Diet Manipulation of Transgenic Zebrafish

Adult double transgenic (mpeg1:mCherry/mpx:eGFPi¹¹⁴) expressing mCherry under the macrophage-specific mpeg1 promotor and GFP under the neutrophil-specific mpx promotor fish were housed and fed as previously described [49 (link)]. Embryos were obtained by natural spawning. Fish were fed as follows: weeks 1 and 2 with rotifers (× 4/day from 5 days post fertilization -dpf-), week 3 with rotifers and Artemia Nauplii 230.000 npg (Nauplii per gram) (Ocean Nutrition Europe, Essen, Belgium) (× 2/day), week 4 with Artemia (× 2/day) and until 40 dpf Artemia and Tetramin Flakes (Tetra, Melle, Germany) (× 2/day). When fish reached the juvenile stage, at 40 dpf [76 (link)], fish were randomly distributed into 6 tanks, 2 per each diet: one was sampled after 1 week and the other tank after 3 weeks of the feeding experiment per each diet. The feeding experiment was performed blind and fish were fed until slightly before satiation twice a day. Each tank received one of the following: a control diet, a saponin-supplemented diet or a butyrate-supplemented diet. Full diet composition is listed in Table 1.Table 1

Formulation of experimental diets analysed once the feed intervention was performed to check which compositions corresponded to the blinded diets

	A: Control diet (%)	B: Butyrate diet (%)	C: Saponin diet (%)
Wheat	7.00	6.99	6.67
Wheat gluten	16.00	16.00	16.00
Sunflower meal	1.68	1.68	1.68
Soy protein concentrate	15.16	15.16	15.16
Fish meal	52.00	52.00	52.00
Fish oil	4.40	4.40	4.40
Rapeseed oil	2.00	2.00	2.00
Vitamin mix	0.35	0.35	0.35
Mineral mix	1.92	1.92	1.92
Butyrate	0.00	0.01	0.00
Saponin	0.00	0.00	0.33
[VOLUME]	100.0	100.0	100.0
Dry matter	92.2	92.0	92.0
Crude protein	56.0	57.4	57.4
Crude fat	13.5	13.8	13.8
Ash	8.9	8.9	8.9

The three diets are similar in composition (dry-matter, protein, fat and ash). 95% ultrapure soy saponin was kindly provided by Trond Kortner NMBU Oslo Norway, origin: Organic Technologies, Coshocton, OH, [40 (link)]