Enhanced biodiversity of gut flora and feed efficiency in pond cultured tilapia under reduced frequency feeding strategies

limited amount of nutrients available [26]. This could play a role in increased feed efficiency in animals initially deprived of specific nutrients or of tilapia fed at reduced frequencies. Here, we evaluated potential underlying mechanisms behind the greater feed efficiency in fish fed on alternate days. It is currently unknown as to how reduced frequency feeding strate- gies may alter the gastrointestinal microbiome and nutrient transporter gene expression of tila- pia resulting in better nutrient utilization and feed efficiencies. Here, we evaluated the practices of alternate-day feeding to both establish whether this practice is effective in other areas of the world and to determine if changes in the tilapia gastrointestinal microbiome and nutrient trans- porter gene expression might account for improved feed efficiency under the management prac- tice. We also sought to assess if reducing the frequency further to every third day might further improve production efficiency of tilapia. We found no difference in growth parameters or sur- vival in tilapia fed on alternate days versus those fed daily. Nile tilapia fed on alternate days had improved feed efficiency and a greater diversity of gut prokaryotic and eukaryotic microorgan- isms, while transcript levels of select nutrient transporters changed little in fish fed on alternate days versus those fed daily, every third day or not at all. Taken together, this is the first study to suggest that enhanced gut microbial diversity may contribute to improved feed utilization under

reduced frequency feeding strategies in the culture of Nile tilapia and perhaps other finfish. Materials and methods All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at North Carolina State University (approval number: 13-139-0). All animals were euthanized using buffered tricaine methanesulfonate (MS-222) followed by cervical transection following standard procedures. Fish and pond sampling The tilapia growth trial was performed at the Fisheries Field Laboratory, Bangladesh Agricul- tural University, Mymensingh, Bangladesh. All-male sex reversed Nile tilapia (~3.5 g) were stocked at 5 fish/m2 in 16 ponds (0.1 ha; 4 replicates per treatment), with weekly pond fertiliza- tion at a rate of 28 kg N and 7 kg P/ha/week for all treatment groups. Twenty ponds were ran- domly divided into one of five treatments (4 ponds per treatment). The treatments were: (T1) commercial diet feeding daily with weekly pond fertilization, (T2) commercial diet feeding every other day with weekly pond fertilization, (T3) commercial diet feeding every third day with weekly pond fertilization, (T4) weekly pond fertilization with no supplemental feeding of commercial diet, and (T5) daily feeding with commercial diet with no pond fertilization. Fish were fed with formulated feed (CP Bangladesh, 30% crude protein) initially at 10% and then down to 3% body weight/day based on a standard tilapia feed schedule. Growth (length and weight) was monitored at 2-week intervals by subsampling of fish over

the twelve-week growing trial. Feed rates were adjusted accordingly based on this biweekly random sampling of 50 tilapia from each pond. After approximately 12 weeks (day 114 of the trial), all fish were harvested by seining the fish from the ponds. All fish were collected, counted, and weights and lengths taken to assess final weights and lengths, survival rate and total production. Samples of tilapia anterior intestinal tissue and fecal material from the colon were collected for analysis of nutrient transporter gene expression and gut prokaryote and eukaryote fauna at North Carolina State University, Raleigh, NC (NCSU; see below). Five fish were randomly collected from each pond, euthanized with buffered tricaine methanesulfonate (MS-222) and cervical transection for gene expression analysis. These samples were taken from a 1 cm section of the anterior intestine 5 cm posterior to the duodenal bulb (stomach sphincter where intestine meets the stomach) and placed in RNALater (Thermo Fisher PLOS ONE Enhanced biodiversity of gut flora and feed efficiency in pond cultured tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0236100 July 23, 2020 3 / 23