Towards the Identification of a Suitable Commercial Diet for Carpione (Salmo carpio, Linnaeus 1758): A Multidisciplinary Study on Fish Performances, Animal Welfare and Quality Traits.

Carpione (Salmo carpio, Linnaeus 1758) is an endangered precious endemism of Lake Garda (Northern Italy), the largest Italian lake. To date, several bottlenecks about its culture remain unsolved, including the identification of a proper growth-out diet. The aim of the present study was to test four different grossly isolipidic, isoproteic, and isoenergetic diets in which the main ingredients had a different origin. Specifically, a diet currently used by local farmers for carpione culture, largely based on marine ingredients, was used as control (CTRL), while the other three diets were formulated by partially replacing marine ingredients with plant ones (VEG) or with different percentages of processed animal proteins (PAP1 and PAP2). The feeding trial was run in triplicate, over a three-month period. No significant differences in growth performance among the experimental groups were observed. However, remarkable histological alterations and inflammatory markers upregulation were observed in VEG group, while PAP inclusion played a role in attenuating inflammation and improving nutrient uptake. Fillet analyses highlighted significant differences in marketable traits and flesh fatty acid composition among the experimental groups, including the reduction of polyunsaturated fatty acids related to PAPs inclusion. In conclusion, PAPs used in the present study promoted S. carpio gut health and absorption capacity, while further studies are required to maintain proper quality traits of the final product.

Uterine leiomyoma as useful model to unveil morphometric and macromolecular collagen state and impairment in fibrotic diseases: An ex-vivo human study.

Collagen is one of the main components of the extracellular matrix (ECM), involved, among all, in the maintenance of the structural support of tissues. In fibrotic diseases, collagen is overexpressed, and its production determines the formation of a significantly stiffer ECM. The cross-linking of high-resolution analytical tools, able to investigate both the tridimensional organization and the secondary structure of collagen in fibrotic diseases, could be useful to identify defined markers correlating the status of this protein with specific pathological conditions. To this purpose, an innovative multidisciplinary approach based on Phase-Contrast MicroComputed Tomography, Transmission Electron Microscopy, and Fourier Transform Infrared Imaging Spectroscopy was exploited on leiomyoma samples and adjacent myometrium to characterize microstructural collagen features. Uterine leiomyoma is a common gynecological disorder affecting women in fertile age. It is characterized by a massive collagen production due to the repairing processes occurring at myometrium level, and, hence, it represents a valuable model to investigate collagen self-organization in a pathological condition. Moreover, to evaluate the sensitivity of this multidisciplinary approach, the effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) omega-3 fatty acids in collagen reduction were also investigated.

Low inclusion levels of Tenebrio molitor larvae meal in laying Japanese quail (Coturnix japonica, Gould, 1837) diet improve the intestinal morphometry, enzymatic activity and caecal short chain fatty acids profile.

One hundred twenty, 12 weeks old laying Japanese quails (JQ) were equally divided into 4 groups (6 replicates of 5 birds/group). The control group (CON) fed a corn-soybean diet; TML1.4, TML2.8, and TML5.6 groups fed a diet where a Tenebrio molitor larvae meal (TML) was included at 1.4, 2.8, and 5.6%, respectively. The trial lasted 54 days. The villi height (VH) and the crypt depth (CD) linearly decreased from the control to the TML5.6 group (P < 0.01) in the duodenum, while an opposite trend was observed for the Ab+ mucous cells count (P < 0.01). The highest VH/CD ratio was found in the TML1.4 group (P < 0.01). In the jejunum a lower VH was observed in the TML1.4 compared to the CON group; the Ab+ mucous cells increased (P < 0.01) according to the increase of the TM inclusion in the diet, while the highest VH/CD ratio (P < 0.01) was recorded in the TML2.8 group. In the duodenum only the L-ANP has been affected by TML (P < 0.001), with a quadratic and linear effect. The L-ANP is affected by the dietary treatment in the jejunum (P < 0.05), where showed a quadratic effect with the highest value in TML2.8. The % of butyric acid is maximized (P < 0.05) with the TML1.4 diet and that of the isobutyrate and valeric acids increased (P < 0.01) from TML1.4 to TML5.6. The use of TML at 1.4% in laying quail diets can be considered as a way to improve the intestinal health of the birds.

Effects of Di-Isononyl Phthalate (DiNP) on Follicular Atresia in Zebrafish Ovary.

Di-isononyl phthalate (DiNP) is a plasticizer reported to elicit hormone-like activity and disrupt metabolism and reproduction in fish and other vertebrates. In general, phthalates have been used at high concentrations beyond reported environmental levels to assess their adverse effects on fish gonadal physiology. The present study exposed adult female zebrafish to a wide range of DiNP concentrations [0.42 µg L-1 (10-9 M), 4.2 µg L-1 (10-8 M), and 42 µg L-1 (10-7 M)] for 21 days. We evaluated gene expression profiles related to apoptosis, autophagy, and oxidative stress; DNA fragmentation (TUNEL assay: terminal deoxynucleotidyl transferase dUTP nick end labeling) and caspase activity (CAS3) were also examined. Exposure to 0.42 and 4.2 µg L-1 upregulated the genes coding for tnfa and baxa, sod1, prkaa1, respectively. CAS3 immunohistochemistry revealed a higher number of positive vitellogenic oocytes in ovaries exposed to 0.42 µg L-1. Subsequently, we examined the relationship between CAS3 signaling and DNA fragmentation. Accordingly, DNA fragmentation was observed in vitellogenic follicles of fish exposed to 0.42 and 4.2 µg L-1. Our results demonstrate that follicular atresia can occur after exposure to environmental levels of DiNP for 21 days, which may adversely affect the reproductive performance of female zebrafish in a non-monotonic manner.

Appetite Regulation, Growth Performances and Fish Quality Are Modulated by Alternative Dietary Protein Ingredients in Gilthead Sea Bream (Sparus aurata) Culture.

By answering the need for increasing sustainability in aquaculture, the present study aimed to compare growth, gene expression involved in appetite regulation, physical characteristics, and chemical composition of Sparus aurata fed alternative protein sources. Fish were fed ten iso-proteic, iso-lipidic, and isoenergetic diets: a vegetable-based (CV) and a marine ingredient-rich (CF) diet were set as control diets. The others were prepared by replacing graded levels (10, 20 or 40%) of the vegetable proteins in the CV with proteins from a commercial defatted Hermetia illucens pupae meal (H), poultry by-product meal (PBM) singly (H10, H20, H40, P20, P40) or in combination (H10P30), red swamp crayfish meal (RC10) and from a blend (2:1, w:w) of Tisochrysis lutea and Tetraselmis suecica (MA10) dried biomasses. The increase in ghre gene expression observed in MA10 fed fish matched with increased feed intake and increased feed conversion ratio. Besides, the MA10 diet conferred a lighter aspect to the fish skin (p < 0.05) than the others. Overall, no detrimental effects of H, PBM, and RC meal included in the diets were observed, and fish fatty acid profile resulted as comparable among these groups and CV, thus demonstrating the possibility to introduce H, PBM, and RC in partial replacement of vegetable proteins in the diet for Sparus aurata.

Evaluation of the hair cell regeneration and claudin b and phoenix gene expression during exposure to low concentrations of cadmium and zinc in early developing zebrafish larvae.

Zebrafish possess hair cells on the body surface similar to that of mammals inner hear, in particular in the neuromasts, and due to its ability in regenerating damaged hair cells, is regularly used as a powerful animal model to study in vivo cytotoxicity. Among the factors leading to hair cell disruption, metal ions are of particular concern since they are important environmental pollutants. To date, several studies on zebrafish hair cell regeneration after metal exposure exist, while no data on regeneration during continuous metal exposure are available. In the present study, neuromast hair cell disruption and regeneration were assessed in zebrafish larvae for the first time during zinc (Zn) and cadmium (Cd) continuous exposure and a visual and molecular approach was adopted. Fluorescent vital dye DASPEI was used to assess hair cell regeneration and the gene expression of claudin b (cldnb) and phoenix (pho), was analyzed. Metallotionein-2 (mt2) gene expression was used as standard molecular marker of metal toxicity and confirmed the higher toxicity of Cd compared to Zn. In addition, Cd caused a delay in hair cell regeneration compared to Zn. Molecular analysis showed cldnb gene expression increased in relation to the metal concentrations used, confirming the involvement of this gene in hair cell regeneration. On the contrary, a dramatic decrease of pho gene expression was observed in Cd exposed groups, suggesting a negative impact of Cd on pho expression, thus negatively interfering with hair cell regeneration in zebrafish larvae exposed to this metal.

Possible Dietary Effects of Insect-Based Diets across Zebrafish (Danio rerio) Generations: A Multidisciplinary Study on the Larval Phase.

Insects represent a valuable and sustainable alternative ingredient for aquafeed formulation. However, insect-based diets have often highlighted controversial results in different fish species, especially when high inclusion levels were used. Several studies have demonstrated that nutritional programming through parental feeding may allow the production of fish better adapted to use sub-optimal aquafeed ingredients. To date, this approach has never been explored on insect-based diets. In the present study, five experimental diets characterized by increasing fish meal substitution levels with full-fat Black Soldier Fly (Hermetia illucens; BSF) prepupae meal (0%, 25%, 50%, 75% and 100%) were used to investigate the effects of programming via broodstock nutrition on F1 zebrafish larvae development. The responses of offspring were assayed through biometric, gas chromatographic, histological, and molecular analyses. The results evidenced that the same BSF-based diets provided to adults were able to affect F1 zebrafish larvae fatty acid composition without impairing growth performances, hepatic lipid accumulation and gut health. Groups challenged with higher BSF inclusion with respect to fish meal (50%, 75% and 100%) showed a significant downregulation of stress response markers and a positive modulation of inflammatory cytokines gene expression. The present study evidences that nutritional programming through parental feeding may make it possible to extend the fish meal substitution level with BSF prepupae meal in the diet up to almost 100% without incurring the well-known negative side effects of BSF-based diets.

Hermetia illucens and Poultry by-Product Meals as Alternatives to Plant Protein Sources in Gilthead Seabream (Sparus aurata) Diet: A Multidisciplinary Study on Fish Gut Status.

The attempt to replace marine-derived ingredients for aquafeed formulation with plant-derived ones has met some limitations due to their negative side effects on many fish species. In this context, finding new, sustainable ingredients able to promote fish welfare is currently under exploration. In the present study, poultry by-products and Hermetia illucens meal were used to replace the vegetable protein fraction in diets totally deprived of fish meal intended for gilthead seabream (Sparus aurata). After a 12-week feeding trial, a multidisciplinary approach including histological, molecular, and spectroscopic techniques was adopted to investigate intestine and liver responses to the different dietary formulations. Regardless of the alternative ingredient used, the reduction in dietary vegetable proteins resulted in a lower incidence of intestine histological alterations and inflammatory responses. In addition, the dietary inclusion of insect meal positively affected the reduction in the molecular inflammatory markers analyzed. Spectroscopic analyses showed that poultry by-product meal improved lipid absorption in the intestine, while insect meal induced increased liver lipid deposition in fish. The results obtained demonstrated that both poultry by-products and H. illucens meal can successfully be used to replace plant-derived ingredients in diets for gilthead seabream, promoting healthy aquaculture.

Physiological responses of Siberian sturgeon (Acipenser baerii) juveniles fed on full-fat insect-based diet in an aquaponic system.

Over the last years, the potential use of Black Soldier Fly meal (BSF) as a new and sustainable aquafeed ingredient has been largely explored in several fish species. However, only fragmentary information is available about the use of BSF meal-based diets in sturgeon nutrition. In consideration of a circular economy concept and a more sustainable aquaculture development, the present research represents the first comprehensive multidisciplinary study on the physiological effects of a BSF diet during sturgeon culture in an aquaponic system. Siberian sturgeon (Acipenser baerii) juveniles were fed over a 60-days feeding trial on a control diet (Hi0) and a diet containing 50% of full-fat BSF meal respect to fish meal (Hi50). Physiological responses of fish were investigated using several analytical approaches, such as gas chromatography-mass spectrometry, histology, Fourier Transformed Infrared Spectroscopy (FTIR), microbiome sequencing and Real-time PCR. While aquaponic systems performed optimally during the trial, Hi50 group fish showed lower diet acceptance that resulted in growth and survival reduction, a decrease in hepatic lipids and glycogen content (FTIR), a higher hepatic hsp70.1 gene expression and a worsening in gut histological morphometric parameters. The low feed acceptance showed by Hi50 group sturgeon highlighted the necessity to improve the palatability of BSF-based diet designed for sturgeon culture.

Polydatin Beneficial Effects in Zebrafish Larvae Undergoing Multiple Stress Types.

Polydatin is a polyphenol, whose beneficial properties, including anti-inflammatory and antioxidant activity, have been largely demonstrated. At the same time, copper has an important role in the correct organism homeostasis and alteration of its concentration can induce oxidative stress. In this study, the efficacy of polydatin to counteract the stress induced by CuSO4 exposure or by caudal fin amputation was investigated in zebrafish larvae. The study revealed that polydatin can reduced the stress induced by a 2 h exposure to 10 µM CuSO4 by lowering the levels of il1b and cxcl8b.1 and reducing neutrophils migration in the head and along the lateral line. Similarly, polydatin administration reduced the number of neutrophils in the area of fin cut. In addition, polydatin upregulates the expression of sod1 mRNA and CAT activity, both involved in the antioxidant response. Most of the results obtained in this study support the working hypothesis that polydatin administration can modulate stress response and its action is more effective in mitigating the effects rather than in preventing chemical damages.

Anti-inflammatory effect of a flavonoid-rich extract of orange juice in adult zebrafish subjected to Vibrio anguillarum-induced enteritis.

Inflammation-related pathologies remain a serious health problem with high costs for the community. Citrus flavonoids are known to possess important pharmacological properties, including anti-inflammatory activity. In this study we evaluated the effects of a flavonoid-rich extract of orange juice (OJe) in an experimental model of enteritis induced by Vibrio anguillarum in adult zebrafish (Danio rerio). Administration of V. anguillarum through live feed (Artemia nauplii) for three consecutive days caused evident signs of enteritis in zebrafish. Three days of treatment with OJe before the pathogenic insult resulted in a remarkable reduction of tissue inflammatory events as well as a molecular down-regulation of the inflammatory genes such as IL-1ß, IL-6 and TNFα. Our data suggest that OJe counteracts the inflammation of zebrafish intestinal mucosa, indicating that the pool of flavonoids present in orange juice could be useful for the prevention of enteritis.

Can Insect-Based Diets Affect Zebrafish (Danio rerio) Reproduction? A Multidisciplinary Study.

Black Soldier Fly (BSF) meal is considered an alternative, emerging, and sustainable ingredient for aquafeed formulation. However, results on fish physiological responses are still fragmentary and often controversial, and no data are available on the effect of insect meal-based diets on fish reproduction. On this regard, zebrafish, with its relatively short life cycle, represents an ideal experimental model to explore this topic. In this study, female zebrafish were fed for 12 months on a control diet based on fish meal (FM) and fish oil and two experimental diets with full-fat BSF (Hermetia illucens) prepupae meal inclusion, to replace 25% and 50% of FM (BSF25 and BSF50). All diets were isonitrogenous, isolipidic, and isoenergetic. The effects of these two experimental diets on female's reproduction were investigated through a multidisciplinary approach, including the evaluation of growth, gonadosomatic index, spawned/fertilized eggs and hatching rate, adult female carcass and fertilized egg fatty acid composition, histological analysis of the ovary, spectroscopic macromolecular composition of class IV oocytes, and expression of genes involved in fish lipid metabolism in the liver. Results showed that while fish were perfectly able to cope with a 25% insect meal dietary inclusion, a 50% inclusion level caused the overexpression of genes involved in lipid metabolism, a general reduction in the number of spawned eggs, and differences in the frequency rate of previtellogenic oocytes, class III, IV, oocytes and postovulatory follicles and atretic oocytes, in the macromolecular composition of class IV oocytes, and in the fatty acid composition of the fertilized eggs, respect to control and 25% group.

Zebrafish (Danio rerio) physiological and behavioural responses to insect-based diets: a multidisciplinary approach.

Black Soldier Fly (BSF) meal is considered as an alternative, emerging and sustainable ingredient for aquafeed production. However, results on fish physiological responses are still fragmentary and often controversial, while no studies are available on fish behavior in response to these new diets. The present work represents the first comprehensive multidisciplinary study aimed to investigate zebrafish physiological and behavioural responses to BSF-based diets. Five experimental diets characterized by increasing inclusion levels (0, 25, 50, 75 and 100% respect to fish meal) of full fat BSF prepupae meal were tested during a 2-months feeding trial. Prepupae were cultured on coffee silverskin growth substrate enriched with a 10% Schizochytrium sp. to improve insects' fatty acids profile. The responses of zebrafish were assayed through biometric, histological, gas chromatographic, microbiological, spectroscopic, molecular and behavioural analyses. Results evidenced that BSF-based diets affected fish fatty acid composition, while behavioural tests did not show differences among groups. Specifically, a 50% BSF inclusion level diet represented the best compromise between ingredient sustainability and proper fish growth and welfare. Fish fed with higher BSF inclusions (75 and 100%) showed hepatic steatosis, microbiota modification, higher lipid content, fatty acid modification and higher expression of immune response markers.

Honey Bee Pollen in Meagre (Argyrosomus regius) Juvenile Diets: Effects on Growth, Diet Digestibility, Intestinal Traits, and Biochemical Markers Related to Health and Stress.

This research aimed to evaluate the effects of the inclusion of honey bee pollen (HBP) in meagre (Argyrosoumus regius) juveniles' diets on growth performance, diet digestibility, intestinal morphology, and immunohistochemistry. Furthermore, stress-related molecular markers and biochemical blood profile of fish were evaluated, together with mineral trace and toxic element concentration in pollen and diets. Specimens of meagre (360) of 3.34 ± 0.14 g initial body weight, were randomly allocated to twelve 500 L circular tanks (30 fish per tank). Four diets were formulated: a control diet and three experimental diets with 1%, 2.5%, and 4% of HBP inclusion. All the growth parameters and crude protein and ether extract digestibility coefficients were negatively linearly affected by increased HBP inclusion (p < 0.0001). Histology of medium intestine showed slight signs of alterations in group HPB1 and HPB2.5 compared to control. Fish from HBP4 group showed severe alterations at the intestinal mucosa level. Immunohistochemical detection of TNF-α in the medium intestine showed the presence of TNF-α+ cells in the lamina propria, which resulted in accordance with the increased level of the TNF-α protein detected by immunoblotting in the liver. This stress situation was confirmed by the increased hepatic level of HSP70 (p < 0.05) in fish fed the HBP4 diet and by the linear decrease of total serum protein levels in HBP-containing diets (p < 0.0001). These negative effects can be related to the ultrastructure of the bee pollen grain walls, which make the bioactive substances unavailable and can irritate the intestine of a carnivorous fish such as meagre.

Dietary diisononylphthalate contamination induces hepatic stress: a multidisciplinary investigation in gilthead seabream (Sparus aurata) liver.

In this study, adult gilthead seabream (Sparus aurata) were exposed for 21 days to Di-iso-nonylphthalte (DiNP at 15 and 1500 µg kg-1 bw day-1) via the diet. This plastic additive has been recently introduced to replace the di-(2-ethylhexyl)phthalate, the toxicity of which has been demonstrated conclusively both in vivo and in vitro trials. An analysis of a set of biomarkers involved in stress and immune response provides evidence of hepatic toxicity by DiNP in the present study. Both hsp70 and gr mRNA levels were upregulated significantly by DiNP, while plasma cortisol increased only in fish fed with the lowest DiNP dose. The oxidative stress markers g6pdh, glut red, gpx1 and CAT were upregulated by DiNP; gst mRNA was induced by the high dose and gck mRNA was downregulated significantly by the low dose. The mRNA levels of genes involved in the immune response, such as pla2, 5-lox, tnfa and cox2, were upregulated significantly only by the high dose of DiNP, while il1 mRNA increases in both doses. These molecular evidences were complemented with features obtained by Fourier Transform Infrared Imaging (FTIRI) analysis regarding the hepatic distribution of the main biological macromolecules. The FTIRI analysis showed an alteration of biochemical composition in DiNP samples. In particular, the low dose of DiNP induced an increase of saturated and unsaturated lipids and phosphorylated proteins, and a decrease of glycogen levels. The levels of caspase did not change significantly in the study, suggesting that DiNP does not activate apoptosis. Finally, the results also suggested the onset of hepatic oxidative stress and the activation of immune response, adding new knowledge to the already described hepatic DiNP toxicity.

A six-months study on Black Soldier Fly (Hermetia illucens) based diets in zebrafish.

Intensive fish farming relies on the use of feeds based on fish meal and oil as optimal ingredients; however, further development of the aquaculture sector needs new, nutritious and sustainable ingredients. According to the concept of circular economy, insects represent good candidates as aquafeed ingredients since they can be cultured through environmental-friendly, cost-effective farming processes, on by-products/wastes, and many studies have recently been published about their inclusion in fish feed. However, information about the physiological effects of insect-based diets over the whole life cycle of fish is presently missing. At this regard, the present study investigated, for the first time, the effects of Black Soldier Fly based diets (25 and 50% fish meal substitution) administration for a six months period in zebrafish (Danio rerio), from larvae to adults. A multidisciplinary approach, including biometric, biochemical, histological, spectroscopic and molecular analyses was applied. Aside a general reduction in fish growth and lipid steatosis, six-months feeding on Black Soldier Fly based diets did not show major negative effects on zebrafish. Gut histological analysis on intestine samples did not show signs of inflammation and both stress markers and immune response markers did not show significant differences among the experimental groups.

Safety assessment of antibiotic administration by magnetic nanoparticles in in vitro zebrafish liver and intestine cultures.

Different in vitro models have been suggested to replace in vivo studies. In vitro studies are of great interest and give the opportunity to analyze cellular responses in a closed system with stable experimental conditions and to avoid direct animal exposure and distress during the experiments. These methods are useful to test drugs and chemicals toxicity in order to better understand their environmental impact. In the present study, fish organ cultures have been used to test different oxytetracycline exposure methods, including oxide nanoparticles (IONPs), using zebrafish as experimental model. Results showed that oxytetracycline accumulation at the end of the experiment (24 h) in the exposed organs did not show any significant difference in the analyzed samples and was not dependent on the exposure way (free or IONPs-bound oxytetracycline). However, as regards molecular analysis, the different exposure ways tested in this study showed some differences in the expression of genes involved in stress response. The present data did not completely agree with a previous in vivo study performed in zebrafish using IONPs, underlying that replacement of in vivo models with in vitro studies cannot always represent the complexity of interactions typical of a biological system.

Effects of Graded Dietary Inclusion Level of Full-Fat Hermetia illucens Prepupae Meal in Practical Diets for Rainbow Trout (Oncorhynchus mykiss).

This study investigated the effects of dietary inclusion levels of full-fat Hermetia illucens prepupae meal (H) on growth and gastrointestinal integrity in rainbow trout (Oncorhynchus mykiss). A 98-day study was conducted using triplicate groups of trout (initial body weight, 137 ± 10.5 g) kept in 1-m3 tanks in a flow-through well water system. Three dietary treatments were prepared: one based on fishmeal and purified protein-rich vegetable ingredients (H0), and two experimental diets including graded levels of H meal (25% and 50%, referred to as H25 and H50, respectively). At the end of the feeding trial, no differences were observed in growth performance and plasma metabolite levels, with the biometric data confirmed by the liver expression of the genes involved in somatic growth regulation (igf1 and mstn1a). In the H50 group, a three-fold up regulation of liver hsp70 was observed. An activation of the stress/immune response (il-10, tnf-α, and tlr-5) was observed in medium intestine in the H25 and H50 groups (p < 0.05) together with a villi length reduction detected through histological analyses. Liver histology and Fourier Transform Infrared Imaging (FTIRI) spectroscopy highlighted an increase in lipid deposition. These findings suggest that caution should be taken into account when 50% replacement of conventional ingredients with H is selected.

Intestinal Morphometry, Enzymatic and Microbial Activity in Laying Hens Fed Different Levels of a Hermetia illucens Larvae Meal and Toxic Elements Content of the Insect Meal and Diets.

To evaluate the effects of feeding a Hermetia illucens (HI) larvae meal on the different intestinal traits of hens, and to determine the toxic elements' concentration in the insect meal and diets, 162 hens were randomly allotted to three groups. The control received a corn-soybean meal-based diet (SBM); the HI25 and HI50 groups received two diets in which the 25% and 50% of the dietary protein were replaced by the HI protein, respectively. The duodenal and jejunal villi height and villi/crypt were higher (p < 0.01) in the SBM than in the HI groups. The ileal villi height was higher (p < 0.05) in the SBM and HI25 groups than the HI50. The HI50 group exhibited a lower duodenal maltase activity. The intestinal alkaline phosphatase (IAP) activity linearly decreased in the duodenum and jejunum as the dietary insect meal inclusion increased. The HI50 group had a higher acetate and butyrate level than the SBM. The levels of cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As) in the diets and insect meal were lower than the maximum values established by the EU Commission. The 25% soybean protein replacement with Hermetia illucens larvae meal in the diet of laying hens was more suitable and closer to the optimal level than 50%.

Presence and distribution of leptin and its receptor in the gut of adult zebrafish in response to feeding and fasting.

Leptin is an anorectic hormone secreted mainly by peripheral adipocytes but also by other central and peripheral tissues. It acts by means of a receptor called OB-R, influencing not only appetite and body mass but being also involved in many fields like endocrinology, metabolism and reproduction. Immunohistochemistry and qRT-PCR techniques were, respectively, used to demonstrate the presence of leptin and its receptor in the gut of adult zebrafish and to evaluate the leptin gene expression response to feeding and fasting. Immunoreactivity for the antibodies utilized was demonstrated in feeding but not in fasting fish, and the gene expression analysis corroborates the data obtained by immunohistochemistry. Therefore, all the obtained results support the hypothesis of the role of this hormone in food regulation in zebrafish.