Influence of dietary inclusion of black soldier fly larvae protein hydrolysates on nutritional indicators and cardiac-related biomarkers of adult beagle dogs

Abstract

Protein hydrolysates derived from insects have been explored in recent years for their bioactive functionalities, namely antioxidant and antihypertensive activities, although the investigation of in vivo effects remains scarce. The present study aimed to assess the effects of dietary inclusion of black soldier fly (Hermetia illucens) larvae (BSFL) hydrolysates on nutritional status and cardiac biomarkers of adult Beagle dogs. Three complete diets were used: a control diet containing 3% shrimp hydrolysate (D_Shrimp), and two experimental diets in which the shrimp hydrolysate was replaced by BSFL hydrolysates obtained through the enzymatic hydrolysis with either ALCALASE 2.5 L (D_BSFL-A) or Corolase PP (D_BSFL-C). Two-bowl tests were performed to determine palatability by pairwise comparison between all diets. A feeding trial designed according to four 3 × 3 Latin squares with twelve adult Beagle dogs, three periods of 28 d each, and the three complete diets were performed. A cardiac evaluation was performed to all dogs before the trial. Diet digestibility, fecal characteristics and metabolites, skin and coat quality, and cardiac biomarkers were assessed at the end of each period. No significant differences were observed on first approach, first-taste, and intake ratio. Experimental diets increased organic matter (P = 0.038) and crude protein (P = 0.048) digestibility compared to control, and dry matter digestibility was greater (P = 0.048) for D_BSFL-A than D_BSFL-C. No significant differences were observed in serum levels of C-reactive protein, cardiac troponin I, and NT-proBNP among diets, but dogs fed D_BSFL-A tended to have a lower (P = 0.086) serum insulin-like growth factor 1 level compared to dogs fed D_BSFL-C. Although all echocardiographic measurements remained within normal reference ranges, experimental diets promoted a lower (P = 0.030) interventricular septum diameter in diastole compared to the control, and a lower pulmonary vein diameter was noted for D_BSFL-C compared to D_BSFL-A (P = 0.049). Also, dogs fed D_BSFL-C showed a tendency to lower blood pressure (P = 0.102), serum angiotensin converting enzyme (P = 0.092), and left atrial-to-aortic root ratio (P = 0.062) compared to dogs fed D_BSFL-A. In conclusion, diets containing BSFL hydrolysates are well accepted and digested by dogs and might influence mechanisms involved in blood pressure regulation and potentially modulate cardiac function, although further studies are warranted.

Type of enzymatic hydrolysis of black soldier fly larvae (BSFL) differently affected diet digestibility or mechanisms regulating blood pressure. Dietary inclusion of 3% of BSFL hydrolysates prior to extrusion, replacing shrimp hydrolysate, shows potential for use in highly digestible diets or as a novel ingredient in formulations targeting cardiac health and blood pressure regulation in dogs.

Introduction

The demand for sustainable and health-promoting ingredients in companion animal nutrition keeps increasing, with insects emerging as a highly efficient alternative for both humans and animals due to their high protein content, low water and land requirements for production, and a lower feed conversion ratio (Pinney and Costa-Font 2024). Specifically, black soldier fly (Hermetia illucens) larvae (BSFL) is gaining industrial relevance in the pet food sector for its nutritional composition and suitability for large-scale production, with several brands already including BSF meals in diet formulations (Kotob et al. 2022; Klinmalai et al. 2025). Apart from being nutritious, insects possess bioactive properties that have been explored in recent years.

When it comes to bringing functionality to pet food formulations, peptides present a promising way to deliver those health-promoting effects (Ribeiro et al. 2025). Protein hydrolysates from chicken and fish are widely used in pet food formulations as highly digestible and palatable protein sources and are commonly incorporated into diets intended for dogs with adverse food reactions, allergic skin conditions, and gastrointestinal disorders, as the hydrolysis process may reduce the antigenicity of dietary proteins (Nagodawithana et al. 2008; Szczepanik et al. 2022; Jackson 2023). Besides those applications, protein hydrolysates containing peptides with 2 to 20 amino acids (AA), with a reduced molecular weight, were also reported to modulate oxidative stress, inflammation, and immune response and can be used as functional ingredients (Rivera-Jiménez et al. 2022). Protein hydrolysates from insects such as Tenebrio molitor, H. illucens, Musca domestica, and Alphitobius diaperinus have already demonstrated in vitro antioxidant, antihypertensive, and anti-inflammatory activities in a variety of studies (Mouithys-Mickalad et al. 2020; Sousa et al. 2020; Brai et al. 2022; Teixeira et al. 2023; Sánchez-Estrada et al. 2024).

Previous studies have shown that pet foods containing BSFL meals or hydrolysates derived from BSFL are well accepted by dogs (Bosch and Swanson 2021; Wei et al. 2024; Leal et al. 2025). However, the commercialization of insect-based pet food products still faces several challenges. In particular, the lack of formal Association of American Feed Control Officials (AAFCO) definitions for many insect-derived ingredients represents an important regulatory barrier to market entry in the United States. In addition, although consumer perceptions of insect-based pet foods are generally positive and many dog owners express willingness to feed such products to their pets (Baptista da Silva et al. 2024; Triggs et al. 2025), concerns regarding product acceptance and willingness to pay may still limit broader market adoption (Fantechi et al. 2024; Ai et al. 2025). Indeed, although pet owners might perceive insects as nutritious and an alternative protein source for their pets (Godgluck et al. 2026), social and cultural factors, along with a sense of disgust influenced by a perception of food safety risks, still have a significant impact on customers’ choices (Pinney and Costa-Font 2024; Nikravech et al. 2025; Kong et al. 2026).

Studies on the potential health benefits of insect hydrolysates for dogs remain scarce. Most in vivo studies have focused on whole or defatted insect meals, generally reporting good acceptance and digestibility compared to chicken meal (Freel et al. 2021; Jian et al. 2022; Li et al. 2023). Recently, an in vivo study reported no adverse effects on nutrient digestibility, fecal characteristics, or metabolites when Beagle dogs were fed complete diets for 10 d containing 3% T. molitor or BSFL hydrolysates added as a powder on top of the diet, replacing shrimp hydrolysate (Leal et al. 2025). It is known that oxidative stress and chronic activation of the renin-angiotensin system, accompanied by hypertension, are key drivers of myocardial remodeling and progression of cardiac disease in dogs (Larouche-Lebel et al. 2019; Rubio et al. 2020). Cardiovascular disorders are among the most common health conditions in aging dogs, representing approximately 10% of diagnosed diseases (Pasławski et al. 2021; Bagardi et al. 2022), with other common pathologies being also accompanied by hypertension, such as chronic kidney disease (Martello et al. 2021). In light of the previously reported in vitro antioxidant capacity and angiotensin converting enzyme (ACE) inhibitory potential of BSFL hydrolysates (Leal et al. 2025), the present study aimed to assess the effects of incorporating BSFL hydrolysates obtained from different enzymatic hydrolysis processes into an extruded canine kibble diet, compared with a control diet containing shrimp hydrolysate. However, the extent to which insect‑derived protein hydrolysates can influence systemic physiological markers in dogs under controlled dietary conditions remains largely unexplored. It was hypothesized that inclusion of BSFL hydrolysates could positively impact blood pressure and cardiac biomarkers in healthy Beagle dogs, without adversely affecting nutrient digestibility, fecal characteristics and metabolites, and skin and coat quality.

Materials and methods

All procedures were approved by the Animal Ethics Committee of School of Medicine and Biomedical Sciences, University of Porto, and licensed by the Portuguese General Directorate of Food and Veterinary Medicine (permit no. 0421/000/000/2021). Trials were conducted in accordance with European Union Directive 2010/63/EU and performed by trained scientists certified under FELASA (Category C). Before starting the trial, all dogs enrolled in the study underwent a complete physical examination, as well as echocardiographic and electrocardiographic evaluations at the Veterinary Hospital of School of Medicine and Biomedical Sciences, University of Porto, to confirm health status and ensure suitability for participation in the trials.

Animals and housing

Twelve healthy Beagle dogs (7 males and 5 females; 5.3 ± 0.45 years old; 13.2 ± 2.05 kg body weight, BW; 5 out of 9 body condition score, BCS, Freeman et al. 2011) were used in the trials. The number of animals followed the recommendations for a minimum sample size number (FEDIAF 2025). Animals were housed in pairs, at the kennel of the School of Medicine and Biomedical Sciences, University of Porto, in communicating boxes with interior and exterior areas of 1.8 and 3.5 m², respectively. Dogs had free access to an outdoor park area between daily meals to exercise and socialize, and were leash-walked once a day for at least 30 min. During the feces collection period, dogs were housed individually at night, having supervised access to the outdoor park between daily meals and daily leash walking.

Experimental diets

Three extruded complete diets were formulated to meet the nutrient requirements for adult dogs according to FEDIAF (2025): a control diet with 3% shrimp hydrolysate (D_Shrimp) and two experimental diets with 3% BSFL hydrolyzed enzymatically with ALCALASE 2.5 L (D_BSFL-A) or Corolase PP (D_BSFL-C) in substitution of shrimp hydrolysate (Table 1). The basal diet was an economy-grade commercial diet for adult dogs (Sorgal Pet Food, Ovar, Portugal), widely available in supermarkets and hypermarkets. This commercial formulation served as the basis for all experimental diets, with shrimp hydrolysate in the control diet being replaced by BSFL hydrolysates in the experimental diets. The basal diet was selected to represent a commercially relevant feeding scenario. As is typical of economy-grade formulations, it contained a relatively high inclusion of plant-derived ingredients, which contributed to its dietary fiber content. Shrimp hydrolysate was selected as the control ingredient based on its previous evaluation in canine feeding trials, in which it was shown to be highly digestible and palatable and to possess bioactive properties, including antioxidant activity and prebiotic effects (Guilherme-Fernandes et al. 2024; Guilherme-Fernandes 2025; Leal et al. 2025). In addition, shrimp hydrolysate was used as the control ingredient in our previous study evaluating insect hydrolysates added on top of canine extruded diets (Leal et al. 2025). All diets were manufactured by extrusion at Sparos feed mill (Olhão, Portugal). All powder ingredients (including the basal commercial mix and the control or the experimental hydrolysates) were mixed according to the target formulation in a double-helix mixer (model 500 L, TGC Extrusion, France) and ground (below 400 µm) in a micropulverizer hammer mill (model SH1, Hosokawa-Alpine, Germany). Diets (pellet size: 10.0 mm) were manufactured with a twin-screw extruder (model BC45, CLEXTRAL, France) with a screw diameter of 55.5 mm. Extrusion conditions: feeder rate (90 kg/h), screw speed (260–265 rpm), water addition (340 mL/min), temperature barrel 1 (45–47 °C), temperature barrel 2 (88–89 °C), temperature barrel 3 (105–107 °C). Extruded pellets were dried in a vibrating fluid bed dryer (model DR100, TGC Extrusion, France). After cooling, poultry fat and fish oil were added by vacuum coating (model PG-10VCLAB, Dinnissen, The Netherlands).

Table 1.

Ingredient composition (% as-is) of diets containing hydrolysates fed to dogs during the palatability and feeding trials.

Ingredient	Diet1
	D_Shrimp	D_BSFL-C	D_BSFL-A
Basal diet2	89	89	89
Shrimp hydrolysate	3.0	—	—
Black soldier fly hydrolysate
Hydrolyzed with corolase PP	—	3.0	—
Hydrolyzed with ALCALASE 2.5 L	—	—	3.0
Poultry fat	5.0	5.0	5.0
Fish oil	3.0	3.0	3.0

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L

²Economy-grade commercial diet for adult dogs (Sorgal Pet Food, Ovar, Portugal) including, in descending order, pea starch, poultry protein meal, corn grain, broken rice, pea protein concentrate, wheat grain, beet pulp, alfalfa meal, vitamin–mineral premix, Ascophyllum nodosum, salt, monoammonium phosphate, and DL-methionine.

Shrimp hydrolysate was supplied by Symrise Aqua Feed (Elven, France) and was produced through enzymatic hydrolysis of heads and cephalothoraxes of Litopenaeus vannamei. Solid residues were removed by centrifugation at the end of the hydrolysis process. Insect hydrolysates were provided by ETSA-SGPS, S.A. (Loures, Portugal) and were obtained from enzymatic hydrolysis of defatted BSFL meal (Hermetia Baruth GmbH, Germany) using either ALCALASE 2.5 L (BSFL-A) or Corolase PP (BSFL-C). ALCALASE 2.5 L and Corolase PP were supplied by Univar Solutions Inc. (Illinois) and AB Enzymes (Darmstadt, Germany), respectively. Hydrolysis with ALCALASE 2.5 L was conducted at a 1% (v/w) enzyme/substrate ratio at 65 °C for 4 h, whereas hydrolysis with Corolase PP was performed at a 2% (w/w) enzyme/substrate ratio at 60 °C for 4 h. After hydrolysis, enzymes were inactivated by heating the mixtures at 90 °C for 10–15 min. The hydrolysates were then passed through a 450 µm sieve to remove solid material and centrifuged at 5,394 × g at room temperature to separate solids, lipids, and proteins. The protein fraction was collected using a separatory funnel, concentrated with a rotary evaporator, and subsequently spray-dried using a Büchi B-290 Spray Dryer (Büchi, Switzerland).

Palatability

Three two-bowl tests were conducted to evaluate palatability through pairwise comparison of all diets (Aldrich and Koppel 2015). On two consecutive days, dogs (n = 12) were offered a choice between two diets presented in separate bowls placed 45 cm apart during both daily meals. To control side bias, bowls’ position was alternated between meals. Each bowl contained the amount of food calculated to meet the metabolizable energy (ME; FEDIAF 2025) requirement for a single meal; thus, a total of twice the dogs’ ME requirements was offered at each feeding. Trials ended after 30 min or when all food from one bowl had been consumed. The first bowl approached and the first food tasted were recorded. Intake of each diet was measured to calculate the intake ratio for each pairwise comparison.

Experimental design

The feeding trial was designed according to four 3 × 3 Latin squares, with 3 diets, 12 adult Beagle dogs, and 3 experimental periods of 28 d each. Consequently, all dogs were fed all diets over the course of the study. Prior to the study, dogs were fed the same commercial diet for 3 months to standardize their nutritional status. Number of animals used exceeded the minimum recommended by FEDIAF (2025) and AAFCO (2020) for carrying out feeding trials. Each experimental period lasted 28 d, with measurements performed during the final week after a 21-d adaptation period. No separate washout period was included between treatments, as each experimental period incorporated an extended adaptation phase before measurements were obtained. Body weight and BCS were assessed before the morning meal on one day in the middle of each experimental period and on one day during the last week of each experimental period. Daily food allowance was determined to ensure the daily requirements of ME according to the BW of the animals (110 × BW^0.75kcal, FEDIAF 2025), and adjusted to the ideal BCS (Freeman et al. 2011). Dietary ME was estimated using modified Atwater factors based on the predicted nutrient composition of the formulation matrices and used to determine food allowance. Daily food was individually distributed to each animal in two equal meals (8.30 h and 16.30 h) with dogs having free access to fresh water at all times. Food intake and refusals were recorded daily. All measurements were performed at consistent times of day to minimize potential circadian variability.

Apparent total tract digestibility and fecal characteristics

The total fecal collection method was used to determine the apparent total tract digestibility (ATTD) of the diets. Feces were collected for five consecutive days during the final week of each experimental period. The number of defecations was recorded, and individual fecal samples were weighed and scored for consistency using a five-point scale, where 1 represents watery feces and 5 represents hard feces, according to Carciofi et al. (2008). Samples were then stored at −20 °C for subsequent analysis.

Chemical composition of diets and feces

Diets were dried in an air-forced oven at 65 °C until constant weight, ground (1 mm), and analyzed according to official methods (AOAC, 2005) for dry matter (DM; ID 934.01), ash (ID 942.05), Kjeldahl N (ID 990.03), ether extract (EE; ID 920.39), and total dietary fiber (TDF; ID 991.43). Crude protein (CP) was calculated as Kjeldahl N × 6.25. Neutral detergent fiber (NDF; with α-amylase and without sodium sulfite, expressed as exclusive ash) and starch (0.5-mm milled) were analyzed according to Van Soest et al. (1991) and Salomonsson et al. (1984), respectively. Gross energy (GE) analysis was performed with an adiabatic bomb calorimeter (Werke C2000, IKA, Staufen, Germany).

The dietary AA profile was determined after acid hydrolysis (Wang et al. 2016). Samples were derivatized with iodoacetic acid and o-phthaldialdehyde (OPA), and AA were separated by reverse-phase high-performance liquid chromatography (RP-HPLC, Beckman Coulter, CA) as described by Oliveira et al. (2022), and quantified using calibration curves prepared from pure standards (Pripis‐Nicolau et al. 2001). Mineral composition of diets was determined by inductively coupled plasma mass spectrometry (ICP-MS; iCAP Q instrument, Thermo Fisher Scientific, Waltham, MA), in dried samples solubilized by microwave assisted acid digestion (MLS 1200 Mega, Milestone, Sorisole, Italy) as described by Cabrita et al. (2016).

Feces were thawed and homogenized, and representative samples were immediately analyzed for N (CP calculated as N × 6.25). Feces were then dried (65 °C), milled (1-mm), and pooled per dog and period, and ash, EE, NDF, starch (0.5-mm milled), and GE were determined according to the methods described for diet samples.

All analyses were performed in duplicate.

Fecal end-fermentation products

The pH of thawed and homogenized fecal samples was measured using a potentiometer (sensION+ PH3, Hach-Langue, S.L.U., Barcelona, Spain). Fecal ammonia-N concentration was determined by diluting thawed feces 1:10 (w/v) in 2 M KCl, followed by centrifugation at 12,000 × g for 20 min at 4 °C. The supernatant was filtered through a 0.45 μm polyethersulfone syringe filter (FILTER-LAB, Barcelona, Spain). An aliquot of 40 μL of filtrate was mixed with 40 μL of water, followed by the addition of 2.5 mL of phenol solution and 2 mL of 0.37% alkaline hypochlorite solution. Samples were incubated for 10 min at 37 °C and then for 40 min at 22 °C in the dark. Absorbance was read at 550 nm using a Synergy™ HT Multimode plate reader (BIOTEK Instruments Inc., Winooski, VT). An ammonia solution (32 mg/dL) was used as the standard (Chaney and Marbach 1962). Short-chain fatty acids (SCFA) concentrations were determined by diluting 1 g of feces in 10 mL of 25% orthophosphoric acid solution containing an internal standard (4 mM 3-methyl valerate, Sigma-Aldrich, Saint Louis, MO). Samples were centrifuged at 18,000 × g for 16 min at 4 °C, and the supernatant was stored at −20 °C until analysis by gas chromatography (Pereira et al. 2021). All analyses were performed in duplicate.

Flatulence evaluation

Flatulence evaluation was conducted over 5 h after the morning meal (Pereira et al. 2020) in one day of the last week of each experimental period. Briefly, a custom-designed canine vest was used to hold a device hardware, which consists of an air pump with an H₂S sensor connected to a microcontroller powered by a power bank. A plastic tube (length: 55 cm; inner diameter: 0.5 cm) was connected to the pump-sensor box and an O-ring was placed at the base of the tail. Gases were detected by the device, and the H₂S concentration was measured in parts per million (ppm) every 2 s. The number of flatulence episodes and the maximum and average intensity of H₂S detected in each episode were recorded. H₂S values higher than 5 ppm and lasting at least 10 s were considered the threshold for flatulence detection.

Skin and coat quality evaluation

Prior to the start of the study, all dogs underwent a complete physical examination performed by a veterinarian and no skin or coat abnormalities were identified in any of the animals. Visual and sensorial evaluation of skin and coat quality was performed during the final week of each experimental period by a naïve panel of eight individuals (dog owners, animal science researchers, and veterinarians), who were blinded to the dietary treatments. The evaluation was conducted indoors under natural daylight. All dogs were bathed the day before the assessment and groomed on the day of evaluation. A five-point scoring scale was used to assess coat glossiness, greasiness, softness, scaling, and general quality of the coat, where 1 represented the least desirable condition and 5 represented the most desirable condition (Devriendt et al. 2021).

Blood pressure, electrocardiogram, and echocardiogram

Blood pressure measurement, electrocardiogram (ECG), and echocardiogram were performed at the beginning of the experimental trial and during the final week of each experimental period. All procedures were conducted with the dogs fully conscious and without sedation. Systolic, diastolic, and mean arterial blood pressures were obtained using a veterinary oscillometric non-invasive blood pressure monitor (Vet20 SunTech, SunTech Medical Inc., Morrisville, NC). The first reading was discarded, and the mean of the subsequent five measurements for each dog was calculated, following the recommendations of Acierno et al. (2018).

For ECG, dogs were placed in right lateral recumbency, and the electrodes were attached to the skin using alligator clips and alcohol to ensure proper electrical contact. A six-lead ECG (leads I, II, III, aVR, aVL and aVF) was recorded for 60–120 s at a paper speed of 50 mm/s and a sensitivity of 10 mm/mV. The amplitude and duration of the P wave, PR interval duration, QRS complex duration and amplitude, QT interval duration, ST segment elevation, and T wave amplitude were manually measured from printed ECG tracings. Heart rate (HR) was calculated from the inter-beat (R-R) intervals.

For echocardiographic assessment, dogs were partially shaved to create a window for echocardiogram. Echocardiographic examinations were performed in accordance with the guidelines of the Echocardiography Committee of the Specialty of Cardiology of the American College of Veterinary Internal Medicine (Thomas et al. 1993). After a 15-min resting period, B-mode (2D), M-mode, color Doppler, and spectral Doppler echocardiography were conducted with simultaneous single-lead ECG monitoring, using an M5S phased-array transducer (1.5–4.5 MHz) coupled to a GE Logiq S8 XDclear ultrasound system (GE HealthCare, Chicago, IL). To minimize measurement variability, a single trained operator obtained three consecutive recordings for each echocardiographic parameter, and all data were digitally stored for subsequent analysis.

Pulmonary vein diameter (PVd) and pulmonary artery diameter (Apd) were measured from the right parasternal long axis 4-chamber view at end-systole in 2D mode, and the PVd/Apd ratio was subsequently calculated (Kim et al. 2024). M-mode imaging from the right parasternal short-axis view at the level of the left ventricular papillary muscles was used to measure interventricular septum thickness (IVS), left ventricular internal diameter (LVID), and left ventricular posterior wall thickness (LVPW) in both diastole (d) and systole (s). The diastolic LVID was normalized to BW (LVIDdN, Cornell et al. 2004), and fractional shortening (FS) and ejection fraction (EF, Flores Dueñas et al. 2023) were calculated using the following formulas:

$$\begin{array}{c}L\mathit{VIDdN}=\frac{\mathit{LVID}\mathrm{ }\left(\mathrm{c}m\right)}{BW\mathrm{ }\left(kg\right)^0.294}\\ \begin{array}{c}FS\mathrm{ }\left(\mathrm{\%}\right)=\frac{\mathit{LIDd}\mathrm{ }-\mathrm{ }\mathit{LVIDs}}{\mathit{LVIDd}}\mathrm{ }\times \mathrm{ }100\\ EF\mathrm{ }\left(\mathrm{\%}\right)=\frac{\mathit{LVIDd}^3\mathrm{ }-\mathrm{ }\mathit{LVIDs}^3}{\mathit{LVIDd}^3}\times \mathrm{ }100\end{array}\end{array}$$

E-point-to-septal separation (EPSS) was obtained in the mitral valve plane from the right parasternal short-axis view using 2D-guided M-mode imaging. The diameters of the left atrium (LA) and aorta (Ao) were measured from the right parasternal short‑axis view at the level of the aortic commissure between the non‑coronary and left coronary cusps, during early diastole, and the LA to Ao ratio (LA/Ao) was calculated. Pulmonary flow velocities were assessed from the right parasternal short-axis view using pulsed-wave Doppler. Aortic and mitral inflows were measured using pulsed-wave Doppler from the left parasternal apical 5 and 4 chamber views, respectively. Transmitral flow was evaluated during diastole, and peak velocities of early diastolic filling (E wave) and atrial contraction (A wave) were recorded.

Echocardiographic examinations were performed by a veterinarian with extensive experience in companion animal cardiology. All echocardiographic measurements were subsequently reviewed and analyzed by the same operator, who was blinded to the dietary treatments. In addition, all dogs were clinically assessed prior to enrolment, including assessment of hydration status based on skin turgor, mucous membrane moisture, and capillary refill time. All dogs were considered adequately hydrated. This was also indirectly supported by routine hematology and serum biochemistry results, which remained within reference intervals throughout the study (data not shown).

Cardiac biomarkers

Blood samples were collected before the morning meal on the last day of each experimental period. Blood was collected from the jugular vein into EDTA BD VACUTAINER (VWR International, Carnaxide, Portugal) and VACUETTE BD VACUTAINER SST II tubes with clot activator and separating gel (Becton, Dickinson and Company, Franklin Lakes, NJ). Blood samples were refrigerated at 4 °C and analyzed on the same day for all parameters except ACE activity, which was analyzed from serum stored at −80 °C. C-reactive protein (CRP), troponin I, and insulin-like growth factor 1 (IGF-1) were measured in serum, and the NT-proBNP was measured in EDTA plasma. Briefly, ACE activity was measured by molecular absorption spectrometry (Siemens Atellica, Siemens Healthineers, Erlanger, Germany), CRP was analyzed by immunoturbidimetric assay (Gentian Canine CRP Immunoassay, Gentian Diagnostics ASA, Moss, Norway, adapted to be analyzed on Cobas c501, Roche Diagnostics, Indianapolis, IN) and Troponin I and IGF-1 were analyzed by chemiluminescence immunoassay (Siemens Immulite 2000, Siemens Healthineers, Erlanger, Germany). NT-proBNP was quantified through the ELISA canine cardiopet proBNP assay (IDEXX Laboratories, Westbrook, ME).

Calculations and statistical analysis

Fecal production (%) was calculated as:

$$\mathit{Fecal}\mathrm{ }\mathit{production}=\mathrm{ }\frac{\mathrm{ }\mathit{dried}\mathrm{ }\mathit{fecal}\mathrm{ }\mathit{output}\mathrm{ }\left(\frac{g}{d}\right)\mathrm{ }}{DM\mathrm{ }\mathit{intake}\mathrm{ }\left(\frac{g}{d}\right)}\mathrm{ }\times \mathrm{ }100$$

ATTD (%) was calculated as:

$$\mathit{ATTD}=\frac{\mathit{nutrient}\mathrm{ }\mathit{intake}\mathrm{ }\left(\frac{g}{d}\right)-\mathrm{ }\mathit{fecal}\mathrm{ }\mathit{nutrient}\mathrm{ }\left(\frac{g}{d}\right)\mathrm{ }}{\mathit{nutrient}\mathrm{ }\mathit{intake}\mathrm{ }\left(\frac{g}{d}\right)}\mathrm{ }\times \mathrm{ }100$$

ME (MJ/kg of DM) was calculated according to Hall et al. (2013):

$$ME=\frac{[(GE\mathrm{\hspace{0.17em}}\mathit{intake}\mathrm{\hspace{0.17em}}\left(\frac{kJ}{d}\right)-\mathit{fecal}\mathrm{\hspace{0.17em}}GE\mathrm{\hspace{0.17em}}\left(\frac{kJ}{d}\right)]-[CP\mathrm{\hspace{0.17em}}\mathit{intake}\mathrm{\hspace{0.17em}}\left(\frac{g}{d}\right)-\mathit{fecal}\mathrm{\hspace{0.17em}}CP\mathrm{\hspace{0.17em}}\left(\frac{g}{d}\right)]\times \mathrm{\hspace{0.17em}}5.23}{DM\mathrm{\hspace{0.17em}}\mathit{intake}\mathrm{\hspace{0.17em}}\left(\frac{g}{d}\right)}$$

All data were analyzed using SAS. OnDemand for Academics (SAS Institute Inc., Cary, NC, 2026). Diet first-approach and first-taste results were submitted to the Chi-square test and the intake ratio to the Student’s t-test. Data from the feeding trial were analyzed using the general linear model with the fixed effects of the square, dog within square, period, diet, and residual error. When differences were significant (P < 0.05), contrasts were performed to compare the means of diets containing insect protein hydrolysates obtained under different enzymatic hydrolysis conditions, as well as to compare the means of the diets with insect hydrolysates with the control diet. Data on fecal consistency and skin and coat quality were analyzed following a Kruskal–Wallis test to compare scores between dogs fed different diets (Scheff 2016). For all analyses, significance was set at P < 0.05 and a trend when 0.05 ≤ P < 0.10.

Results and discussion

Experimental diets and palatability

The proximate chemical composition and the AA and mineral profile of the control and experimental diets are presented in Table 2. Diets had similar CP (30.1–30.6%, DM basis), TDF (10.2–10.7%, DM basis) and GE (21.3-21.5 MJ/kg DM) contents, whereas EE content was slightly lower in D_BSFL-A and D_BSFL-C than in D_Shrimp. As the diets differed only in the type of hydrolysate included at 3% prior to extrusion, the differences observed can be attributed to the composition of the respective hydrolysates. The chemical composition of the hydrolysates added to the complete diets has been previously described (Leal et al. 2025), with shrimp hydrolysate showing higher CP (70% DM basis) and EE (8.85%, DM basis) contents than BSFL hydrolyzed with ALCALASE 2.5 L (66% CP and 1.62% EE, DM basis) and with Corolase PP (58% CP and 2.29% EE, DM basis).

Table 2.

Proximate chemical composition and amino acid and mineral composition of diets containing hydrolysates fed to dogs during the palatability and feeding trials (expressed as g/100 g of dry matter; mg/100 g of dry matter for trace elements and heavy metals).

	Diet1
	D_Shrimp	D_BSFL-C	D_BSFL-A
Dry matter, as is	95.7	96.4	96.5
Ash	7.51	7.45	7.45
Crude protein	30.2	30.1	30.6
Ether extract	14.1	13.0	12.1
Total dietary fiber	10.2	10.7	10.5
Neutral detergent fiber	9.82	9.02	9.25
Starch	32.0	30.5	31.0
Gross energy, MJ/kg	21.3	21.5	21.4
Essential amino acids (EAA)
Arginine	1.84	1.83	1.81
Histidine	0.70	0.69	0.69
Lysine	1.57	1.60	1.58
Threonine	1.23	1.24	1.22
Isoleucine	1.52	1.52	1.49
Leucine	2.65	2.76	2.66
Valine	1.51	1.57	1.52
Methionine	0.87	0.85	0.83
Phenylalanine	1.71	1.72	1.71
Cysteine	0.86	0.85	0.86
Tyrosine	1.60	1.57	1.53
Total EAA	16.1	16.1	15.9
Non-essential amino acids (NEAA)
Aspartic acid + Asparagine	2.41	2.53	2.49
Glutamic acid + Glutamine	4.57	4.87	4.70
Alanine	1.85	1.96	1.92
Glycine	1.99	1.98	1.97
Proline	2.05	2.11	2.05
Serine	1.46	1.47	1.43
Taurine	0.14	0.13	0.13
Total NEAA	14.5	15.0	14.7
Essential macro minerals
Calcium	1.16	1.21	1.22
Phosphorous	0.78	0.68	0.72
Magnesium	0.17	0.16	0.17
Sodium	0.45	0.43	0.43
Potassium	0.59	0.57	0.58
Calcium:Phosphorous ratio	1.48	1.77	1.71
Trace elements
Iron	34.0	32.2	33.6
Copper	1.92	1.82	1.90
Manganese	4.30	4.20	4.24
Zinc	16.8	16.1	16.6
Cobalt	0.06	0.06	0.07
Selenium	0.06	0.05	0.05
Molybdenum	0.09	0.09	0.09
Heavy metals
Arsenic	0.05	0.05	0.05
Cadmium	0.01	0.01	0.01
Lead	0.03	0.03	0.03

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

The AA content of diets was similar, with all diets meeting the nutritional requirements of adult dogs (FEDIAF 2025). Arginine and leucine are the essential AA present in higher amounts in all diets, whereas glutamic acid and glutamine are the non-essential AA found in higher amounts. Arginine contributes to cardiovascular function by promoting vasodilation through nitric oxide synthesis, while leucine is a ketogenic and branched-chain AA, promoting protein synthesis, and is also a precursor for glutamine synthesis (Neinast et al. 2019; Murphy et al. 2020; Li and Wu 2023). Glutamic acid and glutamine may increase diet palatability and support intestinal health. While glutamic acid acts as a stimulator of umami receptors, thus enhancing food palatability (Li and Wu 2023; Watson et al. 2023; Klinmalai et al. 2025), glutamine is widely used by canine small intestine for energy and metabolism, promotes glutathione synthesis and acts as an antioxidant in the gastrointestinal mucosa and, thereby, supports gastrointestinal immune response and a healthy gut barrier (Li and Wu 2023; Melo et al. 2026). Although minor numerical differences in mineral composition were observed among diets, all mineral concentrations and Ca: P ratios remained within recommended ranges for adult dogs (FEDIAF 2025).

No differences between diets were observed in the frequency of first approached, first tasted, or the intake ratio, except for a tendency to a preference for D_Shrimp over D_BSFL-A (P = 0.085; Table 3). Earlier studies have reported positive effects of dietary inclusion of insect hydrolysates on diet palatability. Dietary inclusion of a commercial mixture of BSFL hydrolysate with Schizochytrium (mixture included at 5%, 10%, and 15% levels of inclusion in the diet) improved palatability in Beagle dogs (Wei et al. 2024). Similarly, BSFL hydrolysates added at 3% as a powder top-dressing were well accepted with no differences over the control diet with the same inclusion level of shrimp hydrolysate (Leal et al. 2025). Moreover, the inclusion of 2% cricket protein hydrolysate in dog diets significantly increased the intake ratio (57% of intake ratio) compared to the control diet (0% inclusion), whereas higher inclusion levels (4% and 6%) reduced intake (Saejiem et al. 2025). The absence of significant effects observed in the present study may be attributed to the low dietary inclusion level of the hydrolysates, their incorporation into the kibble rather than being added as a powder on top of the food, and their similar content of umami-associated AA (Watson et al. 2023). Nevertheless, conclusions on the influence of hydrolysates on palatability should be interpreted with caution due to the limited number of animals used in this study (Aldrich and Koppel 2015).

Table 3.

Frequency of diet first approached and tasted, and the intake ratio of diets containing hydrolysates.

	Diet1
	D_Shrimp	D_BSFL-A	P-value
n	12	12
First approach	48%	52%	0.773
First taste	50%	50%	1.000
Intake ratio	54%	46%	0.085
	D_Shrimp	D_BSFL-C	P-value
n	12	12
First approach	50%	50%	1.000
First taste	50%	50%	1.000
Intake ratio	50%	50%	0.923
	D_BSFL-A	D_BSFL-C	P-value
n	12	12
First approach	56%	44%	0.386
First taste	58%	42%	0.248
Intake ratio	50%	50%	0.882

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

Diet intake, digestibility, and fecal characteristics

The dogs remained healthy throughout the feeding trial. The daily food offered was slightly adjusted to maintain ideal BW and BCS, which remained stable throughout the study, with no food refusals or significant episodes of emesis or diarrhea. The median BCS of dogs during the study was 5 out of 9. No significant differences were observed in food intake among diets, but dogs fed the control diet (D_Shrimp) presented a higher intake of EE (P < 0.001), NDF (P < 0.001), and starch (P = 0.005) compared to experimental diets (Table 4). Intake of EE was higher when dogs were fed D_BSFL-C over D_BSFL-A (P = 0.002). Despite significant differences in EE, NDF, and starch intake, the magnitude of change was considered too small to be physiologically relevant.

Table 4.

Body weight and diet, nutrient, and energy intake of dogs fed diets containing hydrolysates.

	Diet1			SEM	P-value	Contrasts
	D_Shrimp	D_BSFL-C	D_BSFL-A			D_Shrimp vs Insect diets	D_BSFL-C vs D_BSFL-A
n	12	12	12
Body weight, kg	13.0	13.0	12.9	0.09	0.789
Diet intake
g diet/d, as is	211	209	209	2.3	0.736
g diet/d, dry matter	202	201	209	2.2	0.973
Nutrient intake, g/d
Organic matter	187	186	188	2.1	0.846
Crude protein	61.0	60.6	61.8	0.69	0.496
Ether extract	28.5	26.1	24.6	0.30	<0.001	<0.001	0.002
Total dietary fiber	20.6	21.5	21.2	0.20	0.051
Neutral detergent fiber	19.8	18.1	18.6	0.21	<0.001	<0.001	0.106
Starch	64.5	61.2	62.4	0.70	0.011	0.005	0.255
Gross energy, MJ/d	4.31	4.32	4.31	0.479	0.974

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

Fecal output, characteristics, and metabolites are presented in Table 5, and fecal consistency scores in Figure 1. Fecal output tended to be higher (P = 0.055) in dogs fed the control diet, followed by D_BSFL-C, whereas D_BSFL-A resulted in lower fecal production. Fecal consistency did not differ between diets (P = 0.164), with all diets promoting hard, formed, and dry stools (median scores ranging from 3.6 out of 5 for D_BSFL-C and 3.8 out of 5 for D_BSFL-A). Fecal output, number of defecations per day, and DM content of feces observed in the present study are aligned with the expected outcomes for dry foods with moderate fiber contents (Montserrat-Malagarriga et al. 2024). Gut transit time, microbiota composition, water-holding capacity of ingredients, and nutrient digestibility are known to play a role in stool consistency and overall fecal characteristics (Pilla and Suchodolski 2019; Godgluck et al. 2025).

Table 5.

Fecal output, pH, ammonia-N concentration, and concentrations of short-chain fatty acids of dogs fed diets containing hydrolysates.

	Diet1			SEM	P-value	Contrasts
	D_Shrimp	D_BSFL-C	D_BSFL-A			D_Shrimp vs Insect diets	D_BSFL-C vs D_BSFL-A
n	12	12	12
Fecal output
g/d, as is	132	127	111	5.9	0.055
g/d, DM	37.6	36.2	32.0	1.65	0.069
Number of defecations/d	1.90	2.00	1.70	0.078	0.100
Feces DM, %	28.9	28.6	29.5	0.28	0.113
Fecal production, %	18.9	18.2	15.8	0.78	0.032	0.067	0.048
Fecal pH	6.34	6.51	6.51	0.049	0.039	0.012	0.915
Ammonia-N, g/kg, DM	247	231	243	7.5	0.314
SCFA, µmol/g, DM
Total	422	442	408	14.6	0.283
Acetate	237	256	236	8.6	0.211
Propionate	129	136	124	5.0	0.233
Iso-butyrate	5.20	5.60	5.30	0.240	0.505
Butyrate	38.8	32.3	31.3	2.89	0.165
Iso-valerate	7.50	7.60	7.50	0.344	0.983
Valerate	2.70	2.10	2.10	0.422	0.637
Iso-caproate	1.30	1.20	1.20	0.090	0.630
Caproate	0.80	0.90	0.90	0.097	0.906

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L. DM, dry matter; SCFA, short-chain fatty acids. Fecal production = (g feces/d [DM basis]/DM intake) ×100.

Figure 1.

Fecal score results of Kruskal–Wallis test between dogs (n = 12) fed diets containing hydrolysates. The violin plot illustrates the distribution of the scores. Wider sections of the plot indicate a higher concentration of data points, and narrower sections indicate fewer data points. The median scores are represented by the line within the plot. D_Shrimp, complete diet with shrimp hydrolysate; D_BSFL-C, complete diet with black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

Dogs receiving the control diet exhibited a lower fecal pH compared with dogs fed the experimental diets (P = 0.012), while no differences were observed in fecal ammonia-N or SCFA concentration. A higher fecal pH was previously observed in dogs fed diets with BSF meals due to a decrease in total SCFA and branched-chain fatty acids production (Jian et al. 2022). In dogs, fecal pH usually ranges from 6 to 7, with an acidic pH being associated with the production of SCFA and the inhibition of pathogenic bacteria growth (Do et al. 2021). On the other hand, excessive protein intake can lead to a higher availability of non-digested protein in the colon, promoting fermentation by intestinal bacteria, which increases intestinal ammonia-N concentration, thus increasing fecal pH (Godgluck et al. 2025). In this study, the observed differences in fecal pH between diets cannot be attributed to protein intake nor to the production of SCFA or ammonia-N, which did not differ among diets. Moreover, although protein intake remained unaffected by diet, the higher CP digestibility observed for experimental diets did not result in a lower fecal pH. Indeed, the observed results may be attributed to the interaction of different peptides reaching the colon among diets with microbiota (Santos et al. 2026). It is also important to consider that SCFA and ammonia-N are rapidly absorbed, and their fecal concentrations may not reflect the true colonic concentration (Pinna et al. 2018).

The highest ATTD of DM was observed with D_BSFL-A (84.2%), whereas diets including insect hydrolysates resulted in higher ATTD of OM (P = 0.038) and CP (P = 0.048) than the control diet (Table 6). These differences in digestibility may be partially related to the enzymatic hydrolysis applied to insect hydrolysates. Hydrolysis can produce peptides with different functional properties and sizes, impacting nutrient digestibility by interfering with nutrient absorption and gut health (Vasconcellos et al. 2024). Corolase PP is a porcine pancreatic enzyme preparation composed of trypsin and chymotrypsin, mimicking digestive processes occurring naturally in the small intestine, and can produce peptides with different molecular weights (Coscueta et al. 2016). ALCALASE 2.5 L is a protease derived from Bacillus licheniformis, known for its efficiency in hydrolyzing proteins at alkaline pH and achieving a higher degree of hydrolysis, resulting in peptides of lower molecular weight (Tacias-Pascacio et al. 2020). A higher degree of hydrolysis tends to produce small peptides with a low molecular weight and including free AA that are rapidly absorbed in the intestinal tract, contributing to a better digestibility (Borges et al. 2022; Vasconcellos et al. 2024). Nevertheless, Sousa et al. (2020) observed a greater degree of hydrolysis of Alphitobius diaperinus for Corolase PP compared to ALCALASE 2.5 L, although both enzymatic hydrolysis resulted in the production of small peptides. Despite this, it is hypothesized in the present study that hydrolysis with ALCALASE 2.5 L produced smaller peptides compared to hydrolysis with Corolase PP, which potentially contributed to the higher digestibility observed for D_BSFL-A. These observations are consistent with previous evidence supporting enhanced protein utilization from hydrolyzed protein sources.

Table 6.

Apparent total tract digestibility and metabolizable energy content of diets containing hydrolysates.

	Diet1			SEM	P-value	Contrasts
	D_Shrimp	D_BSFL-C	D_BSFL-A			D_Shrimp vs Insect diets	D_BSFL-C vs D_BSFL-A
n	12	12	12
Dry matter, %	81.1	81.8	84.2	0.78	0.032	0.067	0.048
Organic matter, %	85.8	86.6	88.3	0.59	0.027	0.038	0.065
Crude protein, %	81.5	82.8	85.3	0.98	0.037	0.048	0.080
Ether extract, %	96.7	96.6	96.6	0.16	0.948
Neutral detergent fiber, %	52.0	50.1	56.3	2.08	0.122
Starch, %	99.6	99.6	99.7	0.04	0.077
Gross energy, %	88.0	88.7	89.8	0.53	0.080
Metabolizable energy, MJ/kg, dry matter	17.5	17.8	17.8	0.10	0.059

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

The relatively moderate ATTD coefficients observed in the present study may be partly explained by the fiber content of the commercial basal diet, reflecting the characteristics of the selected economy-grade formulation, which included a relatively high inclusion of plant-derived ingredients compared with many premium commercial diets. Because all experimental diets were based on the same commercial formulation, differing only in the source of hydrolysate, the background fiber content was consistent across treatments and should therefore be considered when interpreting the overall digestibility values. The ME content was similar among diets and was consistent with the pre-trial estimate calculated using modified Atwater factors (17 MJ/kg DM).

No differences among diets were observed in the number of flatulence episodes or in their average and maximum intensity (H₂S ppm detected; Table 7). Individual variability in flatulence frequency and intensity was noted, as already reported in other studies in dogs (Pereira et al. 2020; Cabrita et al. 2024b), likely reflecting differences in gut microbiota and the resulting production of gas from fermentable fibers and undigested protein reaching the colon (Pilla and Suchodolski 2021; Cai et al. 2022). Further investigation, including gut microbiome analysis, is needed to clarify the observed results.

Table 7.

Number of episodes, average and maximum intensity (H₂S detected) of flatus of dogs fed diets containing hydrolysates.

	Diet1			SEM	P-value
	D_Shrimp	D_BSFL-C	D_BSFL-A
n	12	12	12
Number of episodes	2.67	11.4	16.5	5.02	0.169
Average intensity, ppm	8.67	5.97	8.44	1.174	0.303
Maximum intensity, ppm	17.0	6.79	14.1	3.37	0.208

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L

Blood pressure, ECG, echocardiogram, and cardiac biomarkers

Serum NT-proBNP and cardiac troponin I, together with blood pressure, ECG, and echocardiographic measurements, are typically used in dogs to assess myocardial inflammation or injury, as well as structural or functional cardiac alterations (Leach et al. 2023; Owens et al. 2023; Cabrita et al. 2024a). Although antihypertensive peptides have not yet been identified in BSFL derived hydrolysates (Psarianos et al. 2025), those produced via enzymatic hydrolysis using Corolase PP and ALCALASE 2.5 L have shown in vitro ACE-inhibitory activity, suggesting the potential antihypertensive effect of insect-derived dietary hydrolysates (Leal et al. 2025). To the authors’ knowledge, despite the promising in vitro antihypertensive properties reported for insect hydrolysates (Nongonierma and FitzGerald 2017), no in vivo studies have been conducted in dogs to date. In the present study, cardiac evaluations were performed to assess the in vivo effects of diets containing insect hydrolysates, compared with a control diet formulated with shrimp hydrolysate (Table 8).

Table 8.

Systolic, diastolic, mean blood pressure, heart rate, and echocardiographic and electrocardiographic measurements of dogs fed diets containing hydrolysates.

	Diet1			SEM	P-value	Contrasts
	D_Shrimp	D_BSFL-C	D_BSFL-A			D_Shrimp vs Insect diets	D_BSFL-C vs D_BSFL-A
n	12	12	12
SBP, mmHg	130	124	131	2.4	0.102
BP, mmHg	81.6	82.0	80.5	1.69	0.823
MBP, mmHg	92.1	91.2	91.5	1.63	0.914
HR, bpm	88.9	89.9	87.0	3.83	0.862
Echocardiographic measurements2
PVd	10.7	10.0	10.6	0.213	0.047	0.111	0.049
PAd	8.41	8.00	7.95	0.226	0.307
PVd/PAd	1.29	1.26	1.35	0.045	0.383
IVSd, cm	0.99	0.97	0.90	0.018	0.012	0.030	0.028
IVSs, cm	1.34	1.40	1.25	0.052	0.147
LVIDd, cm	3.12	3.09	3.07	0.049	0.744
LVIDdN	1.47	1.49	1.46	0.026	0.774
LVIDs, cm	2.05	2.07	2.08	0.054	0.920
LVPWd, cm	0.93	0.95	0.90	0.034	0.626
LVPWs, cm	1.22	1.17	1.20	0.050	0.750
EF, Teich, %	61.8	62.9	61.0	1.559	0.694
FS, %	32.5	33.3	31.9	1.197	0.718
LA, cm	2.61	2.47	2.61	0.072	0.289
Ao, cm	1.76	1.80	1.75	0.027	0.395
LA/Ao	1.48	1.37	1.49	0.038	0.062
EPSS, cm	0.20	0.24	0.23	0.034	0.693
PV, m/s	0.75	0.77	0.72	0.028	0.365
PV, mmHg	2.36	2.53	2.11	0.207	0.348
AV, m/s	1.05	1.08	1.02	0.044	0.633
AV, mmHg	4.69	5.02	4.35	0.382	0.490
MV E, m/s	0.63	0.64	0.63	0.017	0.762
MV A, m/s	0.40	0.41	0.41	0.020	0.872
MV E/A	1.63	1.60	1.59	0.069	0.917
Electrocardiographic measurements
P wave, ms	39.2	40.0	39.2	1.48	0.900
P wave, mV	0.20	0.20	0.20	0.008	1.000
QRS complex, ms	60.8	56.7	56.7	3.55	0.638
QRS, complex mV	1.45	1.59	1.60	0.050	0.095
PR interval, ms	110	113	118	2.4	0.110
QT interval, ms	158	172	158	10.3	0.581
ST elevation, mV	0.28	0.25	0.27	0.024	0.620
T wave, mV	0.38	0.35	0.36	0.032	0.838

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

²Ao, aorta; AV m/s, aortic peak systolic velocity; AV mmHg, aortic systolic pressure gradient (mmHg); BPM, beats per minute; DBP, diastolic blood pressure; EF, ejection fraction; EPSS, E-point to septal separation; FS, fractional shortening; HR, heart rate; IVSd, interventricular septum thickness in diastole; IVSs, interventricular septum thickness in systole; LA, left atrium; LA/Ao, left atrial to aorta diameter, short axis; LVIDd, left ventricular internal diameter in diastole; LVIDdN, left ventricular internal diameter in diastole normalized to body weight; LVIDs, left ventricular internal diameter in systole; LVPWd, left ventricular posterior wall thickness in diastole; LVPWs, left ventricular posterior wall thickness in systole; MBP, mean blood pressure; MV E m/s, mitral valve peak E wave velocity; MV A m/s, mitral valve peak A wave velocity; MV E/A, mitral valve E wave velocity to A wave velocity; Pad, pulmonary artery diameter; PVd, pulmonary vein diameter; PV m/s, pulmonary artery peak systolic velocity; PV mmHg, pulmonary artery systolic pressure gradient (mmHg); PVd/PAd ,pulmonary vein diameter to pulmonary artery diameter; SBP, systolic blood pressure.

Blood pressure did not differ between control and experimental diets; however, a trend towards lower systolic blood pressure was observed in dogs fed D_BSFL-C compared with those fed D_BSFL-A (P = 0.102). Dogs receiving the D_BSFL-C diet also tended to have lower LA/Ao ratio (P = 0.062) and significantly reduced PVd (P = 0.049) compared with those fed D_BSFL-A. Additionally, dogs on experimental diets showed a lower interventricular septum thickness in diastole (IVSd) than those on the control diet (P = 0.030). Despite these findings, they are unlikely to be biologically relevant, as all echocardiographic parameters remained within established reference ranges for adult dogs. Although all dogs exhibited mild echocardiographic thickening of the mitral valve leaflets consistent with early myxomatous mitral valve disease (ACVIM Stage B1, Keene et al. 2019), none showed evidence of cardiac remodeling or clinical signs of heart disease. Accordingly, circulating concentrations of CRP, NT-proBNP, and cardiac troponin I, as well as ECG measurements, remained within reference ranges for healthy dogs (Cornell et al. 2004; Oyama 2015).

Although all values remained within normal reference ranges, the tendency toward lower systolic blood pressure in dogs fed D_BSFL-C may be related to their significantly lower PVd and the trend toward a reduced LA/Ao ratio. Despite the modest magnitude of these changes, their consistent direction across multiple parameters indicates potential biological coherence. Enlargement of PVd has been described as an early marker of cardiac disease, preceding changes in the LA/Ao ratio (Merveille et al. 2015; Kim et al. 2024). A lower LA/Ao ratio may correlate with reduced blood pressure and can be modulated by targeted dietary interventions in cardiac patients, as described by Li et al. (2019). In addition to the tendency toward lower systolic blood pressure, dogs fed D_BSFL-C also showed a trend toward reduced serum ACE levels compared with those fed D_BSFL-A (P = 0.092; Table 9). Because inhibition of ACE is known to decrease blood pressure, the potential presence of ACE-inhibitory peptides in D_BSFL-C could help explain these findings. Compared with ALCALASE 2.5 L, hydrolysis with Corolase PP generally yields peptides enriched with aromatic and hydrophobic residues at the C-terminal segment, structural features associated with ACE‑inhibitory activity (CunhaNeves et al. 2022; Daza-Rodríguez et al. 2023; Vasconcellos et al. 2024). Peptides from hydrolysates are being investigated in both human and animal models for their capability to regulate blood pressure also through gut microbiome modulation and consequent metabolite production, with the Firmicutes/Bacteroidetes ratio playing a role (Maiuolo et al. 2022; Tsafack et al. 2022).

Table 9.

Blood results of dogs fed diets containing hydrolysates.

	Diet1			SEM	P-value
	D_Shrimp	D_BSFL-C	D_BSFL-A
n	12	12	12
Angiotensin converting enzyme activity, U/L	37.2	38.8	42.5	1.58	0.092
Insulin-like growth factor, ug/L	248	249	200	16.7	0.086
Troponin I, ng/mL	0.04	0.05	0.04	0.007	0.443
C-reactive protein, mg/L	7.26	6.87	5.10	0.659	0.659
NT-proBNP, pmol/L	299	302	305	14.1	0.954

¹D_Shrimp, complete diet with 3% shrimp hydrolysate; D_BSFL-C, complete diet with 3% black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with 3% black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

Diet with BSFL-A tended to decrease IGF-1 levels compared with D_BSFL-C (P = 0.086; Table 9). Elevated IGF-1 concentrations have been associated with increased IVSd in humans (Chen et al. 2022) and, in dogs, higher IVSd values are linked to left ventricular hypertrophy (Schober et al. 2022). Although evidence in dogs regarding the direct cardiovascular effects of IGF-1 is limited, IGF-1 is known to regulate blood pressure through modulation of endothelial nitric oxide synthase, promoting vasodilation (Macvanin et al. 2023). However, increased IGF-1 levels have also been associated with shorter lifespan and a potentially higher incidence of neoplastic conditions (Greer et al. 2011; Song et al. 2021). In the present study, the higher IGF-1 levels observed with D_BSFL-C align with the tendency for lower systolic blood pressure detected in these dogs, whereas the lower IGF-1 levels in D_BSFL-A are in accordance with a lower IVSd observed. Indeed, effects observed on blood pressure might be attributed to ACE inhibition, as discussed earlier, and to modulation of IGF-1 levels. Although the mechanisms behind the influence of protein hydrolysates on IGF-1 production were not assessed in the present study, studies have been conducted in humans and animals investigating the effects of diet on IGF-1. Diet can stimulate or inhibit IGF-1 production through the activation or suppression of pathways such as the mammalian target of rapamycin (mTOR), modulation of circulating insulin levels and inflammatory mediators, as well as overall energy and protein intake (Grzeczka et al. 2025).

Further studies evaluating the presence of ACE inhibitory peptides in the BSFL hydrolysates studied here, as well as the effects of insect hydrolysates on gut microbiomes, may help elucidate the mechanisms underlying the observed trends in blood pressure. Based on current results, dietary inclusion of BSFL-C or BSFL-A hydrolysates tended to modulate serum ACE and IGF-1 levels, respectively. While these findings are promising, they require confirmation in longer term feeding trials, lasting three to twelve months (Haimovitz et al. 2022; Leach et al. 2023), and in dogs with naturally occurring cardiac disease.

Skin and coat quality

Skin and coat condition are highly responsive to dietary nutrients, with essential fatty acids (e.g. linoleic acid), AA (e.g. methionine and cystine), and key vitamins and minerals (e.g. vitamin A, zinc, and biotin) playing central roles in maintaining epidermal barrier integrity (Marchegiani et al. 2020). The effect of diet on skin and coat quality is outlined in Figure 2. All diets kept a good quality of skin and coat, with no adverse effects observed. The median scores of diets for gloss, greasiness, softness, scale, and general evaluation ranged between 3.5 and 4.8 out of 5, with greater scores being observed for scale in all diets. There were no differences between diets for the parameters evaluated except for greasiness, with D_BSFL-C showing a greater median score (4 out of 5) compared to dogs fed D_Shrimp (median score of 3.8 out of 5; P = 0.012). The mechanisms underlying the difference observed in greasiness remain unclear.

Figure 2.

Violin plot results of Kruskal–Wallis test comparing skin and coat quality parameters between dogs (n = 12) fed diets containing hydrolysates. The violin plot illustrates the distribution of the scores. Wider sections of the plot indicate a higher concentration of data points, and narrower sections indicate fewer data points. The median scores are represented by the line within the plot. D_Shrimp, complete diet with shrimp hydrolysate; D_BSFL-C, complete diet with black soldier fly (Hermetia illucens) larvae hydrolyzed with Corolase PP; D_BSFL-A, complete diet with black soldier fly (H. illucens) larvae hydrolyzed with ALCALASE 2.5 L.

However, it is essential to note that the duration of feeding trials is a relevant factor when assessing skin and coat quality. Previous feeding trials suggest that visible changes in skin and coat parameters may require feeding trials lasting 12 wk (Watson et al. 2018; Park et al. 2025). Also, analyzing other parameters to assess the impact of diet on skin health, such as measuring the transepidermal water loss, can help better elucidate the potential effects on skin and coat quality (Lee et al. 2021; Geary et al. 2022).

Conclusion

The present study provides novel insights into the functional potential of BSFL protein hydrolysates as dietary supplements for adult dogs, highlighting that their effects may vary according to the hydrolysis method employed. While D_BSFL-A improved diet digestibility and decreased fecal production, D_BSFL-C tended to decrease systolic blood pressure and serum ACE. However, these findings should be interpreted in light of several limitations. The number of animals was relatively limited for the assessment of palatability, and the duration of the experimental periods may have been insufficient to fully assess long-term effects on skin and coat quality and cardiovascular responses. In addition, all dogs enrolled in the study were clinically healthy at baseline, limiting the extrapolation of the findings to dogs with underlying health conditions, particularly cardiovascular disease. Further investigations in dogs with spontaneous cardiac disease or hypertension are warranted to determine clinical applicability. Future studies should also establish optimal dietary inclusion levels and characterize the bioactive peptide fractions to better elucidate the mechanisms underlying the functional effects of BSFL protein hydrolysates. Overall, this work supports the concept that enzymatically tailored insect protein hydrolysates may represent a versatile component of future functional canine nutrition.

Glossary

Abbreviations

AA

amino acid

ACE

angiotensin converting enzyme

Ao

aorta

Apd

pulmonary artery diameter

ATTD

apparent total tract digestibility

aV

augmented vector

BCS

body condition score

BSFL

black soldier fly larvae

BW

body weight

CP

crude protein

CRP

C-reactive protein

DM

dry matter

ECG

electrocardiogram

EE

ether extract

EF

ejection fraction

EPSS

E-point-to-septal separation interval

FS

fractional shortening

GE

gross energy

HR

heart rate

IGF-1

insulin-like growth factor 1

IVS

interventricular septum thickness

La

left atrium

LVID

left ventricle internal diameter

LVPW

left ventricle posterior wall

ME

metabolizable energy

NDF

neutral detergent fiber

NO

nitric oxide

PVd

pulmonary vein diameter

SCFA

short-chain fatty acids

TDF

total dietary fiber

Author contributions

Daniela Leal (Formal analysis, Investigation, Writing—original draft), Carolina Barroso (Formal analysis, Investigation, Writing—review & editing), Ana P. Fontes-Sousa (Formal analysis, Investigation, Writing—review & editing), Sandra Borges (Investigation, Writing—review & editing), André Almeida (Funding acquisition, Resources, Writing—review & editing), Manuela Pintado (Funding acquisition, Supervision, Writing—review & editing), António J.M. Fonseca (Funding acquisition, Project administration, Resources, Supervision, Writing—review & editing), and Ana Rita J. Cabrita (Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing—review & editing).

Funding

This work received financial support from the PT national funds (FCT/MECI, Fundação para a Ciência e Tecnologia and Ministério da Educação, Ciência e Inovação) through the project UID/50006 - Laboratório Associado para a Química Verde—Tecnologias e Processos Limpos, and project Bugs@Pets (POCI-01-0247-FEDER-047042) supported by Portugal 2020 through European Regional Development Fund. Sandra Borges was supported by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under contract ref. 2023.15056.TENURE.063. Additional support was provided through projects UID/50016/2025 and LA/P/0076/2020 (https://doi.org/10.54499/LA/P/0076/2020).

Conflict of interest statement. The authors declare no real or perceived conflicts of interest.

Data availability statement

The data underlying this article will be shared on reasonable request to the corresponding author.