Effects of a high-protein, high-fiber diet rich in antioxidants and l-carnitine on body weight, body composition, metabolic status, and physical activity levels of cats after spay surgery

Abstract

Spay and neuter surgeries are useful in controlling pet populations, but increase obesity risk due to increased appetite, decreased metabolic rate, and decreased energy expenditure. Dietary management may help limit post-spay weight gain, but few research studies have been conducted in cats. Therefore, the objective of this study was to evaluate the effects of a high-protein, high-fiber diet (HPHF) compared to a moderate-protein, moderate-fiber diet (MPMF) in female cats following spay surgery. Twenty healthy female cats (9.5 ± 0.1 mo) were used. After a 4-wk baseline phase with cats fed MPMF to maintain body weight (BW), 16 cats were spayed and allotted to MPMF (n = 8) or HPHF (n = 8), with the remaining cats being sham-operated and fed MPMF (n = 4). Cats were fed to maintain BW for 12 wk and then allowed to eat up to twice that amount for another 12 wk. Daily food intake, twice weekly BW, and twice weekly body condition scores (BCS) were assessed. Back fat thickness (BF) using ultrasound, body composition using dual-energy X-ray absorptiometry (DEXA), feline body mass index (fBMI), body fat percentage estimates using zoometry measurements, serum metabolites, and voluntary physical activity levels were measured prior to spay (week 0) and every 6 wk post-spay. A treatment*time effect was observed for food intake (g/d), but not caloric intake (kcal ME/d). Caloric intake was affected by time and treatment, being reduced over the first 12 wk and reduced at higher amounts in HPHF and MPMF cats vs. sham cats. BW, BCS, and body fat percentage were affected over time. Treatment*time effects were observed for blood urea nitrogen, alkaline phosphatase, and fructosamine, whereas blood triglycerides, total cholesterol, creatinine, total protein, phosphorus, and bicarbonate were affected by time. Physical activity was reduced over time. Our results demonstrate that spay surgery affects food intake, BW, metabolism, and physical activity of cats. Dietary intervention in this study, however, led to minor changes.

Lay Summary

Spay surgery helps control pet populations, but increases obesity due to increased appetite, decreased metabolic rate, and decreased energy expenditure. Our objective was to evaluate the effects of high-protein, high-fiber diet (HPHF), and moderate-protein, moderate-fiber diets (MPMF) in female cats following spay surgery. Of the 20 cats used, 16 were spayed and fed MPMF (n = 8) or HPHF (n = 8) and four were sham-operated and fed MPMF. Cats were fed to maintain body weight (BW) for 12 wk and then allowed to overeat for 12 wk. Food intake, BW, body condition scores (BCS), back fat thickness, body composition, feline body mass index, body fat percentage estimates, serum metabolites, and physical activity levels were measured. Over the first 12 wk, caloric intake was reduced at higher amounts in spayed versus sham cats. BW, BCS, body fat percentage, and physical activity levels were altered over time. Our results demonstrate that the diets tested had minor effects, but spaying affected cat food intake, BW, metabolism, and physical activity.

Our results demonstrate that although the diets tested in this study had minor effects on the post-spay weight gain and metabolism of cats, spay surgery affected food intake, body weight, metabolism, and physical activity levels.

Introduction

Obesity is a global health problem in cats. Since 2000, the prevalence of feline obesity has fluctuated between 20% and 60%, depending on population studied (Colliard et al., 2009; Courcier et al., 2010; Cave et al., 2012; Association for Pet Obesity Prevention, 2016). Many factors contribute to pet obesity, including overfeeding, lack of exercise, and gonadectomy. The majority of cats in the United States undergo gonadectomy for population control, but it is known to increase food consumption, reduce physical activity, and alter metabolism, leading to weight gain and obesity (Belsito et al., 2009; Vester et al., 2009; Allaway et al., 2017).

The effects of spay or neuter on appetite, metabolism, body weight (BW), and body composition are highly complex and not well understood in cats. It has been reported that sex hormones (i.e., testosterone or estrogen) not only impact reproductive function, but also control appetite via the hypothalamic-pituitary-gonadal axis (de Godoy, 2018). Post-gonadectomy increases in BW and adiposity due to increased food consumption (Belsito et al., 2009; Backus, 2011; Wei et al., 2014), decreased energy requirements (Flynn et al., 1996; Harper et al., 2001; Kanchuk et al., 2003; Belsito et al., 2009; Backus, 2011; Wei et al., 2014), and decreased voluntary physical activity (Belsito et al., 2009; Vester et al., 2009; Allaway et al., 2017) has been reported in cats.

Overall caloric intake is important to control BW, but so is the specific dietary formulation. Diets high in fiber (Liu et al., 2003; Bermudez Sanchez et al., 2021) and high in protein (Weigle et al., 2005; Soenen et al., 2012; Bermudez Sanchez et al., 2021), for instance, are known to be effective in obesity management in humans and dogs. Dietary fiber reduces caloric content and may increase satiety, whereas high protein concentrations may increase satiety and help prevent the reduction in lean mass (LM) that is common with weight loss (Laflamme and Hannah, 2005). l-carnitine has been shown to increase weight loss and enhance fatty acid metabolism in cats (Center et al., 2000; Blanchard et al., 2002). Finally, because obesity is associated with oxidative stress (Laflamme, 2012), diets enriched in antioxidants may be protective. Dietary concentrations of protein, fiber, antioxidants, and l-carnitine have been tested for their positive effects on feline appetite, body weight maintenance, and/or metabolism individually, but not in combination so testing a diet formulation of this type was of interest.

Although measuring and controlling food intake and monitoring BW over time are important in weight management, body composition (e.g., fat vs. lean mass) is another important measure because it is a more accurate predictor of metabolic status. Research trials often use computed tomography, magnetic resonance imaging, deuterium oxide, or dual-energy X-ray absorptiometry (DEXA) to measure fat and lean mass of cats, but these techniques are expensive, may require anesthesia, and are not available in many areas. Body condition scores (BCS) are commonly used to assess body composition because they are cheap and relatively easy to conduct (Laflamme, 1997), but are subjective and owners often under-estimate their pet’s BCS (Gerstner and Liesegang, 2017; Lee et al., 2021). Other cheap, but more objective methods for assessing the body composition of pets are needed. In previous studies, morphometric measures and/or a feline body mass index (fBMI) has been used to diagnose feline obesity (Witzel et al., 2014; Iwazaki et al., 2015). Like BCS, cats with a higher fBMI had higher plasma triglyceride and non-esterified fatty acid concentrations (Iwazaki et al., 2015). Subcutaneous adiposity and muscularity may also be assessed using ultrasonography, which was recently done in cats (Iwazaki et al., 2018; Iwazaki and Nade, 2020). Because these recent techniques may allow veterinarians to evaluate body composition in daily clinical practice without the need for expensive or invasive methods, they were also included to assess body condition in this study. The objective of this study was to determine whether a dietary formula rich in protein, dietary fiber, antioxidants, and L-carnitine would limit changes in BW, body composition, serum metabolites, and physical activity levels in cats following ovariectomy.

Materials and Methods

All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation (IACUC #17264).

Animals and experimental design

Twenty healthy female domestic shorthair cats [mean age = 9.5 ± 0.1 mo old; mean BW = 3.0 ± 0.1; mean body condition score (BCS) = 5.3 ± 0.1] were used in a completely randomized design consisting of 28 wk. Cats were housed individually in cages (1.02 × 0.76 × 0.71 m³) during two 1-h feeding times each day (8–9 a.m.; 3–4 p.m.) in a humidity- and temperature-controlled room with a 14-h light:10-h dark cycle in the Edward R. Madigan Animal Facility at the University of Illinois at Urbana-Champaign, Urbana, IL. At other times, cats were group-housed and able to socialize and exercise outside their cages. Cats were allowed access to various toys and scratching poles for environmental enrichment and were socialized with humans regularly. Fresh water was available ad libitum.

The experiment contained three phases: a 4-wk baseline phase prior to ovariectomy, a 12-wk restricted phase following ovariectomy, and a 12-wk ad libitum phase. During the baseline period, a moderate-protein, moderate-fiber (MPMF) diet was fed to support normal growth. At that point, 16 cats underwent ovariectomy and 4 cats underwent a sham operation. After ovariectomy, spayed cats were allotted to the MPMF diet (n = 8) or a high-protein, high-fiber (HPHF) diet (n = 8) based on BW and BCS to evenly distribute groups. The sham-operated cats were fed the MPMF diet. During the first 12 wk following ovariectomy, cats were fed to maintain their BW. During the second 12 wk after ovariectomy, cats were fed twice the amount of the diet needed to maintain BW at the end of the weight maintenance phase to allow cats to overconsume and gain weight if they chose.

Diets

Dry, extruded diets formulated to meet all Association of American Feed Control Officials (AAFCO, 2017) nutrient recommendations for growing and reproducing cats were fed (Table 1). Both diets were based on chicken by-product meal, wheat flour, and wheat gluten. The MPMF diet was designed to contain moderate protein, moderate fat, and low fiber concentrations, whereas the HPHF diet was designed to contain a moderate fat concentration and be rich in crude protein, dietary fiber, antioxidants, and l-carnitine.

Table 1.

Dietary ingredient and analyzed chemical composition of diets fed to cats

Ingredient			MPMF1	HPHF
			% as-is
Chicken by-product meal			24.738	44.244
Wheat flour			23.662	7.449
Wheat gluten			19.029	28.544
Brewer’s rice			14.272	–
Chicken fat			11.417	–
Fish powder (palatant; AFB)			1.903	1.903
Cellulose			1.903	11.418
Sunflower oil (high oleic)			–	2.854
Fish oil			0.951	–
DHA (LG-Max; Alltech)			–	0.951
Salt (NaCl)			0.476	0.276
Potassium chloride			0.428	0.847
Methionine			0.352	0.352
Vitamin and mineral premix2			–	0.285
Taurine			0.19	0.19
Vitamin premix3			0.171	–
Mineral premix4			0.171	–
Choline chloride			0.124	0.257
Antioxidant (Naturox; Kemin)			0.095	0.095
Calcium carbonate			0.095	0.095
L-carnintine (l-carnitine tartrate; Frontier Foods)			–	0.095
Vitamin C (Rovimix Stay-35; DSM)			–	0.048
Vitamin E (Rovimix E50 adsorbate; DSM)			0.019	0.095
Selenium (Selplex; Alltech)			0.001	0.001

Chemical Composition	MPMF (% DM)	MPMF (per1,000 kcal ME)	HPHF (% DM)	HPHF (per1,000 kcal ME)
Dry matter	94.27	–	95.26	–
Organic matter	94.55	–	92.57	–
Crude protein	37.16	97.28 g	52.32	176.74 g
Acid-hydrolyzed fat	16.18	42.36 g	12.34	41.68 g
Omega-3 fat	0.29	0.76 g	0.32	1.08 g
Total dietary fiber	14.3	37.44 g	25.6	86.48 g
Gross energy, kcal/g DM	5.09	–	5.04	–
Metabolizable energy (ME); kcal/g	3.82	–	2.96	–
Vitamin C, mg/100 g	–	–	14.78	49.93 mg
Tocopherol, IU/100 g	39.40	103.2 IU	73.60	248.6 IU
Carnitine, mg/100 g	2.13	5.58 mg	49.63	167.65 mg

MPMF, control diet containing a moderate amount of crude protein and fiber; HPHF, diet containing a high amount of protein and fiber.

Provided per kg diet: vitamin A, 12.8 mg; vitamin D3, 0.04 mg; vitamin E, 114 mg; vitamin K, 1.9 mg; vitamin B2, 12.5 mg; vitamin B6, 12.4 mg; vitamin B12, 0.3 mg; nicotinic acid, 38.9 mg; pantothenic acid, 5.5 mg; folic acid, 1.3 mg; Na (as NaCl), 0.3 mg; Zn (as ZnSO4), 538.2 mg; Ca (as Ca(IO3)2), 30.9 mg; Cu (as CuSO4), 28.5 mg; tryptophan, 101.8 mg; taurine, 940.5 mg; oligosaccharide, 99.8 mg; γ‐linolenic acid, 10.0 mg; de-fatted rice bran, 846.6 mg.

Provided per kg diet: Mn (as MnSO4), 66.00 mg; Fe (as FeSO4), 120 mg; Cu (as CuSO4), 18.00 mg; Co (as CoSO4), 1.20 mg; Zn (as ZnSO4), 240 mg; I (as KI), 1.80 mg; Se (as Na2SeO3), 0.24 mg.

Provided per kg diet: vitamin A, 5.28 mg; vitamin D3, 0.04 mg; vitamin E, 120.00 mg; vitamin K, 0.88 mg; thiamin, 4.40 mg; riboflavin, 5.72 mg; pantothenic acid, 22.00 mg; niacin, 39.60 mg; pyridoxine, 3.52 mg; biotin, 0.13 mg; folic acid, 0.44 mg; vitamin B12, 0.11 mg.

Ovariectomy

Food was withheld for at least 8 h before spay surgery. Water was provided until cats were sedated for the surgery. The operations were done in the Edward R. Madigan Laboratory on the University of Illinois campus. Cats were pre-medicated with Ketathesia (ketamine HCL; Henry Schein, Melville, NY; 6.25 mg/kg BW), Torbugesic (butorphanol tartrate; Zoetis, Parsippany-Troy Hills, NJ; 0.25 mg/kg BW), and Dexdomitor (dexmedetomidine; Zoetis; 0.006 mg/kg BW) via intramuscular injection. Metacam oral suspension (meloxicam; Boehringer Ingelheim Vetmedica, Inc., Saint Joseph, MO; 0.2 mg/kg BW) was given just prior to anesthesia. Anesthesia was maintained with 1% to 3% isoflurane in 100% oxygen inhalation. Cats were positioned in dorsal recumbency and ovariectomy was performed using standard techniques. A 2–3 cm ventral midline skin incision was made halfway between the umbilicus and the pubis. The ovarian pedicles were autoligated or clamped and ligated with absorbable suture using a Miller’s knot. An additional clamp was added near the proper ligament of the ovary. The ovarian pedicle was transected between the clamp on the pedicle and the ovary. The uterine body and associated vessels were clamped together. A Miller’s knot ligature, using absorbable suture, was placed near the uterine body bifurcation. The uterus was transected distal to the ligature, and the uterus with ovaries was removed. The incision was closed in three layers. Cats were monitored for 5 consecutive d for pain, lethargy, and inappetence, and the incision site was monitored for any sign of infection or dehiscence.

Food intake, BW, and BCS

Cats were fed twice daily, with food offered and refusals measured at each feeding to calculate food and calorie intake. BW was measured twice weekly and BCS (9-point scale; Laflamme, 1997) was measured weekly before the morning feeding.

Blood collection, DEXA analysis, ultrasonography, and zoometry measurements

Fasted blood samples were collected and DEXA scans, ultrasonography, and zoometry measurements were performed after the baseline phase (wk 0; prior to ovariectomy) and 6, 12, 18 and 24 wk after ovariectomy. Before blood collection and DEXA scans, cats were fasted overnight. Just prior to blood collection, cats were sedated by an intramuscular injection of Ketathesia (ketamine HCL; Henry Schein; 6.25 mg/kg BW), Torbugesic (butorphanol tartrate; Zoetis; 0.25 mg/kg BW), and Dexdomitor (dexmedetomidine; Zoetis; 0.006 mg/kg BW). Blood samples were collected via the jugular vein and placed into #367985 BD Vacutainer tubes for serum separation (Becton Dickinson, Franklin Lakes, NJ) for serum chemistry profiles. After blood collection, body composition was evaluated using a Hologic model QDR04500 fan beam x-ray bone densitometer and the accompanying software (Hologic Inc., Waltham, MA) at the University of Illinois Veterinary Teaching Hospital. DEXA scans were performed under ventral recumbency, with LM, fat mass, and bone mineral content determined. After blood collection and DEXA scans had been completed, an injection of Antisedan (atipamezole HCl; Zoetis; 0.06 mg/kg BW) was administered intramuscularly.

Prior to ultrasonography, the back on the left side of the 6–7th and 13th ribs and the pit of the stomach were shaved with a commercial hair clipper. Cats were laid in left lateral recumbency. Halfway down the thorax, a cross-sectional image on the 6–7th rib and just caudal to the 13th rib was captured by ultrasound (Exago, Echo Control Medical, Angoulême, France) with a linear transducer at 10 MHz. The thickness of subcutaneous adipose tissue just caudal to the 13th rib and back fat thickness (BF) and the thickness of epaxial muscle (EM) on the 6–7th rib and just caudal to the 13th rib were determined by free software (DataPicker Version 1.2, Blue Moon Factory, Tokyo, Japan) in a manner similar to that performed in cats previously (Iwazaki et al., 2018; Iwazaki and Nade, 2020). All ultrasounds measures were taken by a single investigator (E.I.).

Zoometry measurements were performed, with the head and body length being measured from the top of the nose to the joint between the sacrum and the coccyx. The patella to calcaneus length (PCL) was measured. The circumferences on the 9th and 13th ribs were determined as chest girth (CG) and abdominal girth, respectively. fBMI was determined by dividing BW (kg) by PCL (m) as reported by Iwazaki et al. (2013, 2015, 2018).

Calculated fat percentage (CFP) using equation 1 was determined as follows: $\{1.54\times CG(cm)\}-\{1.58\times PCL(cm)\}-8.67$(Hawthorne and Butterwick, 2000).

CFP using equation 2 was determined as follows: $[\{CG(cm)/0.7067\}-PCL(cm)]/0.9156-PCL(cm)$(Butterwick, 2000).

Voluntary physical activity level

Voluntary physical activity levels were measured after the baseline phase (week 0; prior to ovariectomy) and 8, 16, and 24 wk after ovariectomy using Actical devices and computer software (Mini Mitter, Bend, OR). During activity monitoring periods, Actical devices were attached to collars worn around the neck for 6 consecutive d. Mean activity was presented in activity counts per epoch (epoch length = 15 s), with total daily activity, 14 h of light activity, and 10 h of dark activity being measured. The ratio of light to dark activity was also calculated.

Blood metabolite analysis

Blood tubes for serum isolation were centrifuged at $1300\times g$and 4 °C for 10 min (Beckman CS-6R centrifuge; Beckman Coulter Inc., Brea, CA). Serum chemistry profile was analyzed using a Hitachi 911 clinical chemistry analyzer (Roche Diagnostics, Indianapolis, IN) at the University of Illinois Veterinary Diagnostic Laboratory (Urbana, IL).

Chemical analysis of diets

Diets were subsampled and ground through a 2-mm screen using a Wiley mill (model 4, Thomas Scientific, Swedesboro, NJ). Diets were analyzed according to procedures of the Association of Official Analytical Chemists (AOAC) for dry matter (DM; 105 °C) and ash (organic matter was calculated from ash) (AOAC, 2006; methods 934.01, 942.05). Crude protein content was calculated from Leco total N values (TruMac N, Leco Corporation, St. Joseph, MI; AOAC, 2006). Total lipid content (acid-hydrolyzed fat) of the samples was determined according to the methods of the American Association of Cereal Chemists (AACC, 1983) and Budde (1952). Gross energy of the samples was measured using an oxygen bomb calorimeter (model 1261, Parr Instruments, Moline, IL). Total dietary fiber content was determined according to Prosky et al. (1985).

Statistical analysis

The Mixed Models procedure of SAS (version 9.4; SAS Institute, Cary, NC) was used to identify statistical significance based on treatment, time, or treatment × time effects. Daily food intake, daily calorie intake, calorie intake/BW (CI/BW), calorie intake/fat mass (CI/FM), calorie intake/LM (CI/LM), BW, BCS, fBMI, body length, body composition measures, serum metabolites, and physical activity levels were evaluated based on the change from baseline data. These response criteria were analyzed using repeated measures analysis. All values are expressed as the least squares mean ± pooled standard error of the means. Statistical significance was set at P < 0.05, with trends set at P < 0.10.

Results

All baseline data (week 0) were analyzed among groups. Animals were allotted to treatment groups based on BW and BCS and although differences were small and within normal ranges, baseline food intake was slightly higher (P < 0.05) in cats later allotted to the HPHF diet (Table 2). None of the other baseline measures were different (P > 0.05) among groups.

Table 2.

Baseline data of female cats

Item	Dietary treatment group1			SEM	P
	MPMF (n = 8)	HPHF (n = 8)	Sham (n = 4)
Diet
Daily calorie intake, kcal/d	257.85	281.6	204.9	18.989	0.982
Daily food intake, g/d	67.50	73.73	53.63	4.953	0.05
Zoometry measurement
Body weight, kg	3.00	3.22	2.82	0.181	0.36
Body condition score, 9-point scale	5.31	5.56	5.00	0.187	0.17
Feline body mass index, kg/m	22.50	23.55	20.65	1.112	0.26
Body composition
Calculated body fat percentage (equation 1), %	11.85	13.93	11.05	1.017	0.14
Calculated body fat percentage (equation 2), %	13.85	15.79	13.03	1.025	0.17
Body fat thickness (13th rib), cm	0.27	0.29	0.24	0.020	0.30
Epaxial muscle thickness (13th rib), cm	1.53	1.47	1.39	0.075	0.48
Epaxial muscle thickness (6–7th ribS), cm	1.41	1.42	1.31	0.031	0.06
Total fat mass, g	513.4	601.2	426.6	71.8	0.21
Fat percentage	17.7	19.8	16.3	1.5	0.31
Total lean mass, g	2304.7	2365.5	2118.0	116.0	0.39
Total bone mineral content, g	33.2	33.2	31.3	2.8	0.89

MPMF, cats assigned to a control diet containing a moderate amount of crude protein and fiber; HPHF, cats assigned to a diet containing a high amount of protein and fiber; Sham, cats assigned to the sham-operation group fed the control diet.

During the restricted phase, a treatment × time interaction (P < 0.0001) was noted for food intake and calorie intake (Figure 1a and 1b). Mean food and calorie intake decreased (P < 0.0001) for all cats, but was greater for spayed cats when compared with sham cats (food intake: P = 0.05; calorie intake: P < 0.0001). During the ad libitum phase, food intake again had a treatment × time interaction (P < 0.0001), with all groups having increased intake but at different levels. Calorie intake during the ad libitum was increased (P < 0.0001) over time and tended to be higher (P = 0.097) in sham cats and spayed cats fed MPMF than spayed cats fed HPHF.

Figure 1.

Change of food intake (a), caloric intake (b), calorie intake/body weight (c), body weight (d), body condition score (e), and feline body mass index (f) from baseline in female cats fed different diets during a restricted phase and an ad libitum phase after spay surgery. MPMF, cats fed a control diet containing a moderate amount of crude protein and fiber (black circles solid line). HPHF, cats fed a diet containing a high amount of protein and fiber (black squares discontinued line). Sham, sham-operated cats fed the control diet (gray triangles solid line). Data are presented as change from baseline (week 0) least squares ± SEM. Data were divided into restricted phase (1–12 wk) and ad libitum phase (13–24 wk). Statistical significance was set at P < 0.05.

The CI/BW, CI/FM, and CI/LM responses were similar to that of calorie intake. During the restricted phase, CI/BW was decreased (P < 0.0001) over time and decreased (P < 0.01) at a greater extent in spayed cats fed HPHF and MPMF than sham cats (Figure 1c). CI/BW increased (P < 0.0001) over time in the ad libitum phase, but was not affected by diet. CI/FM was decreased (P < 0.01) over time in the restricted phase, but not affected by diet (Supplementary Figure 1). During the ad libitum phase, CI/FM was relatively stable for spayed cats fed HPHF and sham cats, but decreased at week 24 in spayed cats fed MPMF. CI/LM was decreased (P < 0.0001) over time in the restricted phase, with spayed cats being decreased (P < 0.01) to a greater extent than sham cats (Supplementary Figure 2). During the ad libitum phase, CI/LM increased in all groups, but tended to vary depending on diet.

Even though cats were fed to maintain BW during the restricted phase, BW did fluctuate (P < 0.0001) over time and tended to be higher (P = 0.054) in spayed cats fed HPHF than the other groups (Figure 1d). In the ad libitum phase, a treatment*time interaction was observed (P < 0.01), with all BW increasing over time but to a lower extent in sham cats compared to spayed cats fed HPHF or MPMF. BCS was affected over time, being slightly increased (P < 0.0001) over time in the restricted phase and dramatically increased (P < 0.0001) over time in the ad libitum phase, but was not affected by diet (Figure 1e). Body length was not altered during the restricted phase, but increased (P < 0.05) during the ad libitum phase (Supplementary Figure 3). The PCL was lower (P < 0.050) in sham cats than spayed cats during the restricted phase (Supplementary Figure 4). During the ad libitum phase, PCL was constant for sham cats, but increased (P < 0.01) in spayed cats. Chest girth was variable over time (P < 0.05) but relatively constant during the restricted phase, but dramatically increased (P < 0.0001) over time during the ad libitum phase (Supplementary Figure 5). Chest girth was also increased (P < 0.05) to a greater extent in spayed cats fed MPMF than the other cats during the ad libitum phase. Abdominal girth tended to be decreased (P = 0.074) in spayed cats fed HPHF during the restricted phase (Supplementary Figure 6). During the ad libitum phase, abdominal girth increased (P < 0.0001) over time, but was not affected by diet. A product of the body measurements, fBMI fluctuated over time (P < 0.0001) in the restricted phase and was increased (P < 0.0001) over time in the ad libitum phase (Figure 1f).

Similar to fBMI, total fat mass (P < 0.01; Figure 2a), body fat percentage (P < 0.01; Figure 2b), BF (P = 0.01; Figure 2c), and calculated fat percentages (P < 0.05, Figure 2d; P = 0.073, Figure 2e) fluctuated over time in the restricted phase and was increased (P < 0.001) over time in the ad libitum phase. Total fat mass, body fat percentage, and BF were not affected by diet in restricted or ad libitum phases. Total LM increased (P < 0.01) over time and increased to a higher (P < 0.05) extent in spayed cats fed HPHF during the restricted phase (Figure 2f). During the ad libitum phase, total LM increased in all groups, but was lower (P < 0.0001) in sham cats than spayed cats. EM thickness at the 13th rib (Supplementary Figure 7) and 6–7th rib (Supplementary Figure 8) was not different in the restricted phase, but increased (P < 0.05) over time in the ad libitum phase. Total bone mineral content increased (P < 0.05) over time in the restricted and ad libitum phases (Figure 2g).

Figure 2.

Change of total fat mass (a), body fat percentage (b), back fat thickness (c), calculated fat mass 1 (d), calculated fat mass 2 (e), total lean mass (f), and total bone mineral content (g) from baseline in female cats fed different diets during a restricted phase and an ad libitum phase after spay surgery. MPMF, cats fed a control diet containing a moderate amount of crude protein and fiber (black circles solid line). HPHF, cats fed a diet containing a high amount of protein and fiber (black squares discontinued line). Sham, sham-operated cats fed the control diet (gray triangles solid line). Data are presented as change from baseline (week 0) least squares ± SEM. Data were divided into restricted phase (1–12 wk) and ad libitum phase (13–24 wk). Statistical significance was set at P < 0.05.

Total voluntary physical activity levels were numerically, but not statistically lower over time (Supplementary Figure 9). 
Activity during the light period (P = 0.090; Supplementary Figure 10) and dark period (P = 0.080; Supplementary Figure 11) 
tended to be lower over time. The light to dark activity ratio was different (P < 0.05) over time, but was not affected by diet (Supplementary Figure 12).

Blood creatinine fluctuated and was different (P < 0.001) over time, but was not affected by diet (Supplementary Figure 13). Blood urea nitrogen was affected by a treatment*time interaction during the restricted phase, being relatively stable in sham cats and spayed cats fed MPMF but increased in spayed cats fed HPHF (Supplementary Figure 14). During the ad libitum phase, blood urea nitrogen was higher (P < 0.05) in spayed cats fed HPHF than those in the other groups. Blood albumin tended to be higher (P = 0.077) in spayed cats fed HPHF than those in the other groups during the restricted phase, but was not different in the ad libitum phase (Supplementary Figure 15). Blood globulin decreased (P < 0.05) over time in the restricted phase, but increased (P < 0.01) over time in the ad libitum phase (Supplementary Figure 16). Blood globulin was not affected by diet. Blood glucose tended to increase (P = 0.097) over time in the restricted phase and was increased (P < 0.01) over time in the ad libitum phase (Supplementary Figure 17). Serum alkaline phosphatase decreased (P < 0.0001) over time in the restricted phase, but was not affected by diet (Supplementary Figure 18). During the ad libitum phase, serum alkaline phosphatase was stable in spayed cats, but was decreased (P < 0.01) in sham cats. Serum gamma-glutamyl transferase was not different during the restricted phase, but fluctuated over time (P < 0.0001) during the ad libitum phase (Supplementary Figure 19). Blood cholesterol increased (P < 0.001) over time during the restricted phase, but was not altered during the ad libitum phase (Supplementary Figure 20). Blood triglycerides decreased (P < 0.05) over time during the restricted phase, but tended to increase (P = 0.080) in the ad libitum phase (Supplementary Figure 21). Blood fructosamine increased (P < 0.05) in the restricted and ad libitum phases, but was not affected by diet (Supplementary Figure 22). Serum non-esterified fatty acids tended to be higher (P = 0.068) in spayed cats fed HPHF than cats in the other groups during the restricted phase, but were not affected by time or diet in the ad libitum phase (Supplementary Figure 23).

Discussion

It is well known that spaying and neutering helps control cat populations and some negative behaviors, but also increases obesity risk by increasing food consumption, decreasing energy requirements, and decreasing physical activity levels (Belsito et al., 2009; Vester et al., 2009; Backus et al., 2011; Allaway et al., 2017). The data from the current study confirmed these previous findings, demonstrating that cats required a reduced caloric intake to maintain BW after spay and a level of food lower than that of sham-operated cats. In this study, the level of calorie reduction required to maintain BW post-spay (~50%) was greater than that reported in previous studies (~15%–35%; Flynn et al., 1996; Hoenig et al., 2002; Belsito et al., 2009). We believe that although it was our goal to feed cats to maintain BW at baseline, some of the cats were fed at a level a bit too high. Because the baseline phase was only a few weeks in length, cats did not gain significant weight during that time, but this slight over-feeding amplified the post-spay response over a longer period of time (12 wk). All cats dramatically increased food and caloric intake during the ad libitum phase, but the increase in BW was greater in spayed cats than sham-operated cats. Increases in BCS and fat mass also increased in all cats once they were allowed to over-consume their food. Physical activity was not statistically different over time. As expected, blood triglyceride and fructosamine concentrations increased during the ad libitum phase while cats gained weight.

The HPHF diet was formulated to contain high concentrations of protein and fiber, have lower caloric content, and include elevated concentrations of l-carnitine and antioxidants. Dietary fiber reduces caloric content (kcal/g), which was apparent in the diets tested (Table 1). High dietary protein is beneficial in that it may not only aid in satiety, but is known to have a much higher thermic effect (20%–30%) than carbohydrate (5%–15%) or fat (0%–3%) (Tappy, 1996), with both effects aiding in weight maintenance. l-carnitine aids in long-chain fatty acid transport and the beta oxidation of fatty acids and has been shown to be effective in maintaining BW or aiding weight loss in cats (Center et al., 2000; Blanchard et al., 2002). Vitamin C and vitamin E are important antioxidants that protect against oxidative damage and maintain metabolism even during weight gain (Jewell et al., 2000; Gordon et al., 2020).

Even though the HPHF diet was formulated to promote satiety and improve metabolism, it did not result in as many differences as expected. High-protein diets are known to maintain lean mass in cats (Nguyen et al., 2004; Vasconcellos et al., 2009), but we were unable to detect differences in the current study. The lack of diet effects may relate to the study design, the relatively short amount of time that cats were allowed to overeat and gain weight (12 wk), and the restriction placed on ad libitum feeding (capped at twice that needed to maintain BW). At the end of the study, body fat percentage was still only ~25% for many of the cats. Cats were gaining weight, but were still in an early and moderate stages of obesity at that time. Therefore, most blood lipids and hepatic injury markers were still within the normal ranges and no symptoms of ectopic fat accumulation in the liver were apparent. The benefits of this diet may require study over a longer period of time and/or the study of animals with a greater body condition and dysfunctional metabolism. A weight loss study of obese cats may have provided a better opportunity to demonstrate benefits, as markers of hepatic stress are more frequent in that population (Ibrahim et al., 2003; Iwazaki et al., 2016). In such a situation, the protective effects of vitamin E, vitamin C, and l-carnitine may be easier to demonstrate.

In conclusion, our results demonstrate that the diets tested in this study had minor effects, but spaying affected cat food intake, body weight, metabolism, and physical activity. As expected, spayed cats required lower food and caloric intake than sham-operated cats to maintain body weight following surgery and gained body weight, body condition score, and fat mass when allowed to overeat.

Glossary

Abbreviations

AAFCO

Association of American Feed Control Officials

BCS

body condition score

BF

back fat thickness

BW

body weight

CFP

calculated fat percentage

CG

chest girth

CI/BW

calorie intake/body weight

CI/FM

calorie intake/fat mass

CI/LM

calorie intake/lean mass

DEXA

dual-energy x-ray absorptiometry

EM

epaxial muscle

fBMI

feline body mass index

HPHF

high-protein, high-fiber diet

LM

lean mass

MPMF

moderate-protein, moderate-fiber diet

PCL

patella to calcaneus length

Conflict of Interest Statement

E.I. is employed by Nippon Pet Food Co. Ltd. All other authors have no conflicts of interest.