Paper III and IV present results of an experiment conducted to study effects in Atlantic salmon fed commercially relevant diets containing high levels of alternative protein sources on health indicators of the DI including microbiota. The results presented in Paper III report characteristics of the diets regarding production performance, nutritional quality, histomorphological changes, water-content of the DI digesta, plasma osmolality and aquaporin, ion transporter, tight junction and adherence junction expression in the DI tissue. The results presented in Paper IV comprise effects of the alternative diets on immune, cellular stress and cell proliferation responses in the DI tissue, as well as mucosa and digesta-associated DI microbiota. The fish used were Atlantic salmon post-smolt kept in a seawater recirculating system and fed one of five experimental diets for 48 days. The control diet (FM) contained fishmeal as the sole source of protein, while the fishmeal was partially replaced by the following alternative ingredients in four substitution diets:
poultry meal diet (PM); a mix of soybean meal and wheat gluten (SBMWG); a mix of soy protein concentrate and poultry meal diet (SPCPM); or a mix of guar meal and wheat gluten diet (GMWG).
General results
Specific growth rate presented no significant differences (Paper III) between the fish fed FM and the fish fed the substitution diets. However, the substitution diets presented significantly higher feed conversion ratio, possibly reflecting the higher fiber content of these diets.
Regarding the general characterization of the microbiota present in the DI (Paper IV), the general results showed differences between the fish fed the FM and the substitution diets and between digesta and mucosa compartments. The alpha diversity index observed species were significantly different between digesta and mucosa, showing lower richness for the mucosa compared to the
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digesta samples. Alternative ingredients caused differences in DI digesta and mucosa-associated microbiota compared to FM fed fish (beta diversity). The results from LEfSe indicated significant differences in DI microbiota between the fish fed FM diet and the fish fed the diets containing alternative proteins in both digesta and mucosa. A higher number of OTUs showed significant differences between the dietary groups in digesta compared to mucosa. Regardless of the diet, the DI mucosa showed lower abundance of Firmicutes and Fusobacteria and higher abundance of Proteobacteria, Bacteroidetes and OD1in comparison with digesta. For all samples, the core microbiota consisted in 19 OTUs.
Diet specific results
SBMWG diet
Compared to the fish fed FM, the fish fed SBMWG showed the largest number of differences in all the evaluated histomorphological and physiological parameters analyzed in plasma and the DI tissue and chyme. These included increased fecal water content of DI chyme and higher plasma osmolality (Paper III). The histomorphological evaluation (Paper III) showed alteration in the normal morphology of the intestine indicating moderate enteritis (SBMIE), although gene expression of various immune-markers that have previously been differentially expressed in salmon with SBMIE were not differentially expressed in the current study (Paper IV). The latter could be the result of low levels of saponins in the soybean meal used in the diet or a chronic stage of the inflammatory process with an attempt to repair the damage produced by the diet. In line with these findings, the gene expression profile suggested that the diarrhea and high plasma osmolality could be at least partially explained by the alterations observed in several genes related to water regulation, transport and intestinal permeability, i.e. genes coding for aquaporins, ion transporters, tight junction and adherence junction proteins in the DI tissue (Paper III). Among the studied aquaporins, aquaporin 8ab (aqp-8), which has been suggested as a marked gene for the study of SBMIE (Kortner et al., 2012), was markedly reduced. Together, the gene expression profile suggested reduced transcellular transport of water. Moreover, the expression of a gene related with cellular stress, i.e. shock protein 70 (hsp70), was increased in fish fed the SBMWG diet. In addition, fish fed the SBMWG diet presented high cell proliferation as seen by high
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expression of the pcna gene and confirmed by immunohistochemical detection of Pcna protein tissue expression and distribution (Paper IV).
Fish fed SBMWG diet showed differences in the abundances of the DI bacterial communities.
These fish also showed a different mucosal-associated microbiota compared to that of FM fed fish. Weighted and unweighted UniFrac PCoA plots supported the previous results, showing a clear separation between the digesta and mucosa compartments, as well as between diets. Results from the LEfSe analysis indicated that in digesta, the fish fed SBMWG diet presented significantly higher abundance of several members of the LAB group compared to the FM-fed fish. In the mucosa, SBMWG fed fish presented higher abundance of one LAB member, namely Weissella (Paper IV).
GMWG diet
Similarly to the SBMWG-fed fish, the fish fed GMWG diet also presented certain alterations in the expression profile of genes related to water transport such as aqp-8ab and aqp-10 (Paper III), frim, pcna and cat, as well as an increased expression of Pcna-positive cells in the immunohistochemical detection (Paper IV). However, fish fed GMWG diet not showed concomitant alterations in the chyme water content or histomorphological signs of inflammation (enteritis) in the DI tissue. Guar meal is known to contain antinutrients such as saponins, which may explain the alteration in the water transport genes, as well as the cellular stress and increased number of Pcna positive cells. Fish fed the GMWG also presented high abundance of LAB in digesta and mucosa compared to FM fed fish. Together these results for fish fed GMWG diet may indicate that even when antinutrients may be altering the gene functionality and other functional components of the intestine, other mechanisms or mucosal elements may need to be altered in order to cause diarrhea and inflammation. Guar saponins, for example, may not be as concentrated in guar meal and/or as potent in their biological/antinutritional effects as soya saponins.
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Fish fed the SPCPM and the PM diet presented the least changes in the evaluated characteristics compared to the FM-fed salmon. The fish fed SPCPM diet showed elevated water content of the DI chyme and a trend towards lower gene expression in aqp-8 and aqp-10, but otherwise no signs of inflammation. On the other hand, fish fed PM diet showed a decrease in the chyme water content, which did not appear to be related to alteration in gene expression or histomorphological disruption of the DI. This decrease in chyme water content was most likely an indirect result of high chyme lipid content in the chyme, as a result of low lipid digestibility.
In conclusion, the study presented in Paper III and IV clearly showed that the diet containing soybean meal negatively affected the gut health of salmon, causing diarrhea and inflammation with the concomitant changes in gene expression and morphology of the DI. In addition, fish fed SBMWG presented the largest modulation in the microbial-associated community of the intestine with high relative abundance of LAB in both digesta and mucosa compared to fish fed FM diet.
The GMWG diet also presented some modifications in gene expression, cell proliferation and microbial modulation in the DI. However, as the health of the gut appeared unaffected, as indicated by the histological appearance, the effects observed in fish fed the GMWG diet most likely were a result of normal physiological adaptations to the dietary composition. The other two substitute diets produced only minor changes in the DI functionality, but otherwise were apparently not harmful for the health and functionality of Atlantic salmon.
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Based on the summary above regarding the results of this thesis work, the following overarching topics appear to deserve discussion beyond what is already presented in Papers I-IV:
Proof of saponins as inducers of enteritis allow further applications of the enteritis model Importance and implications for the host of digesta and mucosa-associated microbiota Mucosa-associated microbiota is less affected by diet than the digesta-associated - implications for intestinal function
Soybean affect functional characteristics of the intestine but may not always significantly affect the immunological response as assessed by gene expression analysis
Lactic acid bacteria dominate digesta-associated microbiota of fish fed SBM and GM
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