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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%.

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.

In the present study, the immune-stimulatory effect of two levels of honey bee pollen (5 and 10%, P5 and P10 treatment, respectively) and its supercritical fluid extract (0. 5 and 1%, E0.5 and E1, respectively) included in the diet, was tested in gilthead seabream (Sparus aurata). The in vivo trial was preceded by the evaluation of antioxidant properties of three different bee pollen extracts obtained by water, ethanol 80%, and Supercritic Fluids Extraction (SFE). The preliminary evaluation attested that the SFE showed the lowest extraction yield (10.47%) compared to ethanol 80% (48.61%) and water (45.99%). SFE extract showed good antioxidant properties with high polyphenol content (13.06 mg GAE/g), radical scavenging activity (3.12 mg/ml), reducing power (38.68 mg/mL EC50). On the contrary, the water extract showed the significantly lowest polyphenol content (2 mg GAE/g; P < 0.05). The results of in vivo trial demonstrate that the diets supplemented with SFE bee pollen extract had a stimulatory effect on fish serum immunity, respect to the inclusion of raw pollen, this latter revealing some inhibitory effects in the immune response, such a decrease of serum peroxidase and lysozyme activities, particularly in P10 group significantly different (P < 0.05) from the control group. On the contrary, serum peroxidase, protease, antiprotease, were significantly increased in fish fed the diets supplemented with supercritical fluid extract, respect to the fish fed on control and on diets supplemented with 5 and 10% of raw pollen. For what concerns the bactericidal activity against Vibrio harveyii, all the treatments containing bee pollen regardless of the type showed their serum bactericidal activity significantly increased with respect to the control groups (p < 0.05). Given its high antioxidant properties, the absence of toxic solvents and the positive action carried out on improving the humoral response in gilthead seam bream, honey bee pollen SFE extract can be taken into account in the formulation of fish feeds.

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