The feed legislation allows the use of fish protein hydrolysates in feed for the same species in which it came from, since enzymatic hydrolysis degrades the proteins and eliminates potential prions, which have caused disease in mammals, but not in fish. In this trial, we investigated the effects of partially replacing dietary fishmeal (FM) with salmon protein hydrolysate (FPH) on the intestinal gene expression and microbiota. Atlantic salmon post smolts were either fed a control diet containing 30% fishmeal (FM), a 20% FM diet with 9% salmon hydrolysate (FPH-09) or a 10% FM diet with 18% salmon hydrolysate (FPH-18), until doubling of weight. Gene expression analysis by RNA sequencing of pyloric caeca (PC), midgut (MG) and hindgut (HG) revealed a downregulation of immunological genes involved in inflammation in the intestine of FPH-18 fed salmon compared to salmon fed the FM control. The gene expression of paralogous peptide transporters (PepT) was analyzed by real time quantitative PCR in PC, anterior midgut (AMG), posterior midgut (PMG) and HG of salmon fed all the three diets. The PepT1b paralog had highest relative expression levels in PC and AMG, suggesting that PepT1b is most important for peptide uptake in the anterior intestine. PepT1a was also mainly expressed in the PC and AMG, but at lower levels than PepT1b and PepT2b in the AMG. The PepT2b paralog had high levels of expression in AMG, PMG and HG indicating that it contributed significantly to peptide uptake in the posterior part of the gastrointestinal tract. The gut microbiota in the mucosa and digesta of the MG and HG, were dominated by the phyla Cyanobacteria and Proteobacteria, but also Firmicutes were present. The only dietary effect on the microbiota was the higher prevalence of the phyla Spirochaetes in the mucosa of FPH-18 fed salmon compared to the FM fed salmon. In conclusion, replacing FM with salmon hydrolysate reduced the expression of inflammatory markers in the Atlantic salmon intestine suggesting improved health benefits. The reduced inflammation may be related to the reduced FM content, potentially bioactive peptides in the hydrolysate and/or the altered gut microbial composition.
The need for novel and sustainable feed ingredients for cultivated fish and land animals is increasing with the growing human population. By-products from marine food production are sustainable alternatives for production of feed ingredients (Rustad et al., 2011; Almås et al., 2020). In Norway a significant biomass of by-products is produced from farmed Atlantic salmon amounting to > 0.5 million tons in 2021 (Myhre et al., 2022). However, the feed legislation prohibits the reuse of proteins from Atlantic salmon into feed of the same species, unless it is hydrolyzed. Complete processing by enzymatic hydrolysis will degrade potential prions and is therefore an exception from the transmissible spongiform encephalopathy (TSE)-legislation (Regulation (EC) No 999/2001; Commission Regulation (EU) No 142/2011; Sandbakken et al., 2023).
Novel feed ingredients should be nutritious, palatable, digestible, and not have any negative impact on the immune response and health of the animal (Glencross, 2020). Protein hydrolysates are mixtures of peptides and free amino acids, which are nutritious, highly digestible and known to be palatable (Kristinsson and Rasco, 2000; Kousoulaki et al., 2013; Khosravi et al., 2015). Hydrolysates may also contain bioactive peptides (Zamora-Sillero et al., 2018; Gao et al., 2021; Siddik et al., 2021). Marine peptides in fish protein hydrolysates (FPH) have shown beneficial effects including antimicrobial, anti-inflammatory and immunomodulatory activities when consumed in vivo (Kang et al., 2019). Dietary inclusion of FPH in aquaculture has induced growth performance, digestibility and altered the immune response (Zheng et al., 2014; Siddik et al., 2018).
Optimized diets can improve the general health and immune functions of fish, thereby reduce losses due to diseases, as well as improving recovery after diseases in aquaculture (Waagbø, 1994; Dawood, 2020). Therefore, nutritional status has a major impact on the ability of fish to resist pathogens and cope with stress (Martin et al., 2016). Some feed additives have immunomodulatory properties, which either activates the immune system or downregulates immunological genes. Activating the innate immune system can be beneficial when the fish experiences pathogenic threats, but a general activation can be energy consuming and have negative impact on other processes like growth (Tacchi et al., 2011). A downregulation of genes related to inflammation saves energy for the organism to grow and thrive.
Intestinal protein absorption is performed by different transporters in the brush border membrane of absorptive epithelial cells (Evans and Claiborne, 2006; Kiela and Ghishan, 2016). Peptide transporters (PepT) provide a more efficient uptake mechanism for protein building blocks than amino acid transporters since they transport di- and tripeptides instead of single amino acids (Wang et al., 2017). Due to the whole genome duplication event, Atlantic salmon has at least 4 genes encoding paralogous PepT which are part of the solute carrier 15a (Slc15a) family of transporter molecules. These 4 transporters are slc15a1a, slc15a1b, slc15a2a and slc15a2b, also known as PepT1a, PepT1b, PepT2a and PepT2b, respectively. PepT1 paralogs are low affinity/high-capacity transporters that serve a key role in peptide absorption, mainly in the proximal part of the intestine (Verri et al., 2003; Rønnestad et al., 2007; Ostaszewska et al., 2010; Wang et al., 2017). Most research available is on the PepT1b paralog, since PepT1a was identified and characterized in Atlantic salmon (Salmo salar) quite recently by Gomes et al. (2020). PepT2 paralogs are high affinity/low-capacity transporters which recently were characterized in Atlantic salmon by Vacca et al. (2022). Whereas PepT2b mainly is expressed in the kidney and midgut (MG) to hindgut (HG) (Del Vecchio et al., 2021; Vacca et al., 2022), PepT2a is more abundant in brain and gills, and appears to have a minor role in peptide uptake along the gastrointestinal tract (Del Vecchio et al., 2021). There is however little information on how the expression of peptide transporters is regulated by dietary proteins, and especially available peptides and free amino acids in Atlantic salmon.
Intestinal microbiota composition depends on the diet, environmental factors, and host selection. The microbiota affects the nutrient absorption, immune system, and disease resistance of the host (Luan et al., 2023). Mucosal microbiota is colonizing the mucus layer on the intestinal epithelial cells and is more stable and less affected by diet than the microbiota found in the digesta (feces) (Gajardo et al., 2016). This was clearly shown in Atlantic salmon fed diets with and without insect meal (Li et al., 2021), where the digesta-associated microbiota also showed the highest microbial richness and diversity. Dietary protein can affect the intestinal microbiota composition and function, and interactions between proteins and microbiota can impact the host health (Bartlett and Kleiner, 2022). Furthermore, if the protein fraction is absorbed much faster than in normal diets, it is possible that remaining bacteria in midgut and hindgut sections will rely more on utilization of feed components reaching there in larger amounts such as fiber and carbohydrates. Such changes in available nutrients for the microbiota can have profound effect on fermentation patterns altering profiles of short chain fatty acids and other products of fermentation (Bartlett and Kleiner, 2022), and on production of inflammatory/anti-inflammatory components.
The current study aimed at assessing the effects of replacing 2/3 of the fishmeal (FM) in a standard diet, with salmon protein hydrolysate (FPH) on intestinal microbiota and health parameters by RNA sequencing analysis. Furthermore, we also report on the response of intestinal peptide transporter expression when the salmon were fed diets with 1/3 and 2/3 of the FM protein replaced with salmon hydrolysate.
A detailed description of the feed composition, experimental design and fish facilities have been reported in Sandbakken et al. (2023). In brief, Atlantic salmon (Salmo salar) post smolts were fed one of three diets with spray dried salmon hydrolysate (Nutrimar AS) replacing parts of the fish meal: a control diet with 30% fish meal (FM), a 9% hydrolysate diet with 20% fish meal (FPH-09) and a 18% hydrolysate diet with 10% fish meal (FPH-18) (Supplementary Table 1). The diets were designed to be iso-energetic, iso-nitrogenous, iso-lipidic as well as having the same amount of EPA, DHA and phospholipids. Salmon (mean start weight 141.5 ± 23.6 g (standard deviation)) were PIT-tagged and randomly distributed into 9 tanks (24 fish/tank, ~ 300 L, 10-14 L min-1 water flow, > 80% O 2 saturation in the outlet water, 8.2-8.8°C seawater, continuous light) and fed for 2 h twice a day until satiety. After 9 weeks of feeding, the mean salmon weight was 313.8 ± 50 g at sampling. The feeding trial was approved by the Norwegian Food Safety Authority (FOTS ID:23021) and was conducted at NTNU Sealab (Department of Biology, Trondheim, Norway).
Final samples were taken 60 and 62 days after the experimental start, giving RNA samples and microbiota samples, respectively. Before the first sampling day, the fish were not fed for 48 hours to ensure empty intestines for RNA analysis. Five fish from each tank were randomly sampled and euthanized in seawater with Finquel, MS-222 (800 mg/l). Weight, length and PIT-ID was registered before the 5 fish were put on ice and processed immediately. The sampling was performed one tank at a time.
The fish abdomen was opened, and fish were sexed by looking at the gonads. For each fish, 5 intestinal samples were collected for RNA analysis, by cutting ~2 mm of pyloric caeca, 3 segments of the midgut (anterior, mid, and posterior) and a middle segment of the hindgut (HG). Tissue samples for RNA analysis were put in 1 ml cold RNA later (RNA stabilization solution, Ambion Inc.), and stored for 24 h at 4°C before the samples were transferred to -20°C.
The remaining fish (19 per tank) were fed five 2-hour meals over the next 42 hours, before the next sampling two days later. Five random fish from the tanks fed FM control and FPH-18 were euthanized (MS-222) to collect microbiota from midgut (MG) and hindgut (HG) digesta and mucosa. The fish were se
