Introduction
Recently, several products have emerged on the market as new sustainable fish feed ingredients with presumable health promoting effects. One such product is Coryne Pro (Agrifeed), a single cell protein (SCP) of bacterial nature as by-product from fermentation of Corynebacterium glutamicum for industrial production of threonine. Coryne Pro has a high protein level (~70%) and is described to constitute a good protein source for growth, as well as having beneficial health effects. While in vivo trials are the best way to evaluate new ingredients, it would be helpful to develop complementary in vitro methods to reduce the number of animals involved according to 3R principles. The H2020-FETOPEN project Fish-AI, aims to develop such a platform but it requires an experimental set-up that facilitates the in vivo vs. in vitro correlation. To this purpose, as part of this project, we tested Coryne Pro at two different inclusion levels in the feed for rainbow trout and compared it to three reference feeds: one presumably healthy fish meal-based feed, one challenging feed with high soybean meal content and one feed with high inclusion of feather meal with presumable low protein digestibility value. Rainbow trout were fed the five different feeds for nine weeks, and growth performance, organ indices, histology, gene expression, blood values as well as digestive enzyme levels were investigated.
Material and methods
Rainbow trout (~160 g) were fed five different diets; fish meal diet (FM), soybean meal diet (SBM), feather meal diet (FTHM) or diets containing single cell protein at 10% and 20% inclusion levels (SCP-10 and SCP-20, respectively) (Table 1). Feeds were produced by extrusion with pellet size 2mm, and yttrium was added to calculate nutrient digestibility. Macro and micronutrient content were adjusted to meet the NRC standards when formulating the feeds and kept constant in protein and lipid level.
The feeding trial was run at Skretting Aquaculture Innovation’s research facility in Mozzecane, Italy. Rainbow trout were randomly distributed in 0.6 m3 tanks, 35 fish per tank and three replicate tanks per diet. The feeding regime was two meals per day with overfeeding. Fish were held at 15°C water temperature and 24-hours light regime. After 9 weeks of feeding, 6 fish per tank were randomly sampled and euthanized with an overdose of MS-222. Weight, length and organ indices were recorded from individual fish. Blood samples were collected in heparinized syringes from the caudal vein and analyzed for plasma indicators of nutrient metabolism. The intestinal tract (posterior to the stomach) was dissected from the same fish, and divided into pyloric intestine, mid intestine and distal intestine. The intestinal sections were opened longitudinally, and intestinal content collected for analyses of bile salt and trypsin activity. Tissue samples were fixed in formalin for histology, RNA later for gene expression and snap frozen in liquid nitrogen for enzyme analyses. Fecal samples were pooled by tank.
Results and discussion
The SCP diets performed equal to the fish meal diet concerning fish growth, feed intake, and feed conversion ratio, indicating that the SCP may partially replace fish meal as a protein source in diets for rainbow trout. Results indicated however some immune stimulation from the SCP diets by slightly higher numbers of mucosal immune cells in the distal intestine and elevated levels of certain immune genes. Increased levels of liver enzymes in the blood from SCP were moreover observed, although large variation within the groups requires further analyses to conclude. As expected, the SBM diet induced intestinal inflammation in the distal portion of the rainbow trout gut evidenced by infiltration of inflammatory cells in the gut mucosa, reduced intestinal fold height, loss of supranuclear vacuoles, as well as reduced expression of functional brush border genes and reduced organ indices. Further, results indicated that the FTHM diet had a lower protein digestibility compared to fish meal, as feed intake and final body weight were significantly higher in this diet group, although there was no difference in feed conversion ratio. Also, increased organ indices of the pyloric intestine was observed in this diet group possibly from lipid accumulation as known from choline deficiency in salmon.
Conclusions
The SCP diets performed equal to the fish meal diet concerning growth and organ indices. Gene expression results indicated however slight immune stimulation from the SCP. Results confirmed intestinal inflammation in the distal intestinal region from the SBM diet and indications of sub-optimal digestibility from the FTHM diet. The experimental set-up used in the current study with three contrasting reference diets with known characteristics, was thus successful for testing of new feed ingredients and will provide a clear reference for testing the predictive ability of the in vitro platform that is under development by the Fish-AI consortium.
Acknowledgements
This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 828835.