Hydrolyzed chicken liver and increasing crude protein levels on palatability, digestibility, and intestinal fermentation products of cats

Abstract

Feeding companion cats with diets based on hydrolyzed proteins may improve palatability and digestibility due to the presence of peptides and free amino acids. Furthermore, using highly digestible protein sources can decrease the undigested protein content that reaches the hindgut, thereby modifying fermentation end metabolites. This study aimed to evaluate the effects of replacing poultry byproduct meal (PBM), as the most traditional protein source for cats, with hydrolyzed chicken liver (HCL) added at 24%, 32%, and 40% of crude protein (CP) in the diet, replacing carbohydrates from brewers rice. The combination between 2 ingredients and 3 inclusions of CP permitted to test 6 extruded diets: PBM24, PBM32, PBM40; HCL24, HCL32, and HCL40. Thirty-six adult cats were arranged in a complete randomized block design to evaluate the apparent total tract digestibility (ATTD) of nutrients and energy, fermentative end products, and palatability. Crude protein ATTD increased at higher protein concentrations (P = 0.036). Fat ATTD was lower in cats fed HCL40 (P = 0.046). Carbohydrate digestibility decreased with high protein intake (P < 0.05) as a result of replacing carbohydrates with protein in the experimental diets. Gross energy ATTD was lower in cats fed HCL32 (P = 0.027). Similarly, cats fed HCL and HCL32 had lower metabolizable energy (P < 0.05) when compared to PBM. Fecal dry matter (P = 0.005) and fecal scores (P = 0.014) were lower with the inclusion of HCL (P < 0.05). Also, cats fed HCL32 had looser stools (2.61 out of a 0 to 5 score; P = 0.025), but within the acceptable range, compared to the other diets. Urinary pH increased with HCL inclusion and at higher protein concentrations (P < 0.05). Fecal acetate, propionate, valerate, total short-chain fatty acids (SCFA), isovalerate, and total volatile fatty acids (VFA) were higher in cats fed HCL compared to those fed PBM (P < 0.05). In the palatability test, cats mostly preferred diets based on HCL and with higher protein concentrations, mainly HCL40, over those based on PBM (Power test >0.75). The replacement of PBM by HCL in diets was highly accepted and improved the fermentative end metabolites associated with intestinal functionality in cats, especially SCFA.

Hydrolyzed chicken liver increased fecal concentrations of short-chain fatty acids in adult cats compared with those fed diets based on poultry by-product meal.

Hydrolyzed chicken liver, as the only source of animal protein, improved the palatability of diets for adult cats.

Introduction

Protein sources used in pet food come from plant or animal sources and their byproducts. Both sources may be used in their original form or submitted to enzymatic hydrolysis before being used, producing low molecular weight peptides rich in ingredients, with high availability of amino acids (Cave, 2006). Protein hydrolysates have been widely used as dietary protein in therapeutic diets for companion animals with food hypersensitivity and gastrointestinal disorders (Verlinden et al., 2006; Rudinsky et al., 2018). Dogs and cats with adverse food reactions and enteropathies may benefit from diets formulated with hydrolyzed proteins. The low molecular weight of the protein fraction may reduce the chances of stimulation of the immune system; also, hydrolysis facilitates digestion and absorption of amino acids, an important aspect for animals with a compromised gastrointestinal tract (Cave, 2006). A recent trial conducted at our facility showed that dogs fed diets with hydrolyzed chicken liver (HCL) and increasing crude protein (CP) concentrations had higher protein digestibility than those fed poultry byproduct meal (PBM) based diets (Pinto et al., 2023). Conversely, a previous trial demonstrated that inclusion of HCL at 25.8% of the complete diet promoted similar protein digestibility and adequate fecal scores compared to a diet based on a mix of PBM and meat and bone meal in dogs (Pinto et al., 2021). PBM is commonly used in commercial diets for companion animals, but the digestibility of protein varies depending on the raw material quality and rendering process (Volpato et al., 2022). Hydrolyzed proteins, however, are submitted to a more controlled manufacturing process (Hou et al., 2017), resulting in a possible increase in protein digestibility and a reduction in the amount of undigested protein available for fermentation reaching the colon. Bypassing protein to the hindgut, from the food or from endogenous content, results in the synthesis of nitrogenous metabolites, such as ammonia, hydrogen sulfide, branched-chain fatty acids (BCFA), biogenic amines, phenols, and indoles (Blachier et al., 2007). In cats, proteolytic fermentation can lead to the formation of potential uremic toxins that may affect the progression of kidney disease (Chen et al., 2018; Summers et al., 2019). Since even highly digestible protein sources can escape digestion in the small intestine, it is important to assess the impact of different protein sources and concentrations on digestibility and hindgut fermentation in cats. We hypothesized that replacing PBM with HCL in feline diets improves protein digestibility, enhances palatability, and positively modifies fermentative end products in the hindgut, leading to increased short-chain fatty acids (SCFA) production and improved intestinal functionality with minimal effects on fecal characteristics. Therefore, the aim of this study was to investigate the effects of replacing PBM with HCL, in diets with different CP concentrations (24%, 32%, and 40%), on the macronutrient and energy digestibility, fecal and urinary characteristics, fecal fermentation products, and food preference of cats.

Materials and Methods

This study included 2 experiments with the same dietary treatments. The first experiment aimed to assess the effects of diets on apparent total tract digestibility (ATTD) of macronutrients and energy, fecal and urinary characteristics, and fecal fermentative products, and the second analyzed the effects of diets on palatability. All procedures used in this study were approved by the Ethics Committee on the Use of Animals at the Universidade Estadual de Maringá, under protocol n. 1110190121.

Animals

All cats used in this study belonged to the colony of the Centro de Ensino e Estudos Nutricionais em Felinos-CEENUFEL, Universidade Estadual de Maringá, Maringá, Brazil. Cats were free of endo- and ectoparasites, regularly immunized, and submitted to clinical and laboratory tests to measure complete blood count and biochemistry before starting the study.

Diets

Six complete dry diets for adult cats were formulated, varying in animal protein source (PBM or hydrolyzed chicken liver [HCL—PROSURANCE CHX Liver HD, Kemin Industries, Vargeão, Santa Catarina, Brazil]) and protein concentration (24%, 32%, and 40% CP). Thus, the study presented 6 treatments (PBM24, PBM32, PBM40, HCL24, HCL32, and HCL40) as a result of a combination between 2 factors: protein source and concentration (Table 1). Diets were extruded in a single screw extruder (model 2000, TNL Tecnal, Ourinhos, Brazil) and coated with poultry fat (5%) and palatant enhancer (1.5%). Palatability assay was evaluated with diets coated with poultry fat only. All diets were analyzed and met the minimum amount of essential nutrients based on FEDIAF recommendations for adult cats (FEDIAF, 2024). Before starting the study, all cats received a commercial diet for 14 d during a wash-in period.

Table 1.

Ingredients and chemical composition of experimental diets.

Ingredients, g/kg	Treatments1
	PBM24	PBM32	PBM40	HCL24	HCL32	HCL40
Brewers rice	54.9	44.9	34.6	52.5	42.2	30.5
HCL2	—	—	—	14.4	21.9	31.0
Poultry byproduct meal	12.5	20.3	28.7	—	—	—
Maize gluten feed	11.6	15.9	20.0	11.6	15.9	20.0
Porcine plasma spray dried	1.95	2.66	3.38	1.95	2.66	3.38
Sugarcane fiber	3.20	3.11	3.00	3.65	3.80	4.00
Poultry fat	4.87	3.33	1.69	3.85	2.02	—
Salt	0.50	0.50	0.50	0.50	0.50	0.50
Premix mineral/vitamin3	0.40	0.40	0.40	0.40	0.40	0.40
Limestone	0.08	—	—	1.53	2.11	2.41
Dicalcium phosphate	2.05	1.14	0.08	1.56	0.65	0.16
Choline chloride	0.32	0.25	0.16	0.47	0.48	0.50
Potassium chloride	0.65	0.55	0.46	0.55	0.42	0.27
Phosphoric acid	0.50	0.50	0.50	0.50	0.50	0.50
Added by coating after extrusion, g/kg
Poultry fat	5.00	5.00	5.00	5.00	5.00	5.00
Palatant enhancer4	1.50	1.50	1.50	1.50	1.50	1.50
Analyzed chemical composition, % DM basis
Dry matter	93.6	92.6	93.6	92.5	93.3	93.5
Ash	6.50	5.91	6.85	6.45	6.05	6.79
Crude protein	24.2	32.6	41.1	25.2	31.5	39.3
Acid hydrolyzed fat	13.4	13.8	12.3	13.0	11.9	13.2
Crude fiber	10.2	7.83	9.04	5.80	9.88	6.15
Nitrogen free extract	55.9	47.7	39.8	55.3	50.6	40.8
Starch	47.7	37.9	31.0	44.5	39.2	30.4
Gelatinization index of starch	92.7	99.0	90.7	97.6	93.3	91.7
Gross energy, kcal/kg	4921	5164	5246	5023	4961	5166
GMD5, μm	339	399	378	348	324	300
Peroxide value, mEq/kg	16.37	9.65	6.31	5.84	6.68	7.07

¹PBM24 - diet based on PBM with 24% of protein; PBM32 - diet based on PBM with 32% of protein; PBM40 - diet based on PBM with 40% of protein; HCL24 - diet based on HCL with 24% of protein; HCL32 - diet based on HCL with 32% of protein; HCL40 - diet based on HCL with 40% of protein.

²HCL, hydrolyzed chicken liver (PROSURANCE CHX Liver HD, Kemin Industries, Vargeão, Santa Catarina, Brazil).

³Premix mineral/vitamin (supplied per kilogram of diet): vitamin A (10,800U), vitamin D3 (980 U), vitamin E (60 mg), vitamin K3 (4.8 mg), vitamin B1 (8.1 mg), vitamin B2 (6.0 mg), vitamin B6 (6.0 mg), 12 vitamin (30 mcg), pantothenic acid (12 mg), niacin (60 mg), folic acid (0.8 mg), biotin (0.084 mg), manganese (7.5 mg), zinc (100 mg), iron (35 mg), copper (7.0 mg), cobalt (10 mg), iodine (1.5 mg), selenium (0.36 mg), choline (2.400 mg), taurine (100 mg), and, antioxidant BHT (150 mg).

⁴DTECH 8L, S.P.F. Argentina S.A., Argentina, only in the diets for the digestibility and fermentation end products.

⁶GMD, geometric mean diameter.

Experiment 1

The aim of this experiment was to evaluate the effect of diets varying in protein source (PBM or HCL) and concentration (24%, 32%, and 40% CP) on digestibility, fecal and urinary characteristics, and fecal fermentative metabolites. Thirty-six, intact adult cats, between 3 and 4 yr old, weighing 3.5 ± 4.0 kg and a body condition score of 6 out of 9 points (Laflamme, 1997) were used. Cats were weighed weekly until the end of the experimental period to maintain body weight. Cats were housed in groups with free access to a solarium, except during the total feces and urine collection period. Two hours per day, they were stimulated with scratches, toys, and brushing. Cats were distributed in a completely randomized block design, with 3 blocks of 12 cats each and 2 cats per diet in each block, totaling 6 cats (repetitions) per diet (treatment). Each block lasted 30 d, and the ATTDs of macronutrients and energy, fecal and urinary characteristics, and fecal fermentation end products were determined.

Experiment 2

The experiment was conducted to verify the effect of dietary treatments on palatability. Twenty, intact adult cats, between 3 and 4 yr, weighing 3.5 ± 4 kg and a body condition score of 6 out of 9 points (Laflamme, 1997) were used. Prior to the assay, diets were analyzed for peroxide value (PV; AOCS, 2017). Diet PBM24 was excluded from the palatability assessment because PV was higher than 10 mEq/kg, which is not acceptable for pet food (Table 1). Based on this, only 4 diets were tested: PBM32, PBM40, HCL32, and HCL40. The assay was conducted as a completely randomized design, with 4 diets in 6 combinations, 2 by 2, provided to 20 cats 4 times a day, resulting in 80 observations for each combination. The palatability assay lasted 8 d.

Digestibility of macronutrients and energy assay

During the digestibility assay, cats were allocated into individual stainless steel metabolic cages (80 × 70 × 90 cm) equipped with feeders, drinkers, feces and urine collectors, in a controlled room at 22 °C. Digestibility was carried out as a total fecal collection method. At the beginning of each period, cats were adapted to the diets and cages for 5 d, followed by 7 d of total feces and urine collection. After sample collection, cats were maintained on the diets to complete 30 d of ingestion. The amount of food offered was calculated to maintain body weight. Cats were fed twice a day (0800 and 1600 hours). Water was available ad libitum.

During 7 consecutive days, feces were collected and scored by a single trained person using the following system: 1 = watery—liquid that can be poured; 2 = soft, unformed—stool assumes shape of container; 3 = soft, formed, moist—softer stool that retains shape; 4 = hard, formed, dry stool—remains firm and soft; 5 = hard, dry pellets—small, hard mass (Carciofi et al., 2008). Feces were weighed and stored in a freezer at −20 °C each day until analysis. Total urine collection was performed daily for 3 consecutive days (at the end of the 7 d of collection) and then stored in plastic bottles containing 1 g of thymol/100 mL of urine (Synth, Diadema, Brazil). Urinary total volume and pH were measured daily after collection using a bench pH meter (Kasvi, model k39-2014B, São José dos Pinhais, Brazil).

At the end of the assay, stools from each cat were thawed, homogenized, and dried in a forced-air oven at 55 °C for 72 h, according to the recommendations of the AOAC (1995). Feces and diets were ground through a 1 mm screen in a Wiley hammer mill and analyzed for dry matter (DM—AOAC 934.01), CP (AOAC 954.01; model TE 036/2, Tecnal, Piracicaba, Brazil), acid hydrolyzed fat (AHF—AOAC 954.02; model 170/3, Fanem, São Paulo, Brazil), crude fiber (CF—AOAC 962.10; model MA 450/8, Marconi, Piracicaba, Brazil), and ash (AOAC 985.01 [modified]). Based on the results, the ATTD of nutrients and energy of the diets were calculated using the following equation:

$${\displaystyle \begin{array}{lll}& {\displaystyle \mathrm{A}\mathrm{T}\mathrm{T}\mathrm{D}(\mathrm{\%})=}& \frac{g\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-g\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d}}{g\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}\times 100\end{array}}$$

Diets were analyzed for starch, according to Karkalas (1985), and the gelatinization index of starch was calculated as:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{G}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}=\frac{(\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{c}\mathrm{h}-\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{c}\mathrm{h})}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{c}\mathrm{h}}\times 100}\end{array}}$$

Dietary and fecal gross energy were determined using isoperibolic bomb calorimetry (Parr Instrument Company, model 6200, Moline, USA) and used to calculate metabolizable energy (ME) as proposed by FEDIAF (2024):

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{M}\mathrm{E}=\frac{[g\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{E}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-g\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{E}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d}]-[g\mathrm{o}\mathrm{f}\mathrm{C}\mathrm{P}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-g\mathrm{o}\mathrm{f}\mathrm{C}\mathrm{P}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d}]\times 0.86}{g\mathrm{o}\mathrm{f}\mathrm{D}\mathrm{M}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}e}}\end{array}}$$

All analyses were performed in duplicate, and the coefficient of variation was <1% for energy and <5% for the other analyses.

Fecal fermentation products

On day 30 of each period, fresh fecal samples were collected immediately after defecation, and then, pH, SCFA, BCFA, ammonia, and lactate were analyzed. Approximately 5 g of fresh feces was homogenized and mixed with 15 mL of 16% (vol/vol) formic acid solution and precipitated to determine the volatile fatty acids (VFA). Then, the mixture was centrifuged (Thoth, model 9500RI, São Paulo, Brazil) 3 times at 4,500 g for 15 min at 4 °C. The supernatant was retained, and the pellet was discarded. Fecal SCFA and BCFA of the supernatant were determined by gas chromatography (Shimadzu, model GC-2014, Kyoto, Honshu, Japan) as previously described (Erwin et al., 1961). Fecal ammonia was determined using the same extracts prepared for the VFA. The extracts were thawed at room temperature, and 3 mL of each extract was diluted in 9 mL of distilled water and submitted to distillation in a nitrogen system (TECNAL, model TE-036/1, São Paulo, Brazil). Fecal lactate was measured by mixing 3 g of fresh feces with 9 mL of distilled water. Then, the solution was centrifuged 3 times at 4,000 g for 15 min at 4 °C. The supernatant was retained, and the pellet discarded. The extract was analyzed by spectrophotometry (Molecular Devices, model SpectraMax Plus 384, California, USA) as described by Pryce (1969), and samples were quantified by comparing them with a standard curve for lactic acid. Fecal pH was determined with the same extracts prepared for lactic acid, using a bench pH meter (Kasvi, model k39-2014B, São José dos Pinhais, Brazil).

Palatability assay

Palatability was determined using the 2-pan choice method (Griffin et al., 2003). Cats were kept in individual stainless steel metabolic cages (80 × 70 × 90 cm) and 2 diets were offered simultaneously. They were challenged 4 times a day. During the rest of the day, cats were housed in groups, in a controlled room at 22 °C, with access to a solarium. Water was provided ad libitum.

During the testing phase, 70 g of each diet used in the comparison was offered side by side, simultaneously, in identical feeders for 20 min at 0800 hours, 1100 hours, 1400 hours, and 1600 hours. The feeders were alternated for every meal to eliminate any bias effects related to laterality. The leftovers were collected, weighed, and discarded. During the trial, the first choice and intake ratio were observed. The first choice was determined by the number of times that a given diet was chosen first out of the total observations, multiplied by 100. While the intake ratio was calculated as:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}=\frac{\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{r}\mathrm{s}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}(g)}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{o}\mathrm{t}\mathrm{h}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{s}(g)}}\end{array}}$$

Statistical analysis

Data from experiment 1 were tested for homogeneity of variances and normality of errors and then analyzed using 2-way ANOVA complemented by Tukey’s test (P < 0.05) to compare the effects of protein source (PBM and HCL), concentration (24%, 32%, and 40% CP), and the interaction between protein source and concentration. The analysis also included block as a fixed effect and cat as a random effect in Minitab 18 (Minitab Inc., State College, PA).

Data from experiment 2 were analyzed for means and standard deviations using Microsoft Office Excel 2013, and a power test was applied using the software Jamovi (version 2.3). Mean food intake ratio values >0.75 were considered significant, according to the statistical procedures of Cohen (1988).

Results

Diets were considered well processed during extrusion based on starch gelatinization index, which was >90% for all diets (Table 1). All cats remained healthy and maintained their body weight throughout the study and accepted their daily ration without refusals, leftovers, or vomiting (Table S1).

The ATTD of CP increased as cats were fed higher CP concentrations (P = 0.036; Table 2). Although protein source and CP concentration did not impact fat digestibility, cats fed HCL40 had lower fat digestibility compared to HCL24 (P = 0.046). Carbohydrate digestibility was lower in cats fed higher CP (P = 0.025). Also, cats fed HCL32 showed the lowest ATTD of carbohydrates (P = 0.042) and the lowest ATTD of gross energy (P = 0.027), leading to a reduction in ME content (P = 0.032).

Table 2.

Apparent total tract digestibility (ATTD) of macronutrients and energy, and metabolizable energy (ME; mean ± mean standard error) of cats fed a poultry byproduct meal (PBM) or a hydrolyzed chicken liver (HCL) diet at 3 crude protein concentrations (24%, 32% or 40% CP).

		CTTAD, %						ME, kcal/kg
		DM	OM	CP	Fat	NFE	GE
PS1	PBM	84.3 ± 2.94	87.3 ± 2.72	85.5 ± 3.89	93.4 ± 1.48	86.4 ± 3.32	87.8 ± 2.55	4248 ± 160a
	HCL	83.1 ± 4.15	86.2 ± 3.81	85.6 ± 3.84	93.8 ± 2.37	84.2 ± 4.89	86.2 ± 3.81	4151 ± 206b
CPC2	24%	84.3 ± 3.58	87.4 ± 3.31	83.7 ± 4.37b	94.1 ± 1.65	87.4 ± 3.42a	87.8 ± 3.05	4190 ± 164
	32%	83.4 ± 3.92	86.4 ± 3.72	85.5 ± 3.18ab	94.0 ± 1.24	85.1 ± 5.01ab	87.2 ± 3.38	4180 ± 229
	40%	83.4 ± 3.53	86.4 ± 3.07	87.4 ± 3.12a	92.5 ± 2.50	83.5 ± 3.56b	87.0 ± 3.49	4229 ± 178
PBM	24%	83.5 ± 3.46	86.5 ± 3.55	82.2 ± 4.45	93.1 ± 1.32ab	86.9 ± 3.84ab	86.7 ± 3.06ab	4096 ± 142bc
	32%	85.5 ± 3.30	88.5 ± 2.65	86.8 ± 3.00	93.7 ± 1.41ab	88.2 ± 2.93a	89.2 ± 2.39a	4364 ± 115a
	40%	83.9 ± 1.95	86.9 ± 1.72	87.5 ± 1.60	93.3 ± 1.87ab	84.2 ± 2.14ab	87.6 ± 1.78ab	4285 ± 88.7ab
HCL	24%	85.2 ± 3.82	88.2 ± 3.12	85.3 ± 4.03	95.2 ± 1.17a	87.9 ± 3.22a	89.0 ± 2.83a	4284 ± 133ab
	32%	81.2 ± 3.43	84.3 ± 3.59	84.2 ± 3.01	94.3 ± 1.07ab	82.0 ± 4.89b	85.2 ± 3.09b	3997 ± 146c
	40%	82.8 ± 4.77	85.9 ± 4.15	87.3 ± 4.34	91.8 ± 3.00b	82.7 ± 4.67ab	86.5 ± 4.79ab	4173 ± 233abc
P-value	PS	0.240	0.246	0.911	0.438	0.056	0.293	0.032
	CPC	0.694	0.632	0.036	0.050	0.025	0.738	0.625
	PSxCPC	0.089	0.060	0.114	0.046	0.042	0.027	< 0.0001

NM, natural matter; DM, dry matter; OM, organic matter; CP, crude protein; NFE, nitrogen-free extract; GE, gross energy; ME, metabolizable energy.

¹Protein source: PBM (poultry byproduct meal); HCL (hydrolyzed chicken liver).

²Crude protein concentration: 24%, 32%, and 40%.

^a-cMeans in the same column with different lowercase letters are significantly different (P < 0.05).

Cats fed HCL had lower fecal DM (P = 0.005) and fecal score (P = 0.014; Table 3). In addition, the lowest fecal score was observed in cats fed HCL32 (P = 0.025), although it was within acceptable characteristics (ranging between 2.5 and 3.5). Urinary pH was affected by both protein source (P = 0.002) and CP concentration (P = 0.012), with cats fed HCL and higher CP concentrations having the highest urinary pH. Cats fed HCL had greater fecal concentrations of acetate, propionate, valerate, total SCFA, isovalerate, and total VFA (P < 0.05; Table 4).

Table 3.

Fecal and urinary characteristics (mean ± mean standard error) of cats fed a poultry byproduct meal (PBM) or a hydrolyzed chicken liver (HCL) diet at 3 crude protein concentrations (24%, 32%, or 40% CP).

		Fecal characteristics						Urinary characteristics
		Fecal output, g/d	Fecal output, g/d DM	Fecal DM, %	Fecal score1	Fecal pH	Number of defecations/d	Volume, ml/d2	Urine pH
PS3	PBM	19.3 ± 6.03	7.41 ± 1.70	39.7 ± 5.07a	3.47 ± 0.50a	6.01 ± 0.39	0.70 ± 0.20	250 ± 115	6.32 ± 0.19b
	HCL	22.8 ± 7.98	7.69 ± 2.18	34.7 ± 4.74b	3.06 ± 0.57b	6.02 ± 0.36	0.78 ± 0.20	244 ± 118	6.63 ± 0.39a
CPC4	24%	21.2 ± 7.09	7.89 ± 2.28	37.9 ± 4.25	3.42 ± 0.50	6.08 ± 0.33	0.72 ± 0.20	253 ± 126	6.28 ± 0.20b
	32%	21.2 ± 8.46	7.39 ± 1.63	37.0 ± 6.03	3.07 ± 0.64	6.11 ± 0.35	0.73 ± 0.22	220 ± 91.8	6.52 ± 0.49ab
	40%	20.7 ± 6.56	7.38 ± 1.97	36.8 ± 6.25	3.31 ± 0.55	5.86 ± 0.40	0.77 ± 0.20	268 ± 128	6.64 ± 0.20a
PBM	24%	21.9 ± 6.51	8.13 ± 1.78	38.2 ± 5.60	3.33 ± 0.66ab	5.98 ± 0.14	0.74 ± 0.21	266 ± 111	6.21 ± 0.15
	32%	15.8 ± 4.09	6.42 ± 1.31	41.3 ± 3.07	3.53 ± 0.16a	6.20 ± 0.40	0.67 ± 0.19	247 ± 87.1	6.26 ± 0.19
	40%	20.1 ± 6.41	7.67 ± 1.76	39.6 ± 6.37	3.55 ± 0.60a	5.86 ± 0.52	0.69 ± 0.21	237 ± 157	6.48 ± 0.12
HCL	24%	20.5 ± 8.19	7.64 ± 2.84	37.7 ± 2.86	3.51 ± 0.29a	6.19 ± 0.43	0.69 ± 0.21	240 ± 149	6.36 ± 0.23
	32%	26.7 ± 8.37	8.35 ± 1.38	32.6 ± 4.99	2.61 ± 0.60b	6.03 ± 0.30	0.79 ± 0.23	193 ± 96.0	6.74 ± 0.57
	40%	21.4 ± 7.25	7.10 ± 2.30	34.0 ± 5.15	3.07 ± 0.39ab	5.86 ± 0.29	0.86 ± 0.16	300 ± 95.4	6.79 ± 0.11
P-value	PS	0.118	0.646	0.005	0.014	0.932	0.249	0.894	0.002
	CPC	0.979	0.748	0.823	0.179	0.168	0.766	0.620	0.012
	PS × CPC	0.077	0.189	0.132	0.025	0.399	0.417	0.487	0.318

DM, dry matter.

¹Scored as follows: 1 = watery—liquid that can be poured, and 5 = hard, dry pellets—small, hard mass.

²During 7 d.

³Protein source: PBM (poultry byproduct meal); HCL (hydrolyzed chicken liver).

⁴Crude protein concentration: 24%, 32%, and 40%.

^a-cMeans in the same column with different lowercase letters are significantly different (P < 0.05).

Table 4.

Fecal concentrations of short-chain fatty acids (SCFA), branched-chain fatty acids (BCFA), volatile fatty acids (VFA), ammonia and lactate (mean ± mean standard error) of cats fed a poultry byproduct meal (PBM) or a hydrolyzed chicken liver (HCL) diet at 3 crude protein concentrations (24%, 32% or 40% CP).

		SCFA, mmol/g fecal DM				Total SCFA1	BCFA, mmol/g fecal DM		Total BCFA1	Total VFA1	Ammonia, mmol/kg fecal DM	Lactate, mmol/kg fecal DM
		Acetate	Propionate	Butyrate	Valerate		Isobutyrate*	Isovalerate
PS2	PBM	669 ± 227b	176 ± 56.5b	86.4 ± 28.5	44.3 ± 14.2b	975 ± 304b	40.4 ± 14.8	26.2 ± 6.02b	67.1 ± 17.9	1042 ± 314b	124 ± 36.9	0.30 ± 0.18
	HCL	878 ± 434a	255 ± 132a	103 ± 35.7	55.1 ± 18.3a	1300 ± 616a	42.6 ± 18.8	32.3 ± 8.20a	74.9 ± 24.4	1375 ± 632a	147 ± 44.4	0.40 ± 0.38
CPC3	24%	723 ± 226	206 ± 59.8	91.6 ± 24.3	47.5 ± 11.6	1070 ± 299	40.2 ± 18.0	28.5 ± 4.02	68.7 ± 20.8	1139 ± 313	125 ± 28.8	0.39 ± 0.28
	32%	771 ± 385	215 ± 101	82.8 ± 26.2	45.2 ± 17.5	1129 ± 546	35.5 ± 10.6	28.4 ± 9.65	64.6 ± 17.4	1194 ± 561	144 ± 49.3	0.28 ± 0.25
	40%	824 ± 449	225 ± 151	108 ± 42.1	56.4 ± 20.0	1213 ± 645	48.9 ± 18.7	30.9 ± 8.99	79.8 ± 24.3	1293 ± 659	137 ± 46.4	0.38 ± 0.37
PBM	24%	733 ± 233	194 ± 50.1	94.8 ± 26.5	48.7 ± 12.4	1070 ± 301	41.8 ± 13.3	28.2 ± 4.39	70.0 ± 16.4	1140 ± 308	114 ± 15.2	0.30 ± 0.15
	32%	622 ± 223	169 ± 63.8	76.2 ± 24.9	37.9 ± 14.6	905 ± 291	33.7 ± 6.84	22.3 ± 1.46	58.4 ± 8.13	964 ± 293	127 ± 43.9	0.37 ± 0.29
	40%	652 ± 250	164 ± 60.5	88.1 ± 35.1	46.3 ± 15.3	950 ± 349	45.6 ± 20.8	27.4 ± 8.56	73.0 ± 24.8	1023 ± 368	131 ± 47.6	0.25 ± 0.10
HCL	24%	718 ± 240	217 ± 70.8	88.4 ± 24.0	46.2 ± 11.7	1070 ± 327	38.6 ± 23.0	28.8 ± 4.02	67.4 ± 26.1	1138 ± 347	138 ± 35.1	0.48 ± 0.36
	32%	919 ± 474	262 ± 116	90.6 ± 28.2	52.5 ± 18.3	1353 ± 671	37.2 ± 13.9	33.5 ± 10.8	70.8 ± 22.6	1424 ± 693	161 ± 52.2	0.19 ± 0.18
	40%	997 ± 556	286 ± 194	128 ± 41.7	66.6 ± 20.0	1476 ± 793	52.1 ± 17.6	34.5 ± 8.64	86.6 ± 23.9	1563 ± 804	142 ± 48.9	0.51 ± 0.49
P-value	PS	0.036	0.009	0.098	0.044	0.023	0.834	0.019	0.240	0.024	0.070	0.416
	CPC	0.701	0.860	0.171	0.184	0.688	0.067	0.600	0.159	0.662	0.503	0.684
	PS × CPC	0.261	0.359	0.174	0.186	0.244	0.431	0.248	0.530	0.246	0.747	0.236

DM, dry matter.

¹Total SCFA = acetate + propionate + butyrate + valerate; total BCFA = isobutyrate + isovalerate; total VFA = total SCFA + total BCFA.

²Protein source: PBM (poultry byproduct meal); HCL (hydrolyzed chicken liver).

³Crude protein concentration: 24%, 32%, and 40%.

^*Values transformed to log(x) + 1 for statistical analysis.

^a,b,cMeans in the same column with different lowercase letters are significantly different (P < 0.05).

Before starting the palatability assay, diets were evaluated for PV. The PBM24 diet had more than 10 meq/kg of PV, a result considered unacceptable, so the 2 diets with 24% CP (PBM24 and HCL24) were excluded from the assay. In addition, cats were checked for bias prior to palatability assay with a blank diet, which showed that the cats were well trained (P = 0.0688; Figure 1). Among all the contrasts performed, results >0.75 were considered statistically different. Diets containing HCL were preferred compared to PBM (Power test >0.75), regardless of the CP content. Also, diets with higher CP were more preferred and consumed by cats, especially HCL40 (Power test = 1.0000).

Figure 1.

Test power based on cat preference with diets varying in protein source (poultry byproduct meal [PBM] or a hydrolyzed chicken liver [HCL]) and crude protein concentrations (32 or 40% CP). Blank Diet: Commercial diet provided to cats kept in the collective cattery. Diets: PBM32—diet based on PBM with 32% of protein; PBM40—diet based on PBM with 40% of protein; HCL32—diet based on HCL with 32% of protein; HCL40—diet based on HCL with 40% of protein. Results above 0.75 were considered significant.

Discussion

Over the past decade, the search for alternative protein sources with both high nutritional quality and functional properties has been a key focus in companion animal nutrition. There has been a growing trend towards replacing traditional protein sources, such as poultry and bovine byproducts, with hydrolyzed proteins in canine and feline diets. This shift is not only due to the clinical benefits of hydrolyzed proteins for diagnosing and managing adverse food reactions and gastrointestinal disorders (Cave, 2006; Ing & Steiner, 2024), but also for their potential nutritional and functional advantages for healthy animals (Vasconcellos et al., 2024). Considering the chemical composition and degree of hydrolysis, hydrolyzed proteins are more easily digested and absorbed, which may result in reduced undigested protein content available to microbial proteolysis in the hindgut. Therefore, choosing hydrolyzed proteins may offer significant benefits for companion animals, including enhanced digestibility and potential reduction of detrimental fermentative end products, such as ammonia, phenols, indoles, sulfides, and amines. Based on this understanding, we hypothesized that the replacement of PBM by HCL, along with increasing dietary CP, could increase nutrient digestibility and thus reduce undigested protein reaching the colon, thereby decreasing the production of fermentative metabolites in healthy adult cats, and enhance the overall palatability of the diet.

Interestingly, replacing PBM with HCL did not affect macronutrient digestibility in healthy adult cats, except for a reduction in ME content. Previous studies have also observed similar digestibility coefficients among diets based on hydrolyzed PBM and a conventional PBM (Miltenburg et al., 2021; Scarpim et al., 2024). Our initial hypothesis was that HCL-based diets would be superior in terms of digestibility due to their greater bioavailability as a result of the previous hydrolysis process (Cave, 2006; Martínez-Alvarez et al., 2015). In accordance with our findings, a recent study also found no differences in nutrient and energy digestibility when replacing a low ash PBM with hydrolyzed PBM in extruded cat diets (Scarpim et al., 2024).

Despite the high content of small peptides with lower molecular weight, which are linked to enhanced digestion and improved absorption efficiency (Gilbert et al., 2008; Pinto et al., 2021), the protein digestibility of HCL did not differ from that of PBM. However, the apparent digestibility of CP was increased as cats were fed higher CP concentrations. Although PBM is a highly available raw material, it is known that its protein quality varies depending on the parts of the chicken used, the freshness of the raw material, and the processing conditions that directly affect protein digestibility (Murray et al., 1997; Volpato et al., 2022). Similarly to our results, Badri et al. (2021) also observed that cats fed dry extruded diets with higher CP concentration (54.8% protein in DM) had greater protein digestibility than those fed lower CP concentration (28.3% protein in DM), both diets based on dried chicken and pea protein. Another study also reported greater protein digestibility in cats fed diets with higher CP concentrations (34.7% protein in DM vs. 43.8% to 57.4% protein in DM; Paβlack et al., 2014).

In an initial trial conducted with the same HCL, no differences in protein digestibility were observed in dogs fed diets with 24.8% protein and based on HCL (25.8% inclusion) or a mixture of PBM and bovine meat and bone meal (18.6% and 5% inclusion, respectively; Pinto et al., 2021). However, a more recent evaluation using the same diets as the present study demonstrated that dogs fed diets with HCL and higher CP concentrations had better protein digestibility than those fed PBM-based diets (Pinto et al., 2023). Based on this, it appears that, unlike dogs, the protein source alone does not act as a decisive factor in protein digestibility when dietary protein concentration is provided in required amounts for adult cats. In a previous study evaluating protein quality, an assessment that considers protein source, amino acid composition, and protein digestibility, the authors observed no difference in the apparent protein digestibility in cats fed diets with similar CP concentrations (57.4% vs. 54.7% protein in DM) but with varying levels of collagen-rich greaves meal (11% vs. 34% greaves meal; Paβlack et al., 2014).

Cats fed the highest protein concentrations tended to present reduced fat digestibility, possibly because of the higher amount of protein passing through the intestinal lumen that could interfere, at least to some extent, with fat absorption. Higher protein intake also reduced the digestibility of carbohydrates, an expected result since diets were formulated by replacing carbohydrate (NFE) with protein levels. Similar findings were demonstrated in our previous study (Pinto et al., 2023), in which adult dogs fed the same experimental diets used in the present study also showed reduced fat and carbohydrate digestibility as protein intake was increased. No studies were found on the effects of increased protein intake on the reduction of the digestibility and metabolism of fats and carbohydrates. As suggested in the referenced study, the impact on fat digestibility may be associated with the temperature conditions applied during the extrusion and drying processes. Still, it is important to note that all experimental diets resulted in an ATTD of fat above 90%, indicating that diets were well-cooked and carbohydrates would be evaluable. Digestible energy also increased as protein concentration increased, but urinary losses were not accounted. Ultimately, the ME content for the HCL32 diet was lower, probably due to its lower fat content compared to the other diets. Also, despite the high degree of gelatinization all diets reached, the HCL32 diet showed one of the lowest gelatinization indexes, and it may have some impact on digestibility.

The HCL affected some fecal and urinary characteristics in cats. Feeding cats HCL-based diets, especially HCL32 and HCL40, turned the feces softer close to the limit acceptable for fecal score, as a response to the lowest fecal DM. Some protein hydrolysates possess high osmolarity, which leads to water attraction into the intestinal lumen, resulting in softer feces and, in severe cases, diarrhea depending on the degree of hydrolysis (Cave, 2006). Pinto et al. (2021; 2023) also observed reductions in fecal DM and fecal score in dogs fed diets based on HCL compared to conventional animal protein sources. Despite these changes, represented by slightly moist and less consistent stools, the final fecal score remained within the acceptable range (Carciofi et al., 2008).

The increase in urinary pH observed in cats fed diets with HCL and higher CP concentrations differs from previous findings in which high CP diets resulted in a decrease in urinary pH in adult cats (Funaba et al., 2003; Badri et al., 2021). This effect could be explained by the increase in dietary macroelements, specifically cations, which directly affect urinary pH in cats. The urinary pH values ranged from 5.90 to 7.57, it is possible that higher HCL inclusions increased the cation concentration resulting in greater base excess and, consequently, higher urinary pH, which in the long-term should be carefully monitored in cats with a predisposition to lower urinary tract disease (Jeremias et al., 2013). Although there are well-established differences in urinary characteristics between cats and dogs, previous tests with the same HCL in adult dogs showed no difference in urinary pH compared with diets containing a mixture of PBM and bovine meat and bone meal or PBM only (Pinto et al., 2021, 2023). This reinforces the importance of balancing cations and anions during diet formulation for cats to adjust urinary pH to the ideal range between 6.2 and 6.8 (Allen and Kruger, 2000).

In terms of fecal bacterial metabolites, the HCL had a greater impact on acetate, propionate, valerate, and isovalerate concentrations than PBM. Although the ATTD of protein did not differ between the 2 protein sources (numerical ranges of 79.2% to 92.4% in cats fed HCL diets and 78.2% to 91.9% in cats fed PBM diets), it appears that HCL promoted an increase in the amount of bypass nutrients, such as undigested proteins, that entered the hindgut and were metabolized by microbial populations into valerate and isovalerate, both relevant protein-derived metabolites (Blachier et al., 2007). Scarpim et al. (2024) also observed increased fecal valerate, isovalerate, and total BCFA in cats fed a diet with 20% hydrolyzed PBM. Our initial hypothesis was that HCL would increase protein digestibility even at higher protein concentrations, thus resulting in less undigested protein reaching the large intestine of cats. Although there were no significant differences, it was notable that feeding cats with both protein sources and higher CP concentration increased the fecal isobutyrate, isovalerate, and total BCFA after 30 d of trial. BCFA are metabolites of the bacterial amino acid degradation, specifically branched-chain amino acids, that are considered as markers of protein fermentation in the hindgut (Blachier et al., 2007), and are dose-dependently higher with increasing CP in diets for adult cats (Badri et al., 2021). Notably, increases in the concentrations of these metabolites did not impact fecal pH between experimental diets. Changes in fecal pH indicate the production of specific metabolites in response to increased microbial activity on substrates in the colon (Ren et al., 2025). These observations, combined with fecal score, which was kept within the ideal range for all diets, indicate that both protein sources were able to maintain a consistent colonic environment, but further investigations can assess the long-term effects.

Along with carbohydrates, undigested proteins and peptides also contribute to SCFA production through microbial activity in the hindgut (Macfarlane and Macfarlane, 2003; Pilla and Suchodolski, 2021). However, the increased concentrations of acetate, propionate, and total SCFA—metabolites of the fermentation of indigestible polysaccharides—observed in cats fed HCL, should be attributed to the NFE and fiber content of the diets. Although CF analysis is not the gold standard methodology to determine the nutritional value of fibers for companion animals (de-Oliveira et al., 2012), variation in CF content between diets must be considered for these results. Acetate and propionate are the primary SCFAs produced in the colon, followed by butyrate and valerate, and are used as fuels mainly by muscles and the liver, respectively (Cummings and Macfarlane, 1997). Furthermore, SCFAs are essential for intestinal homeostasis, stimulate colonocyte cell proliferation and differentiation, and contribute to immune response regulation (Ma et al., 2021). Sugarcane fiber, an insoluble and low fermentable fiber, was added to all diets at different inclusions and is not related to SCFA production in cats (Fischer et al., 2012). These results may be related to polysaccharides present in other ingredients of the diets, such as brewers’ rice. Thus, the combination of peptides and dietary fiber from carbohydrate sources could explain the increased synthesis of acetate, propionate, and valerate in cats fed HCL diets.

Overall, cats preferred diets based on HCL and high CP concentrations over PBM-based diets. The test power, which was above 0.999 for all comparisons, indicates high reliability of the data given the test recommendation of 0.75 (Cohen, 1988). Protein hydrolysates are commonly applied in pet food kibble as palatant enhancers. During hydrolysis, large proteins are broken into small peptides and free amino acids, which, when combined with reducing sugars, undergo the Maillard reaction to generate flavor compounds (Nagodawithana et al., 2010). According to Chen et al. (2021), peptides with a molecular mass of <1 kDa provide flavor are easier to generate more volatile compounds as precursors of flavor in meat. In a previous study, it was demonstrated that the HCL had 57% of its molecular weight <10 kDa in comparison to 41% of PBM (Pinto et al., 2021). Thus, it is important to consider that the improvement in palatability observed in HCL-based diets may be related to the increased availability of free amino acids and Maillard reaction products that are related to increased acceptability in companion animals (Samant et al., 2021). In addition to the preference for HCL-based diets, cats also preferred diets with higher dietary CP levels, which may be related to the higher inclusion of HCL but also to their strict carnivorous preferences, since adult cats usually prefer high protein and low carbohydrate diets (Hewson-Hughes et al. 2011; Salaun et al., 2017).

Overall, this study had some limitations. The diets were formulated to achieve a balanced amino acid profile, with maize gluten and porcine plasma included as protein sources. However, as the protein level increased, the contribution of these ingredients became less significant in the formulation. Questions remain about how their presence may have influenced the results, particularly in terms of protein digestibility, although diets with higher digestibility were those with the greatest levels of HCL and less inclusion of maize gluten and porcine plasma proportionally to HCL. Additionally, analyzing total dietary fiber could have provided insights into SCFA formation and its possible correlations in HCL-containing diets. Furthermore, assessing urinary nitrogen excretion could have helped clarify HCL’s contribution to nitrogen retention and improve the evaluation of apparent digestibility corrected for nitrogen loss.

Conclusion

HCL protein is a well-digestible protein source for healthy adult cats, offering high palatability and adequate digestibility of nutrients and energy. While HCL did not improve macronutrient digestibility over PBM, higher CP levels enhanced protein digestibility. Cats fed HCL diets had softer feces and increased fecal SCFA and isovalerate related to indigestible carbohydrates and protein that must have escaped digestion. Urinary pH increased with HCL and higher CP, requiring careful formulation. Overall, HCL enhances diet acceptance in cats, but further research is needed to assess its long-term metabolic effects.

Supplementary Data

Supplementary data are available at Journal of Animal Science online.

Glossary

Abbreviations:

ATTD

apparent total tract digestibility

BCFA

branched-chain fatty acid

BCS

body condition score

CF

crude fiber;

CP

crude protein

DM

dry matter

GE

gross energy

HCL

hydrolyzed chicken liver

ME

metabolizable energy

NFE

nitrogen free extract

PBM

poultry byproduct meal

PV

peroxide value

SCFA

short-chain fatty acid

VFA

volatile fatty acid

Conflict of interest statement

Caroline Fredrich Dourado Pinto, Marcelino Bortolo, and Fábio Ritter Marx are employees of Kemin Industries. No potential conflict of interest was reported by the other authors.

Author Contributions

Pamela Sezerotto (Investigation, Methodology, Project administration, Supervision, Writing - original draft, Writing - review & editing), Caroline Fredrich Dourado Pinto (Formal analysis, Methodology, Software, Writing - original draft, Writing - review & editing), Ariane Miranda da Silva (Investigation), Joyce Cristina Paiva Francisco (Investigation), Luciano Trevizan (Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing - review & editing), Marcelino Bortolo (Resources, Writing - review & editing), Fabio Marx (Resources, Writing - review & editing), and Ricardo Vasconcellos (Methodology, Supervision, Writing - review & editing)