reported to be about 30-35oC, which suggests that cats are burning energy when kept at normal room temper atures. Most laboratory cats are kept at a normal room temperature of 20-26oC. In the current study, cats were on average kept at room temperature controlled between 20-24oC which was quite similar to the temperature reported in the past. Therefore, the difference in ME in take between the current study and the studies reported in the past is not likely to be an ambient temperature effect. Activity was thought to be another important f actor influencing ME requirements. Riond et al. measured activity-induced heat production in a respiration chamber when movements of the animals induced a change in wavelength of the reflected radio-wave b ecause of the Doppler effect (36). They showed that activity affects ME and induced a 6 kcal kg BW-1day-1 or 13.5% increase in total daily heat pr oduction in cats that were housed in cages. In the current study, however, there was no evidence of an effect of activity on ME intake. On e explanation could be that most cats in the current study were relative ly inactive. Cats spent 80% of time resting and <1% of time participating in hi gh intensity activity. Moreover, cats tend to indulge in a more sprint-like type of exercise, which may have less of an effect on ME requirements than the

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34 sustained exercise performed by dogs. More studi es are needed, therefore, to clarify whether activity affects ME intake in cat s. Cats with a wider range of activity should be recruited, including outdoor cats and cats from multi-cat hous eholds that are likely to be more active than pet cats from single-cat households. The methodology of calculating ME intake varies among studies th at calculate energy intake from food intake. The high values for ME in take reported in the past may have resulted from the use of Atwater factors to calculate the ME density of the food. Digestibility trials in both dogs and cats indicate that Atwater factors tend to overestimate ME intake because the digestibility of many pet food ingr edients is lower than the diges tibility of most foods consumed by humans (57). On the other hand, modified Atwater factors recommended by the NRC in 1985 tend to underestimate both the dige stibility and the ME density of food (58). Some studies also report digestible energy (DE) rather ME (18, 19) Results should be interpreted with caution, therefore, when comparing among studies. In th e current study, ME was calculated using the latest NRC recommendation published in 2006 (1), which results in an es timate of ME density that lies between values estimated using modified Atwater factors and origin al Atwater factors. In the current study, we used two methods to evaluate the body composition of our cats. The first, body condition scoring by visual assessm ent and palpation, is the most widely used, accepted and practical method in a clinical setti ng. Assessment of BCS is quick and simple, but is subjective and, therefore, can result in inter-observer variation especially if assessors do not have some degree of expertise. To increase accu racy, two scorers (CC & ST) assessed every cat in the current study. A second more objective method of estimating %BF using morphometric measurements was also used. This method uses m easurements of the circumference of the chest at 9th rib (CIRC) and the length betw een the patella and calcaneal t uberosity (LIMB) to calculate

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35 %BF using the so called, FBMI. The FBMI equa tion was established from the correlation of CIRC and LIMB with %BF measured by DEXA in laboratory cats (R2=85%, p<0.0001) (43). In the current study, the measurements of CIRC were not consistent during the winter study. This may be because the body composition of the cat had changed over the four week study period or because the operator did not measure CIRC consistently. Maintaining consistency when measuring CIRC is difficult because the cat is covered in fur and the measurement of CIRC can be affected by the tension applied to the tape Nevertheless, when measurements of %BF and assessed BCS were averaged, the BCS assessments were highly correlated to %BF estimated from the FBMI. The %BF increased about 5% w ith each increase of BCS by one unit. This relationship between BCS and %BF can thereby pr ovide a quick estimate of %BF from BCS for use in daily practice. In conclusion, pet cats in our study only required two thirds of the ME intake recommended by the NRC. This might be because th ey were neutered or because they were more overweight than laboratory cats used in past stud ies. Secondly, the variabili ty of ME intake was not eliminated in the current study despite controlli ng for factors such as age, neutered status and health status. Of the factors that were evalua ted, body composition and age were found to affect ME intake, whereas activity had no evidence of an effect on ME intake. Thirdly, the exponent of the relationship between ME intake and BW appeared to decrease as cats become overweight so overweight cats require proportio nately less energy to mainta in BW. Lastly, morphometric measurements proved a good method to estimat e %BF but CIRC and LIMB should be measured on several occasions to improve accuracy. The daily ME requirement for cats with a BCS < 6, should be calcula ted, therefore, using the equation:

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36 ME (kcal/day) = 38 x BW (kg) For cats with a BCS of 6 or great er, FFM should be used rather than BW to eliminate the effect of fat mass on ME intake. The FFM can be estimat ed morphometrically from the FBMI using a simplified equation, which is easier to calculate in daily practice: %BF = [(CIRC/0.7062)-LIMB]/0.9156-LIMB = 1.5466 x CIRC 2.0922 x LIMB 1.5 x CIRC 2 x LIMB Average daily ME requirements can then be calc ulated using an exponent of 0.75 that can be easily calculated using a simple calculator in daily practice: ME (kcal/day) = 74 x FFM 0.75 (kg) Part II Seasonal Changes in ME Intake of Pet Cats On average, the cats in the current study m aintained weight while consuming 13% less energy in summer than in winter. Age and ac tivity did not change from summer to winter whereas ambient temperature, body composition and thyroid hormone concentrations did change from summer to winter. The ME difference fou nd between the two seasons could be due to a change in any one of these factors or a combination of these factors. There was a 2-3oC decrease in ambient temperature from summer to winter. It was originally intended to conduct the study in July or August when the weather is hottest in Florida, but most of the cat owners were veterinary stud ents and left town for their summer vacation and did not return until the last we ek of August. Nevertheless, cha nge in ambient temperature was still observed with the average am bient temperature recorded from both seasons be ing lower than the lower critical temperature reported for adult cats (30-35oC) (1). This suggest s that cats were burning energy both in winter and summer. Adams et al. reported that th e energy consumption of adult, shorthaired, unacclimatized cats increas ed linearly by approximately 5 kcalkg BW-1day-

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37 1oC-1 below 35oC (59, 60)., whereas we found that the difference in ME intake due to the change of ambient temperature in the 12 households where temperature decreased from summer to winter was on average 2 kcalkg BW-1day-1oC-1. Nevertheless, pet cats in the current study had adapted to the change in temperature over a longe r period of time and ma y have adapted to the change of temperature more effectively by m oving around the house to find the warmest place to lie, whereas laboratory cats housed in cages were not adapted to the new temperature and their activity was severely restricted. Another factor that might cont ribute to the difference found in ME intake is the change in thyroid hormones concentratio ns. Catecholamine and thyroi d hormones influence heat production in response to cold in many mamma lian species (60). Catecholamine-induced thermogenesis mitigates cold stress by mobilizing and oxidizing free fatty acids to generate heat and by inducing peripheral vasoconstriction to reduce heat loss. Thyroid hormones affect thermogenesis in response to th e change of ambient temperatur e by potentiating the effect of catecholamines on beta-adrenergic receptors (61) Mean concentrations of TT4, TT3, fT4, fT3 were on average 18%, 15%, 52% and 41% higher, respectively, in winter than in summer, though all values remained with in the normal range. Thus, the changes in thyroid hormone concentrations with season could have been re sponsible for the change of energy consumption. Nevertheless, TSH was found to be 49% lower in the winter than in the summer in the current study suggesting that the thyroid was more se nsitive to TSH in producing TT4 and TT3 in winter. The difference in ME intake could also be associated with changes in body composition. The FFM is primarily responsible for energy utilization. In the current study, FFM was found to be significantly higher in summ er but cats were consuming less energy in the summer. The

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38 energy utilized by the increased FFM in summer could have moderated the temperature effect on ME requirements between the two seasons. Alternatively, morphometric measurements (CIRC in winter and LIMB in summer) were not measured consistently so the change in FFM might have been due to a measurement error. Another interesting finding was that the ME as from winter to summer was larger in lean cats than overweight cats. It is possible, therefore, that overwei ght cats are protected from the effects of the cold by the insu lation provided by thei r higher amount of body fat. Although hair coat also provides insulation, we were not able to show that hair coat has an effect on ME intake. This study lacked a control population for whic h temperature or season did not change. Thus, the changes observed could have been due to factors such as day length that were not controlled. Controlled studies inve stigating one factor at a time are needed to clarify whether temperature or season are truly responsible fo r the change in energy consumption observed.

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39 A B Figure 1-1. Devices used in the food intake study. A) The ther mometer and food scale, B) The accelerometer-based activity monitor assigne d to each cat attached to a collar. The arrow was directed rostrally on each cat. A B Figure 1-2. Morphometric measurement performed in the food intake study. A) Measurement of the circumference (CIRC) of the chest at the level of the cranial 9th rib. B) Measurement of the leg index (LIMB) from the patella to the calcaneal tuberosity (43).

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40 Figure 1-3. Flow sheet showing the recruitment of participan ts for the food intake study.

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41 0 5 10 15 20 25 30 35 40 45 123456789 BCS (9-point scale)Body fat (%) R2=0.82 Figure 1-4. Correlation between body condition score (BCS) and percent body fat (%BF) of the 31 cats in the food intake study. The diamonds represent values for individual cats; the line represents the equation that best describes the relationship: %BF=1.6 + 4.6 x BCS, with a correlation of variation (R2) of 82%.

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42 0 50 100 150 200 250 300 0123456789BW (kg)ME intake (kcal/day) Figure 1-5. Regression of metabo lizable energy (ME) intake agai nst body weight (BW) of the 31 cats in the food intake study. The diamonds represent values from individual cats; the line represents the equation that best describes the relationship: ME=76 x BW0.568.

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43 0 50 100 150 200 250 300 0123456 FFM (kg)ME intake (kcal/day) Figure 1-6. Regression of metabo lizable energy (ME) intake against fat free mass (FFM) of the 31 cats in the food intake study. The diamonds represent values from individual cats; the line represents the equation that best describes the relationship: ME =71 x FFM0.78.

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44 0 50 100 150 200 250 300 0123456789BW (kg)ME intake (kcal/day) Figure 1-7. Regression of metabo lizable energy (ME) intake agai nst body weight (BW) of the 31 pet cats in the food intake study. Blue diamonds represent lean cats with a body condition score (BCS) <6; pink squares represent overweight cats with a BCS 6, and squares surrounded by a circle represent cats with a BCS 7using a 9 point scale for BCS. The solid line represents the equati on that best describe the relationship in the lean cats: ME= 38 kcalkg BW-1.067day-1. The dashed line re presents the equation that best describe the relationship in the overweight cats: ME= 87 kcalkg BW0.494day-1.

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45 45 50 55 60 65 70 75 80 85 1357911131517192123252729 Cat IDTemperature (F ) Figure 1-8. Average winter ambient temperatur es of the households of 30 cats during the food intake study. Blue diamonds with the blue line represent the mean maximum temperatures ( F) for each cat household averaged over th e study period; pink squares with the pink line represent mean midpoint temperatures ( F) averaged over the study period; yellow triangles w ith yellow line represent m ean minimum temperature ( F) averaged over the study period. The lo w m inimum temperature found in the household of cat 29 occurred because this cat had access to a screened porch.

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46 Figure 1-9. Histogram showing th e average activity counts per minute of 30 cats over a 7-day period in winter. The y-axis represents th e number of cats in each activity level and the x-axis represents activity levels expressed as averag e activity counts per minute. There was a skewed distributi on in the activity levels of the cats enrolled with the majority of the cats being relatively inactive and only a few cats being relatively active.

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47 Figure 1-10. The total activity counts for each minute for the mo st active (left), moderately active (middle), and least activ e (right) cat on a Sunday. The y-axis represents activity counts/minute while the x axis repres ents every minute of the day. Monitors showed that both active and inactive cats i ndulged in short episodes of more intense activity at irregular intervals both day and night. Hours 04812162024 Hours 04812162024 Hours 04812162024 Activity Counts/minute 0 500 1000 1500 2000 2500

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48 0 50 100 150 200 250 300 0123456789 BW(kg)ME intake (kcal/ day) Figure 1-11. Seasonal difference in metabolizab le energy (ME) intake of the 14 cats that participated during both th e winter and summer of 2008. The diamonds represent measurements from the winter with the dotted line representi ng the best fitted equation that describes the re lationship: ME=97 kcalkg BW-0.445day-1. The triangles represent measurements from the summer with the solid line representing the best fitted equation that describes the relationship: ME=53 kcalkg BW-0.698day-1.

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49 A B Figure 1-12. Regression of the difference in metabolizable energy intake (diffME) where diffME= [(MEw-MEs)/MEw] x 100 against average body condition score from the two seasons (averageBCS) (A) and averag e percentage of body fat from the two seasons (averageBF) (B).

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50 Table 1-1. Characteristics of the pet cats enrolled in the food intake study (n=31). Characteristics Mean +/-SD Range Age, years 5 +/2.5 2-10 BW, kg 5.1 +/1.3 3.39-8.46 BCS 6.1 +/1.0 4.4-8.1 %BF, %BW 29.3 +/5.1 19.5-42.1 FFM, kg 3.6 +/0.7 2.6-5.3 Activity counts / min* 64 +/26 25-143 Midpoint Temperature** ,F 71 +/3 57-77 Hair coat density ***(mg/cm2) 35 +/9 22-56 BW: body weight BCS: body condition score %BF: percentage of body fat FFM: fat free mass *Mean calculated from data obtaine d by 7 contiguous days in 30 cats ** Mean calculated from data obtained in 30 cats ***Mean calculated from data obtained in 28 cats

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51 Table 1-2. Body weight, body condition score, and % body fat at week 0, week 2 and week 4 in winter food intake study Phase Week 0 Week 2 Week 4 BW, kg 5.09 +/1.31 (3.5-8.5)a 5.12 +/-1.33 (3.3-8.47)a 5.10 +/-1.31 (3.36-8.4)a BCS 6.0 +/1.0 (4.3-8.3)a6.1 +/1.0 (4.5-8.0)a6.1 +/1.0 (4.5-8.0)a%BF*, %BW 29 +/6 (19-43) a 29 +/5 ( 20-39)a 30 +/6 (20-44) b CIRC*, cm 38.5 +/4.9 (28.5-50.0)a 38.3 +/3.9 (31.0-47.0)a 38.9 +/4.2 (30.5-49.0) b LIMB*, cm 14.6 +/1.3 (12.0-17.5) a 14.5 +/1.3 (12.5-18.0) a 14.3 +/-1.1 (12.5-17.5)a BW: body weight BCS: body condition score %BF: percentage of body fat FFM: fat free mass *%BF was calculated from measurements of the chest circumference (CIRC) at the level of the cranial 9th rib and the distance between the patella and calcaneal tuberosity (LIMB) Values are means +/SD (range), n=31. a b Mean values of the same row sharing different superscripts are significantly different (p<0.05)

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52 Table 1-3. Diets fed to the 31 cats enro lled in the winter food intake study. Cat ID Dry Wet Treat Human food B1 1Science Diet Ocean Seafood C1 1Science Diet Senior Hairball Control C2 2Tiki Cat Chicken and Egg 3Chicken soup 3Chicken,Potato,Pea & Carrot 3Chicken in Gravy 3Chicken with Egg and Peas 3Chicken and Chicken Liver 3Mackerel and Aloe 3Calamari, shrimp and mussels 3Mackerel and Skipjack 3Chicken and Duck D1 1Science Diet Light D2 1Science Diet Adult Hairball Control Feline Greenies F1 1Prescription Diet t/d F2 4Weight and Hairball Control 5Whisker Lickins & 6Wholesome Delights G1 5Purina One Urinary Tract 6Sheba Premium Cuts Feline Greenies & Pounce H1 1Science Diet Natures Best Seafood 5Whisker Linkins H2 1Science Diet Hairball Control Light J1 Natura EVO 5Friskies & 5Proplan & Innova EVO K1 1Science Diet Hairball Control Light K2 1Prescription Diet c/d 5Whisker Lickins Publix Deli Turkey meat K3 5Proplan Indoor Turkey and Rice 6WhiskasTemptation Seafood medley Flavor L1 1Prescription Diet t/d M1 1Science Diet Hairball Control Light 5Whisker Lickins Home cooked chicken M2 1Science Diet Hairball Control Light M3 1Science Diet Indoor Cat N1 7NaturalChoice Indoor Cat Weight Management N2 Mixed 1Prescription Diet c/d & 5Friskies Urinary Health N3 5Light Weight Management Q1 1Science Diet fish and rice & 1Science Diet Hairball Control Light

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53 Table 1-3. Continued Cat ID Dry Wet Treat Human food S1 5Adult Urinary Tract Health 6Whiskas choice cuts chicken gravy S2 1Prescription Diet k/d S3 1Science Diet Light S4 Innova Adult Feline Greenies S5 1Science Diet Adult Optimal Care T1 5Hairball Control 5Proplan chicken and rice T2 1Science Diet Adult Light Feline Greenies Z1 1Prescription Diet t/d Z2 1Science Diet Adult Light Cosmic cat Tuna Flavor 1Hills 2Petropics 3Weruva 4Iams (Eukanuba) 5Purina 6Mars7Nutro

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54 Table 1-4. Proportion of the meta bolizable energy from different types of food fed to each cat Cat ID Dry (%) Wet (%) Tr eat (%) Human food (%) B1 100 0 0 0 C1 100 0 0 0 C2 0 100 0 0 D1 100 0 0 0 D2 98.5 0 1.5 0 F1 100 0 0 0 F2 99.7 0 0.3 0 G1 97.1 0.4 2.5 0 H1 99.7 0 0.3 0 H2 100 0 0 0 J1 93.7 6.3 0 0 K1 100 0 0 0 K2 99.8 0 0.1 0.1 K3 99.7 0 0.3 0 L1 100 0 0 0 M1 99.8 0 0.1 0.02 M2 100 0 0 0 M3 100 0 0 0 N1 100 0 0 0 N2 100 0 0 0 N3 100 0 0 0 Q1 100 0 0 0 S1 93.6 6.4 0 0 S2 100 0 0 0 S3 100 0 0 0 S4 0 96.3 3.7 0 S5 100 0 0 0 T1 89 11 0 0 T2 99.8 0 0.2 0 Z1 100 0 0 0 Z2 98.2 0 1.8 0

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55 Table 1-5. Average of the guaranteed analyses for all dry foods wet foods, cat treats, and human foods in the food intake stu dy. Type of Food % moisture % DM* % CP* as fed % CFi* as fed % CF* as fed % NFE* as fed Kcal ME/g as fed Dry (n=23) 8 +/1 92 +/1 35 +/5 4.4 +/3 14 +/5 33 +/7 3.8 +/0.3 Wet (n=18) 81 +/4 19 +/4 12 +/3 0.3 +/1 4 +/4 2 +/2 0.9 +/0.2 Treats (n=7) 11 +/6 89 +/6 33 +/6 2 +/1 15 +/4 32 +/5 3.8 +/0.4 Human food (n=2) 71 +/6 29 +/6 26 +/8 0.03 +/0.04 1.6 +/0.1 0.3+/-0.5 1.2 +/0.3 Values are means +/SD DM=Dry Matter, CP=Crude Protein, CFi=Crude Fiber, CF=Crude Fat, NF E=Nitrogen free extract

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56 Table 1-6. The effect of including the ener gy associated with changes in body weight on estimated daily metaboli zable energy (ME). Daily metabolizable energy requirements per kg body weight (kcal/kg) pe r kg free-fat mass (kcal/kg FFM) ME1* 38 +/8 (24-57) 54 +/10 (36-79) ME2* 38 +/6 (26-55) 53 +/7 (39-76) p-value** 0.7 0.7 *ME1: ME calculated from food intake; ME2: ME calculated from food intake and taking account of BW change **There was no evidence of an effect on ME requirement when BW change was taken into account Values are means +/SD (range), n=31.

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57 Table 1-7. Daily metabolizable en ergy (ME) intake of the 31 cats in the winter food intake study expressed with different exponents. ME Range Coefficient of Variation (CV) kcal, daily 191 +/46 96-281 24% kcalkg BW-1 daily 38 +/8 24-57 21% kcalkg BW-0.4 daily 100 +/20 59-143 20% kcalkg BW-0.67 daily 65 +/-13 42-93 19% kcalkg BW-0.75 daily 57 +/-11 37-83 20% kcalkg FFM-1 daily 54 +/10 36-79 19% Values are means +/SD (range), n=31.

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58 Table 1-8. Daily metabolizable energy intake of adult cats maintaining body weight. Reference Subjects Metaboli zable Energy Requirements NRC Cats, leana 100 kcalkg BW-0.67day-1 Cats, overweight b 130 kcalkg BW-0.4day-1 Chen et al. Pet cats, leanc 38 kcalkg BW-1.067day-1 Pet cats, overweight d 87 kcalkg BW-0.494day-1 Overall (n=31) 76 kcalkg BW-0.568day-1 Overall (n=31) 71 kcalkg BW-0.78day-1 a, b Body condition score cutoff point of 5 (lean: BCS 5; fat: BCS>5) c,d Body condition score cutoff point of 6 (lean: BCS<6; fat: BCS 6)

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59 Table 1-9. Percentage of time cats spent unde rtaking activity of different intensities. Activity Intensity Activity counts/min % time Resting <100 85.3 (69.2-92.6) Low 100-499 12.1 (6.8-20.9) Medium 500-999 2.4 (0.6-8.7) High >1000 0.5 (0-1.5) Values are median with range in parentheses, n=30.

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60 Table 1-10. The relationship of metabolizable energy intake (ME) with factors that may affect energy requirements. Factors of interest Equation p-value FFM, kg ME= 4+0.88 x FFM <0.0001 BW, kg ME= 4+0.65 x BW 0.0002 %BF, %BW ME= 3+0.67 x %BF 0.01 BCS ME= 4+0.68 x BCS 0.02 Age (y/o) ME= 5-0.18 x Age 0.04 Hair coat density (mg/cm2) ME= 6-0.35 x hair coat 0.06 Activity counts / min 0.18 Midpoint Temperature ,F 0.7 FFM: fat free mass BW: body weight BCS: body condition score %BF: percentage of body fat was calculated by measuring the chest circumference (CIRC) at the level of the cranial 9th rib and the distance between the patella and calcaneal tuberosity (LIMB)

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61 Table 1-11. Seasonal comparison of di ets and their metabolizable energy (M E) density (kcal/g as fed; n=14) Cat ID Winter Diet kcal ME/ g Summer Diet kcal ME/ g C1 1 Science Diet Senior Hairball Control 3.81 Science Diet Sensitive Stomach 4.1 F2 2 Weight and Hairball Control 3 Whisker Lickins 4 Wholesome Delights 3.6 3.9 4.2 2 Indoor Weight and Hairball Control Chicken breast tender (Dog treat) 3.6 3.5 H1 1 Science Diet Natures Best Seafood 3Whisker Lickins 4.2 3.9 3Friskies signature blend 3.4 H2 1 Science Diet Hairball Control Light 3.4 (Mixed) 4 Royal Canin Intense Hariball 34 1 Prescription diet t/d 3.6 K2 1 Prescription diet c/d 3Whisker Lickins Publix Deli Turkey meat 3.9 3.9 1.0 1 Prescription diet c/d 4.0 M1 1 Science Diet Hairball Control Light 3 Whisker Lickins Home cooked chicken 3.4 3.9 1.4 1Science Diet Hairball Control Light Home cooked chicken 3.3 1.4 M2 1Science Diet Hairball Control Light 3.41Science Diet Hairball Control Light 3.2 N1 5 Indoor Cat Weight Management 3.75 Indoor Cat Weight Management 3.6 N2 (Mixed) 1 Prescription diet c/d 3 Friskies Urinary Health 3.81 Prescription diet c/d 3 UR 4.0 3.7 Q1 1 Science Diet Fish and Rice 1 Science Diet Hairball Control Light 4.2 3.4 1 Science Diet Adult Light 3.3 S1 3 Adult Urinary Tract Health 4 Whiskas choice cuts chicken gravy 3.9 0.9 3 Friskies Indoor Delight 3 Whiskas Tender Bites Meow Mix Indoor Formula 3.5 0.8 3.5 S2 1 Prescription diet k/d 4.11 Prescription diet k/d 4.1 S4 Innova Adult Feline Greenies 1.3 3.8 Innova Adult Feline Greenies 1.4 3.7

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62 Table 1-10. Continued Cat ID Winter Diet kcal ME/ g Summer Diet kcal ME/ g T1 3 Hairball Control 3 Proplan chicken and rice (different varieties) 3.7 1.0, 0.7,0.9 3 Hairball Control 3 Proplan chicken and rice 3.8 0.9 1Hills 2 Iams (Eukanuba) 3 Purina 4 Mars5 Nutro

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63 Table 1-12. Comparison of the metabolizable en ergy (ME) intake relativ e to body weight (BW) and fat-free mass (FFM) between winter and summer Metabolizable energy Winter Summer ME (%) p-value Total ME intake (kcalday-1) 208 +/41 (136-281) 182 +/47 (100-267) 13% 0.004 Total ME intake/ BW (kcalkg BW-1day-1) 38 +/7 (24-49) 32 +/5 (21-42) 15% 0.001 Total ME intake/FFM (kcalkg FFM-1day-1) 55 +/8 (38-64) 45 +/6 (34-55) 17% 0.0001 ME% = [(MEw-MEs)/MEw] x 100 Values are means +/SD (range), n=14.

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64 Table 1-13. Seasonal comparison of the aver age ambient temperature within households. Season Maximum Temperature F (C) Mid-point Tem perature F(C) M inimum Temperature F (C) Winter 67.7 +/3.0 (19.8) 71.6 +/ 2.7 (22.0) 75.6 +/3.2 (24.2) Summer 73.6 +/2.4 (23.1) 76.4 +/ 2.2 (24.7) 79.2 +/2.6 (26.2) p-value <0.0001 <0.0001 0.001 Values are means +/SD n=14

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65 Table 1-14. Comparison of body weight, body condition score, fat free mass, percentage of body fat and morphometric measurements between winter and summer Winter Summer p-value BW, kg 5.72 +/1.54 (3.59-8.46 ) 5.84 +/1.63 (3.47-8.82) 0.08 BCS 6.47 +/1.04 (4.42-8.08) 6.50 +/1.03 (5-8.25) 0.8 FFM, kg 3.8 +/0.8 (2.7-5.3) 4.1 +/0.9 (2.6-5.6) 0.005 %BF*, % BW 31.6 +/5.79 (19.51-42.09) 29.6 +/5.13 (21.8-37.78) 0.01 CIRC*, cm 40.2 +/5.2 (30.0-48.7) 40.0 +/4.5 (32.8-47.7) 0.7 LIMB*, cm 14.6 +/1.3 (12.8-17.7) 15.4 +/1.2 (13.8-17.5) <0.0001 BW: body weight BCS: body condition score, %BF: percentage of body fat FFM: fat free mass *%BF was calculated from measurements of the ch est circumference (CIRC) at the level of the cranial 9th rib and the distance between the pate lla and calcaneal tuberosity (LIMB) Values are means +/SD (range), n=14.

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66 Table 1-15. Seasonal comparison of thyr oid hormone concen trations (n=14) Season Thyroid panel Winter Summ er Ref. range p-value TT4 (nmol/l) 37+/8 (27-47) 30+/-7 (22-48) 10-55 <0.005 TT3 (nmol/l) 0.95+/0.22 (0.6-1.3) 0.80+/0.17 (0.5-1.1) 0.6-1.4 <0.005 fT4 (pmol/l) 20+/5 (13-31) 10+/2 (7-15) 10-25 <0.005 fT3* (pmol/l) 3.6+/1.0 (2-4.5) 2.18+/0.81 (1.3-4) 1.5-6.0 <0.005 TSH (mU/l) 8.3+/4.8 (0-14) 12.4+/3.5 (7-19) 0-21 <0.002 Values are means +/SD (range), n=14 Mean was the average from 12 cats because two cats did not have sufficient blood for the analysis.

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67 CHAPTER 2 VALIDATION OF THE DOUBLE-LABLED WAT ER METHOD BY COMPARING AGAINST FOOD INTAKE MEASUREMENTS Introduction Energy requirem ents have been measured in laboratory cats eith er by measuring food intake, by using indirect calorimetry, or by usi ng isotope dilution such as the double-labeled water (DLW) method. Indirect calorimetry relie s on the principle that energy released from nutrients oxidized in the body can be deduced from the rate of oxygen (O2) consumption and carbon dioxide (CO2) production. Indirect calorimetry is th e gold standard for estimating energy expenditure (EE), but is impractical in free-living cats because animals either have to be kept in individual enclosed boxes or have to wear masks. It is also very diffi cult to document individual food intake in multi-animal households or when cats roam. The DLW method has the advantage that it allows energy expenditure to be measur ed in multi-cat households and in cats which roam. The DLW method relies on the same principle as indirect calorime try except that only CO2 production is measured. The DLW method measures the decline in enrichment of the stable isotopes deuterium (2H) and oxygen (18O) in the body after water enriched with these two isotopes (as H2 180 and 2H2O) has been administered. While 18O is lost as both water and CO2, 2H is only lost as water. The difference between the disappearance rates of the two isotopes provides a measure of CO2 production. Nevertheless, the chance of error in calculating EE from CO2 production alone is increased compared to indir ect calorimetry because the energy equivalence of each liter of CO2 varies to a much greater extent than that of O2. This weakness can be minimized if the respirat ory quotient (RQ) (the ratio of the volume of CO2 released to the volume of O2 consumed by a body tissue) is known because the RQ can then be used to calculate the rate of O2 consumption from the rate of CO2 production.

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68 The DLW method has been validated for measur ing energy utilizatio n in free-living dogs but not in cats (62). The DLW methodology has not been standardized in cats with respect to the timing of the first blood collection, the route of administering th e isotopic solutions, and the dose administered. Equations used to estimate EE from CO2 production have been derived for humans and other animals such as reptiles, mammals, mars upial, and birds, and ha ve not been validated for cats (63). No studies have assessed th e accuracy and precision of the DLW method. Five studies have used the DLW method to measure the ener gy expenditure of cats; two were reported only as abstracts (26, 27) and three were published in detail (25, 28, 29). All have been performed in laboratory settings using labo ratory cats and not in homes using pet cats. Ballevre et al. compared ME intake (EI) calculat ed from food measurements directly with EE measured using the DLW method in cat s living in a communal room (24 m2) with fenced outside runs (25). Although they concluded that the DLW method is feasible in cats, only three cats were studied. Energy expenditure was on average 17% a nd 25% higher than the ME intake measured by food consumption depending on the e quation they used for calculating CO2 production (25). Two studies used DLW to measure the effect of neutering cats on EE intake. Kanchuk et al. measured EE before and after neutering in 32 cats housed in individual cages (29). Martin et al. compared EE in 19 neutered and 23 intact freeliving cats (28). Nevertheless, the cats in both these studies were not ma intaining their body weight so a dire ct comparison of EE with EI was not possible (28, 29). Thus, the purpose of this study was to evaluate the DLW method in pet cats that were not gaining or losing weight by comparing energy expenditure measured using the DLW method with ME intake estimated from daily measurement of food intake.

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69 Methods and Materials Cat owners were recru ited and cats were evalua ted as described previously (in Chapter 1) with the exception that cats from multi-cat as well as single-cat households were enrolled. Owners were only recruited if they could guarantee that their pets would be eating separately and would not have access to each others food. Also, blood parameters were not measured in cats that had participated previ ously in the feeding study. Experimental Design Energy expenditure (E E) was measured us ing the DLW method and compared to ME intake determined by documenting food consump tion over a two week period in pet cats claimed by their owners to be maintaining body weight (B W). Each cat was injected subcutaneously with saline containing double-labeled water and the decline in enrichment of the two isotopes in blood was measured over 2 weeks. Food intake was meas ured as described previously (in Chapter 1) over the same 2 weeks. The BW and morpho metrics of each cat were measured and body condition score (BCS) was assessed at the start of the study and after one and two weeks, using methods described previously (in Chapter 1). The saline solution containi ng DLW was prepared on the day of administration using sterile technique. Water enriched with deuterium (99.9 atom % 2H2O; Cambridge Isotope Laboratories, Inc, Andover, MA) was mixe d 50:50 (v/v) with water enriched with 18O (97 atom % H2 18O; Cambridge Isotope Laboratorie s, Inc). Sodium chloride (NaC l, Fisher Chemicals, Fair Lawn, NJ) was then added to obtain a 0.9% so lution, which was then passed through a 0.2 um filter (Sigma Chemical Co. Ltd, St. Louis, MO) and stored in a sterile 3 ml syringe until injection. Food was withheld for approximately 12 hours before the injection of saline containing DLW and for 2.5 hours after the injection. Access to water was not allowed for 2.5 hrs before

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70 and after the injection. E ach cat was injected subcutaneously between the shoulder blades with 0.3 g of this saline solution per kg BW. Syringe s were weighed before and after injection to determine the weight of solution injected. Blood samples (about 2 ml) were collected from the jugular or saphenous vein in vacutainer tubes (BD Vacutainer SST Tubes) immediately before the injection (background assessm ent), 2.5 h, 7 and 14 days after the injection. Serum was separated immediately by centrifuga tion and stored in cryovials at -20 C until shipment on dry ice for analysis. The enrichment of 18O and 2H in serum was measured using Europa 20/20 and Europa Hydra isotope ratio mass spectrometer respectively (Europa Scientific, LTD., Crewe, U.K.) by a commercial laboratory (Metabolic So lutions, Inc., Nashua, NH). The dose of DLW and timing of sample collections were based on a previous study with a slight modification in the dose of isotope administered (25). Data Calculation The serum enrichments of 18O and 2H were measured relative to a standard (SMOW) and corrected for baseline enrichment. The enrichme nts were then transformed to their natural logarithms and a linear regression was performed against time assuming that the disappearance of each isotope followed first order kinetics (64). The enrichment of each isotope at the time of injection was obtained by extrapolating the regre ssion line of each isotope back to the time of injection. The pool sizes for 18O and 2H (NO and NH, respectively) were derived as the reciprocal of the intercept for the regression line for 18O and 2H, respectively. The elimination rate constants for 18O and 2H (kO and kH, respectively) were obtained from the slopes of these regression lines.

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71 The mean daily CO2 production (V CO2) in moles/day was calculated from the difference of 18O and 2H elimination rates corrected for isotope fractionation (65) by assuming oxygen and hydrogen in water exist in more than one pool in the body (two pool model): V CO2= (N/2.196) x (kO-1.0427kH) (2-1) where N= [NO + (NH/1.0427)]/2 (2-2) N is the corrected average pool size for the two isotopes, 18O and 2H. Mean daily CO2 production in moles/d from (2-1) was converted to L/d by multiplying by 22.4 L/mol, assuming that one mole of an ideal gas at standard temperature and pre ssure (STP) occupies 22.4 liters. The EE was then determined using the modifica tion of the Weir equation developed by Mansell and MacDonald, which adjusts for changes in the amount of protein in the diet (66): EE (kcal/day) = K x V O2 (2-3) where K= (3.799 + 1.248 x RQ) x (1-0.04 x P) (2-4) K is the energy equivalent of each unit volume of oxygen consumed during the oxidation of a mixture of nutrients; V O2 is the rate at which O2 is consumed in L/d; RQ is the respiratory quotient which represents the ratio of the volume of CO2 released (V CO2) to the volume of O2 (V O2) consumed over time. P represents the fraction of the total ME of the diet contributed by protein. As cats were in a state of energy balance, the food quotient (FQ) was used in place of respiratory quotient (RQ) to calculate EE (67) The FQ was calculated for each cat from the composition of the food consumed: FQ= 0.81 x P + 0.71 x F + 1 x C (2-5)

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72 in which P, F, and C represent the fraction of the total ME of the diet co ntributed by protein, fat and carbohydrate, respectively, and the constants are the classical values for the FQ of the individual fuels (67). By assuming FQ=RQ, V O2 can then be obtained from the equation: V O2=V CO2/RQ=V CO2/FQ (2-6) Thus, EE was calculated by the equation re vised by Mansell and MacDonald (66): EE(kcal/day)= K x V O2 = (3.799 + 1.248 x RQ) x (1-0.04 x P) x V O2 = (3.799 + 1.248 x RQ) x (1-0.04 x P) x (V CO2/FQ) = (3.799 + 1.248 x FQ) x (1-0.04 x P) x (V CO2/FQ) = (3.799/FQ + 1.248) x (1-0.04 x P) xV CO2 (2-7) FQ could be calculated using (2-5) and V CO2 calculated using (2-1). Food and energy intake (EI) were calculated as described in the food intake study. The total daily ME intake was also adjust ed for any BW gain or loss (EIBW) observed during the 2-week study period, assuming 7 kcal of energy was associated with each gram of tissue loss or gain. Body Composition Total body water (TBW) was estim ated from the corrected average 18O and 2H pool size (N, in mols) multiplied by the molecular weight of unenriched natural water (g/mol): TBW (kg) = (18.02 x N)/1000 (2-8) The FFM (in kg) was calculated from the TBW using the mean ratio of water to FFM (0.732) reported for various species (68): FFM (kg) = TBW (kg)/0.732 (2-9)

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73 The fat mass (FM) and the percent body fat (%BF) were then derived fr om BW by difference: FM (kg) = BW (kg) FFM (kg) (2-10) %BF = FM (kg) / BW (kg) (2-11) Statistical Analysis All procedures were perf ormed with a comput er statistics progra m (SAS for Windows 9.2, SAS Institute Inc., Cary, NC). Body weight, BCS, CIRC, LIMB and %BF were tested for consistency of measurement during the stu dy using the glimmix function (46). Energy expenditure measured by the DLW method for each cat was regressed agai nst energy intake as described previously (in Chap ter 1) (45). The FFM and % BF derived using DLW method was also regressed against the corresponding measure of FFM or %BF estima ted morphometrically from the FBMI (45). The slopes and intercepts of the natural logarithm of the enrichment of the stable isotopes of hydrogen and oxygen in water over the first and second week and over both weeks after injection of double-labeled water were compared using proc ANOVA (45). The level of significance for all statistical tests was set at p <0.05. Results are reported as means +/one standard deviation unless otherwise stated. Results Animals Twelve cats were initially enrolled in the DLW validation study: six, which had previously participated in the food intake study and six newly recr uited. Two of the 12 cats did not complete the study: one cat resented further blood collection after the initial day, and the other cat became ill, unrelated to the study (Figure 2-1). Data from the remaining ten cats (6 males, 4 females, all neutered) were used for analysis. These cats were 4.8 +/2.8 years of age (Table 2-1). Two were from the same home but fed separately, the remainder were from single-cat households. Two

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74 were pure breed cats (one Russian blue and one Persian), whereas the other eight were mixed breed domestic cats. Four cats were living with dogs. Body Weight, Body Condition Sco re and Morphometric Measurements Mean BW of the 10 cats was 5.14 kg and mean BCS was 6.1 (Table 2-1). There was no evidence of a change in BW over 2 weeks but a differen ce in BCS was found between week 0 and 2 (p<0.05). The mean CIRC and LIMB were 40.2 cm and 15.0 cm, respectively, and there was no evidence of a change dur ing the study. Mean %BF was 31% of BW and mean FFM was 3.51 kg (Table 2-1). BCS was positively correlated (p<0.001) with %BF, with a coefficient of determination (R2) of 61%. Energy intake (EI) and Energy Expenditure (EE) Food intake was docum ented on a median a nd mean of 89% (range 71-100%) of days during the 2-week study period. On average, 182 kcal ME wa s taken in daily (range 72-331), whereas average EE measured using DLW was 192 kcal ME daily (range 157-252) (Figure 2-2). The correlation between EI and EE had an R2 of 62% and was best described by the equation: EE = 122 + 0.38 x EI where the units of EE and EI were expressed as kcal per day (Figure 2-3 A). There were two outliers: one with a high EI and the other with a low EI comp ared to the EE measured with DLW (Figure 2-3 A). After removing the two outl iers, the correlation between EI and EE was improved with an R2 of 69% and can be best described by the equation below, where the units of EE and EI were also expressed as kcal per day (Figure 2-3 B): EE = 57 + 0.76 x EI The total daily ME intake was also adjusted fo r any BW gain or loss observed during the 2-week study period, assuming 7 kcal of energy was associ ated with each gram of tissue loss or gain

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75 (EIBW). The correlation between EIBW and EE had an R2 of 48% and was best described by the equation: EE = 104 + 0.41 x EIBW where the units of EE and EIBW were expressed as kc al per day (Figure 24 A). After taking the BW change into account, the outli er with the low EI in Figure 2-3 A no longer differed greatly from EE, whereas the outlier with the high EI still remained much higher than EE. After excluding the outlier with the hi gh EI, the correlation between EI BW and EE had an R2 of 42% and can be best described by the eq uation, where the units of EE and EI BW were also expressed as kcal per day (Figure 2-4 B): EE = 74 + 0.57 x EI BW Body Composition Comparison Using Two Methods The average FFM for the 10 cats obtained using FBMI and DLW was 3.51 and 3.52 kg, respectively. There was a high correlation (R2=87%, Figure 2-5) betw een the FFM calculated using FBMI and that measured using DLW but th e correlation between %BF calculated using the two methods was not as strong (R2 =54%; Figure 2-6). Elimination of the Isotopes Administered (18O and 2H) No evidence of a difference in either slope or intercept of the two stable isotopes was detected between the first and second week a nd over both weeks after in jection of double-labeled water (Table 2-2). Discussion In the current study, EE was on average 7% higher than EI and there was a wide variation in the difference between EI and EE with EE ranging from 42% lower to 54% higher than EI. The regression line of EI against EE did not intersect zero and did not coincide with the expected relationship where EI=EE. Neverthe less, with the exception of two potential outliers, one with a

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76 low EI and the other with a high EI, most data poi nts were close to the expected regression line (EI=EE) (Figure 2-3 B). Change in BW could explain the outlier with the low EI because the difference between EI and EE was reduced from 85 kcal to 5 kcal after taking account of the change of BW. Nevertheless, change in BW did not explain the other outli er with the high EI (Figure 2-4 A). Once the outlier with the high EI is discounted, the regressi on line lies closer to the expected regression line (EI=EE) (Figure 24 B). The influence of a change of BW was magnified because the energy from the change in body mass was made available over a short time period (only 14 days instead of the 28 da ys of the previous food intake study). An explanation for the other outlier wi th the high EI compared to EE may be that either EI or EE was measured incorrectly. Since the cat in questi on was living with a dog, it is very possible that the dog was eating the cats food without the owners knowledge. Several models and equations have been used to estimate body water pool size and CO2 production and EE in cats (Table 2-3). Some assume that the two isotopes flux within the same single body water pool as described by Lifson a nd McClintock (69). Others used a two pool model initially proposed by Schoeller (70), which assumes that both isotopes exist in more than one pool in the body. In the current study, a revised equation proposed by Speakman was used because it has been found to improve accuracy and precision compared with the existing equation proposed by Schoeller (71). Neverthele ss, the equations used to calculate CO2 production were designed for humans (69-71). In addition, most studies have used the equation proposed by Weir to calculate of EE from V CO2 (72) without taking account of the increased protein metabolism in cats. This study used th e revision of the Weir equation developed by Mansell and MacDonald to adjust for changes in the amount of pr otein in the diet (66). Since there is currently no validated equations for cats, the ultimate choice of equation can only be

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77 made when the estimate of CO2 production can be compared with CO2 production measured directly during indirect calorimetry or EE is compared to energy intake obtained from food intake in cats with stable body weight. The time when the first sample was collected has also varied among previous studies. Isotope enrichments rise to a peak after equili brating within the body. Es timates of the time for isotopes to reach equilibrium vary from minutes to hours depending on the species and route of administration (63). Collecting samples too soon or too late would underestimate or overestimate the elimination rate of the isot ope, respectively, and could also affect the calculated pool size when the regression of enrichment is extrapolated to the time of injection. Ballevre and Martin selected the same route (subcutan eous injection) for isotope ad ministration as in the present study, but the first sample colle ction time varied between 2-4hrs (25, 28). Speakman suggested an equation for intraperitoneal and intramuscula r injection to estimate equilibration time using BW based on previous studies summ arized from different species of animals, such as reptiles, mammals, marsupial, birds (63): Equilibration time = 2.555 + 0.360 x loge BW (2-11) where the equilibration time is estimated in hour s, and BW is in kilograms. Speakman also suggests an alternative method whereby the eq uilibration time is obtained by adding 1 h for every 10 kg of BW to an initial value of 1 h (63). In the cu rrent study, the mean BW of cats was 4.8 kg, so the first sample time of 2.5h was simila r to that suggested using equation 2-11, i.e. 2.8h, and slightly higher than that suggested by the alternative method (1.5h) (Table 2-3). The need for frequent blood sampling may limit owners participati on. Therefore, it may be possible to recruit more cats if the DLW method could be si mplified by obtaining measurements from one rather than two weeks. In the current study, the slope and intercept of

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78 both isotopes were not different between 1st and 2nd weeks or the two weeks as a whole. This supports the assumption that the disappearance of each isotope follows single order kinetics (64) and suggests that it might be sufficient to perform the DLW study over one week only. The feline body mass index (FBMI) develope d by Waltham had been shown to be well correlated to the %BF measured by DEXA (R2=85%, p<0.0001) (43). In the current study, we showed that the %BF calculated by FBMI was well correlated to %BF estimated by the DLW method in a group of pet cats with a wide range of BCS, thereby sugges ting that FBMI is a practical method which could be applied in daily practice. In conclusion, although DLW is a useful technique for es timating EE in free living animals, its accuracy in predicting energy requirements is still questionable. More cats are still needed to clarify whether there is indeed a good corre lation between EI and EE. Equations used to determine V CO2 and EE need to be validated in cats.

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79 Figure 2-1. Flow sheet showing the recruitment of participants in the double-labeled water (DLW) study.

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80 0 50 100 150 200 250 300 350 12345678910 Cat IDME intake (kcal /day) Figure 2-2. Comparison between the metabolizable energy (ME) intake estimated from food intake (blue columns) and energy expend iture calculated us ing the double-labeled water method (purple co lumns) in ten cats.

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81 A 0 50 100 150 200 250 300 350 050100150200250300350 EI (kcal/day)EE (kcal/day ) B 0 50 100 150 200 250 300 350 050100150200250300350 EI (kcal/day)EE (kcal/day ) Figure 2-3. Regression of ener gy intake (EI) against energy expenditure (EE). The x-axis represents EI measured from food intake and the y-axis represents EE assessed using the double-labeled water method. A) The pi nk line is the expected line EI=EE, whereas the blue line is th e regression line that best describes the data points: EE=122+0.38 x EI with a R2 of 62%. The blue diamonds with circles around are the two outliers found in the study. B) After removing the two ou tliers, the blue line is quite close to the expected line (pink line).

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82 A 0 50 100 150 200 250 300 350 050100150200250300350400 EI (kcal/day)EE (kcal/day ) B 0 50 100 150 200 250 300 350 050100150200250300350 EI (kcal/day)EE (kcal/day ) Figure 2-4. Regression of ener gy intake (EI) against energy expenditure (EE) after taking account of the change in body weight (BW). Th e x-axis represents EI measured from food intake and the y-axis represents EE assessed usi ng the DLW method. A) The pink line and the blue line are as describe in Figure 2-2. The yellow triangles are the data points derived after taking account of the BW changes over the 2-week study period and the yellow line is the equation that best describe these data points. The yellow triangle with circle remains an outlier B) The yellow line is quite close to the expected line (pink line) if the outlier is excluded.

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83 0 1 2 3 4 5 6 012345 FFM-FBMI (kg)FFM-DLW (kg ) R2=0.87 Figure 2-5. Correlation of the fat free mass (F FM) derived using two methods. The x-axis represents FFM calculated using morphom etric measurements (the feline body mass index or FBMI) and the y-axis represents FFM estimated using isotope dilution of double-labeled water (DLW). Strong correlation was observed with a R2 of 87%.

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84 0 5 10 15 20 25 30 35 40 45 051015202530354045 %BF, %BW (FBMI)%BF,%BW (DLW ) R 2=0.54 Figure 2-6. Correlation of the percentage body fat (%BF) derived by two method. The x-axis represents %BF of %BW calculated using mo rphometric measurements (the feline body mass index or FBMI) and the y-axis re presents %BF of %BW estimated using isotope dilution of double-labeled water (D LW). Moderate correlation was observed with a R2 of 54%.

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85 Table 2-1. Characteristics of the pet cats enrolled in the double -labeled water (DLW) validation study (n=10) Characteristics Means +/SD Range Age, years 4.8 +/2.8 2-10 BW, kg 5.14 +/1.28 3.32-7.92 BCS 6.1 +/1.0 5.0-8.0 %BF*, %BW 31+/5 22-40 FFM, kg 3.51 +/0.66 2.59-4.76 CIRC, cm 40.2 +/3.8 32.8-46.2 LIMB, cm 15.0 +/0.8 13.8-16.0 BW: body weight BCS: body condition score %BF: percentage of body fat FFM: fat free mass *%BF was calculated from measurements of the chest circumference (CIRC) at the level of the cranial 9th rib and the distance between the pate lla and calcaneal tuberosity (LIMB)

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86 Table 2-2. The slopes and intercepts of the natu ral logarithm of the enri chment of the stable isotopes of hydrogen and oxygen in water over the first and second week and over both weeks after in jection of double-labeled water. Isotope 1s t wk 2n d wk Both wks p-value Deuterium (2H) Slope Intercept -0.0522 +/-0.008 -4.9999 +/0.2 -0.0492+/-0.007 -5.0207+/0.2 -0.0507 +/-0.007 -5.0036 +/0.2 0.7 1.0 18O Slope Intercept -0.0782 +/-0.009 -4.9243 +/-0.2 -0.0750+/-0.010 -4.9463+/0.2 -0.0766 +/0.009 -4.9281 +/0.2 0.8 1.0 Values are means +/SD n=10

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87 Table 2-3. Sources for the equations used for calculating carbon dioxide production (V CO2) and energy expenditure (EE) Primary author (ref.) V CO2 (ref.) EE (ref.) Ballevre (25) Single pooled model: Lifson and McClintock (69) Two pooled model: Schoe ller and Coward (70) Elia (73) Nguyen (26) Nguyen (27) Not specified Weir (72) Martin (28) Single pooled model:Lifs on and McClintock (69) Weir (72) Kanchuk (29) Not specified Weir (72) Chen (current study) Two pooled model: Speakman and Nair (71) Mansell & MacDonalds (66) Table 2-4. Comparison of publishe d methodologies that have used the double-labele d water to measure energy expenditure in cats Primary author (ref.) Route of isotope administration Time of first sample collection after isotope administration Sampling frequency Ballevre (25) Subcutaneous injection 2 hrs Multiplea Nguyen (26) Nguyen (27) Not Specified Not Specified 2 hrs 14 days Multiple b Two point c Martin (28) Subcutaneous injection 4 hrs Multiple d Kanchuk (29) Intravenous injection 1 hr Multiplee Chen (current study) Subcutaneous injection 2.5 hrs Multiple f a Blood samples were collected imme diate before injection, 2hrs, 7days and 11 days after the injection b Blood samples were collected imme diate before injection, 2hrs, 7days and 14 days after the injection c Blood samples were collected immediate before injection a nd 14 days after the injection d Blood samples were collected imme diate before injection, 4hrs, 7days and 14 days after the injection e Blood samples were collected immediate before injection, 1hr, 2 days, 5 days,7da ys and 12 days afte r the injection f Blood samples were collected imme diate before injection, 2.5hrs, 7days and 14 days after the injection

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88 APPENDIX A QUESTIONNAIRE FOR STUDY OF ENERGY REQUIREMENTS OF PET CATS 1. Cats nam e _____________________________________________________________ 2. Owners name __________________________________________________________ 3. Owners address _________________________________________________________ 4. Phone numbers __________________________________________________________ 5. Email address ________________________________________________________ 6. Age of cat? ______________yrs and/or DOB___________________________ 7. Gender? _____________________________________________________________ 8. Neutered/spayed? _______________________________________________________ 9. What breed is your cat? ___________________________________________________ 10. Length of hair coat _________________________________________________ 11. Does cat live indoors only? ________________________________________________ 12. Floor area of cats domain? Square feet? _____________________________________ 13. What kind of toys? (can be mu ltiple choices): Balls / Laser po inter / catnip toys / Climbing frame? Other stuffed mice? __________________________________________________ How long and how often do you play with your cat? _____________________ 14. What is your cats usua l activity level? ________________________________ 15. Do you have other pets? Do they play with your cat? If yes, please describe how they interact. ___________________________________________________________________________ 16. If you have other pets in your house, will you be absolutely sure that they wont get the others food? ____________________________________________________________ 17. Is the temperature in your house thermostatically controlled? _______________ 18. To what temperature is the ther mostat set: Winter ____ F Summer____ F 19. Does your cat normally wear a collar? ________________________________ 20. What do you usually feed your cat Manufacturer Brand name Flavor Can/dry Amount Times/d Diet 1 ___________ ___________ ________ ________ _______ ______ Treat ___________ ___________ ________ ________ _______ ______ Human food: _______ ___________ ________ ________ _______ ______

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89 APPENDIX B ACTIVITY MONITOR QUESTIONNAIRE 1. How m any people live in your house? __________________________________________________________________ 2. What time do you usually get up in the morning? Person 1 Person 2 Person 3 Person 4 Weekdays Weekends 3. What time in the day you play w ith your cat? What kind of activity? ___________________________________________________________________________ ___________________________________________________________________________ 4. What time in the day are you most at home? ___________________________________________________________________________ _________________________________________________________ 5. Do you have dogs living with the ca t? Are they playing together? __________________________________________________________________ __________________________________________________________________ 6. What time do you usually go to bed? Person 1 Person 2 Person 3 Person 4 Weekdays Weekends 7. Do you often have companies coming over to your house? __________________________________________________________________ 8. What time do you get out of class/ work? __________________________________________________________________ 9. How long is your cat alone throughout the day? ____________________________________________________

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90 APPENDIX C ATTACHMENT FOOD CONSUMPT ION AND ROOM TE MPERATURE Cats name________________ TITLE OF RESEARCH STUDY: Energy requirements of pet cats 1. Manufacturer_________________ Brand_________________ Type of food______ 2. Manufacturer_________________ Brand_________________ Type of food______ 3. Manufacturer_________________ Brand_________________ Type of food______ 4. Manufacturer_________________ Brand_________________ Type of food______ 5. Manufacturer_________________ Brand_________________ Type of food______ *Please record the Max and Min temperature in the morning everyday before resetting. *Please also record the time when you weigh the food. This is especially important for canned food. Date Type of food Time Wt Fed (g) Time Wt Left(g) Max Temp(F) In Out Min Temp(F) In Out

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91 LIST OF REFERENCES 1. National Research Council Nutrient requi rements of dogs and cats. Washington, DC: National Academy Press; 2006. 2. Nelson RW HC, Feldman EC, Bottoms GD. Gl ucose tolerance and insulin response in normal weight and obese cats. Am J Vet Res. 1990;51:1357-62. 3. Feldhahn JR RJ, Martin G. Insulin sensitivity in normal and diabetic cats. J Feline Med Surg. 1999;1:107-15. 4. Scarlett JM DS. Associati on between body condition and dis ease in cats. J Am Vet Med Assoc. 1998;212:1725-31. 5. Lund EM AP, Kirk CA, Klausner JS. Prevalence and risk factors for obesity in adult cats from private US veterinary practices. Intern J Appl Res Vet Med. 2005;3:88-96. 6. Aub JC FJ, Bright EE. The effect of adre nalectomy upon the total metabolism of the cat. Am J Physiol. 1922;61:326-48. 7. Caldwell GT. Studies in water metabolism of the cat. Physio l Zool. 1931;4:324-55. 8. Benedict FG. Vital Energetics. Washingt on DC: Carnegie Institution of Washington; 1938. 9. Carpenter TM. The effect of sugars on th e respiratory exchange of cats. J Nutr. 1944;28:315-23. 10. Hauschild C. Energetische Untersuchunge n zum Erhaltungsbedar f von adulten Katzen. (Investigations on maintenance energy requireme nts of cats.). Berlin: Freie Universitaet; 1993. 11. Radicke B. Der Einflu-beta unterschiedliche r Naehrstoffgehalte in Alleinfuttermitteln fuer Katzen auf den energetischen Erhaltungsbedarf Ansatz und auf den Rohproteinbedarf von adulten Katzen (Effect of nutrient composition of complete diets on maintenance energy requirements, ener gy accretion and energy utilization for accretion and crude protein requirements of a dult cats). Berlin: Freie Universitaet,; 1995. 12. Tennant B. Assessment of energy expenditure in cats using indirect calorimetry. J Anim Physiol Anim Nutr. 1998;80:60-2. 13. Stiefel M. Einfluss dreier unterschi edlicher Diaeten auf den Energieund Proteinstoffwechsel adulter Ka tzen unter spezieller Be ruecksichtigung der physischen Aktivitaet (Effect of three di fferent diets on energy and prot ein metabolism of adult cats with special consideratio n of physical activity). Un iversity of Zurich; 1999. 14. Laeuger S. Der Energieumsatz von Katern vor und nach der Kastration (The energy expenditure of male cats be fore and after neutering.): University of Zurich; 2001.

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92 15. Krehl WA CG, Whedon D. Non-deleterious effects of polyoxyethylene esters in the nutrition of rats and ca ts. J Nutr. 1955;55:35-61. 16. Miller SA AJ. The dietary nitrogen requirements of the cats. J Nutr. 1958;64. 17. Skultety FM. Alterations of caloric intake in cats followi ng lesions of the hypothalamus and mid brain. Ann NY Acad Sci. 1969;157:867-74. 18. Kendall PT BS, Smith PM. Comparative digest ible energy requirements of adult beagles and domestic cats for body weight ma intenance. J Nutr. 1983;113:1946-55. 19. Earle KE, Smith PM. Digestible energy require ments of adult cats at maintenance. J Nutr. 1991 Nov;121:S45-6. 20. Taylor EJ CA, and R Neville. Some nutritional aspects of ageing in dogs and cats. Proc Nutr Soc. 1995;54:645-56. 21. Flynn MF HE, Armstrong PJ. Effect of ovariohysterectomy on maintenance energy requirement in cats. J Am Vet Med Assoc. 1996;209:1572-81. 22. Parkman AL MK, Erswell KE, Laflamme DP How many calories do pet cats really need? Purina Nutrition Forum Proceedings. 2000;23(9A):85. 23. Laflamme DP BJ. Effect of age on maintenance energy requirements of adult cats. The Purina Nutrition Forum. St.Louis, MO; 2001. 24. Edtstadtler-Pietsch G. Untersuchungen zum Energiebedarf von Katzen (Investigations on energy requirements of cats). Munich : Ludwig-Maximilians-University; 2003. 25. Ballevre O, Anantharaman-Barr G, Gicquello P, Piguet-Welsh C, Thielin AL, Fern E. Use of the doubly-labeled water method to a ssess energy expenditure in free living cats and dogs. J Nutr. 1994 Dec;124:2594S-600S. 26. Nguyen P MS, Martin L, Dumon H, Biourge V, Darmaun D, Robins R, Naulet N. Assessment of energy expenditure with doubly la beled water in adult cats. Supplement to Compendium on Continuing Education for th e Practicing Veterinarian. 1999;22 (9A):96. 27. Nguyen P DH, Frenais R, Siliart B, Martin L, Bleis P, Fregier T. Energy expenditure and requirement assessed using three different methods in adult cats. Supplement to Compendium on Continuing Education for th e Practicing Veterinarian. 2000;23 (9A):86. 28. Martin L, Siliart B, Dumon H, Backus R, Biourge V, Nguyen P. Leptin, body fat content and energy expenditure in intact and gonad ectomized adult cats: a preliminary study. J Anim Physiol Anim Nutr (Berl). 2001 Aug;85:195-9.

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93 29. Kanchuk ML, Backus RC, Calvert CC, Morris JG, Rogers QR. Weight gain in gonadectomized normal and lipoprotein lipase-def icient male domestic cats results from increased food intake and not decreased energy expenditure. J Nutr. 2003 Jun;133:186674. 30. Elliott DA. Techniques to assess body composition in dogs and cats. Waltham Focus. 2006;16(1):16-20. 31. Berman M DI. The social behavior of free-ranging suburban dogs. Appl Anim Ethol. 1983;10:5-17. 32. Sterman MB, Knauss T, Lehmann D, Clemente CD. Circadian sleep and waking patterns in the laboratory cat. Electroencepha logr Clin Neurophysiol. 1965 Nov;19:509-17. 33. Panaman R. Behaviour and ecology of free-ranging female farm cats. Z Tierpsychol. 1981;56:59-73. 34. Kuwabara N SK, Aoki K. Circadian, sleep and brain temperature rhythms in cats under sustained daily light-dark cycles and consta nt darkness. Physiol Behav. 1986;38(2):2839. 35. Houpt KA. Domestic animal behavior for ve terinarians and animal scientists. 3rd ed. Ames, Iowa: Iowa State University Press.; 1998. 36. Riond JL, Stiefel M, Wenk C, Wanner M. Nu trition studies on protein and energy in domestic cats. J Anim Physiol An im Nutr (Berl). 2003 Jun;87:221-8. 37. Yamada M, Mikihiko T. Spontaneous activities measured continuously by an accelerometer in Beagle dogs housed in a cage. J Vet Med Sci. 2000;62:443-7. 38. Lascelles BD, Hansen BD, Thomson A, Pier ce CC, Boland E, Smith ES. Evaluation of a digitally integrated accelerometer-based activ ity monitor for the measurement of activity in cats. Vet Anaesth Analg. 2008 Mar;35:173-83. 39. Burger IH. Energy needs of companion anim als: matching food inta kes to requirements throughout the life cycle. J Nutr. 1994 Dec;124:2584S-93S. 40. Nguyen PG, Dumon HJ, Siliart BS, Martin LJ, Sergheraert R, Biourge VC. Effects of dietary fat and energy on body weight and co mposition after gonadectomy in cats. Am J Vet Res. 2004 Dec;65:1708-13. 41. Laflamme D. Development and Validation of a Body Condition Score System for Cats : a clinical tool. Felin e Pract. 1997;25:13-8. 42. Blaxter K. Energy metabolism in animals and man. New York: Cambridge University Press; 1989.

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94 43. Hawthorne A. Butterwick RB. Predicting the body composition of cats: development of a zoometric measurement for estimation of percentage body fat in cats [abstract]. J Vet Intern Med. 2000;14:365. 44. Freund RJ LR. SAS System for Regre ssion Cary, NC: SAS Institute Inc.,; 2006. 45. Cody RP SJ. Applied Statistics and th e SAS Programming Language. 4th edition ed. Upper Saddle River, NJ: Prentice-Hall, Inc.; 1997. 46. SAS/STAT 9.2 User's Guide: The GLIMMI X Procedure (Book Excerpt) Cary, NC: SAS Institute Inc.; 2008. 47. Hoenig M TK, Waldron M, Ferguson DC,. Insulin sensitivity, fat distribution, and adipocytokine response to differe nt diets in lean and obese cats before and after weight loss. Am J Physiol Regul Integr Comp Physiol. 2007;292:R227-34. 48. Lester T C-MG, Lewis D,. Cats increase fa tty acid oxidation when isocalorically fed meat-based diets with increasing fat content. Am J physiol. 1999;46:R878-R86. 49. Kienzle E, Edtstadtler-Pietsch G, Rudnick R. Retrospective study on the energy requirements of adult colony cat s. J Nutr. 2006 Jul;136:1973S-5S. 50. Cave NJ, Backus RC, Marks SL, Klasing KC. Oestradiol, but not ge nistein, inhibits the rise in food intake following gonadectomy in cat s, but genistein is associated with an increase in lean body mass. J Anim Phys iol Anim Nutr (Berl). 2007 Oct;91:400-10. 51. Fettman MJ SC, Banks LL, et al. Effects of neutering on bodyweight, metabolic rate and glucose tolerance of domestic cats. Res Vet Sci. 1997;62:131-6. 52. Harper EJ SD, Watson TDG, et al,. E ffects of feeding regimens on bodyweight, composition and condition score in cats fo llowing ovariohysterectomy. J Small Anim Pract. 2001;42:433-8. 53. Lauten SD, Cox NR, Baker GH, Painter DJ, Morrison NE, Baker HJ. Body composition of growing and adult cats as measured by use of dual energy X-ray absorptiometry. Comp Med. 2000 Apr;50:175-83. 54. Stanton CA, Hamar DW, Johnson DE, Fettm an MJ. Bioelectrical impedance and zoometry for body composition analysis in domestic cats. Am J Vet Res. 1992 Feb;53:251-7. 55. Anantharaman-Barr HG GP, Rabot R. The effect of age on the digestibility of macronutrients and energy in cats. Proc Br Small Anim Vet Assoc,. 1991:p.164 (abs.). 56. Scarlett JM DS, Saidla J, Wills J. Overwei ght cats: Prevalance a nd risk factors. Int J Obesity. 1994;18:s22-s8.

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95 57. Kendall PT BI, Smith PM. Methods of estima tion of the metabolizable energy content of cat foods. Feline Pract. 1985;15:38-44. 58. National Research Council Nutrient re quirementsof dogs and cats. Washington, DC: National Academy Press; 1985. 59. Adams T MM, Hunter WS, Holmes KR. Temp erature regulation of the unanesthetized cat during mild cold and severe heat stress. J A ppl Physiol. 1970;29:852-8. 60. Gale CC. Neuroendocrine aspects of th ermoregulation. AnnRevPhysiol. 1973;35:391430. 61. Fregly MJ FF, Katovich MJ, Barney CC. Ca techolamine-thyroid hormone interaction in cold-acclimated rats. Federation Proc. 1979;38:2162-9. 62. Speakman JR P-CG, McCappin T, Frankel T, Thompson P. Validation of the doublylabelled water technique in the domestic dog (Canis familiaris). Br J Nutr. 2001;85:7587. 63. Speakman JR. Doubly Labelled Water: Theory and Practice. 1st ed. London: Chapman & Hall; 1997. 64. Lifson N GG, McClintock R. Measurement of total carbon dioxide production by means of D218O. J Appl Physiol. 1955;7:704-10. 65. Speakman JR. The history and theory of th e doubly labeled water technique. Am J Clin Nutr. 1998 Oct;68:932S-8S. 66. Mansell PI MI. Reappraisal of the Weir equation for calculation of metabolic rate. Am J Physiol. 1990;258:R1347-R54. 67. Black AE PA, Coward WA,. Use of food quotie nts to predict respiratory quotients for the doubly-labeled water method of measuring energy expenditure. Human Nutrition: Clinical Nutrtion. 1986;40C:381-91. 68. Rathbun EN PN. Studies on body composition I. The determination of total body fat by means of the body specific grav ity. J Biol Chem. 1945;158:667-76. 69. Lifson N MR. Theory of use of the turnover rates of body water for measuring energy and material balance. J Theor Biol. 1966;12:46-74. 70. Schoeller DA RE, Schultz Y, Acheson KJ, Baertschi P, Jequier E. Energy expenditure by doubly labelled water: valida tion in humans and proposed calculation. Am J physiol. 1986;250:R823-30. 71. Speakman JR NK, Goran MI. Revised e quation for calculating CO2 production from doubly labeled water in humans. Am J Physiol. 1993;264:E912-E7.

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96 72. Weir JB. New methods for cal culating metabolic rate with special reference to protein metabolism. J Physiol. 1949;109:1-9. 73. Livesey G EM. Estimation of energy expenditu re, net carbohydrate ut ilization, and net fat oxidation and synthesis by indi rect calorimetry: evaluation of errors with special reference to the detailed composition of fuels. Am J Clin Nutr. 1988;47:608-28.

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97 BIOGRAPHICAL SKETCH Ching-Ai Chen was born on March 28, 1983 in Kaohsiung, Taiwan. She interacted with com panion animals extensively from a young age b ecause her father is a veterinarian. Ching-Ai studied veterinary medicine a nd graduated from National ChungHsing University in Taiwan after a five year program in 2006. Ching-Ai was admitted the master program in veterinary medical sciences at the Univer sity of Florida in 2007. She is now graduating and would like to bring back the information that she has learned in the United States to Taiwan. Her plan for now is to work as a vete rinary practitioner.