Safety of the two isolates probiotics was studied by using 90 apparently healthy fish that were acclimatized for two weeks in indoor tanks. The fish were divided into 3 equal groups (three replicates each). Fish of the 1st and 2nd groups were i.p. inoculated by 0.3 ml of saline containing 107 cells / ml of 24 hrs M. luteus or Pseudomonas sp., respectively. Fish of the 3rd group were I/P inoculated by 0.3 ml of saline as controls according to Irianto and Austin (2002).
Preparation of probiotics diet:-
The probiotic bacteria were prepared by inoculating the bacterial isolates in TSB and incubated at 30°C for 48 hrs, then centrifuged at 3000 rpm for 30 minutes. After centrifugation, the bacteria was washed twice with saline. The solution was added to the bacterial cells (till one ml saline contained 1010 bacterial cells). The saline containing the probiotic isolates was added to commercial sterilized food to give an initial number of 1010 bacterial cells/ g.
Dry ingredients were sieve and ground to a small particle size in a Thoms-Wiley Laboratory Mill model 4, USA. Ingredients were mixed to formulated basic diet, which consisted of 13% hearring fish meal, 40% soybean meal, 5% wheat bran, 35.94% ground yellow corn, 2.5% fish and corn oil (1:1), 0.06% ascorbic acid, 1.5% vitamins and minerals mixture, 2% carboxymethyl cellulose as a binder. The experimental basic diet formulated to contain 30% crude protein and 421 k cal of gross energy/100g diet on a dry weight basis. Bacteria strains supplemented in base diet to conducted four treatments: base diet untreated (control); base diet mixed with M. luteus (T1), base diet mixed with Pseudomonas sp (T2) and a mixture from M luteu and Pseudomonas sp diets with equal amounts (T3). Experimental diet were passed with clean and sterile auger and diet of Spaghetti Machine, La Parmigiana, Model D45 LE, Italy” and dried at room temperature. Diets transferred to black plastic bags and stored in a freezer (–20 ºC) until immediately prior feeding. The number of probiotic bacterial cells in the prepared pellets were found to be 07 cells/g diet.
Feeding culture system design:
The feeding experiment was conducted in 12 glasses aquaria (75×40×50 cm), at the Center Laboratory for Aquaculture Research, Abbassa, Abou-Hammad, Sharkia, Egypt. The aquaria were supplied with 100 ml well aerated tap water. Each aquarium was supplied by compressed air via-stone. Fresh tap water was stored in cylindrical fiberglass tanks for 24 hours under areation in order to dechlorenated the water. Each aquarium was cleaned daily by siphoning fish feces and remaining feed with 75% of total water volume, then refilled to fixed volume again. Water temperature range was (26- 28ºC).
Nile tilapia fries, O. niloticus with an average weight (2.35 - 2.45 g / fish) were acclimatized in an indoor fiberglass for two weeks. The fish were distributed randomly at a rate of 20 fish / aquarium. Treatments in triplicate were arranged at random. The experimental fish were batch weighed at the beginning of the experimental period. Fish were fed twice daily, 6 days a week, at a fixed feeding rate of 3% body weight of fish per day. The feed given were adjusted at 2-weeks intervals after fish were weigh. The experiment ran for 90 days, after which all the experimental fish were collected , counted and weight. The growth parameters and feed utilization were calculated according to:
Specific growth rate (SGR) = 100 (ln W2 – ln W1) T -1