Wet-food diet promotes the recovery from surgery of castration and control of body weight in adult young cats

Abstract

Inappropriate dietary management may lead to delayed recovery from castration surgery and significant weight gain in cats after castration. Wet canned food often exhibits more advantageous characteristics than dry food (e.g., higher palatability and digestibility, and lower energy density). This study compared the effects of canned and dry food on surgical recovery and weight management in cats after castration. Eighteen healthy cats (weighed 4.33 ± 1.04 kg and aged 18-months old) were allocated to one of the two dietary treatments (N = 9/group), dry (CON) and canned food (CAN) balanced for sex and initial BW. Cats were fed ad libitum for 7 weeks, including one week before surgery (week 0) and 6 weeks after surgery (week 1–6). Daily dry matter intake (DMI), and weekly body weight (BW) and body condition score (BCS) was obtained. Feces were collected for measuring nutrient digestibility and concentrations of short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA). Physical pain and wound surface assessment were performed at week 1. Blood was also collected intermittently for measuring biochemical indices and untargeted metabolomics analysis. Results indicated that BW, BCS and daily DMI in CON group increased (P < 0.05) over time after castration, but were maintained relatively stable in CAN group. Cats in CAN group exhibited less pain-related behavior as reflected by lower score of comfort (P < 0.05) and vocalization (P < 0.10), improved wound surface assessment (P < 0.10), lower level of lipase (P < 0.10) and ratio of blood urea nitrogen/serum creatinine (BUN/SC; P < 0.05), and higher level of superoxide dismutase (SOD; P < 0.05) in week 1 than CON cats. Meanwhile, the CAN group had significantly higher concentration of immunoglobulin G (IgG) on days 5 and 7, and higher level of high-density lipoprotein cholesterol (HDL-C; P < 0.10) but lower triglyceride (TG; P < 0.05) than CON group on day 20 and 48. Fecal total and most individual SCFA increased significantly from week 1 to week 6 regardless of diet, but the increase of butyric acid over time only occurred in CON group (P < 0.05). Also, serum metabolomic analysis revealed differential metabolic pathways between the two groups. Overall, compared with the dry food, the canned food tested in our study promoted cat wound recovery by reducing pain and increasing immune and antioxidative capacity after sterilizing surgery, and helped to maintain healthy body condition in cats after castration.

Wet canned food, even fed at libitum may serve as a more suitable diet for promoting the recovery from castration surgery and management of body condition in cats after castration than dry food, potentially due to the high palatability of the canned food as a result of higher moisture, protein, and fat content, and its lower energy density from water dilution.

INTRODUCTION

Cats are among the most popular pets worldwide (Foreman-Worsley and Farnworth, 2019). Many owners consider their cats as family members (Ines et al., 2021) and pay increased attention to the cat physical and mental health. Meanwhile, pet overpopulation could result in pet ­abandonment and relinquishment, causing overwhelming burden to animal shelters and euthanasia of unwanted pets (Scarlett et al., 2002; Kustritz, 2007). Castration is one of the most common technique for surgically sterilizing dogs and cats, which signifies the removal of reproductive organs (Howe, 2006). Early statistics from 2007 showed that 92.0% of cats aged between 6 and 12 months were castrated in the United Kingdom (Murray et al., 2009), while the prevalence of cat castration was 82.1% in the United States in 2007 (Trevejo et al., 2011). There are behavioral and health benefits to castration, such as decreased urine spraying, roaming, and aggression in male cats, and reduced incidence of reproductive tract neoplasms in females (McKenzie, 2010).

The procedure of castration (e.g., stress from transportation, change of environment and the surgical procedure) poses challenges to cat welfare. Physiological and behavioral indicators of pain, wound healing process, and the overall effects of surgery on cat well-being should be closely monitored and evaluated (Väisänen et al., 2007). Nutritional management is critical in postoperative care (Collins, 2016; Vendramini et al., 2020). Relevant studies in dogs and cats have been focused on nutritional support (Brunetto et al., 2010; Corbee and Kerkhoven, 2014) and the supplementation of bioactive ingredients, such as ginger rhizome powder (Javdani et al., 2021). For example, adequate energy support, even if modest and close to resting energy requirement of dogs and cats during the hospitalization could reduce the length of the hospital stay (Brunetto et al., 2010). This indicates the importance of nutritional support in speeding the recovery of pet cats and dogs from treatment procedures of illness, including surgery. Hyporexia (i.e., reduced appetite) and in the more severe case anorexia (i.e., complete loss of appetite) can occur due to surgery, therefore stimulation of spontaneous food intake shortly after surgery by providing food with high palatability (e.g., canned food with raised moisture, protein and fat content) is important (Corbee and Kerkhoven, 2014). Cats were shown to prefer wet food with a moisture content close to that of raw meat (Zaghini and Biagi, 2005). In addition, food with high water content can contribute to rehydration of animals and reduce the chance of food being vomited or regurgitated due to quicker movement of digesta from stomach to intestinal tract (Sachdeva et al., 2013). Nutrient digestibility of canned food has been shown to be higher than that of dry kibbles in cats (Bermingham et al., 2013, 2018). Collectively, canned food may promote the body recovery for cats after surgical procedure due to its advantages in promoting rehydration and better nutrient support (Corbee and Kerkhoven, 2014).

Castration has been associated with weight gain and obesity in pet cats (Larsen, 2017). The sexual and appetite-related hormonal changes after castration was shown to impact feeding behavior and general activity (Fettman et al., 1997). Castration may induce significant weight gain and obesity by reducing energy expenditure and increasing food intake (Martin et al., 2001). A significant increase in feed intake was observed as early as three days after neutering, along with a body weight (BW) gain of about 28% by week 7 in male cats (Kanchuk et al., 2003). Nutritional management can also be important for the maintenance of normal BW after castration, and existing studies mainly concentrate on the adjustment of energy and nutrients. For instance, previous experiment suggests that diet of low energy and fat can effectively control excessive weight gain in castrated dogs (Schauf et al., 2016). Excessive BW gain can be controlled by limiting the amount of energy supply in food. One major difference between wet and dry pet food is the water content, which contributes to the dilution of energy density in wet food compared to the dry food. A survey on feline obesity identified feeding mainly dry kibbles as a major risk factor for cats at age of 12.5–13 months (Rowe et al., 2015). One study also reported that adding 40% water to commercial dry food could increase voluntary activity and ameliorate regain of BW after caloric restriction in cats (Cameron et al., 2011). Therefore, the diluted energy density of wet food due to higher water content may positively regulate cat BW and body condition (BC) through mainly reducing dry matter and energy intake, and increasing activity to maintain energy balance.

Till date, few studies have compared the effects of dry and wet pet food on the control of BW and body condition score (BCS) in cats after castration. Accordingly, we hypothesized that high-moisture canned food which is often more palatable, and with better nutrient digestibility and lower energy density, might speed the recovery from castration surgery and the long-term BC control after castration in cats when compared to dry food.

MATERIALS AND METHODS

Animal ethics

All experimental procedures were authorized by the Animal Care and Use Committee prior to animal experimentation (Approval number: 2021a030) and were performed following the guidelines of the Laboratory Animal Center at the South China Agricultural University.

Animals and housing

A total 18 healthy Ragdoll cats, 6 males and 12 females, with the mean BW of 4.33 ± 1.04 kg and aged 18 months old were included in this experiment. Cats were housed individually at the laboratory in Qingke Biotechnology Co., Ltd (Guangzhou, China) with relative humidity and temperature of 70% ± 3% and 22°C ± 2°C, respectively. The housing cage (1.1 m × 0.7 m × 0.7 m) included separate areas for feeding, defecation and resting. Cats were also allowed to interact with people for 10 minutes daily and access to toys. All cats were vaccinated and dewormed and no drugs such as antibiotics that may influence the results of this experiment were given one month prior to the trial. Before the experiment, cats were free to eat adequate fresh food and drink clean water.

Diets and experiment design

Cats were allocated to one of the two dietary treatments (N = 9/group), dry food (CON group, 3 males and 6 females) and wet canned food (CAN group, 4 males and 5 females), according to their sex and initial BW. All cats were offered respective diet ad libitum. Specifically, the CON group were fed excess amount of dry food (i.e., 200 g) once daily at 9:00 am. Meanwhile, wet food was provided to the cats in CAN group for four times daily at around 8:00 am, 13:00 pm, 18:00 pm, and 23:00 pm, respectively, every time with a new can that contains 160 g wet food opened. After five days of adaption to the experimental diets, the CON and CAN group were castrated in the same veterinary hospital. All the experimental subjects were then transported back to the lab for the 6 weeks of postoperative recovery and BW monitor ­experiment. The timeline and sampling time points of this experiment are shown in Figure 1.

Figure 1.

Timeline and time points of sample collection.

Recording of Daily Dry Matter Intake (DMI), BW, and BCS

The daily DMI of all experiment subjects was recorded every morning and the BW and BCS which referred to the five-point BCS system in cats (Shoveller et al., 2014) were recorded weekly after overnight fasting during the whole period. Cats all started with a normal BCS that ranged from 2.7 to 3.2.

Diet composition and apparent digestibility of nutrients

The CON group was fed a commercial extruded pet food (Qingke Biotechnology Co., Ltd, Guangzhou, China) and the CAN group was fed a canned pet food manufactured in Guangdong Munchkin Biotechnology Co., Ltd (Shantou, China). Both diets meet the nutrient recommendations of Association of American Feed Control Officials for adult cats. The ingredients, analyzed chemical and energy composition of two diets tested are listed in Table 1.

Table 1.

Major ingredients, analyzed chemical and energy composition of two diets tested

Item	CON	CAN
Ingredients	Chicken (40%), chicken meat powder (33%), chicken fat (6%), tapioca flour (4%), sweet potato flour (3.5%), chicken liver (2%), chicken heart (2%), Brewer’s yeast powder (2%), alfalfa powder (2%), fish oil (1.5%), chicken liver powder (1%)	Water, chicken (55%), swine liver (16%), cornmeal (1.7%), soy protein isolate (0.25%), fish oil (0.2%), chicken offal powder (0.4%)
Analyzed composition
DM (%)	93.43	34.23
OM (% DM)	91.39	94.24
CF (% DM)	1.70	1.90
CP (% DM)	47.09	52.23
EE (% DM)	16.50	20.74
GE (J/g)	21943.00	9375.96
GE (J/g, on DM basis)	23486.03	27391.06

DM, dry matter; OM, organic matter; CF, crude fiber; CP, crude protein; EE, ether extract; GE, gross energy. CON, cats fed dry food; CAN, cats fed wet food.

Total feces from every cat were collected for 5 consecutive days before morning feeding at week 2 to determine the apparent digestibility. The process was conducted in week 2 because cats at week 1 after surgery ate little and feces excreted during this period was not enough for the analysis of nutrient digestibility. The diets and feces samples collected were oven-dried at 65°C until dry and grounded for chemical analysis. Samples were analyzed for dry matter (DM, method No. 934.01), organic matter (OM, method No. 942.05), crude fiber (CF, method No.962.09), crude protein (CP, method No. 954.01) and ether extract (EE, method No.920.39) according to Association of Official Analytical Chemists methods (Horwitz and Latimer, 2007). Gross energy (GE) of diets and feces were determined by oxygen bomb calorimeter (IKA C 200, IKA Guangzhou Instrument Equipment Co., Ltd, Guangzhou, China). Apparent digestibility of certain nutrient was measured using the following formula: apparent digestibility (%) = (1 – A1/A) × 100%, where A is the content of a given nutrient in the diet, A1 is the content of the same nutrient in the feces. And the digestible energy (DE) was measured using the following formula: DE (J/g) = B – B1, where B is the GE (J/g) of diets and B1 is the GE (J/g) of feces.

Evaluation of physical pain and wound surface

A composite pain scale was adopted to evaluate the state of somatic pain in cats with minor modifications (Brondani et al., 2011). The rationality is that cats experiencing different levels of stress and pain would exhibit differences in pain-related behaviors (e.g., vocalization, aggression, body posture) and response to human social interaction (Brondani et al., 2011). The most relevant items selected from the tool were vocalization, posture, and comfort. Besides, wound healing scale containing items of inflammation, swelling, and color of the wound surface was applied to evaluate the wound recovery in cats (Drudi et al., 2018). Results were presented as physical pain score (PPS) and wound surface score (WSS), respectively. Evaluations were performed within week 1 by two experimenters who were blind to the treatments, and an agreed score was given for each item by the two experimenters. The scoring criteria were shown in Tables 2 and 3.

Table 2.

Evaluation standard of physical pain after surgery

Item	Assessment	Score
Vocalization	Cats purr and interact with humans when they are touched.	0
	Cats hiss or groan when people approach. When people petted the cat, it calms down.	1
	Cats hiss or groan when they are petted.	2
	Cats hiss or groan spontaneously.	3
Posture	Cats are relaxed and comfortable.	3
	Cats lie on their sides, stretch limbs and tense muscles.
	Cats lie on their backs with muscular tension and low activity.	2
	Cats are nervous, moving around and trying to find a comfortable place.	1
Comfort	Cats are interested in their surroundings and have exploratory behavior.	3
	Cats are relatively quiet and not interested in external stimuli.	2
	Cats keep lying down and getting up and feeling restless.	1

Table 3.

Evaluation standard of wound surface after surgery

Item	Assessment	Score
Inflammation	A large amount of tissue fluid or pus ooze from the wound with tissue proliferation.	3
	A small amount of fluid oozes from the wound.	2
	The wound is dry without fluid exudation.	1
Swelling	The wound is markedly swollen.	3
	The wound is slightly swollen.	2
	There was no swelling on the wound.	1
Color	The wound is bright red.	3
	The wound is pink.	2
	The wound has the color of normal skin.	1

Blood collection and serum biochemical analysis

As shown in Figure 1, on the day before the surgery, day 1, day 5, day 14 and day 42 after the surgery (i.e., day 5, day 7, day 11, day 20 and day 48), 4 mL blood was collected from each cat via forelimb vein after overnight fasting. The blood samples were then transferred to a pre-cooled serum separator tube, left to stand for 30 minutes before centrifugation at 3,500 rpm at room temperature for 15 minutes. After centrifugation, the supernatants were aliquoted into microcentrifuge tubes and stored at −80°C for further analysis.

Serum albumin (ALB), total protein (TP), globulin (GLOB), albumin/globulin (A/G), glutamyl transferase (GT), alkaline phosphatase (ALP), total bile acid (TBA), lipase, blood urea nitrogen (BUN), and ratio of BUN/SC (blood urea nitrogen/creatinine) on day 5, day 7, and day 11 were measured with commercial kits using an automatic blood biochemical analyzer (Chemray 800, Shenzhen Redu Life Technology, Shenzhen, China). Total antioxidative capacity (T-AOC) and superoxide dismutase (SOD) on day 5, day 7 and day 11 and triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) on day 20 and day 48 were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s protocols. Tumour necrosis factor-α (TNF-α), interleukin 10 (IL-10), interleukin 8 (IL-8), and immunoglobulin (Ig) A, G, and M on day 5, 7 and 11 were evaluated using commercial cat enzyme-linked immunosorbent assay (ELISA) kits (MEIMIAN, Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China). These parameters were chosen to reflect the inflammatory state, and the antioxidative and immune capacity of the body, which can contribute significantly to postoperative recovery (Moldal et al., 2012; Mogheiseh et al., 2019), and might be differentially impacted by the two diets if the wet food showed advantages in rehydrating the body and nutrient support than dry food.

Collection of fresh feces and analysis of short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA)

Fresh feces were collected at week 1 and week 6 for consecutive 3 days as shown in Figure 1, and stored at −80°C for further analysis. Upon processing, samples of feces were placed on ice for thawing, and 1 ml of ultra-pure water was added to 0.2 g fecal sample and vortexed for five minutes. The samples were placed in ice bath for ten minutes of ultrasonic crushing. Then samples were centrifuged at 13,000 rpm for ten minutes at 4°C, whereafter 20 μl of 25% metaphosphoric acid solution and 0.25 g anhydrous sodium sulfate was added to the collected supernatant. After mixing for 2 minutes, 1 ml of methyl tert-butyl ether was added into each sample. The samples were then centrifuged at 13,000 rpm for 5 minutes at 4°C after five minutes of mixing. Supernatant was harvested and filtered through 0.22-μm Millipore pore membrane filter to a sample vial.

The quantitative analysis of SCFA and BCFA of the preprocessed samples were carried out using gas chromatography-MS-QP2020 system (Shimadzu, Tokyo, Japan) following the method previously used in our laboratory (Yang et al., 2022). The gas chromatography was equipped with an auto-injector AOC-20i (Shimadzu) and coupled to a flame ionization detector. The chromatographic separation was performed on a DB-FFAP capillary column (30 m × 0.25 mm × 0.25 mm). Sample (0.6 μl) was injected with a 30:1 split ratio using an autosampler. The injection port was set to a temperature of 250 °C. The initial temperature of the column was 80 °C for 2 minutes. Then the temperature was increased to 150 °C at a rate of 10 °C/minute and maintained for 2 minutes. Finally, the temperature was increased to 180 °C at a rate of 15 °C/minute and maintained for 5 minutes. The total run time was 18 minutes. Helium (He; 99.999%) was the carrier gas with a flow rate of 3 ml/minute. The MS parameters were electron impact mode at ionization energy of 70 eV. The ion source and interface temperatures were 230 °C and 250 °C, respectively. The solvent delay time was 1 minute at the temperature of 230 °C. The acquisition mode was selected at ion monitoring mode with a scan interval of 0.3 second. Fecal SCFA and BCFA were measured with the intention to provide additional explanations for the potentially different impacts of dry and wet food on BW and BC of cats, as studies have correlated gut microbiota, along with their products (e.g., SCFA) with weight gain and the development of obesity in different species (Kieler et al., 2017; Riva et al., 2017; Wei et al., 2021).

Untargeted serum metabolomics analyses

As an advanced method widely applied in the research field of nutrition and metabolism (Carlos et al., 2020), serum metabolomics analyses allows the identification of changes in almost all the small molecules involved in the metabolic processes in serum, and can help to determine the underlying metabolic pathways that mediate the potential differences in the observed physiological responses caused by the two diets.

Serum samples were thawed and 200 μl of each sample was mixed with 800 μl methanol. After two minutes of vortex, the samples were centrifuged at 14,500 rpm for 15 minutes at 4°C. The supernatant was blow-dried with nitrogen. Each sample was then re-dissolved with 200 μl methanol and mixed for two minutes. Ultrasonic crushing was performed at a low temperature for ten minutes and samples were centrifuged at 14,500 rpm for 15 minutes at 4°C. Finally, all samples were filtered through 0.22-mm microporous membranes for UPLC-Orbitrap-MS/MS analysis using the method described previously (Xin et al., 2018).

The Compound Discoverer 2.1 (Thermo Fisher Scientific) data analysis tool was employed to complete raw data preprocessing automatically and was applied to identify metabolites by searching the mzCloud library and mzVault library. Orthogonal partial least-squares discriminant analysis (OPLS-DA) of metabolites was performed with the SIMCA-P 14.1 software. Response permutation test (RPT) was performed to test the accuracy of OPLS-DA model. The metabolites with variable importance in projection (VIP) > 1 and fold change (FC) > 2 or < 0.5 were deemed as the differential metabolites. To explore the changes of metabolic process further, a KEGG pathway analysis of differential metabolites was performed by using the enrichment analysis module on MetaboAnalyst 5.0.

Statistical analysis

SPSS 26.0 and GraphPad Prism 8.0 software were used for statistical analysis and graphic presentation. Independent samples Student’s t-test were performed to compare the difference between the two groups. Two-way repeated measure analysis of variance with Bonferroni adjustment for multiple comparisons was performed to analyze the differences within two groups (i.e., diet) at different time points (i.e., time). Significant differences were set at P < 0.05, and tendencies at P < 0.10.

RESULTS

BW, BCS, and daily DMI

Time and the interaction between diet and time (diet × time) affected BW and daily DMI of cats (P < 0.05; Figure 2). Time but not diet × time significantly affected BCS of cats (Figure 2). No significant differences of BW, BCS, and daily DMI were observed between the two diet-treated groups (Figure 2a).

Figure 2.

The effect of diets and experiment duration on BW, BCS and daily DMI in cats: (a) BW, body weight, (b) BCS, body condition score, (c) daily DMI, daily dry matter intake. CON, cats fed dry food; CAN, cats fed wet food. Week 0 represents the five days before the surgery, week 1, 2, 3, 4, 5 and 6 represent the following six weeks after surgery. Data are presented as mean ± SEM. The increase of BW, BCS, and daily DMI over time occurred in both diet-treated groups, but were at greater rates in the CON group than the CAN group.

Briefly, the increase of BW, BCS, and daily DMI over time occurred in both diet-treated groups, but were at greater rates in the CON group than the CAN group. Specifically, cats of both groups lost weight at week 1 compared with week 0 (P < 0.05, Figure 2a). Increase of BW can be noticed in the CON group at week 5 and 6 when compared with week 0 (P < 0.05, Figure 2a), but no such difference was observed with cats in the CAN group (P > 0.10, Figure 2a). Compared with the CAN group, which showed no significant difference in BCS at different time points, BCS in the CON group at week 3, 4, 5 and 6 was significantly higher than week 1 but not week 0 (Figure 2b). For the CON group, daily DMI from week 3 to week 6 was significantly higher than week 0 (P < 0.05, Figure 2c). Diversely, daily DMI of CAN group maintained at a stable level over the whole period (P > 0.10, Figure 2c).

Apparent digestibility of nutrients and DE

The apparent digestibility of DM, CF, CP, and EE were relatively higher (P ≤ 0.06) in CAN group than CON group, while digestibility of OM and GE between the groups were not different (Table 4). After calculation, DE of wet and dry food was 20022.11 J/g and 8565.80 J/g (as is), respectively.

Table 4.

Effects of diets on apparent digestibility in cats

Item	CON	SEM	CAN	SEM	P-value
DM (%)	87.04	0.01	89.14	0.01	0.02
OM (%)	87.64	0.01	89.17	0.01	0.13
CF (%)	30.08	0.04	61.39	0.05	0.01
CP (%)	90.62	0.01	91.52	0.01	0.06
EE (%)	94.03	0.01	96.60	0.01	0.01
GE (%)	91.25	0.01	91.36	0.01	0.82

DM, dry matter; OM, organic matter; CF, crude fiber; CP, crude protein; EE, ether extract; GE, gross energy. CON, cats fed dry food; CAN, cats fed wet food. Data are presented as mean ± SEM. Statistical difference was calculated by Student’s t-test.

PPS and WSS

Cats in the CAN group had lower score of vocalization than CON group (P < 0.05, Figure 3a), while the CON group had a moderately higher score of comfort than CAN group during the first week after the surgery (P < 0.10, Figure 3a). With regard to the WSS, the score of inflammation and color of the wound in the CAN group is slightly lower than that of the CON group (P < 0.10, Figure 3b).

Figure 3.

The effect of diets and experiment duration on the PPS and WSS in cats at week 1: (a) PPS, physical pain score, (b) WSS, wound surface score. CON, cats fed dry food; CAN, cats fed wet food. Week 1 represents the first week after surgery. Data are presented as mean ± SEM. The symbol (^*) indicates statistically significant differences between two groups (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001), and the symbol (^#) represents difference tendency (P < 0.10).

Serum chemistry

The main effect of diet was significant for lipase and BUN/SC (P < 0.05), while the effects of time and diet × time were not significant (P < 0.10; Figure 4). It’s evident that CON group had higher concentration of lipase and higher ratio of BUN/SC than CAN group on day 5, 7 and 11 (P < 0.10, Figure 4a, b). The effects of diet and diet × time on BUN were not significant (P > 0.10), while time significantly affected BUN. The level of BUN of both groups displayed a conspicuous reduction after surgery on day 7 compared with day 5 (P < 0.001, Figure 4c).

Figure 4.

The effect of diets and experiment duration on serum chemistry in cats on day 5, 7 and 11: (a) lipase, (b) BUN/SC, blood urea nitrogen/serum creatinine, (c) BUN, blood urea nitrogen. CON, cats fed dry f food; CAN, cats fed wet food. Day 5 represents the day before the neutering surgery, and day 7 and day 11 represent the first and fifth day after the surgery. Data are presented as mean ± SEM. The symbol (^*) indicates statistically significant differences between two groups (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001), and the symbol (^#) represents difference tendency (P < 0.10).

Inflammatory cytokines, immunoglobulins and antioxidant parameters

There was significant time effect but no diet and diet × time effect on TNF-α, IL-8 and IgM. Specifically, the level of TNF-α increased on day 7 (P < 0.10, Figure 5a) and decreased on day 11 (P < 0.001, Figure 5a) in both groups. The level of IL-8 decreased on day 7 (P < 0.05, Figure 5b), when compared to day 5. As for immunoglobulins, the level of IgM on day 11 was significantly lower than it was on day 5 and day 7 (P < 0.05, Figure 5c). The effects of diet and diet × time on IgG was significant (P < 0.05). The CAN group had higher level of IgG than CON group on day 5 and day 7, but not on day 11 (P < 0.05, Figure 5d). On day 5, 7 and 11, diet significantly impacted the concentration of SOD in that the SOD level in the serum of CAN group was higher than that of CON group (P <0.05, Figure 5e).

Figure 5.

Effects of diets and experiment duration on the levels of inflammatory cytokines, immunoglobulins and antioxidant parameters in cats on day 5, 7 and 11: (a) TNF-α, tumor necrosis factor-α, (b) IL-8, interleukin 8, (c) IgM, immunoglobulin M, (d) IgG, immunoglobulin G, (e) SOD, superoxide dismutase. CON, cats fed dry food; CAN, cats fed wet food. Day 5 represents the day before the neutering surgery, and day 7 and day 11 represent the firstly and fifth day after the surgery. Data are presented as mean ± SEM. The symbol (^*) indicates statistically significant differences between two groups (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001), and the symbol (^#) represents difference tendency (^#P < 0.10).

Obesity-related indices

As shown in Figure 6, there was a significant effect of diet on the concentration of TG (P < 0.05), but time and diet × time had no effect (P > 0.10). The concentration of TG in CAN group was lower than that in CON group on day 20 and 48 (P < 0.05, Figure 6a). Only the effect of diet on HDL-C was obvious (P < 0.10). Concentration of HDL-C in CAN group tended to be higher than CON group on day 20 and 48 (P = 0.10, Figure 6b).

Figure 6.

The effect of diets and experiment duration on the levels of obesity-related indices in cats on day 20 and 48: (a) TG, triglyceride, (b) HDL-C, high- density lipoprotein cholesterol. CON, cats fed dry food; CAN, cats fed wet food. Day 20 and day 48 represent the 14^th day and 42^th day after the surgery. Data are presented as mean ± SEM. The symbol (^*) indicates statistically significant differences between two groups (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001), and the symbol (^#) represents difference tendency (P < 0.10).

Fecal SCFA and BCFA

The data of SCFA provided is on wet feces basis. Only time had significant effect on the level of total SCFA, acetic acid, and propionic acid (P < 0.05), and diet × time affect butyric acid (P < 0.10; Figure 7). The concentration of total SCFA in feces increased from week 1 to week 6 in both groups (P < 0.05, Figure 7a), and a similar trend could be observed with acetic acid (P < 0.01, Figure 7b) and propionic acid (P < 0.10, Figure 7c). The level of butyric acid was significantly higher at week 6 than week 1 in CON group but not in CAN group (Figure 7d). Besides, the content of butyric acid was marginally lower in CAN group than CON group at week 6 (P < 0.10, Figure 7d).

Figure 7.

The effect of diets and experiment duration on the levels of SCFA and BCFA in feces at week 3 and 6: (a) SCFA, short-chain fatty acids, (b) acetic acid, (c) propionic acid, (d) butyric acid. CON, cats fed dry food; CAN, cats fed wet food. Week 1 and week 6 represent the first week and sixth week after the surgery. Data are presented as mean ± SEM. The symbol (^*) indicates statistically significant differences between two groups (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001), and the symbol (^#) represents difference tendency (^#P < 0.10).

Serum metabolome on day 5, day 7 and day 11

Untargeted serum metabolome was monitored to further explore the effects of time and diet in cats (Figure 8). Results showed that a total of 157 metabolites were detected in both groups on day 5, day 7, and day 11. The OPLS-DA analysis indicated that there was evident separation between the CON and CAN group at different time (Figure 8a, d and g). As shown in Figure 8b, e and h, the RPT method revealed the high reliability of the OPLS-DA models. Details about the differential metabolites at specific time points are shown in Supplementary Table S1. The primary differential metabolites were adhumulone, cohumulone, desaminotyrosine, taurine, and 3,5-dihydroxybenzoic acid on day 5, desaminotyrosine, adhumulone, cohumulone, and 3,5-dihydroxybenzoic acid on day 7, and desaminotyrosine, p-hydroxyphenylacetic acid and cohumulone on day 11, respectively. To further explore the changes of metabolic processes, a KEGG pathway analysis of differential metabolites was performed. The affected metabolic pathways mainly focused on amino acid metabolism (i.e., d-glutamine and d-glutamate, taurine and hypotaurine, and arginine) and energy metabolism, such as nitrogen metabolism on day 5 (Figure 8c). On day 7, diet affected arginine biosynthesis and phenylalanine metabolism (Figure 8f). Amino acid metabolism (i.e., d-glutamine and d-glutamate metabolism, arginine biosynthesis, valine, leucine and isoleucine biosynthesis, and histidine metabolism), nucleotide (i.e., Pyrimidine) metabolism, and energy metabolism involving nitrogen were impacted on day 11 (Figure 8i).

Figure 8.

The effect of diets and experiment duration on the serum metabolome in cats on day 5, day 7 and day 11 and day 20 and day 48: (a, d and g) Score plots from the orthogonal partial least-squares discriminant analysis (OPLS-DA) model among two groups on day 5, 7 and 11. (b, e and h) RPT of the OPLS-DA models among two groups on day 5, 7 and 11. (c, f and i) Bubble chart of the metabolic pathway analysis of differential metabolites among two groups on day 5, 7 and 11. CON, cats fed dry food; CAN, cats fed wet food. Day 5 represents the day before the neutering surgery, and day 7 and day 11 represent the first and fifth day after the surgery.

Serum metabolome on day 20 and day 48

For the analysis of serum metabolome on day 20 and day 48, we used the same analytical process as for data from day 5, 7, and 11(Figure 9). The results showed that 87 metabolites were detected in total and there was obvious separation between two dietary groups at the two selected time points (Figure 9a and d). As shown in Figure 9b and e, the RPT method also revealed the reliability of the OPLS-DA model. The differential metabolites at varying time points are listed in Supplementary Table S2. The major differential metabolites were 2,4-Dihydroxybenzoic acid, desaminotyrosine, cohumulone and lenticin on day 20 and 48. Metabolic pathways that were affected by diet on day 20 were mainly centered on lipid (i.e., arachidonic acid) metabolism and amino acid (i.e., tryptophan) metabolism (Figure 9c), and on day 48 focused on amino acid metabolism (i.e., biosynthesis of phenylalanine, tyrosine, and tryptophan, and tryptophan metabolism) and aminoacyl-tRNA biosynthesis (Figure 9f).

Figure 9.

The effect of diets and experiment duration on the serum metabolome on day 20 and 48. (a and d) Score plots from the orthogonal partial least-squares discriminant analysis (OPLS-DA) model among two groups on day 20 and 48. (b and e) RPT of the OPLS-DA models among two groups on day 20 and 48. (c and f) Bubble chart of the metabolic pathway analysis of differential metabolites among two groups on day 20 and 48. CON, cats fed dry food; CAN, cats fed wet food. Day 20 and 48 represent the 14^th and 42nd day after the surgery.

Discussion

Acute recovery from castration surgery

Previous studies have reported that during the short period after surgical castration, there is a decline in feed intake probably due to systematic inflammation, wound pain, and reduced gastric motility according to previous studies (Kushner et al., 2006; Tewari et al., 2013). Diet with high palatability and moisture, such as canned food can contribute to the recovery of voluntary food intake (Corbee and Kerkhoven, 2014), and is beneficial to rehydration and nutrient absorption (Sachdeva et al., 2013). In contrast, significant differences between the two diet groups regarding BW, daily DMI, and BCS were not observed shortly after surgery in our study. Even though voluntary water intake was not recorded in our study, cats fed dry food generally have less total water intake and decreased urine volume compared to cats fed wet canned diet (Seefeldt and Chapman, 1979; Thomas et al., 2017). In addition, the canned food had higher fat, protein, and energy content on a dry matter basis than the dry food (Table 1). Meanwhile, digestibility of most nutrients in CAN group was at higher rate than CON group (Table 4), which is similar to what has been reported in other studies (Bermingham et al., 2013, 2018). Therefore, even with similar DMI between diet groups, the CAN food could allow for more water, nutrient, and energy intake which is important for the restoration of circulation of biofluids, and promoting the nutrient and energy utilization (Corbee and Kerkhoven, 2014).

Behavioral measures have been used to assess pain of cats in clinical research (Mollenhoff et al., 2005). Cats which are comfortable are more likely to interact with humans and their bodies appear to be more relaxed (Brondani et al., 2011). Cat–human interaction and cat’s behavior observed in our study indicated that the physical pain was weaker for cats in CAN group, as reflected in the lower scores in vocalization and comfort evaluation compared to CON cats. A dry wound surface without obvious inflammation and swelling (i.e., a lower WSS) represents a better wound recovery (Drudi et al., 2018). Wet canned food tended to lower the score level of inflammation and redness of wound color in cats in week 1 after surgery compared to dry food. Therefore, feeding the wet food seems to accelerate postoperative wound recovery in our study.

Higher lipase levels in the CON group may indicate a risk of pancreatic inflammation in cats (Zavros et al., 2008). Collective evidence suggest that moisture content affect the health of animal urinary system. A previous study found that the probability of developing urethral calculi in canines provided diet of moisture (78%–82%) is much lower than subjects fed the dry diet (Lekcharoensuk et al., 2002). Higher ratio of BUN/SC was suggested to be associated with renal dysfunction in cats (Finco and Duncan, 1976), and a lower ratio in CAN group in the current study indicated that wet diet might contribute to the recovery of renal function after surgery.

Exposed wound and compromised health condition after surgery is susceptible to infection by pathogenic microorganisms in the environment. IgG as the major antibody and exclusive antitoxin could contribute to immune response by neutralizing viruses and bacteria (Woof and Kerr, 2006). The difference in IgG levels on day 5 and day 7 between cats in CON and CAN groups indicated that feeding wet diets for a certain period before surgery is conducive to improve the immune function in cats. Higher IgG level is also associated with better surgical outcome (Chen et al., 2016). Accordingly, we estimate that the relief of postoperative pain and recovery of somatic function by wet food in cats may be achieved through the activation of humoral immunity and the alleviation of wound affection. The reason why the level of inflammatory cytokines did not differ between groups may be due to the small incision of the surgery and sufficient postoperative care in both groups of cats. Stressful events such as ovariohysterectomy can trigger oxidative stress in female dogs which could further aggravate the trauma and inflammation (Ali et al., 2020; Sakundech et al., 2020). As an antioxidative enzyme inside cells, SOD could protect cell structures and functions by eliminate free radicals (Ali et al., 2020). In our study, diet of wet food may function better in protecting cats from oxidative damage than dry food since serum SOD level was higher in cats of CAN group than CON group.

The serum metabolome refers to almost all of the small molecules involved in the metabolic processes in serum which is influenced by diets, environment and gut microbiota (Wikoff et al., 2009). The OPLS-DA model revealed an obvious difference in serum metabolome between cats fed wet and dry diet in this experiment, suggesting that diet variation affected the serum metabolites. The differed metabolic pathway mainly focused on arginine biosynthesis in the postoperative period. Plenty of studies have reported that arginine metabolism could regulate the immune response of the body by regulating the differentiation and maturation of macrophages and B lymphocytes (de Jonge et al., 2002; Martí i Líndez and Reith, 2021). The other altered metabolic pathway, glutamine and glutamate metabolism was also shown to be involved in the oxidative stress and inflammation (Wang et al., 2022). So, the differences in the immune response and antioxidant capacity between cats in the two diet groups might be attributed to their metabolomics changes in certain amino acids, such as arginine, glutamine, and glutamate.

Long-term change following castration surgery

Due to changes with sexual and satiety-related hormones (e.g., cholecystokinin, total peptide YY and estrogen) after castration, cats tend to have better appetite and reduced activity level which leads to a positive energy balance that could contribute to massive weight gain and the development of obesity (Schauf et al., 2016; Kutzler, 2020; Phungviwatnikul et al., 2020). Consistently, our results revealed a dramatic increment of daily DMI and BW in cats of the CON group at week 5 and 6 after castration compared to baseline, while the same measurements in cats fed wet food remained at a relatively stable level over the experimental period. The BCS of CON group also followed similar trend as DMI and BW. These differences between cats fed dry and wet food may attribute to the lower energy density and higher satiety of the wet-diet (Rowe et al., 2015). Faster filling of the stomach by the moisture in wet food can activate the stretch receptors and mechanoreceptors to prevent excessive energy intake (Havel, 2001). A relevant study showed that, in the case of ad libitum feeding, wet canned food resulted in BW loss in cats after only 3 weeks in comparison to cats fed the same food but with water being removed beforehand (Wei et al., 2011). Besides, a diet with high moisture content was shown to increase activity level in cats, which also helped to burn energy to maintain a normal body condition in cats (Cameron et al., 2011). Apparent nutrient digestibility of diet is critical for animals to gain the essential nutrition and energy (Schauf et al., 2021). Even though the digestibility of most nutrients (i.e., DM, CF, CP, and EE) in our study was significantly higher for the wet canned food than for the dry food (Table 4), DE of wet food was significantly lower than that of dry food. The dry food of higher energy density is one of the risk factors for obesity in adult cats according to a cross-section study (Öhlund et al., 2018). Therefore, compared to dry kibbles, wet foods of high satiety and low energy density might help neutered cats to control DMI and energy intake, and as a result maintain normal body weight after surgical sterilization.

Obese cats usually have higher TG, as well as lower concentration of HDL in serum which could promote the excretion of excess cholesterol in extrahepatic tissues (Jordan et al., 2008; März et al., 2017). After being castrated, the level of TG in cats fed diet of high energy density (i.e., dry kibbles) increased substantially over time, which corresponded to their changes of BW and BCS. However, lower TG and higher HDL-C concentration was observed in the CAN group compared to CON. The changes of TG and HDL may be correlated to BW and BCS in cats. The results indicated that free feeding of dry food after castration promotes weight gain and may eventually result in the development of obesity in cats, while canned food may reduce this risk by maintaining BW and BCS relatively stable.

As the products of fermentation of polysaccharides by gut microorganism (GM), SCFA mainly include acetic acid, propionic acid, and butyric acid (Tan et al., 2014). The SCFA-producing gut microbiota, along with SCFA are involved in the regulation of many physiological processes, such as inflammation (Suchodolski, 2016) and nutrient metabolism (Wernimont et al., 2020), and were shown closely related to overweight and obesity in pet cats and dogs (Kieler et al., 2016; Wernimont et al., 2020). The results of the current study indicate that SCFA and butyric acid production is closely related to weight gain or obesity in cats. There is evidence that the concentration of acetic acid and propionic acid in gut were negatively associated with the rate of BW loss in dogs (Kieler et al., 2017). In agreement, our study showed that total fecal SCFA, as well as acetic and propionic acid, increased over time (i.e., from week 1 to week 6) after castration, during which time period the increase of BW and BCS was also observed in cats, especially those on dry food. Meanwhile, fecal content of butyric acid exhibited an increase over time only in cats fed dry food. Consistently, concentration of fecal butyric acid in obese children was higher than that in normal-weight children (Riva et al., 2017), and the level of fecal butyric acid was positively correlated with the distribution of body fat in children (Wei et al., 2021). Therefore, increased SCFA and/or butyric acid production, which is likely due to the increased DMI and increased substrate reaching the colon for fermentation, such as in the case of cats fed dry diet, may be closely involved in promoting weight gain and/or the development of obesity in cats. Even though SCFA in colon could provide a small amount of energy for dogs and cats (Suchodolski, 2016), SCFA/butyrate production may not directly contribute to the significant weight gain observed in the cats in our study, due to their limited tolerance of intestinal fermentation.

The metabolomics data on day 20 and 48 may provide information of the mechanisms underlying the long-term effects of the two diets on BW and BCS in cats after castration. The differing metabolites and metabolic pathways between CON group and CAN group were also shown to be related to weight gain and/or obesity in humans. Examples include the differing metabolic pathways of phenylalanine and tyrosine between obese and lean people (Morris et al., 2012), the disorder of tryptophan metabolism in obese adults (Cussotto et al., 2020), and the association of higher levels of arachidonic acid with visceral fat accumulation in man, with higher levels present in adipose tissue in overweight and obese people (Savva et al., 2004; Inoue et al., 2013). Therefore, we speculate that the different changes in BW and BCS between two dietary groups might be related to the metabolic pathways of certain amino acids, such as the biosynthesis of tyrosine and tryptophan, and the metabolism of phenylalanine and arachidonic acid.

In addition to moisture content, the proximate composition (e.g., fat and protein content) of the two diets included in our study differed due to varied ingredients and manufacturing techniques. Food intake in cats was also not restricted. Therefore, a major limitation of our study is that while the current data does indicate a potential benefit of wet-canned food in cats, we could not determine which factor(s) of the diet (e.g., moisture content, nutrient composition or level of caloric intake) contributed to the observed findings. In addition, cats of different sex may respond differentially to castration and experience different severity from the surgery. We did try to balance the sex distribution in two treatment groups to reduce the gender effect. But still sample size differed largely for two sexes, which was also a limitation of this study. We originally included sex in the model for data analysis but sex showed no significant effect on various parameters. Therefore, it was later removed from the final model. Future studies may increase sample size for both sexes to better elucidate the gender effect on cat recovery from castration surgery and the potentiality of dietary regulation on their performance. Moreover, further studies may investigate the correlation of GM and the serum metabolome to further the understanding of how different diets could affect wound recovery and the control of BW and body condition in cats.

CONCLUSION

The dry and wet canned food included in this study had varied effects on cats experiencing castration, probably due to the differences in moisture content, nutrient composition and nutritional support between the diets. The high palatability of canned food as a result of higher moisture content, and protein and fat content on DM basis, might promote wound recovery shortly after sterilizing surgery in cats by increasing their water and energy intake, nutrient digestion, and anti-oxidative and immune capacity. The wet food also helped maintain BW and BC in cats after castration in that cats in CON group gained weight significantly faster than cats in CAN group over the 4 weeks after the castration surgery. This may be primarily due to energy dilution by the high-water content in the wet food when compared to the dry food. The underlining mechanism may involve differing effects of the two diets on SCFA/butyric acid production and certain metabolic pathways (e.g., amino acid metabolism).

Glossary

Abbreviations

A/G

albumin/globulin

ALB

albumin

ALP

alkaline phosphatase

BC

body condition

BCFA

branched-chain fatty acids

BCS

body condition score

BUN/SC

blood urea nitrogen/serum creatinine

BW

body weight

CAN

wet canned diet

CF

crude fiber

CON

control diet (i.e., dry extruded food)

CP

crude protein

DE

digestible energy

DM

dry matter

DMI

dry matter intake

EE

ether extract

ELISA

enzyme-linked immunosorbent assay

FC

fold change

GE

gross energy

GT

glutamyl transferase

HDL-C

high-density lipoprotein cholesterol

IgA

immunoglobulin A

IgG

immunoglobulin G

IgM

immunoglobulin M

IL-10

interleukin 10

IL-8

interleukin 8

OM

organic matter

OPLS-DA

orthogonal partial least-squares discriminant analysis

PPS

physical pain score

PRT

response permutation testing

SCFA

short-chain fatty acids

SOD

superoxide dismutase

T-AOC

total antioxidative capacity

TBA

total bile acid

TG

triglyceride

TNF-α

tumour necrosis factor-α

TP

total protein

VIP

variable importance in projection

WSS

wound surface score

Conflict of Interest Statement

Hongcan Huang was employed by Guangdong Munchkin Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.