Effects of weight loss and feeding specially formulated diets on the body composition, blood metabolite profiles, voluntary physical activity, and fecal metabolites and microbiota of overweight cats

Abstract

Feline obesity is a common and preventable disease, posing a myriad of health risks and detriments. Specially formulated diets and restricted feeding may serve as an intervention strategy to promote weight loss and improve feline health. In this study, our objective was to determine the effects of restricted feeding and weight loss on body composition, voluntary physical activity, blood hormones and metabolites, and fecal microbiota of overweight cats. Twenty-two overweight adult spayed female and neutered male cats [body weight (BW) = 5.70 ± 1.0 kg; body condition score (BCS) = 7.68 ± 0.6; age = 4 ± 0.4 yr] were used in a weight loss study. A control diet (OR) was fed during a 4-wk baseline to identify intake needed to maintain BW. After baseline (week 0), cats were allotted to OR or a test diet (FT) and fed to lose ~1.0% BW/wk for 24 wk. At baseline and 6, 12, 18, and 24 wk after weight loss, dual-energy x-ray absorptiometry scans were performed and blood samples were collected. Voluntary physical activity was measured at weeks 0, 8, 16, and 24. Fecal samples were collected at weeks 0, 4, 8, 12, 16, 20, and 24. Change from baseline data were analyzed statistically using the Mixed Models procedure of SAS, with P < 0.05 considered significant. Restricted feeding of both diets led to weight and fat mass loss, lower BCS, and lower blood triglyceride and leptin concentrations. Cats fed the FT diet had a greater reduction in blood triglycerides and cholesterol than cats fed the OR diet. Restricted feeding and weight loss reduced fecal short-chain fatty acid, branched-chain fatty acid, phenol, and indole concentrations. Fecal valerate concentrations were affected by diet, with cats fed the OR diet having a greater reduction than those fed the FT diet. Fecal bacterial alpha diversity was not affected, but fecal bacterial beta diversity analysis showed clustering by diet. Restricted feeding and weight loss affected relative abundances of 7 fecal bacterial genera, while dietary intervention affected change from baseline relative abundances of 2 fecal bacterial phyla and 20 fecal bacterial genera. Our data demonstrate that restricted feeding promoted controlled and safe weight and fat loss, reduced blood lipids and leptin concentrations, and shifted fecal metabolites and microbiota. Some changes were also impacted by diet, highlighting the importance of ingredient and nutrient composition in weight loss diets.

In this study, we aimed to determine the effects of diet, restricted feeding and weight loss on body composition, voluntary physical activity, blood hormones and metabolites, and fecal metabolites and microbiota populations of overweight cats. Restricted feeding was shown to promote safe weight and fat loss, reduce blood lipids and leptin concentrations, and shift fecal metabolites and microbiota.

Introduction

In 2022, 28% of cats were classified as overweight and 61% were classified as overweight or obese by their veterinary professional in the United States (APOP, 2022). Of the cats that were classified as overweight or obese, 28% of cat owners considered their pet’s body condition to be “normal”. Even more alarming is that 7% of owners with obese cats considered them to have a “normal” body condition score (BCS). Accurately assessing a cat’s BCS can be challenging with their long hair and prominent primordial pouches, commonly referred to as “belly flaps”. Previous literature has shown that owners with long-haired cats were 11.5 times more likely to underscore their cat’s BCS compared to those with short-haired cats (Courcier et al., 2010).

Obesity is multi-factorial disease that plagues the vast majority of pets. Some factors that play a role are lifestyle-specific and include: voluntary engagement in physical activity, living in a captive environment, age, sex, spay and neuter status, feed management techniques, and genetics. Cats that are 2 to 3 yr of age usually have an increased prevalence of obesity, which plateaus between 5 and 11 yr of age, and then declines at 11+ yr of age (Bjornvad et al., 2011). In the first months of life, many cats undergo gonadectomy, or the surgical removal of the testes in males or the ovaries in females. This life-changing event plays a significant role in their predisposition for obesity, as gonadectomy increases appetite, decreases metabolic rate, and decreases energy expenditure (Belsito et al., 2009; Iwazaki et al., 2022). Research shows that following a gonadectomy, the production of sex hormones (i.e., estrogen and testosterone) are greatly reduced, resulting in alterations of the hypothalamic–pituitary–gonadal axis and appetite control (de Godoy, 2018). As a result, spaying female cats has been shown to lower the maintenance energy requirement (MER) and decrease physical activity levels (Courcier et al., 2012).

To compound the issue further, there has been a shift away from allowing cats to have access to the outdoors in recent years. In the United States, 63% of cats are kept exclusively indoors (Foreman-worsley et al., 2021), whether it be for safety, health, or personal reasons. This practice limits a cat’s ability to hunt and subsequently decreases its drive to inadvertently engage in physical activity. A positive correlation has been identified between cats exclusively kept indoors and obesity, showing a reduction in physical activity, lack of enrichment, and/or greater consumption of food as a result of boredom (Robertson, 1999; Rowe et al., 2015; Wall et al., 2019). Numerous metabolic and clinical disorders are directly or indirectly associated with or exacerbated by obesity in domestic cats, such as hepatic lipidosis, diabetes mellitus, exercise intolerance, altered kidney function, urinary tract diseases, and neoplasia (Laflamme, 2006). Therefore, the consequences of obesity have detrimental effects on the health and longevity of cats, undermining their quality of life (Day, 2017; Wall et al., 2019; Ward et al., 2019). The common thread between these diseases and obesity is the excess body fat and alterations to circulating cytokines, hormones, and oxidative stress markers. Adipose tissue is not only an energy storage depot. Rather, the various cell types it contains are able to produce and secrete numerous cytokines and hormones (Miller et al., 1998; Coppack, 2001; Gayet et al., 2004; Toll et al., 2010).

Due to the high incidence of obesity in humans and companion animals, many preventative and management ­strategies have been discussed and implemented over the years. According to APOP (2022), 58% of cat owners in the United States have tried some form of weight loss program for their cat, with only 19% reporting it as being successful. Oftentimes, obesity is best addressed with a proactive, rather than a reactive approach. Dietary intervention is usually the first reactive approach in the management of obesity, with many manufacturers capitalizing on the weight loss diet market. Diets formulated to combat obesity commonly include lower calories [≤3.4 kcal metabolizable energy (ME)/g], lower fat content [≤10% dry matter basis (DMB)], higher total dietary fiber (TDF) content (15% to 20% DMB), higher protein content (≥35% DMB), lower carbohydrate content (≤35% DMB), and the addition of nutraceuticals like L-carnitine (≥500 ppm) (Toll et al., 2010; Hoelmkjaer and Bjornvad, 2014; Shoveller et al., 2016). Purpose-driven formulations, using the key nutritional factors presented above, help to effectively support weight loss and minimize signs of hunger by reducing demanding behaviors and improving owner compliance (West, 1996; Weber et al., 2007; German, 2016). Several previous studies have shown that dietary restriction and the use of specially formulated diets results in controlled weight loss and is the most recommended and feasible method of feline obesity treatment (Butterwick and Markwell, 1996; Nguyen et al., 2002; O’Connell et al., 2018; Zhang et al., 2023).

The gastrointestinal microbiome can change with dietary patterns, feeding behaviors, nutrition, and health status. A balanced population of commensal bacteria can be beneficial to the host by helping with food digestion and nutrient absorption, limiting proliferation of pathogenic bacteria by competing for nutrients and binding sites, producing antimicrobial substances, modulating gut-associated lymphoid tissue, strengthening gap junctions and cells of the intestinal epithelia, and generating metabolites that have beneficial effects on the host (Swanson et al., 2011). Obesity has been reported to cause gut microbiota dysbiosis, altered gut barrier integrity allowing bacterial translocation, and the breakdown of tight junctions (Turnbaugh et al., 2006; Nagpal et al., 2018). In the past decade, many disease states, such as gastrointestinal diseases, cardiovascular diseases, diabetes, and obesity, have been linked to changes in the bacterial diversity, composition, and functionality in companion animals and humans (Meng et al., 2018; Blake et al., 2019; Witkowski et al., 2020; Li et al., 2021). Of interest is the gut microbiota’s potential to impact metabolic dysfunction and obesity in companion animals. It has been reported that there is reduced microbial diversity and an increased Firmicutes/Bacteroidetes ratio in mice and humans with obesity (Turnbaugh et al., 2006; Salas-Mani et al., 2018; Sanchez et al., 2020; Liu et al., 2021). However, those changes are not always observed (Schwiertz et al., 2010; Frost et al., 2019; Magne et al., 2020; Phungviwatnikul et al., 2022). Therefore, while the gut microbiota are linked with obesity, more research is needed to determine if or how they directly or indirectly contribute to the etiology of obesity in companion animals. Currently, proposed mechanisms include increased dietary harvest via the production of fermentative products and other bioactive molecules (Turnbaugh et al., 2006; Tal et al., 2020), changes in the regulation of fat storage and lipid metabolism (Bäckhed et al., 2007; Ghazalpour et al., 2016), altered effects of satiation (Arora et al., 2011), and inflammation from the gut microbiota and its metabolites crossing the intestinal barrier (Leong et al., 2018; Kieler et al., 2019; Wernimont et al., 2020; Liu et al., 2021).

The purpose of this study was to evaluate the effects of restricted feeding and specially formulated diets on weight loss, body composition, voluntary physical activity, blood metabolite profiles, serum markers of oxidative stress, and fecal microbiota populations and metabolites of overweight cats. We hypothesized that closely monitoring body weight (BW) and adjusting dietary intake would result in consistent weight and fat loss and lean muscle preservation, but greater changes will be observed in cats fed the weight loss diet. As a result of restricted feeding and subsequent weight loss, we also hypothesized that diet restriction coupled with weight loss would increase voluntary physical activity, lower blood lipid concentrations, and reduce serum markers of oxidative stress. Lastly, we hypothesized that diet restriction and weight loss would beneficially alter the fecal microbiota community and metabolite concentrations.

Materials and Methods

All procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation (IACUC #21050) and were performed in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Animals, housing, and experimental design

Twenty-two overweight adult spayed female (n = 14) and neutered male (n = 8) domestic shorthair cats [BW = 5.70 ± 1.0 kg; BCS= 7.68 ± 0.6; muscle condition score (MCS) = 3.5 ± 0.4; age = 4 ± 0.4 yr] were used in a longitudinal weight loss study. All cats were housed individually in cages (1.02 × 0.76 × 0.71 m³) during feeding time (08:00 to 09:00 hours) in a temperature- and light-controlled (14-h light: 10-h dark cycle) room in the Edward R. Madigan Animal Facility on the University of Illinois Urbana-Champaign campus. Rooms were cleaned daily and other than feeding times, cats were group-housed and able to socialize and exercise outside their cages. Cats had access to various toys and scratching poles for environmental enrichment and play time with human interaction at least two times per week.

The study consisted of a 4-wk baseline phase followed by a 24-wk experimental phase of diet restriction and weight loss. To confirm health, a physical exam was performed by a veterinarian at baseline and were monitored throughout the study. No medications or treatments were administered at least 4 wk before the baseline phase and throughout the experiment. A control diet (OR; Orijen Original, Champion Petfoods, Edmonton, Canada) formulated to meet all of the Association of American Feed Control Officials (AAFCO, 2021) nutrient profiles for adult cats at maintenance, was first fed during a 4-wk baseline phase to identify the maintenance energy requirement (MER) and food intake needed to maintain BW for all adult cats. After the baseline phase (week 0), cats were allotted to one of two diets: 1) control or 2) a weight control diet [Fit & Trim (FT); Champion Petfoods, Edmonton, Canada]. The FT diet was also formulated to meet all of the AAFCO (2021) nutrient profiles for adult cats, but was formulated to contain higher protein concentrations, higher total TDF concentrations, and lower caloric content to support healthy weight loss.

Cats were initially fed 80% of calories required to maintain baseline BW at the start of the 24-wk weight loss trial and then fed at a rate to lose ~1.0% BW per week, as suggested by the American Animal Hospital Association (AAHA) for weight management (Brooks et al., 2014). To achieve and maintain the desired loss, energy intake was adjusted for each individual cat weekly based upon the amount of BW loss week over week. Food intake was recorded daily and water was available ad libitum. BW, BCS (9-point scale; Laflamme, 1997), and MCS (1 = severe muscle loss, 2 = moderate muscle loss, 3 = mild muscle loss, 4 = normal muscle mass; WSAVA, 2011) were measured weekly prior to feeding. Statistics were performed on MCS data using a 1-4 scale; 1 = severe muscle loss, 2 = moderate muscle loss, 3 = mild muscle loss, 4 = normal muscle mass. As cats reached their target BW, food intake was adjusted for maintenance.

Blood samples were collected at baseline (week 0) and 6, 12, 18, and 24 wk after diet restriction and weight loss for serum chemistry, hematology, oxidative stress marker [malondialdehyde (MDA); superoxide dismutase (SOD)], and leptin measurement. Fecal samples were collected at baseline (week 0) and 4, 8, 12, 16, 20, and 24 wk after diet restriction and weight loss for fecal microbiota and metabolite analyses. One fresh fecal sample from each cat was collected at each time point within 15 min of defecation for measurement of pH, dry matter (DM), and metabolite concentrations [short-chain fatty acids (SCFA), branched-chain fatty acids (BCFA), ammonia, phenol, and indole]. At baseline (week 0), wk 8, wk 16, and wk 24, accelerometers were used to measure voluntary physical activity. At baseline (week 0), and 6, 12, 18, and 24 wk after diet restriction and weight loss, dual-energy x-ray absorptiometry (DEXA) scans were performed to estimate body fat, lean soft tissue, and bone mass.

Chemical analysis of diets and calculations

Subsamples of the experimental diets were collected monthly. A composite sample of all subsamples was ground through a 2-mm screen using a Wiley Mill (Model 4, Thomas Scientific, Swedesboro, NJ). Ash and DM were analyzed in accordance to methods established by the Association of Official Analytical Chemists (AOAC, 2006; ash: method 942.05; DM: method 934.01), with OM calculated. Using acid hydrolysis and extraction methods established by the American Association of Cereal Chemists (AACC, 1983) and Budde (1952), fat content was measured. Crude protein (CP) content was calculated from Leco total nitrogen values (TruMac N, Leco Corporation, St. Joseph, MI; AOAC, 2006) and gross energy was measured using an oxygen bomb calorimeter (Model 6200, Parr instruments, Moline, IL). Total dietary fiber, including soluble and insoluble components, was determined according to Prosky et al. (1985). Dietary nitrogen-free extract (NFE) was calculated using the calculation described by equation (1). The ME in kcal on an “as-is” basis was calculated using predictive equation (2) developed by the National Research Council (NRC, 2006).

$${\displaystyle \begin{array}{lll}{\displaystyle NFE\{\mathrm{\%}\}=}& 100-(Fat\{\mathrm{\%}\}+CP\{\mathrm{\%}\}+Ash\{\mathrm{\%}\}& +\mathrm{T}\mathrm{D}\mathrm{F}\{\mathrm{\%}\})\end{array}}$$ (1)

$${\displaystyle \begin{array}{lll}{\displaystyle ME\left\{\frac{\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}}{100\mathrm{g}}\right\}=}& \mathrm{G}\mathrm{E}\left\{\frac{\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}}{100\mathrm{g}}\right\}\times \left(\frac{95.6-[0.89\times \mathrm{T}\mathrm{D}\mathrm{F}\{\mathrm{\%}\}]}{100}\right)& -(0.77\times \mathrm{C}\mathrm{P}\{\mathrm{\%}\})\end{array}}$$ (2)

Resting energy requirement (RER) was estimated by multiplying the metabolic body weight (MBW; BW^0.67) by a factor of 70 according to the NRC (2006).

Complete blood count and serum chemistry profile, hormones, and oxidative stress markers

On blood collection days, blood samples were collected from overnight fasted cats via jugular puncture. Prior to bleeding, cats were sedated by an intramuscular injection of Dexdomitor (dexmedetomidine; 0.02 mg/kg BW; Zoetis). Blood was immediately transferred to a vacutainer serum tube containing a clot activator and gel for serum separation (BD Vacutainer SST Tube—367988; Becton, Dickinson, and Co., Franklin Lakes, NJ) for serum chemistry profile, oxidative stress markers, and leptin analyses. Serum tubes were centrifuged at 1,000 × g for 15 min at 4 °C to harvest serum. Whole blood tubes containing K₂EDTA (BD Microtainer Tubes-363706, Becton, Dickinson, and Co., Franklin Lakes, NJ) were used for complete blood count. Serum chemistry profile and complete blood count was analyzed at the University of Illinois Veterinary Medicine Diagnostics Laboratory using a Hitachi 911 clinical chemistry analyzer. Concentrations of MDA (MBS2700234; MyBioSource, San Diego, CA), SOD (MBS1602678; MyBioSource, San Diego, CA), and leptin (MBS057075; MyBioSource, San Diego, CA) were measured using feline-specific commercial enzyme-linked immunosorbent (ELISA) assay kits.

Body composition

While cats were still sedated from blood collection, they were positioned in sternal recumbency so that body composition could be measured at the University of Illinois Veterinary Teaching Hospital by DEXA (Hologic X-ray Bone Densitometer QDR 4500 Elite Acclaim Series; Hologic Inc., Waltham, MA). Each cat was scanned individually and regions of measurement included all 4 legs, trunk, and head. All region measurements included fat, lean soft tissue, and bone mineral content. Body fat percentage was calculated by the DEXA algorithm for the entire body and each part measured thereof. After DEXA scans were complete, an injection of the reversal agent for dexmedetomidine, Antisedan (atipamezole HCl; 0.2 mg/kg BW; Zoetis), was given.

Voluntary physical activity

Voluntary physical activity was measured using Actical monitors and analyzed by computer software (Mini Mitter, Bend, OR). Devices were attached to collars that each cat wore around their neck for four consecutive days at baseline (week 0), and 8, 16, and 24 wk after restricted feeding and weight loss. Mean activity was presented in activity counts per epoch (epoch length = 0.25 min), with periods of light (07:00 to 21:00 h) and dark (21:00 to 07:00 h) activity continuously measured.

Fecal sample collection

One fresh fecal sample was collected and processed within 15 min of defection from each cat at baseline (week 0), and 8, 16, and 24 wk after restricted feeding and weight loss. Each fresh sample was subject to measurement of pH, DM, metabolite concentrations, and microbiota composition. Fecal pH was measured using a pH meter (Accumet AP1001 Portable pH Meter; Fisher Scientific, Waltham, MA) equipped with an electrode (InLab Surface pH Electrodes 51343157; Mettler Toledo, Columbus, OH) immediately after collection. Aliquots were then collected for further analyses. Samples for SCFA, BCFA, and ammonia concentrations were mixed with 2 N hydrochloric acid in a 1:1 (weight:weight) ratio and stored at −20 °C until analysis. Samples for phenol and indole concentrations were stored in duplicate at −20 °C until ­analysis. Aliquots for microbiota analysis were immediately frozen on dry ice and stored at −80 °C until analysis.

Fecal scores

All fresh fecal samples were scored using a 5-point scale to determine shape, consistency, and texture. The following scale was used: 1 = very hard and dry pellets; 2 = firm and formed, segmented in appearance and holds shape; 3 = soft, formed, moist stool, retains shape, but little to no segmentation visible; 4 = soft and very moist, has some texture, but no defined shape; 5 = watery, liquid that is present in flat puddles with no texture.

Fecal chemical analyses

Fresh fecal samples were analyzed for DM determination using a 105 °C oven and according to procedures of the Association of Official Analytical Chemists (AOAC, 2006). Fecal SCFA and BCFA concentrations were determined using a gas chromatograph (Hewlett-Packard 5890A series II, Palo Alto, CA) and a glass column (180 cm × 4 mm i.d.) packed with 10% SP-1200/1% H₃PO₄ on 80/100 + mesh Chromosorb WAW (Supelco Inc., Bellefonte, PA) in accordance to methods of Erwin et al. (1961). A flow rate of 75 mL/min was used, with nitrogen as the carrier. The oven, detector, and injector temperatures were 125, 175, and 180 °C, respectively. Fecal ammonia concentrations were determined by methods of Chaney and Marbach (1962). Fecal phenol and indole concentrations were determined using gas chromatography according to methods described by Flickinger et al. (2003).

Fecal microbiota populations

Total DNA was extracted from fecal samples using a DNeasy PowerLyzer PowerSoil Kit (Qiagen, Carlsbad, CA). Extracted DNA concentration was quantified using a Qubit 3.0 Fluorometer (Life Technologies, Grand Island, NY). Using a Fluidigm Access Array (Fluidigm Corporation, South San Francisco, CA) and Roche High Fidelity Fast Start Kit (Roche, Indianapolis, IN), 16S rRNA gene amplicons were generated. Primers 515F (5ʹ-GTGCCAGCMGCCGCGGTAA-3ʹ) and 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ) target a 252-bp fragment of the V4 region of the 16S rRNA gene, which was used for amplification (primers synthesized by IDT Corp., Coralville, IA) and in methods described by Caporaso et al. (2012). A CS1 forward tag and CS2 reverse tag were added in accordance to the Fluidigm protocol. Amplicon quality was assessed using a Fragment Analyzer (Advanced Analytics, Ames, IA) to confirm amplicon regions and sizes. By combining equimolar amounts of the amplicons from each sample, a DNA pool was generated. Pooled DNA samples were then size selected on a 2% agarose E-gel (Life Technologies, Grand Island, NY) and extracted using a Qiagen gel purification kit (Qiagen). Cleaned and size-selected pooled products were run on an Agilent Bioanalyzer to confirm the appropriate profile and average size. Illumina sequencing was performed on a MiSeq using v3 reagents (Illumina Inc., San Diego, CA) at the Roy J. Carver Biotechnology Center at the University of Illinois.

Bioinformatics and statistical analysis for assessing fecal microbial communities

Using the FASTX-Toolkit (version 0.0.14), forward reads were trimmed and sequences were analyzed using QIIME 2.0, version 2021.4 (Caporaso et al., 2012) and the DADA2 pipeline for quality control (Callahan et al., 2016). High-quality (quality value ≥ 20) sequence data, derived from the sequencing process, were demultiplexed. Sample sequences were clustered into operational taxonomic units (OTU) and taxonomic groups with the Silva database (Silva 138 99% OTU from 515F/806R region of sequences, with the QIIME 2 classifier trained on the 515F/806R V4 region of 16S) (Bokulich et al., 2018; Robeson et al., 2021). Singletons (OTU that are observed fewer than two times) and OTU that had <0.01% of total observations were discarded. A total of 6,821,933 reads were obtained, with an average of 44,298 reads (range = 7,923 to 63,020) per sample. The dataset was rarified to 7,923 reads for analysis of diversity and species richness. Principal coordinates analysis was performed using both weighted and unweighted unique fraction metric (UniFrac) distances (Lozupone and Knight, 2005).

Statistical analyses

Except for fecal scores, all data were analyzed using SAS (version 9.4; SAS Institute, Cary, NC) using the Mixed Models procedure, with cat being considered a random effect. Fecal scores were analyzed using SAS, the Mixed Models procedure, and PROC GLIMMIX because they are categorical variables. Cat was again considered to be a random effect. In both cases, baseline values were tested to highlight any differences between groups before treatments were administered. At all other time points, the study was conducted as a repeated measures design that tested the effects of treatment (diet), time, and treatment*time interactions. Using a Fisher-protected LSD change from baseline differences were determined with a Tukey adjustment to control for experiment-wise error. Using the univariate procedure and Shapiro-Wilk statistic, data normality was checked, with logarithmic transformation being applied when normal distribution was not observed. If normality was not achieved after the application of a logarithmic transformation, data were analyzed using the npar1way procedure and Wilcoxon statistic. Data were reported as means, with P < 0.05 considered significant and P < 0.10 considered trends.

Results

Chemical analysis of diets

Dietary chemical composition was analyzed and is provided in Table 1. Diets exceeded all AAFCO (2021) ­nutrient ­recommendations and had similar DM, OM, ash, and insoluble fiber concentrations. The CP percentage (44.4% vs 40.5%, DMB) was slightly higher in the FT diet. Acid-hydrolyzed fat (18.5% vs 22.8%, DMB), NFE (9.47% vs 15.54%, DMB), gross energy (5.35 kcal/g vs 5.57 kcal/g, DMB), and ME (4.76 kcal/g vs 5.01 kcal/g, DMB) were lower in the FT diet. Total dietary fiber (19.33% vs 13.36%, DMB) and soluble fiber (7.54% vs 2.40%, DMB) concentrations were higher in the FT diet.

Table 1.

Analyzed chemical composition of experimental diets

Item	FT5	OR6
Dry matter, %	94.6	93.8
	Dry matter basis
Organic matter, %	91.7	92.2
Ash, %	8.3	7.8
Crude protein, %	44.4	40.5
Acid-hydrolyzed fat, %	18.5	22.8
Total dietary fiber, %	19.3	13.4
Insoluble fiber, %	11.8	11.0
Soluble fiber, %	7.5	2.4
Nitrogen-free extract1, %	9.5	15.5
Gross energy2, kcal/g	5.35	5.57
Metabolizable energy (ME)NRC3, kcal/g	4.76	5.01
MEMA4, kcal/g	3.46	3.90
Crude protein intake (g)/caloric intake (Mcal)NRC	93.3	80.8
Crude protein intake (g)/caloric intake (Mcal)MA	128.3	103.8
Acid-hydrolyzed fat intake (g)/caloric intake (Mcal)NRC	38.9	45.5
Total dietary fiber intake (g)/caloric intake (Mcal)NRC	40.5	26.7
Insoluble fiber intake (g)/caloric intake (Mcal)NRC	24.8	22.0
Soluble fiber intake (g)/caloric intake (Mcal)NRC	15.8	4.8

¹Nitrogen-free extract = 100 − (ash + crude protein + acid-hydrolyzed fat + total dietary fiber).

²Measured by bomb calorimetry.

³ $M{E}_{NRC}\left\{\frac{\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}}{100\mathrm{g}}\right\}=\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\left\{\frac{\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}}{100\mathrm{g}}\right\}\times \left(\frac{95.6-[0.89\times \mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{f}\mathrm{i}\mathrm{b}\mathrm{e}\mathrm{r}\{\mathrm{\%}\}]}{100}\right)-(0.77\times \mathrm{c}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\{\mathrm{\%}\})$

⁴ME_MA (kcal/g) = (3.5 × crude protein) + (8.5 × crude fat) + (3.5 × nitrogen-free extract).

⁵Fit & Trim diet (Orijen; Champion Petfoods, Edmonton, Canada): Chicken, chicken liver, whole herring, turkey, turkey giblets (liver, heart, gizzard), flounder, eggs, cod, dehydrated chicken liver, dehydrated egg, dehydrated sardine, dehydrated chicken, dehydrated turkey, dehydrated herring, whole red lentils, whole pinto beans, whole peas, whole navy beans, chicken fat, natural fish flavor, dehydrated pumpkin, lentil fiber, whole green lentils, whole chickpeas, chicken heart, pea starch, lentil starch, choline chloride, dried kelp, zinc proteinate, whole pumpkin, whole butternut squash, whole apples, whole pears, collard greens, mixed tocopherols (preservative), thiamine mononitrate, niacin, pyridoxine hydrochloride, vitamin E supplement, dried chicory root, turmeric, sarsaparilla root, althea root, rosehips, juniper berries, citric acid (preservative), rosemary extract, dried Lactobacillus acidophilus fermentation product, dried Bifidobacterium animalis fermentation product, and dried Lactobacillus casei fermentation product.

⁶Original diet (Orijen; Champion Petfoods, Edmonton, Canada): Chicken, turkey, whole mackerel, turkey giblets (liver, heart, gizzard), flounder, chicken liver, whole herring, eggs, dehydrated chicken, dehydrated turkey, dehydrated mackerel, dehydrated chicken liver, dehydrated egg, chicken fat, whole red lentils, whole pinto beans, whole peas, whole green lentils, whole chickpeas, natural chicken flavor, whole navy beans, pollock oil, lentil fiber, pea starch, chicken heart, choline chloride, dried kelp, mixed tocopherols (preservative), vitamin E supplement, zinc proteinate, whole cranberries, whole pumpkin, whole butternut squash, collard greens, whole apples, whole pears, copper proteinate, thiamine mononitrate, niacin, pyridoxine hydrochloride, dried chicory root, turmeric, sarsaparilla root, althea root, rosehips, juniper berries, citric acid (preservative), rosemary extract, dried Lactobacillus acidophilus fermentation product, dried Bifidobacterium animalis fermentation product, and dried Lactobacillus casei fermentation product.

Food intake, caloric intake, BW, BCS, body composition, and voluntary physical activity

Baseline food intake, caloric intake, BW, BCS, MCS, body composition measures, and voluntary physical activity levels were not different between groups (Supplementary Tables S1 to S3). Throughout the study, mean weight loss was 1.11 ± 0.51% per wk, as determined by weekly BW records. By the end of the study, cats lost approximately 22% of their baseline BW. Time-related differences were noted on caloric intake (kcal/d), but no diet-related differences or diet × time interactions were observed for cats undergoing restricted feeding and weight loss (Figure 1). At baseline, overweight cats consumed 321.0 ± 41.94 kcal/d or 56.32 ± 3.64 kcal/ kg BW (1.25 ± 0.03 × RMR or 100.32 ± 2.34 × MBW) to maintain BW. At that intake level, 8.11 ± 0.19 g of protein was consumed per kg MBW. Weight loss was initiated at week 1 with a reduction in caloric intake by 63.94 ± 9.49 kcal/d or 9.76 ± 0.81 kcal/kg BW for the FT group and 64.70 ± 7.56 kcal/d or 9.69 ± 1.02 kcal/kg BW for the OR group. Decreasing the intake at week 1 decreased the protein intake to 7.65 ± 0.11 g/kg MBW for the FT group and 6.66 ± 0.21 g/kg MBW for the OR group. Caloric intake was adjusted continually to maintain a mean weight loss of 1.0% BW per week. At the end of the weight loss program, cats were consuming 28.12 ± 4.11 kcal/kg BW or 0.61 ± 0.10 × RMR to continue the weight loss at a ratio of 1.0% per week. Caloric restriction to maintain weight loss was not different in those fed the FT or OR diet (Figure 2).

Figure 1.

Change from baseline caloric intake (kcal/d) and body weight (kg) data of overweight cats during weight loss with those at maintenance removed. Data are change from baseline and presented as least square means ± SEM.

Figure 2.

Change from baseline caloric intake (kcal/body weight^0.67) and body weight (kg) data of overweight cats during weight loss with those at maintenance removed. Data are change from baseline and presented as least square means ± SEM.

Restricted feeding and subsequent weight loss resulted in reduced (P < 0.0001) fat and lean soft tissue mass, and increased (P < 0.0001) lean:fat ratio (Table 2). Body composition measures were not impacted by diet. BCS was decreased (P < 0.0001) with restricted feeding and weight loss, but was unaffected by diet. In contrast, MCS was increased (P < 0.0001) with restricted feeding and weight loss, and increased to a greater (P = 0.0001) extent in cats fed the OR diet (Supplementary Figure 1). Restricted feeding and weight loss did not affect total physical activity counts or activity during the light period (Table 3). However, a significant diet*week interaction was observed for voluntary physical activity during the dark period. During the dark period, physical activity was reduced to a greater (P = 0.048) extent in cats fed the OR diet than those fed the FT diet.

Table 2.

Change from baseline body composition measures of overweight cats during weight loss

Measurements	△ wk0-wk6		△ wk0-wk12		△ wk0-wk18		△ wk0-wk24			P-value
	FT1	OR1	FT1	OR1	FT1	OR1	FT1	OR1	SEM3	Diet	Week	Diet*wk
Body mass, kg	−0.24	−0.20	−0.55	−0.51	−0.70	−0.78	−0.97	−1.09	0.060	0.7290	<0.0001	N/A4
Fat mass, kg	−0.21	−0.12	−0.41	−0.34	−0.52	−0.52	−0.73	−0.78	0.060	0.7136	<0.0001	0.2998
LBM2, kg	−0.04	−0.07	−0.14	−0.17	−0.18	−0.25	−0.25	−0.31	0.042	0.3420	<0.0001	0.8612
BMC2, g	3.12	−0.80	−1.56	−0.50	2.27	−2.08	−0.61	−2.36	2.870	0.5054	0.5010	0.4693
LBM + BMC, kg	−0.03	−0.07	−0.14	−0.17	−0.18	−0.26	−0.25	−0.31	0.041	0.3065	<0.0001	0.8091
BMC, %	0.14	0.04	0.18	0.15	0.32	0.24	0.39	0.35	0.001	0.3290	<0.0001	0.8404
Fat, %	−2.74	−1.40	−5.30	−4.15	−6.92	−6.49	−10.17	−10.66	0.978	0.5932	<0.0001	0.5337
Lean, %	2.60	1.36	5.12	4.00	6.59	6.26	9.79	10.30	0.979	0.6394	<0.0001	0.5228
LBM + BMC, %	2.74	1.40	5.30	4.15	6.92	6.49	10.17	10.66	0.978	0.5930	<0.0001	0.5334
Lean:fat ratio	0.35	0.15	0.75	0.47	0.96	0.82	1.64	1.64	0.186	0.3457	<0.0001	0.2705

¹FT, Fit & Trim (Orijen; Champion Petfoods, Edmonton, Canada); OR, Original (Orijen; Champion Petfoods, Edmonton, Canada).

²BMC, bone mineral content; LBM, lean body mass.

³SEM, pooled standard error of the means.

⁴N/A. Wilcoxon signed ranks test; no interaction effect between diet and week.

Table 3.

Change from baseline voluntary physical activity (counts/epoch) of overweight cats during weight loss

Measurements	△ wk0-wk8		△ wk0-wk16		△ wk0-wk24			P-value
	FT2	OR2	FT2	OR2	FT2	OR2	SEM3	Diet	Week	Diet*wk
Average AC/min1	−0.12	−0.16	0.03	−0.09	−0.01	−0.02	0.08	0.2406	0.3537	N/A4
Average light AC/min1	−0.01	0.08	0.07	0.00	0.12	0.12	0.07	0.9309	0.2331	0.2780
Average dark AC/min1	0.05	−0.18	0.02	−0.24	−0.13	−0.21	0.07	0.1064	0.0223	0.0480
Light:dark	1.45	1.05	−0.61	1.17	0.60	4.75	1.47	0.4531	0.4868	N/A4

¹AC, activity counts; light = light on (14 h/d); dark = light off (10 h/d).

²FT, Fit & Trim (Orijen; Champion Petfoods, Edmonton, Canada); OR, Original (Orijen; Champion Petfoods, Edmonton, Canada).

³SEM, pooled standard error of the means.

⁴N/A, Wilcoxon signed ranks test; no interaction effect between diet and week.

Complete blood count and serum metabolites, hormones, and oxidative stress markers

Other than being overweight, all cats were considered healthy at the start of the experiment. Complete blood count and serum metabolite profiles at baseline showed that many parameters were within normal reference ranges. Those that were outside the normal reference ranges were expected based on previous colony history (e.g., glucose, 21 cats) and overweight status. Blood total cholesterol concentration (20 cats), Na/K ratio (5 cats), and red blood cell count (10 cats) were higher, while mean corpuscular hemoglobin (22 cats) and hemoglobin (16 cats) concentrations were lower than reference ranges.

Baseline serum metabolites were not different between treatment groups at baseline (Supplementary Table 4). Restricted feeding and weight loss, however, affected several serum chemistry marker concentrations from initial baseline measurements (Table 4). Serum triglycerides were the only marker with a significant diet*week interaction. While serum triglyceride concentrations were reduced in both groups over time, the decrease was greater (P = 0.0039) in cats fed the FT diet. Serum creatinine, glucose, total protein, albumin, globulin, sodium, chloride, total cholesterol, total alkaline phosphatase, alanine aminotransferase, creatinine phosphokinase, albumin/globulin ratio, and anion gap were impacted by diet or restricted feeding and weight loss over time. Serum concentrations of creatinine (P = 0.0023), sodium (P = 0.0032), chloride (P = 0.0035), alkaline phosphatase (P < 0.0001), and alanine aminotransferase (P = 0.0412) increased with restricted feeding and weight loss. In contrast, serum total protein (P < 0.0001), albumin (P = 0.0001), globulin (P < 0.001), sodium (P = 0.0032), chloride (P = 0.0035), and creatinine phosphokinase (P = 0.0043) concentrations decreased over time with restricted feeding and weight loss. Change from baseline serum glucose concentrations were affected by diet, being reduced to a greater (P = 0.0227) extent in cats fed the OR diet than those fed the FT diet. Serum cholesterol concentrations were also impacted by diet, but with the opposite effect, with cats fed the FT diet having a greater (P = 0.0290) decrease than those fed the OR diet. Statistical trends were observed for serum calcium and phosphorus concentrations. Restricted feeding and weight loss tended to increase (P = 0.0510) serum calcium and decrease (P = 0.0963) serum phosphorus concentrations.

Table 4.

Change from baseline serum metabolite concentrations of overweight cats during weight loss

	Δ wk0-wk6		Δ wk0-wk12		Δ wk0-wk18		Δ wk0-wk24			P-value
Item	FT1	OR1	FT1	OR1	FT1	OR1	FT1	OR1	SEM3	Diet	Week	Diet*wk
Creatinine, mg/dL	0.11	0.15	0.14	0.09	0.25	0.20	0.14	0.17	0.05	0.9721	0.0023	0.1634
BUN, mg/dL2	1.00	0.36	1.64	0.73	1.09	0.55	0.91	1.18	0.75	0.6350	0.5448	0.4885
Calcium, mg/dL	0.19	0.26	0.25	0.33	0.11	0.24	0.26	0.34	0.07	0.2969	0.0510	0.9187
Phosphorus, mg/dL	−0.10	−0.09	−0.33	−0.30	−0.58	−0.08	−0.46	−0.22	0.15	0.1996	0.0963	0.1434
Glucose, mg/dL	7.91	−23.00	7.64	−30.18	−10.55	−16.36	−5.36	−43.18	20.16	0.0227	0.9140	N/A4
Total cholesterol, mg/dL	−42.09	−5.91	−42.64	1.91	−42.18	−4.55	−48.73	−11.64	9.10	0.0290	0.5381	0.5881
Triglycerides, mg/dL	−3.36ab	3.09a	2.27ab	0.82ab	−11.45ab	−4.73ab	−21.00b	−6.36ab	5.30	0.0474	<0.0001	0.0039
Bicarbonate	−0.82	−0.91	−0.46	−0.82	−0.73	−0.18	−0.27	−0.46	0.30	0.9401	0.2007	0.2316
Total protein, g/dL	−0.06	0.03	−0.15	−0.07	−0.18	−0.13	0.17	0.09	0.08	0.7015	<0.0001	0.3528
Albumin, g/dL	−0.04	−0.02	0	0.03	−0.04	−0.01	−0.11	−0.10	0.04	0.6316	0.0001	0.9945
Globulin, g/dL	−0.03	0.05	−0.15	−0.10	−0.15	−0.12	0.28	−0.18	0.06	0.6988	<0.0001	0.4636
Sodium, mmol/L	1.64	1.64	1.64	1.36	2.18	1.00	0.91	0.27	0.53	0.7652	0.0032	0.5630
Potassium, mmol/L	−0.23	−0.05	−0.14	0.01	−0.07	0.16	−0.12	0.08	0.18	0.4820	0.6989	N/A4
Chloride, mmol/L	1.64	1.82	2.55	2.73	2.91	2.27	1.91	1.64	0.42	0.7672	0.0035	0.4920
Total ALP, U/L2	3.73	5.64	2.64	2.45	1.55	2.09	−2.27	−2.00	1.08	0.6384	<0.0001	0.3809
ALT, U/L2	8.55	11.27	18.73	8.91	9.45	8.27	6.55	9.73	5.44	0.9496	0.0412	0.0671
CPK, U/L2	2.45	−9.73	−18.64	−0.55	−23.36	−10.91	21.27	−10.82	27.05	0.4999	0.0043	0.5550
A/G2 ratio	−0.02	−0.01	0.05	0.06	0.02	0.04	−0.10	−0.05	0.02	0.4673	<0.0001	0.5399
Na/K ratio	3.09	1.18	2.00	0.18	1.36	−1.09	1.27	−1.00	1.94	0.3816	0.7157	0.8666
Anion gap	0.55	0.73	−0.55	−0.36	−0.09	−0.64	−0.73	−0.73	0.46	0.9236	0.0023	0.6950

¹FT, Fit & Trim (Orijen; Champion Petfoods, Edmonton, Canada); OR, Original (Orijen; Champion Petfoods, Edmonton, Canada).

²BUN, blood urea nitrogen; total ALP, total alkaline phosphatase; ALT, alanine aminotransferase; CPK, creatinine phosphokinase; A/G, albumin/globulin.

³SEM, pooled standard error of the means.

⁴N/A, Wilcoxon signed ranks test; no interaction effect between diet and week.

^a,bMean values within the same row with unlike superscript letters differ significantly (P < 0.05) and relate to the interaction.

Baseline complete blood counts were not different between treatment groups at baseline (Supplementary Table 5), but there were a few changes due to diet and restricted feeding and weight loss (Table 5). Red blood cell count, mean cell volume, mean ­corpuscular hemoglobin concentration, and hemoglobin concentration were reduced (P < 0.05) with restricted feeding and weight loss, but unaffected by diet. Blood hematocrit and white blood cell counts were reduced to a greater (P < 0.05) extent in cats fed OR than those fed the FT diet. In contrast, mean platelet volume showed a greater reduction (P = 0.0186) in cats fed the FT diet than those fed the OR diet.

Table 5.

Change from baseline complete blood cell counts of overweight cats during weight loss

	Δ wk0-wk6		Δ wk0-wk12		Δ wk0-wk18		Δ wk0-wk24			P-value
Item	FT1	OR1	FT1	OR1	FT1	OR1	FT1	OR1	SEM3	Diet	Week	Diet*wk
RBC, 106/µL2	0.16	−0.13	0.21	−0.39	0.15	−0.54	−0.30	−0.93	0.28	0.1370	<0.0001	0.4591
Reticulocyte count, /µL	0.00	−0.02	0.01	−0.02	0.00	−0.02	−0.01	−0.02	0.02	0.300	0.7956	0.7010
Platelets, fL	34.73	77.09	−56.18	18.62	−3.55	82.08	−2.64	86.61	14.89	0.1168	0.0888	0.8608
MCV, fL2	−0.85	−0.76	−0.64	−0.45	−0.10	0.07	0.29	−0.07	0.28	0.9351	0.0004	0.4837
MCH, pg2	−0.11	−0.29	−0.21	−0.28	−0.05	−0.17	−0.01	−0.15	0.11	0.2617	0.2231	0.9691
MCHC, g/dL2	0.45	−0.06	0.03	−0.35	0.00	−0.45	−0.27	−0.32	0.20	0.1394	0.0049	0.2510
Hemoglobin, g/dL	0.14	−0.39	0.08	−0.75	0.17	−0.83	−0.40	−1.36	0.35	0.0857	0.0004	0.4805
Hematocrit, %	−0.09	−1.11	0.21	−1.84	0.51	−1.99	−0.92	−3.70	1.06	0.0456	0.2593	N/A4
Lymphocytes, %	−5.63	−2.66	0.15	2.88	−4.64	−0.15	−5.18	−1.15	3.50	0.3749	0.1650	0.9817
Monocytes, %	0.25	−0.52	−0.35	1.13	0.01	−0.55	−0.20	0.58	0.77	0.8060	0.5257	0.0706
Eosinophils, %	−0.98	−1.06	0.42	0.27	1.27	−0.61	−1.92	−0.66	1.56	0.9064	0.3362	0.4510
Basophils, %	0.75	0.44	−0.01	0.43	−0.13	−0.04	0.21	−0.26	0.35	0.5968	0.1615	N/A4
Mean platelet volume, 103/µL	−0.67	−0.15	−0.77	0.24	−0.78	0.46	−1.07	0.09	0.15	0.0186	0.6375	0.7271
WBC2 count, 103/µL	0.32	−1.13	−0.48	−1.99	−0.35	−1.61	0.96	−1.94	0.67	0.0004	0.6345	N/A4
Basophils, 103/µL	0.09	0.03	0.00	0.02	−0.01	−0.01	0.03	−0.03	0.03	0.2432	0.0869	N/A4
Lymphocytes, 103/µL	−0.53	−0.73	−0.21	−0.41	−0.68	−0.55	−0.52	−0.69	0.39	0.7734	0.8597	N/A4
Monocytes, 103/µL	0.02	−0.09	−0.05	0.01	0.00	−0.11	0.03	−0.03	0.07	0.5794	0.5730	0.1285
Eosinophils, 103/µL	−0.07	−0.17	0.05	−0.08	0.13	−0.15	−0.10	−0.18	0.12	0.2927	0.3151	0.5837

¹FT, Fit & Trim (Orijen; Champion Petfoods, Edmonton, Canada); OR, Original (Orijen; Champion Petfoods, Edmonton, Canada).

²RBC, red blood cells; MCV, mean cell volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cells.

³SEM, pooled standard error of the means.

⁴N/A, Wilcoxon signed ranks test; no interaction effect between diet and week.

Serum leptin, MDA, and SOD concentrations were not different between dietary treatment groups at baseline (Supplementary Table 6), but were altered over time with restricted feeding and weight loss (Table 6). Serum leptin concentrations decreased (P < 0.0001), whereas serum MDA concentrations increased (P < 0.0001) with restricted feeding and weight loss.

Table 6.

Change from baseline blood leptin, malondialdehyde (MDA), superoxide dismutase (SOD) concentrations of overweight cats during weight loss

	Δ wk0-wk6		Δ wk0-wk12		Δ wk0-wk18		Δ wk0-wk24			P-value
Item	FT1	OR1	FT1	OR1	FT1	OR1	FT1	OR1	SEM2	Diet	Week	Diet*wk
Leptin, ng/mL	−0.44	−0.24	−0.45	−0.54	−0.91	−0.81	−0.99	−0.77	0.0918	0.2667	<0.0001	0.1659
MDA, nmol/mL	13.46	18.30	7.46	13.70	7.01	11.06	0.62	5.69	2.9674	0.1848	<0.0001	0.9032
SOD, ng/mL	−0.27	0.05	0.26	0.06	−0.23	0.18	−1.12	−0.87	0.3137	0.6161	<0.0001	0.3305

¹FT, Fit & Trim (Orijen; Champion Petfoods, Edmonton, Canada); OR, Original (Orijen; Champion Petfoods, Edmonton, Canada).

²SEM, pooled standard error of the means.

Fecal characteristics and fermentative metabolites

Baseline fecal pH was lower (P = 0.0461) in cats allotted to the OR diet than those allotted to the FT diet, but no other baseline fecal characteristics were different between groups ­(Supplementary Table 7). Fecal total SCFA, acetate, ­propionate, butyrate, isobutyrate, valerate, phenol, indole, and total phenol and indole concentrations decreased (P < 0.05) over time by restricted feeding and weight loss (Table 7). Fecal total BCFA tended to decrease (P = 0.0670) by restricted feeding and weight loss. Fecal valerate concentrations (P = 0.0373) decreased to a greater extent in cats fed the OR diet than those fed the FT diet. In contrast, fecal pH tended to have a greater (P = 0.0505) increase in cats fed the OR diet than those fed the FT diet. No diet*week interactions were observed with fecal characteristics and fermentative metabolite concentrations.

Table 7.

Change from baseline fecal characteristics and metabolites of overweight cats during weight loss

	△wk0-wk4		△wk0-wk8		△wk0-wk12		△wk0-wk16		△wk0-wk20		△wk0-wk24			P-value
Variables	FT2	OR2	FT2	OR2	FT2	OR2	FT2	OR2	FT2	OR2	FT2	OR2	SEM3	Diet	Week	Diet*wk
Fecal score	−0.27	−0.41	−0.27	−0.55	−0.14	−0.59	−0.05	−0.27	−0.14	−0.36	−0.05	−0.32	0.19	0.2437	0.1875	0.8448
Fecal pH	0.09	0.80	0.51	0.93	0.54	0.90	0.54	1.02	0.42	1.06	0.39	1.02	0.29	0.0505	0.7060	0.8053
Fecal DM	−0.194	3.46	5.30	4.66	3.83	6.02	2.74	1.88	3.78	4.75	4.09	1.92	1.89	0.7936	0.3954	N/A4
	μmol/g (DMB)
Total SCFA1	−141	−234	−246	−231	−252	−251	−116	−142	−197	−185	−253	−227	48.3	0.8457	0.0023	0.4971
Acetate	−86.8	−124	−139	−127	−145	−146	−54.9	−69.2	−113	−100	−142	−122	30.0	0.9665	0.0018	0.7615
Propionate	−45.6	−93.4	−96.9	−92.0	−93.8	−92.7	−53.6	−71.3	−75.9	−76.2	−96.6	−93.7	18.0	0.6421	0.0239	0.2317
Butyrate	−8.53	−15.9	−10.1	−11.9	−13.1	−12.5	−7.17	−1.22	−8.70	−8.81	−14.2	−11.0	4.22	0.9855	0.0073	0.1579
Total BCFA1	−0.32	−3.22	1.50	−1.80	−1.97	−5.03	1.37	−0.04	−1.49	−4.29	−2.71	−4.19	2.01	0.4391	0.0670	N/A4
Isobutyrate	−0.56	−0.71	1.30	0.49	−0.61	−0.83	0.14	0.64	−0.83	−0.93	−1.54	−0.99	0.63	0.8413	0.0020	N/A4
Isovalerate	0.38	−1.20	0.13	−1.12	−1.28	−2.07	0.96	1.31	−0.33	−0.93	−0.81	−0.82	1.06	0.7813	0.1532	N/A4
Valerate	−0.15	−1.31	0.07	−1.17	−0.08	−2.13	0.26	−1.99	−0.33	−2.43	−0.35	−2.39	0.88	0.0373	0.0206	0.5117
Ammonia	1.81	−16.0	−12.9	−5.66	−20.6	−17.7	−12.49	−8.86	−25.3	−24.7	−32.6	−27.0	10.08	0.8147	0.1052	N/A4
Total P/I1	3.82	7.35	−2.10	−2.01	−2.35	−2.42	−1.94	−1.42	−1.99	−1.20	−1.74	−1.90	1.52	0.7209	<0.0001	N/A4
Phenol	0.45	0.70	−0.40	−0.20	−0.47	−0.29	−0.32	−0.14	−0.54	−0.12	−0.43	−0.16	0.22	0.1497	<0.0001	N/A4
Indole	2.83	4.46	−0.42	−0.54	−0.63	−0.43	−0.75	−0.29	−0.94	−0.19	−0.66	−0.43	0.86	0.3847	<0.0001	N/A4

¹SCFA, short-chain fatty acids; BCFA, branched-chain fatty acids; P/I, phenol/indole.

²FT, Fit & Trim (Orijen; Champion Petfoods, Edmonton, Canada); OR, Original (Orijen; Champion Petfoods, Edmonton, Canada).

³SEM, pooled standard error of the means.

⁴N/A, Wilcoxon signed ranks test; no interaction effect between diet and week.

Fecal microbiota

Fecal bacterial α diversity is represented by observed OTU and Faith’s phylogenetic diversity (PD; Figure 3). Alpha diversity was not different between dietary treatments or over time with restricted feeding and weight loss. Fecal bacterial β diversity is represented by principal coordinates analysis (PCoA) plots of weighted and unweighted UniFrac distances of fecal microbial communities over time with restricted ­feeding and weight loss (Figure 4). The UniFrac unweighted (P = 0.003) and weighted (P = 0.009) distances showed significant separation between dietary groups.

Figure 3.

Alpha diversity measures of fecal samples collected from overweight cats during weight loss. (a) Alpha-diversity measures by dietary group throughout the study. (b) Alpha diversity measures by time point throughout the study.

Figure 4.

Beta diversity measures of fecal samples collected from overweight cats during weight loss. At baseline (week 0), all animals were fed the same diet (OR; original diet). Principal coordinates analysis (PCoA) plots, including all time points of weighted (a) and unweighted (b) UniFrac distances of fecal microbial communities were performed on the 97% OTU abundance matrix using QIIME2.

Baseline fecal microbial phyla and genera were not different between treatment groups at baseline (Supplementary Table 8). The relative abundances of one fecal bacterial phylum and five fecal bacterial genera were impacted by restricted feeding and weight loss, with the relative abundances of fecal Fusobacteriota, unclassified Erysipelotichaceae, and Peptococcus increasing (P < 0.05) over time (Table 8). Relative abundances of Holdemanella and Lactobacillus decreased (P < 0.05) with restricted feeding and weight loss. Fecal Romboutsia were affected (P < 0.05) by time, but relative abundance tended to vary over time.

Table 8.

Change from baseline fecal bacterial phyla and genera (relative abundance, %) of overweight cats during weight loss

Phyla	Genus	△wk0-wk4		△wk0-wk8		△wk0-wk12		△wk0-wk16		△wk0-wk20		△wk0-wk24			P-value
		FT1	OR1	FT1	OR1	FT1	OR1	FT1	OR1	FT1	OR1	FT1	OR1	SEM2	Diet	Week	Diet*wk
Actinobacteriota		1.37	1.59	−1.45	0.26	−0.58	−3.10	−3.73	−2.68	−1.26	−2.02	−4.14	−1.60	1.53	0.0525	0.3320	0.9627
	Actinomyces	−0.01	0.00	−0.01	0.01	0.01	0.01	−0.02	0.01	−0.02	0.01	−0.01	0.01	0.01	0.0209	0.4883	N/A3
	Bifidobacterium	0.06	0.19	−0.03	0.15	−0.08	0.03	−0.03	−0.06	0.02	−0.05	−0.06	−0.03	0.10	0.0312	0.5526	N/A3
	Uncultured Eggerthellaceae	0.02	0.00	0.01	0.01	0.00	0.01	0.00	−0.01	0.01	0.00	0.01	−0.01	0.01	0.0436	0.8721	N/A3
	Parvibacter	0.03	0.00	0.02	0.00	0.01	−0.01	0.00	−0.01	0.01	−0.01	0.00	0.00	0.01	0.0005	0.2309	N/A3
	Slackia	0.07	0.00	0.09	−0.02	0.04	−0.05	0.02	−0.02	0.08	−0.04	0.01	−0.05	0.03	0.0058	0.0872	0.4835
Bacteroidota		2.58	5.21	6.09	4.76	2.58	6.09	2.86	4.54	4.45	8.36	7.35	10.04	2.25	0.0173	0.3647	N/A3
	Alloprevotella	0.11	0.47	0.26	0.39	−0.05	0.31	0.30	0.47	0.51	0.32	0.75	0.59	0.35	0.0465	0.7101	N/A3
Campilobacterota		0.14	0.53	−0.01	0.30	−0.05	0.28	0.33	0.33	−0.01	0.41	0.22	0.25	0.18	0.0006	0.6987	N/A3
Firmicutes		−5.11	−7.23	−4.27	−6.71	−2.22	−2.38	−2.57	−5.04	−4.33	−7.20	−10.93	−13.60	3.48	0.8327	0.2225	0.3126
	Catenibacterium	−1.10	−1.36	−0.28	−1.17	−0.44	−1.58	−1.20	−1.93	−0.75	−1.10	−0.97	−2.27	0.72	0.0091	0.8660	N/A3
	Lachnospiraceae− CHKCI001	0.00	0.02	0.00	0.03	0.00	0.02	0.00	0.01	0.00	0.01	0.01	0.01	0.01	0.0389	0.9370	N/A3
	Enterococcus	0.16	−0.13	0.25	−0.01	0.25	−0.21	−0.08	−0.14	−0.08	−0.13	−0.05	−0.15	0.18	0.0064	0.4917	N/A3
	Erysipelatoclostridium	0.46	−0.02	0.71	0.03	0.43	−0.03	0.50	−0.06	0.53	−0.04	0.36	−0.02	0.12	<0.0001	0.1534	0.8098
	Unclassified Erysipelotichaceae	0.01	0.01	0.04	0.01	0.01	0.00	0.00	−0.01	0.02	−0.01	0.00	−0.01	0.01	0.0808	0.0408	N/A3
	Eubacterium− Brachy group	0.03	0.00	0.01	−0.01	−0.01	−0.02	0.00	0.02	0.03	−0.04	−0.01	−0.04	0.03	0.0075	0.7671	N/A3
	Eubacterium− Halli group	0.19	−0.54	0.09	−0.71	−0.21	−0.59	−0.04	−0.61	0.10	−0.44	−0.27	−0.81	0.25	0.0103	0.4708	N/A3
	Holdemanella	−0.69	−0.35	0.06	−0.54	−0.74	−1.82	−1.14	−0.45	−0.66	−0.36	−2.46	−2.35	0.81	0.9591	0.0186	0.7629
	Howardella	0.01	−0.02	0.01	−0.02	0.01	−0.02	−0.01	−0.02	−0.02	−0.02	−0.02	−0.02	0.01	0.0052	0.6626	N/A3
	Lachnospiraceae	0.00	0.00	0.01	−0.01	0.02	0.00	0.01	−0.01	0.01	−0.01	0.04	0.00	0.02	0.0379	0.8961	N/A3
	Lactobacillus	−11.92	−5.04	−12.55	−5.10	−8.77	4.20	−7.66	−2.14	−9.56	0.07	−14.61	−6.92	3.24	0.0310	0.0024	0.4450
	Negativibacillus	0.29	0.09	0.40	0.01	0.29	−0.03	0.34	0.00	0.51	0.01	0.28	−0.02	0.08	<0.0001	0.9692	N/A3
	Peptoclostridium	3.22	−0.60	2.03	0.59	2.02	−1.62	0.52	−1.61	0.98	−1.33	−1.29	−0.59	0.93	0.0392	0.0064	0.0262
	Peptococcus	1.06	0.43	0.78	0.08	0.40	−0.05	0.98	0.01	1.01	−0.13	0.33	−0.23	0.34	0.0682	0.0412	0.6252
	Romboustia	−0.06	0.04	0.05	0.14	0.11	0.23	−0.07	−0.04	−0.15	−0.09	−0.13	−0.09	0.12	0.7569	0.0117	N/A3
	Ruminococcus−Gauvreauii group	0.21	0.16	0.31	0.11	0.24	0.12	0.14	−0.01	0.28	0.04	0.21	−0.08	0.08	0.0004	0.1180	N/A3
	Solobacterium	0.05	−0.02	0.13	−0.02	0.02	−0.02	0.09	−0.02	0.16	−0.02	0.00	−0.01	0.05	<0.0001	0.9138	N/A3
	Clostridia−UCG−014	0.01	0.07	0.06	−0.01	0.05	−0.01	0.06	0.01	0.08	0.01	0.04	0.01	0.03	0.0041	0.9274	N/A3
Fusobacteriota		1.01	0.04	1.61	0.62	1.13	−1.10	3.33	2.23	1.35	0.24	5.95	3.50	1.48	0.4146	0.0041	0.9038
Proteobacteria		0.01	−0.13	−0.22	0.79	−0.93	0.20	−0.23	0.64	−0.20	0.17	1.51	1.42	1.19	0.1174	0.8285	N/A3

¹FT, Fit & Trim (Orijen; Champion Petfoods, Edmonton, Canada); OR, Original (Orijen; Champion Petfoods, Edmonton, Canada).

²SEM, pooled standard error of the means.

³N/A, Wilcoxon signed ranks test; no interaction effect between diet and week.

A time*diet interaction (P < 0.05) was observed for the relative abundance of fecal Peptoclostridium. While this taxa tended to be reduced over time in cats fed OR, its relative abundance initially increased but then decreased in cats fed FT. Both diets showed a reduction of fecal Peptoclostridium from baseline measures at week 24. The relative abundances of two fecal bacterial phyla and 19 fecal bacterial genera were impacted (P < 0.05) by diet. At the phyla level, change from baseline relative abundances of fecal Bacteroidota and Campilobacterota were greater (P < 0.05) in cats fed the OR diet than those fed the FT diet. Change from baseline relative abundance of fecal Actinobacteriota tended to be lower (P = 0.0525) in cats fed the FT diet than those fed the OR diet. Six genera had clear changes due to diet. First, the increased change from baseline relative abundance of fecal Alloprevotella were greater (P < 0.05) in cats fed the OR diet than those fed the FT diet. In contrast, increased change from baseline relative abundances of fecal Negativibacillus and Ruminococcus-Gauvreauii group were greater (P < 0.05) in cats fed the FT diet than those fed the OR diet. The reduced change from baseline relative abundances of fecal Catenibacterium and Eubacterium-Halli group were greater (P < 0.05) in cats fed the OR diet than those fed the FT diet. In contrast, the decreased relative abundance of fecal Lactobacillus was greater (P < 0.05) in cats fed the FT diet than those fed the OR diet. The relative abundances of the other 13 fecal genera, including Actinomyces, Bifidobacterium, uncultured Eggerthellaceae, Parvibacter, Slackia, Lachnospiraceae-CHKC1001, Enterococcus, Erysipelatoclostridium, Eubacterium-Brachy group, Howardella, Lachnospiraceae, Solobacterium, and Clostridia-UCG-014, were impacted (P < 0.05) by diet, but changed slightly and were highly variable.

Discussion

Proper feeding management practices are key in achieving and maintaining a healthy BW and corresponding BCS. Taking it a step further, proper feeding practices oftentimes translate to proper nutrition, and proper nutrition is recognized as one of the most important factors in the management of health and disease. Animals that are diagnosed to be overweight or obese commonly are a result of inept feeding management, lack of nutritional, product or feeding knowledge, or simply ‘over-loving’ by pet owners. Recognition of an overweight or obese status is the mainstay of a successful weight loss plan. In the current study, overweight status was determined via a 9-point BCS and a 4-point MCS scale (WSAVA, 2011). These two methods of determination are subjective assessments, but to further confirm BCS and overweight status, DEXA scans were used to estimate the body fat percentage (31.56% ± 3.85) of all subjects at baseline. According to Brooks et al. (2014), this level of body fat is present in cats that are about 20% overweight and corresponds to a BCS 7. In cats, a general classification defines overweight cats as those that weigh 10% to 20% over their ideal BW and those that weigh 20% over their ideal BW are classified as obese (de Godoy, 2018).

While there is not a single optimal nutritional intervention for weight loss in cats, most weight loss products on the market today have a lower caloric density, contain higher concentrations of protein and fiber, and have a greater nutrient:energy ratio. Those specially formulated weight loss diets aim to maintain a product that is safe for adequate weight loss and help preserve lean soft tissue mass. Dietary management through specialized diets and calorie deficits are considered the keystone approaches to weight management in companion animals (German, 2010). To estimate the daily energy requirements for weight loss, it is currently recommended to use the pet’s estimated ideal weight, then feed a percentage of that amount. According to AAHA, daily RER for ideal BW in kg can be calculated as follows: 70 × (ideal BW [kg])^0.75. For neutered and spayed adult cats a daily caloric intake can be calculated using their RER × 1.2 to 1.4. At baseline, the cats in the present study were fed at an RER × 1.4 to maintain BW. To begin a weight loss plan, the feeding of 80% of a pet’s ideal-weight RER has been shown to be effective and well tolerated (German et al., 2011; Wakshlag et al., 2012). Using this reduction, cats in the present study lost an average of 3.47% BW (range of 1.4% to 6.4% BW) in their first week. At that time, the daily energy intake was 100.32 ± 2.34 kcal/kg^0.67. By the end of the weight-loss program and based upon their current weight, those that were still undergoing weight loss (n = 5) were consuming 49.03 ± 8.02 kcal/kg^0.67. By that time, 17 cats had successfully completed the weight loss program and achieved a desired BCS of 5.

The level of caloric restriction needed to achieve desired weight loss results varies widely among cats. Diets that are designed to target weight loss are formulated to contain more protein, vitamins, and minerals than conventional diets to ensure that there is sufficient nutrient intake during caloric restriction. Comparing the nutritional composition of the two experimental diets, the FT diet was higher in CP, TDF, and soluble fiber, but lower in AHF and NFE compared with the OR diet. These nutritional attributes may be advantageous in a weight loss diet, as a higher inclusion of fiber can aid in satiety, help control blood glucose concentrations, and dilute the caloric density of the formulation. Animals that require drastic caloric restriction have been subject to nutrient deficiencies as a result of restricted feeding practices (Linder et al., 2012; Hoelmkjaer and Bjornvad, 2014; Verbrugghe et al., 2021). More specifically, the reduction of non-protein energy intake increases the relative protein requirements for cats to maintain the body’s protein content (Archibald et al., 1983; Laflamme and Hannah, 2005). The NRC recommended allowance for protein in cats is 4.96 g CP/ BW (kg)^0.67 per day. To meet this recommendation during restricted feeding, it is recommended that cats consume diets containing 89 g CP/1,000 kcal when fed at 80% RER and 104 g CP/1,000 kcal when fed at 60% RER (Brooks et al., 2014). In the present study, the FT diet contained 93 g CP/1,000 kcal and the OR diet contained 81 g CP/1,000 kcal. By the end of the study, the few cats that were still undergoing weight loss were consuming 55.14 ± 10.45 kcal × MBW (FT diet) or 44.96 ± 3.64 kcal × MBW (OR diet). This translated to consumption of 5.14 and 3.63 g CP/BW(kg)^0.67 for cats fed the FT and OR diets, respectively. The FT diet was better suited to meet dietary protein needs during restricted feeding for weight loss.

For those still undergoing weight loss at week 24 in the current study, cats fed the OR diet were being fed at RER × 0.6 while those fed the FT diet were fed at RER × 0.7. Currently, AAHA suggests an RER × 0.8 for weight loss. This level of restriction needed highlights the importance of feeding a ­specially formulated diet that may enable weight loss, but minimize the chance of nutritional deficiency. During periods of weight loss it can become challenging to limit the loss of lean muscle mass. Therefore, monitoring lean mass (i.e., MCS) is a vital part of the health assessment during feline weight loss programs. Dietary protein intake is foundational to preserving lean mass and may improve satiety (Ibrahim et al., 2000; Halton and Hu, 2004; Laflamme and Hannah, 2005; Weber et al., 2007; Brooks et al., 2014). The method of muscle condition scoring is considered to be a subjective assessment. Therefore, when cats lost weight, their muscle likely becomes more prominent and easier to palpate, without a subsequent increase of lean soft tissue mass measured by DEXA scans. However, despite the level of restriction required for ­continued weight loss in the current study, cats increased MCS, lean soft tissue mass percentage, and the lean:fat ratio, suggesting that the CP and amino acid concentrations were suitable in both dietary treatments.

Changes in serum biochemistry are expected with restricted feeding and weight loss, with some changes being observed in the present study. Serum concentrations of leptin, MDA, and SOD were significantly impacted with restricted feeding and weight loss, but not diet. Serum leptin decreased over time, showing the greatest decrease after 24 wk of weight loss. MDA concentrations increased with restricted feeding and weight loss, but decreased over time while serum SOD was highly variable over time. Significant reductions in blood cholesterol, triglyceride, and glucose concentrations were observed in cats after weight loss, which has been observed in similar studies during weight loss (Ibrahim et al., 2000; Pallotto et al., 2018). While baseline measures of serum glucose and cholesterol were higher than the reference range, all animals remained healthy. Such measures have been documented in previous obese cat studies (Appleton et al., 2001; Hoenig et al., 2003). Because glucose homeostasis depends on the coordination among pancreatic β-cell function and glucose utilization by peripheral tissues, the reduction in blood glucose with weight loss indicates altered glucose tolerance as in other studies (Hoenig et al., 2003; Jordan et al., 2008). Serum cholesterol decreased with weight loss in both diet groups, but was more pronounced in cats fed the FT diet containing lower fat and greater dietary total and soluble fiber.

In the present study, the most prominent bacterial phyla detected in the feces were Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria, aligning with previous feline studies (Ritchie et al., 2010; Bermingham et al., 2011; Deusch et al., 2015; Fischer et al., 2017). The most dominant bacterial phylum was Firmicutes, with the second most abundant being Bacteroidetes, which is consistent with other 16S rRNA and metagenomics studies (Kieler et al., 2016; Fischer et al., 2017; Ma et al., 2022). Despite their carnivorous nature, the cat fecal microbiome contains many of the same bacteria as other host species, with many responsible for the synthesis of SCFA (acetate, butyrate, and propionate) and BCFA (valerate, isobutyrate, and isovalerate) from carbohydrate and protein fermentation, respectively (Levine et al., 2013; Butowski et al., 2019; Ganz et al., 2022). The synthesis of SCFA provides many benefits to the host, by providing energy to colonocytes, stimulating motility and blood flow, and promoting the growth of commensal bacteria while inhibiting pathogenic bacteria (Koh et al., 2016; Richards et al., 2016; Louis and Flint, 2017; Ganz et al., 2022).

Restricted feeding and weight loss impacted a few fecal microbiota taxa, but widespread differences were not observed. Bacterial alpha diversity was highly variable and was not statistically different over time. Bacterial beta diversity was also unable to identify shifts over time. The ­experimental design of the present study made it impossible to separate the independent effects of restricted feeding and weight loss. While a definitive association and conclusive relationship cannot be established, hypotheses for the bacterial shifts can still be made. Fecal microbial populations are reliant upon the substrate available to them and may be affected by changes in the gastrointestinal environment (e.g., pH, metabolite concentrations, and oxygen tension). The decrease in Holdemanella and Peptoclostridium would appear to be a positive change, as these taxa can be pathogenic and are proteolytic in nature. The large reduction in Lactobacillus, however, is difficult to interpret. Lactobacillus is usually linked with health, as it not only has beneficial effects on gastrointestinal health, but has been negatively associated with metabolic syndrome in humans and mice, improving glucose tolerance and supporting weight loss (Cani et al., 2008; Teixeira et al., 2013; Kathrani et al., 2016). The decrease in Lactobacillus with restricted feeding and weight loss could be due to reduced substrate availability, changes to the gastrointestinal environment, or an indirect effect of weight loss.

Restricted feeding of any diet reduces the available substrate accessible to the microbiota present in the large intestine, but the dietary nutrient concentrations are important too. The results of this study appear to demonstrate that point, with more bacterial changes being observed between dietary treatments than restricted feeding and weight loss. This may not be that surprising, as the FT diet was higher in TDF (19.3% vs. 13.4%) content with most of that coming from the soluble fiber portion (7.5% vs. 2.4%). Differences in dietary protein (44.4% vs. 40.5%) of NFE (9.5% vs. 14.5%) content may have contributed to some of the changes. Of the taxa that shifted due to dietary treatment, many were members of the Firmicutes phylum and are saccharolytic in nature suggesting effects of dietary fiber, resistant starch, or another carbohydrate fraction between the FT and OR diets. A few genera within the Actinobacteriota phylum were also shifted by dietary treatments, but most changes were quite small. Alloprevotella, a member of Bacteroidota, was also altered due to dietary treatment, but the difference was quite small.

In summary, restricted feeding of OR and FT in overweight cats led to a steady loss of weight and fat mass, while maintaining the majority of lean soft tissue mass over a 24-wk period. Restricted feeding and weight loss also reduced circulating leptin, triglyceride, and cholesterol concentrations, with greater reductions in triglycerides and cholesterol in cats fed the weight loss diet. Restricted feeding and weight loss reduced the concentration of fecal metabolites, including short-chain fatty acids, branched-chain fatty acids, and phenols and indoles. Restricted feeding and weight loss had minor effects on the fecal microbiota, altering the relative abundances of 7 fecal bacterial genera. Fecal bacterial beta diversity analysis showed clustering by diet. Dietary intervention affected change from baseline relative abundances of 2 fecal bacterial phyla and 20 fecal bacterial genera. Most changes were to members of the Actinobacteriota and Firmicutes phyla, which were likely due to differences in soluble dietary fiber content.

Glossary

Abbreviations:

AAFCO

Association of American Feed Control Officials

AAHA

American Animal Hospital Association

BCFA

branched-chain fatty acids

BCS

body condition score

BW

body weight

CP

crude protein

DEXA

dual-energy x-ray absorptiometry

DM

dry matter

DMB

dry matter basis

FT

Fit & Trim diet

MBW

metabolic body weight

MCS

muscle condition score

MDA

malondialdehyde

ME

metabolizable energy

MER

maintenance energy requirement

NFE

nitrogen-free extract

OM

organic matter

OR

Original/control diet

OTU

operational taxonomic unit

RER

resting energy requirement

SCFA

short-chain fatty acids

SOD

superoxide dismutase

TDF

total dietary fiber

UniFrac

unique fraction metric

Conflicts of Interest Statement

D. K. and J. K. are employed by Champion Petfoods Holding, Inc. The founding sponsors had no role in the collection, ­analyses, or interpretation of the data. All other authors have no conflicts of interest.