Effects of woody forages on biodiversity and bioactivity of aerobic culturable gut bacteria of tilapia (Oreochromis niloticus)

Materials and methods The study was conducted under the guidelines of the Animal Ethics Committee at the South China Agricultural University, China (approval ID: SYXK-2019-0136). Woody forage The ingredients of the reference diet group (the control group) in this study are shown in Table 1. The reference (Ref) diet was prepared according to the protocol of Pan et al [29]. The nutrient compositions of the five diets were controlled similarly, containing 5.00% moisture, 32.00% of crude protein, 5.20% of crude lipid, 10.40% crude ash and 1.15% phosphorus. In the process of feed fermentation, the activated strains are first expanded and mixed with fermentation materials (leaf powder, starch and other additives), and then the mixed fermentation materials and strains are fully mixed and placed in fermentation bags and placed in an artificial climate room for fer- mentation 9 days. And the mixed all raw materials was extruded into pellet diet with 2 mm in diameter by twin screw extruders (VALVA60-III, Guangzhou, China), placed in an air-condi- tioned room and dried in the air, then sieved and placed in a sealed bag at -20˚C keep in the refrigerator for use. For non-fermented feed, the fermentation process is omitted directly. Experimental fish and feeding management Tilapia were purchased from the National Tilapia Breeding Farm of Guangdong Province, China, and their initial weight was 70.0 ± 4.0 g. The fish were acclimated in large net cages in a pond for one week, after which 1200 fish were randomly stocked into 20 net cages, with 60 fish

Table 1. Feed formulation and proximate composition of the five test feeds (%). Cont. MOL FMOL FM FFM soybean meal 45.00 36.50 36.50 39.00 39.00 rapeseed meal 10.00 14.50 14.50 12.00 12.00 wheat flour 34.99 12.89 7.89 10.89 8.39 starch 5.00 2.50 tapioca flour 5.00 5.00 3.00 3.00 wheat gluten 4.00 4.00 freshwater fish meal 3.00 3.00 3.00 3.00 3.00 fresh water fish oil 2.00 2.00 2.00 2.00 2.00 soybean lecithin 1.00 1.00 1.00 1.00 1.00 mono-calcium phosphate 2.00 2.00 2.00 2.00 2.00 feed additive premix 2.01 3.11 3.11 3.11 3.11 MOL meal 20 FMOL meal 20 FM meal 20 FFM meal 20 Analyzed proximate composition (%) Moisture 5.10 5.40 4.63 6.44 4.52 Crude protein 32.60 32.90 32.59 31.96 32.91 Crude lipid 5.12 5.37 5.25 5.05 5.38 Crude ash 10.17 10.32 10.77 10.26 10.61 Phosphorus 1.22 1.12 1.09 1.14 1.11 The five groups respectively are control group (Cont.), Moringa oleifera Lam group (MOL), fermented Moringa oleifera Lam group (FMOL), Folium mori leaf meal group (FM) and fermented Folium mori leaf meal group (FFM). https://doi.org/10.1371/journal.pone.0235560.t001 PLOS ONE Effects of wood forages on gut bacterial biodiversity and bioactivity in tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0235560 July 2, 2020 3 / 17