Identification of Single-Nucleotide Polymorphisms in Differentially Expressed Genes Favoring Soybean Meal Tolerance in Higher-Growth Zebrafish (Danio rerio).

Genetic variability within the same fish species could confer soybean meal (SBM) tolerance in some individuals, thus favoring growth. This study investigates the single-nucleotide polymorphisms (SNPs) in differentially expressed genes (DEGs) favoring SBM tolerance in higher-growth zebrafish (Danio rerio). In a previous work, nineteen families of zebrafish were fed a fish meal diet (100FM control diet) or SBM-based diets supplemented with saponin (50SBM + 2SPN-experimental diet), from juvenile to adult stages. Individuals were selected from families with a genotype-by-environment interaction higher (170 ± 18 mg) or lower (76 ± 10 mg) weight gain on 50SBM + 2SPN in relation to 100FM. Intestinal transcriptomic analysis using RNA-seq revealed six hundred and sixty-five differentially expressed genes in higher-growth fish fed 50SBM + 2SPN diet. In this work, using these results, 47 SNPs in DEGs were selected. These SNPs were genotyped by Sequenom in 340 zebrafish that were fed with a 50SBM + 2SPN diet or with 100FM diet. Marker-trait analysis revealed 4 SNPs associated with growth in 3 immunity-related genes (aif1l, arid3c, and cst14b.2) in response to the 50SBM + 2SPN diet (p-value < 0.05). Two SNPs belonging to aif1l y arid3c produce a positive (+19 mg) and negative (-26 mg) effect on fish growth, respectively. These SNPs can be used as markers to improve the early selection of tolerant fish to SBM diet or other plant-based diets. These genes can be used as biomarkers to identify SNPs in commercial fish, thus contributing to the aquaculture sustainability.

Enhancing Selenium Accumulation in Rhodotorula mucilaginosa Strain 6S Using a Proteomic Approach for Aquafeed Development.

It is known that selenium (Se) is an essential trace element, important for the growth and other biological functions of fish. One of its most important functions is to contribute to the preservation of certain biological components, such as DNA, proteins, and lipids, providing protection against free radicals resulting from normal metabolism. The objective of this study was to evaluate and optimize selenium accumulation in the native yeast Rhodotorula mucilaginosa 6S. Sodium selenite was evaluated at different concentrations (5-10-15-20-30-40 mg/L). Similarly, the effects of different concentrations of nitrogen sources and pH on cell growth and selenium accumulation in the yeast were analyzed. Subsequently, the best cultivation conditions were scaled up to a 2 L reactor with constant aeration, and the proteome of the yeast cultured with and without sodium selenite was evaluated. The optimal conditions for biomass generation and selenium accumulation were found with ammonium chloride and pH 5.5. Incorporating sodium selenite (30 mg/L) during the exponential phase in the bioreactor after 72 h of cultivation resulted in 10 g/L of biomass, with 0.25 mg total Se/g biomass, composed of 25% proteins, 15% lipids, and 0.850 mg total carotenoids/g biomass. The analysis of the proteomes associated with yeast cultivation with and without selenium revealed a total of 1871 proteins. The results obtained showed that the dynamic changes in the proteome, in response to selenium in the experimental medium, are directly related to catalytic activity and oxidoreductase activity in the yeast. R. mucilaginosa 6S could be an alternative for the generation of selenium-rich biomass with a composition of other nutritional compounds also of interest in aquaculture, such as proteins, lipids, and pigments.

Selenized non-Saccharomyces yeasts and their potential use in fish feed.

Selenium (Se) is a vital trace element, essential for growth and other biological functions in fish. Its significance lies in its role as a fundamental component of selenoproteins, which are crucial for optimal functioning of the organism. The inclusion of Se in the diets of farmed animals, including fish, has proved invaluable in mitigating the challenges arising from elemental deficiencies experienced in captivity conditions due to limitations in the content of fishmeal. Supplementing diets with Se enhances physiological responses, particularly mitigates the effects of the continuous presence of environmental stress factors. Organic Se has been shown to have higher absorption rates and a greater impact on bioavailability and overall health than inorganic forms. A characteristic feature of yeasts is their rapid proliferation and growth, marked by efficient mineral assimilation. Most of the selenized yeasts currently available in the market, and used predominantly in animal production and aquaculture, are based on Saccharomyces cerevisiae, which contains selenomethionine (Se-Met). The object of this review is to highlight the importance of selenized yeasts. In addition, it presents metabolic and productive aspects of other yeast genera that are important potential sources of organic selenium. Some yeast strains discussed produce metabolites of interest such as lipids, pigments, and amino acids, which could have applications in aquaculture and further enrich their usefulness.

Lipid Production from Native Oleaginous Yeasts Isolated from Southern Chilean Soil Cultivated in Industrial Vinasse Residues.

In this research, six strains of oleaginous yeasts native to southern Chile were analyzed for their biotechnological potential in lipid accumulation. For this purpose, the six strains, named PP1, PP4, PR4, PR10, PR27 and PR29, were cultivated in a nitrogen-deficient synthetic mineral medium (SMM). Then, two strains were selected and cultivated in an industrial residual "vinasse", under different conditions of temperature (°C), pH and carbon/nitrogen (C/N) ratio. Finally, under optimized conditions, the growth kinetics and determination of the lipid profile were evaluated. The results of growth in the SMM indicate that yeasts PP1 and PR27 presented biomass concentrations and lipid accumulation percentages of 2.73 and 4.3 g/L of biomass and 36.6% and 45.3% lipids, respectively. Subsequently, for both strains, when cultured in the residual vinasse under optimized environmental conditions, biomass concentrations of 14.8 ± 1.51 g/L (C/N 80) and 15.83 ± 0.57 g/L (C/N 50) and lipid accumulations of 28% and 30% were obtained for PP1 and PR27, respectively. The composition of the triglycerides (TGs), obtained in the culture of the yeasts in a 2 L reactor, presented 64.25% of saturated fatty acids for strain PR27 and 47.18% for strain PP1. The saturated fatty acid compositions in both strains are mainly constituted of fatty acids, myristic C 14:0, heptadecanoic C 17:0, palmitic C 16:0 and stearic C 18:0, and the monounsaturated fatty acids constituted of oleic acid C 18:1 (cis 9) (28-46%), and in smaller amounts, palmitoleic acid and heptadecenoic acid. This work demonstrates that the native yeast strains PP1 and PR27 are promising strains for the production of microbial oils similar to conventional vegetable oils. The potential applications in the energy or food industries, such as aquaculture, are conceivable.

Screening and Identification of Coastal Chilean Thraustochytrids for Arachidonic Acid Production: Biotechnological Potential of Ulkenia visurgensis Lng2-Strain.

Thraustochytrids are unicellular heterotrophic marine protists that have been described as producing a high content of polyunsaturated fatty acids (PUFAs). Among them, arachidonic acid (ARA) stands out as a precursor of several mediators of pivotal importance for the immune system. However, the biotechnological potential of thraustochytrids for ARA production has not been developed. The objective of this study is to isolate and identify native strains from different Chilean coastal environments and evaluate in vitro the effect of culture parameters such as C/N ratio (19 and 33) and temperature (15 °C and 23 °C) on biomass production and arachidonic acid content. A total of nine strains were identified and classified into four genera of the Thraustochitridae family. The Lng2 strain with 99% identity belongs to the species Ulkenia visurgenis and was the most prominent one for ARA production. Temperature had an effect on the PUFA profile but not on the ARA content nor on the biomass yield. Additionally, the C/N ratio has been identified as a key parameter. The ARA productivity increased by 92% (from 0.6 to 8.3 ARA mg/g-DW) and its total biomass by 62.7% (from 1.9 to 5.1 g/L) at a high C/N ratio (33) as compared to the control.

Protist-Lactic Acid Bacteria Co-Culture as a Strategy to Bioaccumulate Polyunsaturated Fatty Acids in the Protist Aurantiochytrium sp. T66.

Thraustochytrids are aquatic unicellular protists organisms that represent an important reservoir of a wide range of bioactive compounds, such as essential polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), which are involved in the regulation of the immune system. In this study, we explore the use of co-cultures of Aurantiochytrium sp. and bacteria as a biotechnological tool capable of stimulating PUFA bioaccumulation. In particular, the co-culture of lactic acid bacteria and the protist Aurantiochytrium sp. T66 induce PUFA bioaccumulation, and the lipid profile was evaluated in cultures at different inoculation times, with two different strains of lactic acid bacteria capable of producing the tryptophan dependent auxins, and one strain of Azospirillum sp., as a reference for auxin production. Our results showed that the Lentilactobacillus kefiri K6.10 strain inoculated at 72 h gives the best PUFA content (30.89 mg g-1 biomass) measured at 144 h of culture, three times higher than the control (8.87 mg g-1 biomass). Co-culture can lead to the generation of complex biomasses with higher added value for developing aquafeed supplements.

Microalgae as Raw Materials for Aquafeeds: Growth Kinetics and Improvement Strategies of Polyunsaturated Fatty Acids Production.

Studies have shown that ancient cultures used microalgae as food for centuries. Currently, scientific reports highlight the value of nutritional composition of microalgae and their ability to accumulate polyunsaturated fatty acids at certain operational conditions. These characteristics are gaining increasing interest for the aquaculture industry which is searching for cost-effective replacements for fish meal and oil because these commodities are one of the most significant operational expenses and their dependency has become a bottleneck for their sustainable development of the aquaculture industry. This review is aimed at highlighting the use of microalgae as polyunsaturated fatty acid source in aquaculture feed formulations, despite their scarce production at industrial scale. Moreover, this document includes several approaches to improve microalgae production and to increase the content of polyunsaturated fatty acids with emphasis in the accumulation of DHA, EPA, and ARA. Furthermore, the document compiles several studies which prove microalgae-based aquafeeds for marine and freshwater species. Finally, the study explores the aspects that intervene in production kinetics and improvement strategies with possibilities for upscaling and facing main challenges of using microalgae in the commercial production of aquafeeds.

Dietary carbohydrate-to-protein ratio influences growth performance, hepatic health and dynamic of gut microbiota in atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) fed a carbohydrate-rich diet exhibit suboptimal growth performance, along with other metabolic disturbances. It is well known that gut microbes play a pivotal role in influencing metabolism of the host, and these microbes can be modified by the diet. The main goal of the present study was to determine the effect of feeding graded levels of digestible carbohydrates to Atlantic salmon on the distal intestine digesta microbiota at 3 sampling times (i.e., weeks 4, 8 and 12), during a 12-week trial. A low carbohydrate-to-high protein diet (LC/HP, 0% wheat starch), a medium carbohydrate-to-medium protein diet (MC/MP, 15% wheat starch) or a high carbohydrate-to-low protein diet (HC/LP, 30% wheat starch) was fed to triplicate fish tanks (27 to 28 fish per tank). We performed an in-depth characterization of the distal intestine digesta microbiota. Further, growth parameters, liver histology and the expression of genes involved in hepatic neolipogenesis in fish were measured. Fish fed a HC/LP diet showed greater hepatosomatic and viscerosomatic indexes (P = 0.026 and P = 0.018, respectively), lower final weight (P = 0.005), weight gain (P = 0.003), feed efficiency (P = 0.033) and growth rate (P = 0.003) compared with fish fed the LC/HP diet. Further, feeding salmon a high digestible carbohydrate diet caused greater lipid vacuolization, steatosis index (P = 0.007) and expression of fatty acid synthase (fas) and delta-6 fatty acyl desaturase (d6fad) (P = 0.001 and P = 0.001, respectively) in the liver compared with fish fed the LC/HP diet. Although, the major impact of feeding a carbohydrate-rich diet to Atlantic salmon in beta diversity of distal intestine digesta microbiota was observed at week 4 (HC/LP vs MC/MP and HC/LP vs LC/HP; P = 0.007 and P = 0.008, respectively) and week 8 (HC/LP vs MC/MP; P = 0.04), no differences between experimental groups were detected after 12 weeks of feeding. Finally, at the end of the trial, there was a negative correlation between lactic acid bacteria (LAB) members, including Leuconostoc and Lactobacillus, with hepatic steatosis level, the hepatosomatic and viscerosomatic indexes as well as the expression of fas and d6fad. Weissella showed negative correlation with hepatic steatosis level and the hepatosomatic index. Finally, further research to explore the potential use of LAB as probiotics to improve liver health in carnivorous fish fed fatty liver-induced diet is warranted.

Analysis of Muscle Lipidome in Juvenile Rainbow Trout Fed Rapeseed Oil and Cochayuyo Meal.

This study aimed to analyze the effects on the lipidome of juvenile Oncorhynchus mykiss muscle fed 90% Brassica napus "rapeseed" oil and different amounts of Durvillaea antarctica "Cochayuyo" meal (1.5, 3 and 6%) as a replacement for cellulose. The analysis allowed for the identification of 329 lipids, mainly represented by phospholipids and fatty esters. The inclusion of Brassica napus oil significantly increased the levels of C18:2 species and fatty esters of hydroxylated fatty acids, which could play a bioactive role in human health. One of the most abundant lipids in all fillets was Phosphatidylcholine 33:6, which, according to the literature, could be considered a biomarker for the identification of Oncorhynchus mykiss. In all experimental diets, the species Phosphatidylethanolamine 15:1-18:24 showed four-fold higher levels than the control; increments of n-3- and n-6-rich phospholipids were also observed. Diets containing Durvillaea antarctica meal did not generate more significant variation in fish muscle phospholipids relative to the muscle of the rapeseed-oil-only group. These lipid species consist of medium- and long-chain fatty acids with different degrees of unsaturation. Still, it appears that the rapeseed oil masks the lipid contribution of the meal, possibly due to the low levels of total lipids in the macroalgae.

Intestinal Transcriptome Analysis Reveals Enrichment of Genes Associated with Immune and Lipid Mechanisms, Favoring Soybean Meal Tolerance in High-Growth Zebrafish (Danio Rerio).

The molecular mechanisms underlying fish tolerance to soybean meal (SBM) remain unclear. Identifying these mechanisms would be beneficial, as this trait favors growth. Two fish replicates from 19 experimental families were fed fishmeal-(100FM) or SBM-based diets supplemented with saponin (50SBM + 2SPN) from juvenile to adult stages. Individuals were selected from families with a genotype-by-environment interaction higher (HG-50SBM + 2SPN, 170 ± 18 mg) or lower (LG-50SBM + 2SPN, 76 ± 10 mg) weight gain on 50SBM + 2SPN for intestinal transcriptomic analysis. A histological evaluation confirmed middle intestinal inflammation in the LG- vs. HG-50SBM + 2SPN group. Enrichment analysis of 665 differentially expressed genes (DEGs) identified pathways associated with immunity and lipid metabolism. Genes linked to intestinal immunity were downregulated in HG fish (mpx, cxcr3.2, cftr, irg1l, itln2, sgk1, nup61l, il22), likely dampening inflammatory responses. Conversely, genes involved in retinol signaling were upregulated (rbp4, stra6, nr2f5), potentially favoring growth by suppressing insulin responses. Genes associated with lipid metabolism were upregulated, including key components of the SREBP (mbtps1, elov5l, elov6l) and cholesterol catabolism (cyp46a1), as well as the downregulation of cyp7a1. These results strongly suggest that transcriptomic changes in lipid metabolism mediate SBM tolerance. Genotypic variations in DEGs may become biomarkers for improving early selection of fish tolerant to SMB or others plant-based diets.

Vitellogenesis in the Patagonian toothfish (Dissostichus eleginoides) conditioned to a recirculating aquaculture system.

The Patagonian toothfish (Dissostichus eleginoides) is a new promising fish species for diversifying the aquaculture industry in Chile because of its high economic value and high international demand. However, when attempting to start aquaculture of a new species, one of the major challenges is successfully achieving conditions to reproduce them. This is particularly difficult when the information on the biology and physiology of the reproduction process of the species in question is scarce, as is the case with D. eleginoides. Additionally, female reproductive dysfunction is more prevalent under culture conditions and it is very important to have tools to evaluate the progress of oocyte maturation. Therefore, evaluation of the vitellogenesis process in addition to measuring gonadosomatic index (GSI) and oocyte diameter is an important parameter for allowing the monitoring of females from a broodstock that will spawn in the reproductive season. This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) specific for the Patagonian toothfish (D. eleginoides) vitellogenine (Vtg) and quantify the plasma level in the fishes, maintained in a recirculation aquaculture system (RAS), throughout their reproductive cycle. A polyclonal antibody was prepared using the isolated major egg protein as antigen. This antibody was specific to the major plasma phosphoprotein identified as Vtg and was used to develop and standardize an indirect ELISA assay. The assay standard curve was linear from 0.1 to 1 µg/ml purified egg yolk protein and the average r2 was 0.995. We corroborated our ELISA assay by demonstrating a strong correlation between high levels of plasma Vtg obtained by the assay and the intensity of the corresponding bands in both SDS-PAGE coomassie stained gels and Western Blot. During the two reproductive seasons analyzed, the highest Vtg plasma level was obtained in the majority of the females in the last three months before spawning (December-January). This differs from the wild population in which the spawning occurs during the austral winter (June-September). Therefore, the RAS condition established to maintain in captivity the D. eleginoides allows females to develop mature oocytes normally, as was evidenced by picks of Vtg plasma levels.

Effect of Dietary Carbohydrate-to-Protein Ratio on Gut Microbiota in Atlantic Salmon (Salmo salar).

Atlantic salmon (Salmo salar) is a carnivorous fish species whose productive performance tends to be suboptimal when fed low-cost carbohydrate rich meals. It is of interest to study the dynamics of gut microbiota communities in salmonids fed high carbohydrate diets since gut microbes are referred to as key players that influence the metabolism and physiology of the host. A study was conducted to determine the effect of feeding a high carbohydrate diet to Atlantic salmon in gut microbiota communities. A medium carbohydrate (15% wheat starch)/medium protein (MC/MP) diet or a high carbohydrate (30% wheat starch)/low protein (HC/LP) diet was fed to triplicate tanks (28 fish each) during four weeks. We conducted an in-depth characterization of the distal intestine digesta microbiota using high-throughput sequencing of the V4 region of the 16S rRNA gene. Firmicutes, Actinobacteria and Proteobacteria were the major phyla determined in either experimental group. Phylum Planctomycetes, class Planctomycetia, order Planctomycetales and genus Lactococcus were significantly more abundant in fish fed the HC/LP diet compared with fish fed the MC/MP diet. Our study suggests feeding a carbohydrate rich meal to salmon exerts a low impact on the structure of gut microbial communities, affecting mostly low-abundance bacteria capable of metabolizing anaerobically carbohydrates as a major energy-yielding substrate.

Lactoferrin Decreases the Intestinal Inflammation Triggered by a Soybean Meal-Based Diet in Zebrafish.

Intestinal inflammation is a harmful condition in fish that can be triggered by the ingestion of soybean meal. Due to the positive costs-benefits ratio of including soybean meal in farmed fish diets, identifying additives with intestinal anti-inflammatory effects could contribute to solving the issues caused by this plant protein. This study evaluated the effect of incorporating lactoferrin (LF) into a soybean meal-based diet on intestinal inflammation in zebrafish. Larvae were fed with diets containing 50% soybean meal (50SBM) or 50SBM supplemented with LF to 0.5, 1, 1.5 g/kg (50SBM+LF0.5; 50SBM+LF1.0; 50SBM+LF1.5). The 50SBM+LF1.5 diet was the most efficient and larvae had a reduced number of neutrophils in the intestine compared with 50SBM larvae and an indistinguishable number compared with control larvae. Likewise, the transcription of genes involved in neutrophil migration and intestinal mucosal barrier functions (mmp9, muc2.2, and ß-def-1) were increased in 50SBM larvae but were normally expressed in 50SBM+LF1.5 larvae. To determine the influence of intestinal inflammation on the general immune response, larvae were challenged with Edwardsiella tarda. Larvae with intestinal inflammation had increased mortality rate compared to control larvae. Importantly, 50SBM+LF1.5 larvae had a mortality rate lower than control larvae. These results demonstrate that LF displays a dual effect in zebrafish, acting as an intestinal anti-inflammatory agent and improving performance against bacterial infection.

Effects of dehulling, steam-cooking and microwave-irradiation on digestive value of white lupin (Lupinus albus) seed meal for rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar).

A digestibility trial was conducted to assess the effect of dehulling, steam-cooking and microwave-irradiation on the apparent digestibility of nutrients in white lupin (Lupinus albus) seed meal when fed to rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Six ingredients, whole lupin seed meal (LSM), dehulled LSM, dehulled LSM steam-cooked for 15 or 45 min (SC15 and SC45, respectively) and LSM microwave-irradiated at 375 or 750 W (MW375 and MW750, respectively), were evaluated for digestibility of dry matter, crude protein (CP), lipids, nitrogen-free extractives (NFE) and gross energy (GE). The diet-substitution approach was used (70% reference diet + 30% test ingredient). Faeces from each tank were collected using a settlement column. Dehulled LSM showed higher levels of proximate components (except for NFE and crude fibre), GE and phosphorus in comparison to whole LSM. Furthermore, SC15, SC45, MW375 and MW750 showed slight variations of chemical composition in comparison to dehulled LSM. Results from the digestibility trial indicated that dehulled LSM, SC15, SC45 and MW375 are suitable processing methods for the improvement of nutrients' apparent digestibility coefficient (ADC) in whole LSM. MW750 showed a lower ADC of nutrients (except for CP and lipids for rainbow trout) in comparison with MW350 for rainbow trout and Atlantic salmon, suggesting a heat damage of the ingredient when microwave-irradiation exceeded 350 W.