Effect of Nutritional Interventions on Longevity of Senior Cats

Carolyn J. Cupp, DVM, MS1 Clementine Jean-Philippe, DVM, PhD2 Wendell W. Kerr, MS1 Avinash R. Patil, BVSc, PhD1 Gerardo Perez-Camargo, PhD, MRCVS2

1Nestlé Purina PetCare Research St. Joseph, Missouri This article was previously published 2Nestlé Purina R&D Centre, Amiens in Volume 4, Number 1. It is being Aubigny, France reprinted as a service to our readers with appended tables. KEY WORDS: aging, feline, nutrition, INTRODUCTION longevity, antioxidants In all mammals, aging is associated with ABSTRACT changes in body condition, body composition, energy requirements, declining organ The objective of this study was to evaluate function and immune status, and other whether antioxidants, alone or in combina- metabolic changes. Nutrition may play an tion with other nutritional supplements, important role in delaying such changes or increase health and longevity in a population preventing their progression.1 In the older of older cats. A group of 90 cats between human population, a link between nutritional the ages of 7 and 17 years was blocked into status, quality of life, and overall physical 3 groups by age, body condition score, and and cognitive health has been demonstrat- gender. Cats were assigned to 1 of 3 diets: ed.2–5 Nutritional and healthy lifestyle modi- control (basal diet of nutritionally complete fications initiated in older human individuals cat food), basal diet with added antioxidants were positively related to reduced mortality (vitamin E and β-carotene), and basal diet risk and delayed deterioration in health sta- with added antioxidants, dried whole chico- tus, indicating that even health promotion at ry root (source of prebiotic), and a blend of older ages can contribute to healthy aging.6,7 supplemental n-3 and n-6 fatty acids. The In dogs, life-long maintenance of ideal body diets were fed exclusively for the remaining condition by calorie restriction delayed the lifetime of each cat. Physical exams, body onset of disease and extended median lifes- condition scores, complete blood count, pan by 15% over full-fed littermates.8 serum chemistries, plasma fatty acids, serum Aging encompasses a complex set of antioxidant status, fecal microflora, urinaly- processes that gradually leads to increased sis, and body composition by dual-energy x- vulnerability and damage at the cellular ray absorptiometry were performed at study and organ level, and eventual death of the initiation and at periodic intervals thereafter. organism. While multiple environmental After 5 years, cats fed the diet with the and genetic factors may be involved, there antioxidants vitamin E and β-carotene, dried is considerable evidence that oxidative chicory root, and a blend of n-3 and n-6 stress plays a major pathophysiologic role in fatty acids lived significantly longer than aging and may explain the pathogenesis of cats fed the control diet. Positive indicators many age-related diseases. With increases of reduced disease incidence and improved in oxidative stress and reduced levels of intestinal health were also observed. circulating antioxidants, a potential link with

Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. 133 longevity would be expected. An increased MATERIALS AND METHODS requirement for dietary antioxidants in aging Animals and Housing individuals is a logical conclusion.9 A multi-year feeding study was initiated in The influence of nutrition on the qual- February 2000 with 90 healthy mixed-breed ity and length of life is of great interest to cats in a controlled research facility setting. pet owners and veterinarians alike. The Several age groups of senior cats were as- presumption that nutritional needs change signed to the trial in an attempt to positively with aging has been addressed through a influence the visible as well as physiological number of approaches to the formulation of changes that occur during aging. The large senior pet foods, but there are no published age range provided an opportunity to study data to date showing that specific nutrient the effect of dietary manipulation initiated in combinations, when given to healthy aging mature as well as older adult animals. pets, increase longevity or delay the onset of Selected cats were at least 7 years of age, disease.10 There are no official guidelines for often considered to be the beginning of the specific nutritional requirements in senior senior life stage.16–18 Cats were divided into pets, nor is there consensus on when a pet 3 chronological age groups of 30 cats each: becomes “senior.” Nutritional therapy is 7 to 9 years, 10 to 12 years, and 13 or more widely practiced for aging pets with specific years. Initial ages ranged from 7 to 17 years. clinical problems such as renal disease,11,12 Each diet group had an equal distribution of the 3 age groups. cardiac disease,13 diabetes,14 and inflammatory bowel disease,15 but in the clinically Prior to initiation of the study, cats were screened to eliminate those with pre-exist- healthy aging pet, dietary modifications also ing clinical disease. The screening consisted may be useful to compensate for declining of complete physical exam by the colony functional systems. veterinarian, complete blood count, serum The primary objective of this study was chemistry, thyroid status, and urinalysis. to evaluate whether antioxidants, alone or in Cats were also required to have normal fecal combination with other nutritional supple- consistency (no loose stools) for inclusion ments, increase longevity in a population of in the study. A body condition score was older cats when compared with cats fed a assigned to each cat by the staff veterinar- nutritionally complete control diet. Two test ian using a numerical classification from diets were chosen, one with supplemental extremely thin (1) to obese (5). Cats were antioxidants vitamin E and β-carotene, and distributed equally among dietary treatment a second diet combining these same antioxi- groups on the basis of age, body condition dants with additional dietary modifications score, and gender. (a prebiotic and supplemental n-6 and n-3 Cats were initially maintained based on fatty acids) that could have a positive impact the housing conditions to which they were on the health of cats. We hypothesized that most accustomed, either individually or in combining several nutritional strategies rooms of 25–27 cats. As group-housed cats became less active and unable to reach the could have synergistic or additive effects elevated perches and feeding stations in not evident in studies investigating only therooms, they were moved to individual- individual nutrient components, and that ized housing to complete the study. Food these potential nutrient interactions could was provided ad libitum with the exception measurably benefit the health and longevity of an overnight fast prior to anesthesia or of the aging cat. Initial results on longevity blood collection. Water was available at all and several of the measured parameters are times. All housing was maintained at a room reported here. temperature of 18°C–24°C and 40%–50% relative humidity, with controlled lighting

134 Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. to provide 12-hour light and dark periods. staff. Any cat exhibiting signs of distress Cats were individually housed on days when or illness was thoroughly examined and a feces were obtained for evaluation. health report generated. Medical treatments Diets were administered according to established colony veterinary procedures. Dietary treat- Cats were assigned to 1 of 3 diets: Diet ments were not adjusted due to illness, and 1 (control): nutritionally complete and the dietary treatment group did not influence balanced adult cat food; Diet 2: control therapeutic regimens. All veterinary person- formula + antioxidants (vitamin E as alpha nel were blinded to dietary treatment groups. tocopheryl acetate and β-carotene); and Diet Humane euthanasia was carried out accord- 3: control formula + antioxidants + prebiotic ing to established colony procedures only (whole chicory root) + blend of oils (supple- after all appropriate diagnostic procedures, mental source of n-6 and n-3 fatty acids). therapeutic regimens, and multiple assess- Diets were produced every 6 to 8 weeks ments failed to show a clinical response, in the Nestlé Purina Product Technology and the staff veterinarian deemed the cat’s Center (PTC, St. Joseph, Missouri, USA). prognosis poor. Typical nutrient comparisons are shown in The test diets were fed as the exclusive Table 1. source of nutrition for the remaining lifetime Animal Care and Health of each cat assigned to the trial. Fresh food Health monitoring and treatment of all was offered daily in stainless steel bowls. If animals were carried out according to a cat refused to eat the test diet and the lack established colony veterinary procedures of food consumption did not appear to be throughout the trial. All cats were observed related to a medical condition, the cat was several times per day for general appearance considered for removal from the study after and behavior by the veterinary and caretaker all avenues to improve consumption were attempted. Table 1. Nutrient Composition. Trial Protocol Diet 1 The study protocol was reviewed and ap- Nutrient (Control) Diet 2 Diet 3 proved by the Nestlé Purina PTC Animal ME, kcal/g 4.8 4.8 4.8 Care and Use Committee. Cats selected for DMB the study were assigned to 3 groups of 30 Protein, % 43.5 43.0 39.3 cats per age level. Each group was divided DMB equally between dietary treatments and Fat, % DMB 35.6 35.9 36.9 started on trial over a 3-month period. If cats Ash, % DMB 9.8 9.8 10.2 had to be removed from trial for low food consumption, replacement cats of compa- Calcium, 3.9 4.1 4.1 rable age, sex, and body condition were g/1000 kcal subsequently started on trial. Phosphorus, 3.1 2.8 3.1 The following measurements were taken g/1000 kcal for all cats at study initiation: complete Vitamin E, 69.9 140.7 149.5 blood count, serum chemistries, plasmafatty IU/1000 kcal acid profile (control and Diet 3 only), serum β-Carotene, ND 5 5 antioxidant status (superoxide dismutase, mg/1000 kcal glutathione peroxidase, vitamin E, β-caro- Linoleic acid, tene), bone density and body composition by % of dietary 10.5 10.5 21.3 dual-energy x-ray absorptiometry (DEXA, fat GE Lunar Prodigy, GE Healthcare, Diegem, ME = metabolizable energy; Belgium), fecal microflora (control and DMB = dry matter basis; ND = not detectable. Diet 3 only), and urinalysis. Cats also were

Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. 135 given complete physical examinations and determinations, standard microscopic sedi- assigned a body condition score. ment analysis, Orion pH meter for urine pH Food consumption was measured daily (Model 620, Thermo Electron Corporation, during the study, and body weights were as- Waltham, Massachusetts, USA) and Reichert sessed weekly. Clinical measurements taken Veterinary refractometer for urine specific at study initiation (time 0 [t0]) were repeated gravity (Model 10436, Reichert Analytical throughout the study duration, as shown in Instruments, Depew, New York, USA). Table 2. Fresh fecal samples were collected and Routine veterinary dental prophylaxis processed for microflora evaluation within was performed every 6 months, when cats 15 minutes of defecation. Samples (5 g) were anesthetized for DEXA scanning. were mixed in a stomacher bag with 10%

Anesthesia was performed according to w/w glycerol, CO2 flushed, and hot sealed. established colony veterinary procedures, Samples were frozen at -80°C, and micro- based on the health status of the cat. Uri- flora analysis was performed in batches by nalysis was performed as part of the routine phase within 3 months of collection. If a cat health monitoring. Urine was evaluated us- was placed on antibiotic therapy during the ing commercial reagent strips for chemical study, fecal collection was not performed

Table 2. Parameter Measurements and Frequency (in months). Procedure Cats t0 3 6 9 12 15 18 21 24 27 30 33 39 42 45 48 51 54 Body condition All X X X X X X X X X X X X X X X X X X score Veterinary physical All X X X X X X X X X X X X X X X X X X exam Body composition All * X X X X X X X X X X X (DEXA) CBC/serum chem- All X X X X X X X X X X X X X istry Diets Plasma 1 and X X X X X fatty acids 3 Antioxidant enzymes All X X X X X X X X X X X SOD, GP Serum All X X X X X X X X X vitamin E Serum β- All X X X X carotene Diets Fecal 1 and X X microflora 3 Urinalysis All X X X X X X X X X X X DEXA = dual-energy x-ray absorptiometry; CBC - complete blood count; SOD = superoxide dismutase; GP = glutathione peroxidase. *In some cases, anesthesia and DEXA were not performed if the cat was considered by the veterinary staff to be too frail to undergo the procedure.

136 Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. until at least 14 days after completion of Hazard ratios along with their 95% confi- antibiotic therapy. Fecal microflora (Bi- dence intervals were estimated. fidobacteria, Lactobacillus, Clostridium To compare the average age at death for perfringens) were quantified using real- the cats on the 3 diets, a censored regression time polymerase chain reaction with Roche analysis was used.22 In the censored regres- LightCycler® System (Idaho Technology sion, the dependent variable was the age of Inc., Salt Lake City, Utah, USA).19,20 death and the independent variables were Blood samples were obtained by jugular initial age and diet. venipuncture. For complete blood count and Analysis of measured parameters, such antioxidant enzymes, whole blood samples as blood values, body condition score, body were collected in EDTA tubes and evaluated composition by DEXA, body weight, and immediately using the Baker 9110 hematol- food consumption, was performed by a lon- ogy analyzer (pre-2002; Bio Chem Immu- gitudinal analysis.23 Results of longitudinal nosystems, Inc., Allentown, Pennsylvania, analysis were expressed as average slopes or USA) or Sysmex KX-21N hematology “predicted means” (LSMEANS from PROC analyzer (Sysmex America Inc., Munde- MIXED in SAS/STAT® Software, SAS In- lein, Illinois, USA) and Randox ELISA kits stitute, Cary, North Carolina, USA) for cats (Ransel and Ransod, Randox Lab, Crumlin, with an initial age of 12 years. The longitu- United Kingdom). For serum chemistry and dinal model allowed for each animal’s trend vitamin analysis, serum was obtained within to be considered over time and an average 30 minutes by centrifugation, and frozen at trend or slope predicted for each group. For -80°C for later batch analysis by test phase. parameters that were only measured at a few Serum chemistries were quantified on a time points, repeated measures analysis of Ciba Corning Express Plus analyzer (Bayer variance was performed. Healthcare, Tarrytown, New York, USA) Fecal microflora data was analyzed us- through mid-2001, and a Hitachi 912 ana- ing Kruskal-Wallis test.24 lyzer (Boehringer Mannheim Corporation, Chi-square contingency table analysis Indianapolis, Indiana, USA) since mid-2001. was used to evaluate the incidence of pa- Samples for antioxidant vitamin analysis thologies at necropsy. In this analysis, 2-way were sent to the Michigan State Diagnostic contingency tables were used to determine Center for Population and Animal Health, the differences between Diets 1 and 3. Be- Nutrition Department. cause most cats died with multiple patholo- Statistical Analysis gies, a separate analysis was performed for Analysis of variance was used to compare each group of pathologies. Cats that had initial parameters across groups to confirm not died at the time of analysis were not that randomization was effective in produc- accounted for, so the analysis is considered ing balance at baseline (t0) in the 3 study preliminary. groups. All statistical calculations were per- Using the number of days a cat was on formed using SAS.25 trial until death, a Cox proportional hazards RESULTS model was used to compare the survival rates of the 3 diets.21 Because there was a Study Population wide range of ages of the cats at trial initia- A total of 15 cats were removed from the tion, the initial age of the cat was used as study for refusal to eat, mostly within the a covariate in the model. Separate adjusted first 6 weeks of the trial. Ten of these cats survival curves were generated for the 3 age were replaced with other cats and started groups of cats. The model used an initial on trial at a later date than the original 3 age of 8, 11, and 14 years, representing the groups. Five cats had to be removed later in average initial age of cats in each age group. the study and were not replaced; 3 of those

Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. 137 cats (1 from each dietary treatment group) on Diet 3 was only 39% of the hazard of were removed by the veterinarian more than dying for the cats on Diet 1. There was no 6 months after trial initiation. These 3 cats significant difference between Diets 1 and 2 have been included in the statistical data set or between Diets 2 and 3. Figures 1, 2, and and are considered “censored” data, along 3 show the adjusted survival curves from the with the remaining cats that are still living. proportional hazard model using initial ages Baseline Data (t0) of 8, 11, and 14 years, respectively, which represent the average initial age of cats in At the time of diet assignment, there were each age group. For all starting ages, the no statistical differences in clinical measures analyses demonstrated that the proportion (age, weight), body composition (body con- of cats surviving was significantly higher on dition score, DEXA parameters), hematolog- Diet 3 than Diet 1 (P < 0.05). ical, or serum biochemical values between The results of censored regressions com- the 3 groups (Table 3). The analysis showed paring the diets for age at death are shown no significant differences between groups in in Table 5. These results show that the cats any of the baseline measures, indicating that on Diet 3 lived significantly longer than the randomization was effective in producing cats on Diets 1 and 2. After calculating the balance at baseline in the 3 study groups. predicted age of death for different initial Longevity ages, it was observed that cats on Diet 3 For the survival analysis, there were 88 cats lived about 1 year longer than cats in the in the data set: 68 had died, 3 were removed other groups (Table 6). early, and 17 had not yet died at the time of Disease Incidence/Pathology analysis. Table 4 gives the results of the Cox Histopathology results from deceased cats proportional hazard model using initial age were recorded and tabulated for 6 general as a covariate. Results for the hazard ratios disease pathologies: renal, pancreas, cancer show that Diet 3 was significantly different of any type, thyroid (hyperplasia or adeno- than Diet 1 (P < 0.01). In this model, the mas), gastrointestinal, and liver. While the hazard ratio of Diet 1 vs. Diet 3 was 0.39, analysis must be considered preliminary meaning that the hazard of dying for the cats because not all cats have died, there was Table 3. Baseline Data (t0). a statistically Variable Diet 1 Diet 2 Diet 3 P-Value significant dif- Initial age (years) 11.7 ± 2.5 12.3 ± 2.4 12.2 ± 2.6 0.589 ference for Initial weight (g) 4529 ± 1411 4118 ± 1195 4032 ± 993 0.254 thyroid hyperplasia/adenomas Body condition score 2.62 ± 1.0 2.42 ± 0.7 2.64 ± 0.6 0.475 (P < 0.05), with Body fat (%) 17.5 ± 12.1 14.9 ± 9.5 12.9 ± 8.2 0.246 Diet 3 cats hav- Body fat (g) 817 ± 792 591 ± 469 501 ± 421 0.117 ing significantly Lean mass (%) 79.7 ± 11.5 82.2 ± 8.9 84.1 ± 7.6 0.227 fewer incidences Lean mass (g) 3164 ± 793 2933 ± 774 2969 ± 627 0.453 of disease. Only RBC (×106 U) 8.86 ± 1.6 8.28 ± 1.7 8.87 ± 1.4 0.254 14% of cats on Albumin (g/dL) 3.34 ± 0.4 3.22 ± 0.5 3.34 ± 0.3 0.433 Diet 3 were affected compared SUN (mg/dL) 23.5 ± 8.9 27.9 ± 14.0 25.4 ± 9.3 0.320 with 43.5% of Calcium (mg/dL) 9.13 ± 1.0 9.15 ± 1.0 9.25 ± 1.2 0.894 cats on Diet 1. Creatinine (mg/dL) 1.42 ± 0.5 1.34 ± 0.4 1.47 ± 0.4 0.467 There was also Potassium (mEq/L) 4.80 ± 0.7 4.85 ± 0.5 5.13 ± 0.9 0.201 a nearly sig- SUN:Creatinine ratio 16.8 ± 4.0 19.9 ± 9.0 17.3 ± 4.9 0.135 nificant trend for Vitamin E (μg/mL) 13.3 ± 8.3 10.6 ± 5.9 12.9 ± 4.8 0.244 gastrointestinal RBC = red blood cell; SUN = serum urea nitrogen. pathologies (P

138 Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. = 0.08), with only 19% of cats on Diet 3 having some form of gastrointestinal disease compared with 43.5% of cats on Diet 1. For the other general pathologies, there were no statistically significant differences between diets (Figure 4). Body Weight Maintenance All 3 groups lost weight over time, on average, an expected result with aging cats. Dietary differences in the average trends Figure 1. Adjusted survival curves starting at (slopes) were statistically significant for age 8 years. body weight (P < 0.05), with cats fed Diet 3 showing less decrease in body weight over time than cats on Diets 1 and 2 (Figure 5). Average food consumption in calories per kilogram of body weight increased over time for all 3 groups. There was a statistically significant difference between the average trends (slopes) for food consumption (P < 0.05). Cats fed Diet 3 showed less of an increase in food consumption over time than cats fed Diets 1 and 2 (Figure 6).

Figure 2. Adjusted survival curves starting at Fecal Microflora age 11 years. Initial and 3-month fecal microflora results are summarized in Table 7. There was a significant increase in Bifidobacteria (P < 0.01) and a significant decrease in Clostrid- ium perfringens bacteria (P < 0.01) for the cats on Diet 3, while cats consuming Diet 1 had no significant changes. Cats on Diet 3 had an overall improved fecal flora profile than cats on Diet 1, as shown by the ratio of Bifidobacteria + Lactobacilli to Clos- tridium perfringens. From the Kruskal-Wal- lis test, the change from initial values was significantly different between the 2 diets Figure 3. Adjusted survival curves starting at age 14 years. for Bifidobacteria, Clostridium perfringens, and ratio of Bifidobacteria + Lactobacilli to Clostridium perfringens (P < 0.01). A total of 77% of cats on Diet 3 responded with either an increase in Bifidobacteria or Lac- tobacillus and/or a decrease in the number of Clostridium perfringens bacteria. Overall, the data showed that cats consuming Diet 3 harbored healthier gut microflora than cats fed the control (chicory-free) diet.

Figure 4. Incidence of pathologies in deceased cats.

Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. 139 Body Composition and Body Condition Scores For all parameters of body composition, including those measured by DEXA as well as body condition scores assigned by the examining veterinarian, longitudinal analysis showed significant decreases over time across all treatment groups (P < 0.05). Dietary differences in the average trends (slopes) were statistically significant (P < Figure 5. Body weights by diet (average trend 0.05) for body condition score, lean body lines or predicted means). mass, bone density, bone mineral content, and total tissue (lean plus fat). Cats fed Diet 3 showed less decrease over time than both Diets 1 and 2 for body condition score (Figure 7), bone density, and bone mineral content, and less decrease over time than Diet 1 for lean body mass and total tissue (Figure 8). There were no significant differences between diets for fat (expressed as total fat, region fat, or tissue fat). Blood Figure 6. Food consumption in kcal/kg body Cats fed Diet 3 had significantly higher weight (average trend lines or predicted levels of serum vitamin E, serum β-carotene, means). and plasma linoleic acid compared to cats fed the control diet. Tables 8 and 9 show the means and P-values from the ANOVA for β-carotene and linoleic acid. After 6 months on the study, cats on Diet 3 had significantly higher blood levels of β-carotene and linoleic acid than cats on Diet 1. Vitamin E levels were evaluated at 0 (t0), 3, 6, 9, 24, 30, 36, 42, 48, and 54 months, and a longitudinal analysis of variance was performed relating vitamin E to Figure 7. Average trend lines (predicted means) time by diet. Figure 9 shows a plot of the for body condition scores. predicted means for each diet for an initial

Figure 8. Average trend lines (predicted means) Figure 9. Average trend lines (predicted means) for total tissue (kg). for serum vitamin E.

140 Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. Table 4. Survival Analysis. and invertebrate species 95% CI for have shown that a variety Variable df P-Value Hazard Ratio Hazard Ratio of genetic and pharma- Diet 1 vs. cologic interventions 1 0.2701 0.712 0.389–1.303 Diet 2 can produce increases in 30 Age 1 <0.0001 1.464 1.253–1.711 longevity. One study in mice showed that the Diet 1 vs. 1 0.0042 0.386 0.201–0.741 life-extending effects of Diet 3 caloric restriction and the Age 1 <0.0001 1.513 1.297–1.765 presence of a “longev- Diet 2 vs. 1 0.2097 0.685 0.379–1.238 ity” gene that blocks the Diet 3 production or response Age 1 <0.0001 1.382 1.197–1.595 to growth hormone were df = degrees of freedom; CI = confidence interval. additive, suggesting that age of 12 years. There was a significant at least 2 mechanisms are 31 diet-time interaction (P < 0.05), and Diet 3 involved. Reducing total fat mass without cats had significantly higher serum levels caloric restriction also increased longevity of vitamin E than cats fed either Diets 1 or in mice, suggesting that leanness rather than food restriction could be the key factor in 2 (P < 0.05). The slopes of the linear and 32 quadratic effects show that vitamin E levels extended longevity. did not change over time in cats fed Diet 2. While it is not known whether caloric For cats consuming Diet 1, vitamin E levels restriction or the maintenance of a more decreased with time, leveling off in later lean body mass throughout life could months. For those fed Diet 3, there was an extend lifespan in cats, there is evidence increase in serum vitamin E with time but that extreme leanness in old cats may actu- also a leveling off for the later months. ally be detrimental. Emaciated cats had a significantly higher risk of death compared DISCUSSION with cats in optimal body condition.33 Perez- The hypothesis that a combination of nutri- Camargo et al34,35 demonstrated that body tional interventions could have a positive weight, lean body mass, and fat mass decline effect on the health and longevity of senior in cats over the age of 12 years, particularly cats was supported by the data reported here. in the last 1 to 2 years of life. Studies have Cats consuming a diet containing supple- also shown an increase in energy require- mental antioxidants, a source of prebiotics, ments despite progressive weight loss in and a blend of oils lived significantly longer than cats fed the control diet, when adjusted Table 5. Comparison of Diets for Age at Death. for their initial age. The hazard of dying for Wald Chi the cats on the supplemented diet was only Effect df Square P-Value 39% of the hazard of dying for the cats on the control diet. A key question is whether All diets 2 6.7917 0.0335 these results can be attributed to just one of Diet 1 vs. 1 0.1222 0.7266 the dietary manipulations or if they reflect Diet 2 an additive or synergistic effect of multiple Diet 1 vs. nutritional interventions. 1 6.8389 0.0089 Caloric restriction has long been known Diet 3 as an intervention to increase longevity in Diet 2 vs. 1 4.2744 0.0387 a number of species.8,26–29 While there are Diet 3 many theories, one mechanism involves a reduction in metabolic rate and reduced Initial age 1 35.9581 <0.0001 oxidative stress. Studies in both vertebrate df = degrees of freedom

Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. 141 Table 6. Predicted Mean Age at Death at healthy lifestyle factors showed a more Different Initial Ages (Years). than 50% reduction in mortality rate from Initial Age = 8 all causes in elderly people who started the Diet Predicted Mean Standard Error study between the ages of 70 and 90 years.7 1 12.9714 0.43624 Thus, even in old age, it appears possible to 2 13.1313 0.46382 positively affect longevity. 3 14.0744 0.47951 In this study, a group of mature and geriatric cats fed a diet consisting of supplemen- Initial Age = 11 tal antioxidants, a prebiotic, and a source of Diet Predicted Mean Standard Error long-chain n-3 and n-6 polyunsaturated fatty 1 14.3526 0.32643 acids lived significantly longer than cats 2 14.5124 0.33221 on a control diet. Studies that have evalu- 3 15.4555 0.34803 ated the influence of similar dietary modi- Initial Age = 14 fications individually have shown various Diet Predicted Mean Standard Error benefits. 1 15.7337 0.35903 Antioxidants 2 15.8936 0.33421 Antioxidants vitamin E and β-carotene were 3 16.8367 0.34411 added to both test diets in this study since oxidative stress is thought to play a critical aging cats.36,37 Slowing down the inexorable role in aging. Antioxidants are believed to body weight loss of aging could theoreti- exert a protective effect in cells by scav- cally help extend the life expectancy of the enging the toxic free radicals that cause elderly cat. In the current study, cats were oxidative stress and DNA damage.9,29,39–42 fed ad libitum, and those on Diet 3 had bet- Antioxidant status is reportedly reduced ter long-term maintenance of body weight, and may even be predictive of mortality in body condition, and body composition mea- elderly humans.39,40,43 However, high levels sures, such as lean body mass. They lived on of circulating antioxidants have been seen in average about a year longer than cats on the the “oldest old,”40,44,45 providing a potential control diet. explanation for the extreme longevity of A multi-factor nutritional approach to some individuals. studying longevity is not uncommon. Recent Vitamin E is a large component of plas- population-based studies in humans have ma membranes and helps protect membrane shown statistically significant increases in phospholipids from peroxidation. Jewell et longevity for individuals eating a Mediter- al46 showed improved antioxidant status with ranean diet.7,38 A combination of diet and Table 7. Fecal Microflora (log cfu/g feces).*

Diet 1 (Control) Diet 3 P-Value, Change from Initial Diet 3 Bacteria Initial 3 Months Initial 3 Months vs. Diet 1 Bifidobacteria 7.45 6.19 6.56a 8.23b 0.002 (Bifido) Clostridium perfrin- 10.70 8.36 10.93a 7.70b 0.000 gens Lactobacilli (Lacto) 5.85 6.96 6.29 6.67 0.629 Bifido + Lacto:Clos- tridium perfringens 1.24 1.57 1.17a 1.93b 0.007 ratio *Means across rows with different letter superscripts are statistically different (P < 0.05).

142 Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. high levels of dietary vitamin E (540 IU/kg) the blood. Supplementation of vitamin E in dogs and cats. Vitamins E and C and in a variety of diet types has been shown β-carotene in combination reduced oxida- to increase serum vitamin E concentra- tive stress and DNA damage in lymphocytes tions.45,53,56,57 Chew et al58 showed that cats in cats with renal insufficiency.47 Vitamin are able to readily absorb β-carotene dosed E also has been reported to be efficient at orally in a water solution, with concentra- preventing oxidation of low-density lipo- tions of plasma β-carotene increasing in a proteins in humans.48 At high doses (5 g/kg dose-dependent manner. Charlton et al59 also diet), aging male mice fed supplemental showed significant increases in plasma β- vitamin E showed a 40% increased median carotene concentrations of cats after feeding lifespan, a 17% increase in maximal lifes- a carotenoid-containing diet. Both moder- pan, and up to 45% improved neurological ate antioxidant supplementation and a diet performance over control mice.49 high in carotenoids elevated serum carot- Epidemiological studies in humans have enoids and antioxidant levels in an older shown mixed results. A large 10-year study human adult population.60 The group eating in women showed no significant effect of more fruits and vegetables increased serum vitamin E on total mortality, but did show a alpha tocopherol over time, and the authors significant reduction in cardiovascular mor- speculated that perhaps concurrent supple- tality, particularly in women over the age of mentation with carotenoids may increase 65 years.50 Other studies have demonstrated nutrient absorption or bioavailability of the that a diet rich in antioxidants reduces the tocopherols. incidence of cancer and chronic disease, and Both test diets in the current study were the maintenance of a strong immune system formulated with the same supplemental may be a key factor.9 In puppies, antioxidant levels of vitamin E and β-carotene, and both supplementation improved immune response groups of cats showed significant increases to vaccination,51 and dietary lutein showed in serum β-carotene over control and positive effects on the immune system in baseline levels. However, only cats fed Diet cats.52 Antioxidant vitamins added to the diet 3 showed a significant increase in serum also increased immunological responses of vitamin E over control and baseline values elderly people53,54; an inverse relationship as well as a significant effect on longevity. between plasma vitamin E and infectious Possible explanations could include higher disease episodes was observed.53 Similarly, levels of oxidative stress and antioxidant supplementation with β-carotene reduced requirements in the Diet 2 cats, reduced the number of infection days in an elderly absorption of vitamin E from the intestinal human population.55 tract, or differences in the intestinal micro- Assessment of antioxidant status in the environment, producing a vitamin E-sparing living system is somewhat difficult, but effect for Diet 3. The nature of the dietary a relatively simple indicator is the mea- lipids accompanying vitamin E and their in- surement of the specific antioxidants in testinal lipolytic products may affect vitamin E absorption.61 Alpha tocopherol absorption Table 8. ANOVA for Plasma Linoleic Acid. across enterocyte cell membranes is P-Values dependent on the for Diet 1 Parameter Time Diet 1 Diet 3 vs. Diet 3 incorporation and solubilization of the Plasma Initial 21.38 ± 0.5 20.04 ± 0.41 0.1738 vitamin into mixed linoleic acid 62 3 Months 19.89 ± 0.5 26.89 ± 0.41 <0.0001 bile salt micelles. (g/100 g fat) Dietary long-chain ± SE 6 Months 19.17 ± 0.52 26.74 ± 0.43 <0.0001 polyunsaturated fat- SE = standard error. ty acid levels have

Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. 143 Table 9. ANOVA for Serum β-Carotene. P-Values Diet 1 vs. Diet 1 vs. Diet 2 vs. Parameter Time Diet 1 Diet 2 Diet 3 Diet 2 Diet 3 Diet 3 0.004 ± 0.028 ± 0.018 ± Initial 0.4312 0.6265 0.7214 Serum 0.022 0.021 0.018 β-carotene 0.067 ± 0.131 ± 0.146 ± 3 Months 0.0413 0.0073 0.5799 (μg/mL) ± 0.023 0.021 0.018 SE 0.036 ± 0.134 ± 0.171 ± 6 Months 0.0021 <0.0001 0.1977 0.023 0.021 0.019 SE = standard error. been shown to have an impact on vitamin E are increased.69 Similar results have been ob- absorption that could be linked to a change served in cats. Results from studies in young 63,64 in size and charge of the mixed micelles. and old cats have shown a significant corre- 65 Wander et al showed that a low n-6 to n-3 lation between age and intestinal flora, with fatty acid ratio in the diet can reduce circu- Bifidobacteria decreasing and Clostridium lating vitamin E levels in dogs, presumably perfringens increasing with advancing age.70 due to increases in lipid peroxidation caused In older animals, positively modifying the by the higher levels of n-3 fatty acids. In intestinal ecosystem could have significant pre-term human infants, however, formula potential anti-inflammatory and immune supplemented with n-3 and n-6 polyunsatu- benefits within the gut.71,72 Chicory is also rated fatty acids resulted in higher plasma reported to have antioxidant properties, due and cellular tocopherols than standard for- to the presence of active compounds such 66 mulas. The authors hypothesized that the as flavonoids and polyphenols that could fatty acids improve tocopherol solubility and contribute to improving health status of ag- stability in biological membranes. ing animals.73 Prebiotics In the current study, 77% of cats on Diet 3 contained whole dried chicory root Diet 3 responded with either an increase as a source of intestinal prebiotic. Prebi- in Bifidobacteria or Lactobacillus and/or a otic fibers are fermented in the colon and decrease in Clostridium perfringens. These selectively stimulate the growth of Bifido- cats had an overall healthier intestinal bacteria and/or Lactobacilli, considered as microflora profile than those fed the control “beneficial” bacteria of the large intestine. diet, as shown by the ratio of Bifidobac- Inulin is an important source of fructo-oligo- teria and Lactobacillus to Clostridium saccharide (FOS), and chicory root contains perfringens. In the long term, a reduction in 50%–55% by weight inulin. Multiple studies inflammatory diseases of the gut could be have demonstrated the efficacy of chicory expected. While preliminary, the analysis in modifying intestinal microflora in dogs of the incidence of pathologies at necropsy and cats. Cats consuming food containing in this study did show a trend for reduced chicory had a significant increase in fecal numbers of cats on Diet 3 with histological Bifidobacteria and a significant decrease evidence of gastrointestinal diseases. in fecal Clostridium perfringens compared In addition to positive effects within with the same cats when fed control prod- the gut, improving the balance of intestinal ucts without chicory.67,68 bacteria in aging cats could have systemic With advancing age, it is generally re- benefits. Reduction in toxic by-products of ported that Bifidobacteria are diminished in bacteria, such as Clostridium perfringens, human feces, while Clostridium perfringens could have far-reaching effects relating to

144 Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. inflammation in a variety of body systems. fatty acids have potential anti-inflammatory, More favorable bacterial fermentative end skin and coat, and digestive health benefits. products could benefit renal health by re- The anti-inflammatory effects of n-3 duced ammonia production and consequent fatty acids have been well studied.83,84 The reduced urea load on the kidneys. oxidation of fatty acids results in forma- Healthy cats without any signs of tion of eicosanoids, such as prostaglandins gastrointestinal tract dysfunction have been and leukotrienes, which have roles in the shown to have high numbers of bacteria, immune system, modulate inflammation, including Clostridium perfringens, in the and maintain normal epidermal integrity. proximal part of the small intestine.74–76 This Reducing inflammation could be of signifi- flora could be responsible for bacterial deg- cant benefit for numerous organ systems in radation of nutrients in the small intestine the aging cat. 76 and for other side effects on digestion. It Older cats often exhibit changes in the has been demonstrated in broiler chickens appearance or consistency of the hair coat that Clostridium perfringens, the primary and skin that could be related to the lipid small intestinal gram-positive bacteria in content of the diet. Essential fatty acid the chicken, have the ability to deconjugate deficiency in cats results in numerous gross bile salts by expressing high levels of bile clinical, pathological, histological, and phys- 77 salt hydrolase activity. One consequence iological changes. Clinical changes reported is an alteration of alpha tocopherol and in cats fed essential fatty acid-deficient lipid absorption, which can be corrected by diets include a rough or greasy hair coat, 78 antibiotic treatment. When incorporated dandruff or scaly skin, failure of wound into the food, alpha tocopheryl acetate must healing, increased wax in ears, and increased be hydrolysed into alpha tocopherol prior to susceptibility to infections.85–89 MacDonald absorption. The main enzyme responsible et al90 noted a marked increase in water loss is a pancreatic esterase that is dependent on through the skin, alopecia, and focal exuda- 79–81 bile salt in numerous species. It could tive dermatitis in cats fed essential fatty be hypothesized that the prebiotic source in acid-deficient diets. Adding essential fatty Diet 3 decreased the population of Clos- acids back into the diet reversed some of tridium perfringens in the small intestine as these negative effects.83 well as in the large intestine, and therefore A number of digestive health benefits reduced the deconjugation of bile salts, their have been reported for the polyunsaturated precipitation, and excretion into the feces. fatty acids. Due to their anti-inflammatory The better efficacy of pancreatic esterases by and antibacterial properties, some polyun- stronger bile salt activation could have led saturated fatty acids have the ability to kill to increased vitamin E absorption with the harmful bacteria that are likely to be present prebiotic supplemented diet. Further studies in the gastrointestinal tract.91 Bomba et al92 would be needed to confirm this hypothesis. proposed that dietary fatty acids affect at- Fatty Acids tachment sites of intestinal flora by modify- An oil blend providing a source of poly- ing the fatty acid composition of the intesti- unsaturated n-3 and n-6 fatty acids was nal wall. Thus, dietary lipids may influence included in Diet 3 for several reasons. The gastrointestinal microflora populations. rate-limiting enzyme in fatty acid metabo- Nutrient digestibility, particularly fat di- lism, delta-6-desaturase, reportedly declines gestibility, is reduced in old cats.1,93,94 Recent in activity with age, while some pro-inflam- studies evaluating cats in several research matory metabolites of eicosanoids have been colonies fed a variety of dry and wet cat shown to increase with age,82 suggesting foods showed that the incidence of low fat that fatty acid requirements may be different digestibility increases with advancing age, in older pets. Additionally, polyunsaturated affecting at least one third of geriatric cats

Intern J Appl Res Vet Med • Vol. 5, No. 3, 2007. 145 over 12 years of age.34,95 There are likely on lifespan in healthy old cats. Whether the multiple mechanisms involved, including results from this study reflect the combined subclinical diseases of the pancreas, liver, or benefits of the different dietary interven- intestinal mucosa, but defects in the absorp- tions or some synergistic effects between the tive capacity of the aging gut may also be nutrients is not known. Certainly there are associated. some documented interactions between anti- Linoleic acid is an essential n-6 fatty oxidants such as vitamin E and polyunsatu- acid in the diet of cats. The National Re- rated fatty acids that may have come into search Council (1986) recommended that play in this study.66,99–101 Additionally, the all feline diets contain a minimum of 1 g of positive effects of chicory on gastrointestinal linoleic acid per 1000-kcal diet.96 The Asso- microflora and perhaps even improvements ciation of American Feed Control Officials in vitamin E and fatty acid absorption may Nutrient Profile for Cats lists the dietary have played a role in this study. requirement for linoleic acid in growing and In summary, senior cats fed a diet con- adult cats as 1.25 g/1000 kcal ME.97 While taining supplemental antioxidants vitamin most commercial pet foods contain well E and β-carotene, dried chicory root, and over the minimum requirement, aging cats a blend of n-3 and n-6 fatty acids lived that suffer from reduced fat absorption or di- significantly longer than cats fed a standard gestibility could be at risk for essential fatty nutritionally complete feline diet. Positive acid deficiency, making the supplementation trends for decreased incidence of thyroid of higher levels potentially beneficial. One and gastrointestinal pathologies suggest that study of human cystic fibrosis patients found the nutrient blend may provide some protec- that increasing dietary linoleic acid helped tion against certain disease states that may improve fat malabsorption.98 contribute to their increased longevity. Addi- CONCLUSIONS tional data collected over the next few years may help further elucidate the mechanisms Results from this study suggest that a diet for both the increased survival and improve- supplemented with a combination of antioxi- ment in health status of these aging cats. dants, prebiotic, and long-chain polyunsaturated fatty acids, at specifically formulated ACKNOWLEDGMENTS levels, can increase lifespan in senior cats. Appreciation is expressed to Lynn Gerheart, It is logical to hypothesize that multiple Dr. Gail Czarnecki-Maulden, and Dr. Linda nutritional approaches may be necessary Young for assistance in diet formulation, to measurably slow the inevitable physi- to James Ambrose for animal care and data ological decline of aging. Because increased collection, to Dr. James Mrkvicka, Ronda oxidative stress and consequent disturbances Lopez, and Adriane Stafford for veterinary in energy metabolism are probably criti- care, and to Rick McKnight, Rachel Ander- cal mechanisms, both dietary interventions son, Kenneth Tomes, and Cynthia Steeby for included elevated levels of antioxidants. An laboratory support. increased incidence of inflammatory condi- REFERENCES tions, renal disease, cancer, and disorders of 1. Taylor EJ, Adams C, Neville R: Some nutritional intestinal function in the aging cat, as well aspects of ageing in dogs and cats. Proc Nutr Soc as observed physical changes, such as a 1995;54:645-656. deterioration in skin and coat condition, led 2. Diehr P, Beresford SA: The relation of dietary patterns to future survival, health, and cardio- to the inclusion of prebiotics and dietary n-3 vascular events in older adults. J Clin Epidemiol and n-6 fatty acids in the second test diet. 2003;56(12):1224-1235. While these dietary interventions have 3. Keller HH, Ostbye T, Goy R: Nutritional risk predicts quality of life in elderly community-liv- shown various health benefits by themselves ing Canadians. J Gerontol A Biol Sci Med Sci in other studies, until now there have been 2004;59(1):68-74. no reports indicating a measurable effect 4. Seccareccia F, Alberti-Fidanza A, Fidanza F,

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