Molecular approach for insect detection in feed and food: the case of Gryllodes sigillatus

The production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method\u2019s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = 123.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food

Pea protein concentrate as a substitute for fish meal protein in sea bass diet

Pea seeds, even if lower in protein than oilseed meals, have been shown to successfully replace moderate amounts of fish meal protein in diets for carnivorous fish species (Kaushik et al., 1993, Gouveia and Davies, 2000). A further processing of such pulses provides concentrated protein products which look very promising as fish meal substitutes in aquafeeds (Thiessen et al., 2003). The aim of the present study was to evaluate nutrient digestibility, growth response, nutrient and energy retention efficiencies and whole body composition of sea bass (Dicentrarchus labrax, L.) fed complete diets in which a pea protein concentrate (PPC) was used to replace graded levels of fish meal protein

Including marine microalgae in European seabass (D. labrax) diets: effects on digestive-absorptive functions

Marine microalgae deserve increasing attention as fish feed ingredients or supplements, due to their nutritional value and functional properties. They were recently shown to improve intestinal morphophysiology, which is often challenged in fish fed diets high in protein-rich plant feedstuffs. The aim of this study was to evaluate the effects of supplemental dried marine microalgae on gut histology and expression of genes encoding brush border membrane enzymes and transporters, in E. sea bass fed diets low in fish meal and supplying substantial levels of plant protein feedstuffs. Two test diets (A1 and A2) were prepared by including a blend of Tisochrysis lutea and Tetraselmis suecica dried biomass (2:1 w:w ratio) to replace 15 and 45% fish meal protein and 10 and 30% fish lipid of a control diet (C+) containing 50:50 fish to vegetable protein and lipid ratios. A negative control (C-) preparation was also formulated with a 30:70 fish to vegetable protein ratio. One hundred and forty four sea bass (mean weight 204.3\ub10.78g) were divided among 12 tanks connected to a recirculating aquaculture system ensuring optimal rearing conditions (T, 23.8\ub0C; Salinity 30 ppt). Fish groups were fed the test diets to visual satiety over 105 days according to a randomized design with 3 replicates per dietary treatment. At the end of the trial, 6 fish per treatment were euthanized; the digestive tract removed, divided into pyloric caeca (PC), foregut (FG) and hindgut (HG) sections, and frozen in liquid N for gene expression analysis. Subsamples of gut tissue were also collected for histological evaluation. From histological analyses fish fed diet C- had the lowest villi thickness (P<0.05) while those given diets including microalgae, irrespective of the inclusion level, resulted in greater villi length than that observed in either positive or negative controls. Irrespective of the dietary treatment, gene expressions of sucrase-isomaltase, PepT1, Na+/K+-ATPase and APN were highest in the foregut. Regardless of the gut section, gene expression of the same enzymes-transporter was higher in fish fed microalgae-containing diets (P<0.05) when compared to both controls. The present results indicate that even at the lower dietary inclusion level investigated here, a mix of T. lutea e T. suecica resulted in potentially improved digestive-absorptive functions. Further studies are needed to understand the mechanisms underlying the observed positive effects of dietary microalgae addition

Compensatory growth following long term multi-phase cyclic feeding in rainbow trout (Oncorhynchus mykiss)

Compensatory growth (CG) during recovery from feed deprivation is a well-known phenomenon in fish, making the practice of cyclic feed restriction-refeeding a possible tool for aquaculturists to optimize growth performance. While earlier studies in this direction focused on relatively short single feed restriction-refeeding protocols, the present trial was designed to evaluate the impact of different repeated cyclic feeding schemes on the zootechnical response of rainbow trout (O. mykiss) over a complete growing phase up to the commercial size. Three hundred trout (body weight 72\ub16 g) were randomly distributed among 12 tanks, each of 0.5 m3 capacity and supplied with 8 L min-1 of well water at a temperature of 12.7\ub10.8\ub0C. Triplicated groups of fish were subjected over 27 weeks to one of the following treatments: C, control, continuous feeding to visual satiety 6 days a week; T1, cyclic feeding regularly alternating 1 week starvation (S) and 3 weeks refeeding (F) (1S+3F); T2, cyclic feeding consisting in 3 consecutive phases: 1S+3F, 2S+6F, and 3S+12F; T3, where a feed restriction (70% of the satiety level observed in the previous week) was applied instead of starvation with the same schedule as T2. The same trout feed (45% crude protein, 28% crude lipid) was used throughout the trial. At the end of the trial the different cyclic feeding protocols resulted in the same zootechnical outcome (P>0.05). A nearly complete convergence of body mass was evident as no significant differences were found among treatments in individual weight (543\ub128g), specific growth rate (1.06\ub10.03%), feed conversion ratio (0.84\ub10.03) and protein efficiency ratio (2.64\ub10.12) despite a lower feed consumption in treatments T1, T2 and T3 relative to controls (357 vs. 390 g fish-1, P<0.05). All protocols imposing fasting or feed restriction resulted in CG at the end of each re-feeding phase. Hyperphagia was a major cause of CG. Both phenomena were emphasized with treatment T3 after recovering from the last 3-week fasting period, when they were associated to a marked improvement of feed conversion ratio relative to controls (0.75 vs. 0.85, P<0.05). The results obtained so far suggest repeated cyclic feeding as a reliable practice in trout farming, provided fasting or feed restriction periods are followed by refeeding phases of suitable length to allow recovery of body mass. This could result in improved profitability and environmental sustainability

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

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

Welfare and quality of farmed trout fed high plant protein diets. 2 innovative killing method effect on stress and quality indicators

AbstractThe fish stunning/slaughtering procedure has an important role both for the ethical aspect related to animal welfare and for the potential quality of the final products. Stressful harvest procedure and killing methods can negatively influence the post mortem biochemical processes with a consequent faster fish freshness loss. In particular, killing procedures causing a long agony are not humane and can shorten fish shelf life; others, more humane, can have risks for the consumers health (anaesthetics or chemical substances) or are not feasible in small/medium size fish industry (spiking and knocking). The aim of this study was to compare the traditional killing method (asphyxia - A) used for rainbow trout (Onchorynchus mykiss) with an innovative stunning/slaughtering method (two-stage electric stun: 2s at a 500Hz electric field of 2.5V/cm and then 58s at a 50Hz electric field of 0.75V/cm - E), through the study of their effects on stress and quality indicators in fish fed with traditional fish meal..

Processed Animal Proteins from Insect and Poultry By-Products in a Fish Meal-Free Diet for Rainbow Trout: Impact on Intestinal Microbiota and Inflammatory Markers

Sustainability of aquaculture is tied to the origin of feed ingredients. In search of sustainable fish meal-free formulations for rainbow trout, we evaluated the effect of Hermetia illucens meal (H) and poultry by-product meal (P), singly (10, 30, and 60% of either H or P) or in combination (10% H + 50% P, H10P50), as partial replacement of vegetable protein (VM) on gut microbiota (GM), inflammatory, and immune biomarkers. Fish fed the mixture H10P50 had the best growth performance. H, P, and especially the combination H10P50 partially restored \u3b1-diversity that was negatively affected by VM. Diets did not differ in the Firmicutes:Proteobacteria ratio, although the relative abundance of Gammaproteobacteria was reduced in H and was higher in P and in the fishmeal control. H had higher relative abundance of chitin-degrading Actinomyces and Bacillus, Dorea, and Enterococcus. Actinomyces was also higher in H feed, suggesting feed-chain microbiome transmission. P increased the relative abundance of protein degraders Paeniclostridium and Bacteroidales. IL-1\u3b2, IL-10, TGF-\u3b2, COX-2, and TCR-\u3b2 gene expression in the midgut and head kidney and plasma lipopolysaccharide (LPS) revealed that the diets did not compromise the gut barrier function or induce inflammation. H, P, and H10P50 therefore appear valid protein sources in fishmeal-free aquafeeds