Carolina Barroso

Industrial processing of tomato generates large amounts of tomato pomace (TP), whose disposal and stabilization are challenging due to its high moisture content. This study evaluated, for the first time, the effects of increasing inclusion levels of enzymatic pre-treatment, vacuum paddle-dehydrated TP (ETP), in extruded diets for adult dogs. Three diets containing 0%, 2% or 4% ETP, replacing wheat bran and sunflower meal, were produced. Three two-bowl tests assessed palatability, and a feeding trial was performed using a four 3 × 3 Latin square design with 12 healthy adult Beagle dogs across three 28-day periods. Inclusion of ETP had negligible effects on the chemical composition of diets and kept unaffected palatability, body weight, food intake, fecal consistency and output. Fecal pH was lower, and valerate proportion was higher, in dogs fed the 2% ETP diet. All diets exhibited high digestibility (>90%) without effects of dietary treatments. Overall, ETP, a locally sourced co-product of the food industry, may represent a more sustainable alternative to imported raw materials, although further studies are needed to explore its effects on fecal microbiota and health-related parameters.

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.

The inclusion of protein hydrolysates from animal by-products in petfood may enhance the industry’s sustainability, while contributing to dogs’ health due to their nutritional and functional properties. However, inconsistent results of protein hydrolysates on gut microbiota, immune responses, among other health parameters, have been observed in dogs. This study aimed to evaluate the effects of diets supplemented with 5% shrimp hydrolysate, compared to non-supplemented diets, on hematological parameters, serum chemistry profile, innate and adaptive immune function, and fecal microbiota composition in adult Beagle dogs during a 12-week feeding trial. Dietary inclusion of shrimp hydrolysate decreased blood eosinophils (P = 0.017) and glucose levels (P = 0.023), while increasing levels of white blood cells (P = 0.002), platelets (P = 0.038), neutrophils (P = 0.036), and CD4⁺ (P < 0.001) and CD8⁺ T cells (P < 0.001) single producers of tumor necrosis factor-alpha. It also led to a more extensive proliferation of CD4⁺ T cells in response to LipL32 antigen (P = 0.020) and to a decrease in superoxide production in stimulated cells (P = 0.002). Additionally, supplemented diets increased Oscillospiraceae (P-adj = 0.005) and Clostridia (P-adj = 0.017) abundance, and decreased such of Sellimonas (P-adj = 0.025). Overall, the results indicate that diet supplementation with shrimp hydrolysate at 5% modulates the immune response and fecal microbiota, highlighting its potential to be included in hypoallergenic and gastrointestinal diets, and in diets for diabetic dogs.

Trends in the pet food industry are driven by the humanization of pets, favoring the inclusion of functional ingredients or supplements that promote animal health. Several commercial diets claim to include supplements with benefits for dogs’ immune function, but in vivo evidence that supports their efficacy remains limited. This literature review aimed to better understand the current knowledge on the effects of vitamins, minerals and phytonutrients on dogs’ immune function. A total of 27 peer-reviewed articles were identified in PubMed and Web of Science databases. Although vitamin supplementation is often claimed to support immune function, only two studies promoting slight benefits of vitamins C and E were found. The limited research on minerals suggests that organic sources promote a better immune response. Studies evaluating the inclusion of different phytonutrients show that these compounds might exert immunomodulatory and anti-inflammatory effects. Despite the increased popularity of commercial diets claimed to support the immune response of dogs, further research is needed in order to substantiate their effects. This knowledge will contribute to the development of effective diets to enhance immune health in dogs.

Locally produced fish hydrolysate and oil from the agrifood sector comprises a sustainable solution both to the problem of fish waste disposal and to the petfood sector with potential benefits for the animal’s health. This study evaluated the effects of the dietary replacement of mainly imported shrimp hydrolysate (5%) and salmon oil (3%; control diet) with locally produced fish hydrolysate (5%) and oil (3.2%) obtained from fish waste (experimental diet) on systemic inflammation markers, adipokines levels, cardiac function and fecal microbiota of adult dogs. Samples and measurements were taken from a feeding trial conducted according to a crossover design with two diets (control and experimental diets), six adult Beagle dogs per diet and two periods of 6 weeks each. The experimental diet, with higher docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids contents, decreased plasmatic triglycerides and the activity of angiotensin converting enzyme, also tending to decrease total cholesterol. No effects of diet were observed on serum levels of the pro-inflammatory cytokines interleukin (IL)-1β, IL-8, and IL-12/IL-23 p40, and of the serum levels of the anti-inflammatory adipokine adiponectin. Blood pressure, heart rate and echocardiographic measurements were similar between diets with the only exception of left atrial to aorta diameter ratio that was higher in dogs fed the experimental diet, but without clinical relevance. Diet did not significantly affect fecal immunoglobulin A concentration. Regarding fecal microbiome, Megasphaera was the most abundant genus, followed by Bifidobacterium, Fusobacterium, and Prevotella, being the relative abundances of Fusobacterium and Ileibacterium genera positively affected by the experimental diet. Overall, results from the performed short term trial suggest that shrimp hydrolysate and salmon oil can be replaced by protein hydrolysate and oil from fish by-products without affecting systemic inflammatory markers, cardiac structure and function, but potentially benefiting bacterial genera associated with healthy microbiome. Considering the high DHA and EPA contents and the antioxidant properties of fish oil and hydrolysate, it would be worthwhile in the future to assess their long-term effects on inflammatory markers and their role in spontaneous canine cardiac diseases and to perform metabolomic and metagenomics analysis to elucidate the relevance of microbiota changes in the gut.

Erythroferrone is a recently identified erythroid regulator produced by erythroblasts in the mammalian bone marrow and extramedullary sites, known to be induced in conditions of anemia or blood loss. Iron metabolism is affected by erythroferrone through its capacity to inhibit hepcidin production, leading to the increase of iron availability required for erythropoiesis. However, little is known about erythroferrone function in other vertebrates, in particular teleost fish, that unlike mammals, present two different functional types of hepcidin, one type mostly involved in iron metabolism and the other in antimicrobial response. The study of erythroferrone evolution and its biological role in teleost fish can give us valuably new insights into its function. To address these questions, we characterized erythroferrone in the European sea bass (Dicentrarchus labrax), a species presenting two hepcidin types, and evaluated variations in its expression levels in response to different experimental conditions. During experimental anemia, erythroferrone responds by increasing its expression and suppressing hepcidin production, following the pattern observed in mammals, but it is not influenced by iron overload. However, during bacterial infection, erythroferrone is downregulated and hepcidin levels increase. Furthermore, administration of Hamp1 but not of Hamp2 peptides suppresses erythroferrone expression. In conclusion, in dual hepcidin teleost fish erythroferrone seems to only interact with type 1 hepcidin, known to be involved in iron homeostasis, but not with type 2, which has an almost exclusive antimicrobial role.

The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (Dicentrarchus labrax), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection.

Beta-defensins consist in a group of cysteine-rich antimicrobial peptides (AMPs), widely found throughout vertebrate species, including teleost fish, with antimicrobial and immunomodulatory activities. However, although the European sea bass (Dicentrarchus labrax) is one of the most commercially important farmed fish species in the Mediterranean area, the characterization of its beta-defensins and its potential applications are still missing. In this study, we characterized two members of the beta-defensin family in this species. Phylogenetic and synteny analysis places sea bass peptides in the beta-defensin subfamilies 1 and 2, sharing similar features with the other members, including the six cysteines and the tertiary structure, that consists in three antiparallel beta-sheets, with beta-defensin 1 presenting an extra alpha-helix at the N-terminal. Further studies are necessary to uncover the functions of sea bass beta-defensins, particularly their antimicrobial and immunomodulatory properties, in order to develop novel prophylactic or therapeutic compounds to be used in aquaculture production.

Fish rely on their innate immune responses to cope with the challenging aquatic environment, with antimicrobial peptides (AMPs) being one of the first line of defenses. Piscidins are a group of fish specific AMPs isolated in several species. However, in the European sea bass (Dicentrarchus labrax), the piscidin family remains poorly understood. We identified six different piscidins in sea bass, performed an in-depth molecular characterization and evaluated their antimicrobial activities against several bacterial and parasitic pathogens. Sea bass piscidins present variable amino acid sequences and antimicrobial activities, and can be divided in different sub groups: group 1, formed by piscidins 1 and 4; group 2, constituted by piscidins 2 and 5, and group 3, formed by piscidins 6 and 7. Additionally, we demonstrate that piscidins 1 to 5 possess a broad effect on multiple microorganisms, including mammalian parasites, while piscidins 6 and 7 have poor antibacterial and antiparasitic activities. These results raise questions on the functions of these peptides, particularly piscidins 6 and 7. Considering their limited antimicrobial activity, these piscidins might have other functional roles, but further studies are necessary to better understand what roles might those be.