Does feeding frequency affect utilization of added amino acids in Nile tilapia?
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- Master's theses (IHA) 
Nile tilapia (Oreochromis niloticus) is one of the major farmed fish species, with main production in Asia, South and Central America that can tolerate a wide range of environmental stress and easily adapt with low quality of feed ingredients. The aims of the experiments were to determine effects of feeding frequency on utilization of protein and energy in Nile tilapia, to quantify differences in excretion of ammonia and ammonium in Nile tilapia fed the same daily ration, distributed over 2 and 4 meals a day. The aims were derived from a hypothesis that frequent feeding facilitates higher overlap in absorption of crystalline dietary amino acid supplement and protein-bound amino acids, since digestion of protein requires time and thereby may improve utilization of dietary protein. Significant differences were indicated for P<0.05. The fish were kept in indoors rearing tank (70×50×50 cm) with equal water flow (180 l h-1) supplied with freshwater from a recirculation system. Each tank contained 30 tilapias (mean weight ± SEM; 721 ± 0.1 g) with an average weight of individual fish 24 g. All tanks had 24 h d-1 light. The water temperature and dissolved oxygen were recorded on a daily basis. The average water temperature was 27.5 °C with 7.5 mg l-1 dissolved oxygen and pH 7 during the experimental period. The fish were fed for 46 days One plant ingredient based diet was prepared with 31% crude protein, 37% starch and 6 % fat in the dry matter. There were two different feeding regimes. One was only twice in a day (at 10:00 and 20:00) and the other was four times per day (at 08:00, 12:00, 16:00 and 20:00). Each meal lasted for 70 min when the fish were fed 2 meals d-1, and 35 min for 4 meals d-1. In three reference tanks feeding was twice per day to satiation level, and the fish in the remaining tanks were pair-fed 90 % of the dry matter consumed by the fish fed to appetite the previous day. No significant differences were found for growth performance, body composition, or feed conversion ratio when the fish received 90% of appetite level in 2 or 4 meals. However, liver weight in percent of whole body weight was significantly higher for 2 than 4 meals. Feeding the fish to satiation in 2 meals resulted in higher feed intake, and lower protein and energy retentions than feeding 90% of satiation in 2 meals. Ammonium (NH4+), ammonia (NH3), total ammonia nitrogen (TAN) and nitrite (NO2-) were measured one hour before the last meal (19:00) and every 2 hours after last meal ( 22:00; 00:00; 02:00; 04:00; 06:00) over night at days of 14, 26 and 39. During this measurement water samples were collected from the inlet and outlet of tanks. At the end of the experiment (day 45) nitrogenous catabolites were allowed to accumulate in a stagnant system (water flow closed), following the same sampling intervals as described for the run-through sampling approach. From the flow-through system, significant differences were observed between feeding frequency, time interval and their interaction in all parameters (TAN, ammonium, ammonia) at days 14, 26 and 39. Similarly, significant differences were seen for nitrite measurement at different period of time. However, no significant difference was seen for feeding frequency and the interaction between feeding frequency and measurement at different period. In the flow-through system, the highest TAN was found at 4 hours after feeding in 2 meals (90% and appetitive) while for 4 meals (90%) it was at 2 hours after feeding during all the sampling days. The maximum TAN at 588 µg l-1 water was measured at day 39 in 2 meals (appetitive), 4 h after feeding. The lowest value at 89 µg l-1 was measured at day 14 in 4 meals (90%), 10 h after last feeding in the day. TAN was consistently highest for to 2 meals (appetitive) and lowest for 4 meals (90%). The TAN excretion started immediately after feeding, then reached peak value, and thereafter gradually declined to the based level. Nitrite accumulation did not follow any particular pattern in the flow-through system, bur tended to reach peak value 2 hours after feeding. The accuracy of the results obtained from the stagnant system was high, and metabolic nitrogen excretion (with or without values for nitrite) over time was described (R2=0.98-0.99) by 3rd degree polynomial patterns. Maximum nitrogen excretion, with nitrite was maximum at 4.59, 4.61 and 2.88 h after feeding in 2 meals (appetitive), 2 meals (90%) and 4 meals (90%) respectively. The estimates without nitrite included showed maxima at 4.59, 3.64 and 4.02 h after feeding in 2 meals (appetitive), 2 meals (90%) and 4 meals (90%) successively. NO2-, which was not a catabolite increased linearly over time. A probable reason for the observed increase in nitrite may be oxidation of TAN from metabolism and/or microbial oxidation of nitrogen in intestinal contents and faeces. The nitrogen excretion rate over gills (not including excretion of urea via the kidneys) 4 h after last feeding was the highest (150 µmoles (kg body weight (BW)* h)-1) for 2 meals (90%) and the lowest (113µmoles (kg BW * h)-1) for 4 meals (90%). Whereas for 2 meals (appetitive) showed very close value 145 µmoles (kg body weight * h)-1 to 2 meals (90%). Meanwhile, 2 meals (appetitive) reached at peak (149 µmoles (kg BW * h)-1) at 6 h after last feeding and the lowest (88 µmoles (kg BW * h)-1) for 4 meals (90%). Therefore, it was seen in all the observations that the maximum level of nitrogen excretion was found in 2 meals (appetitive) and lowest in 4 meals (90%). In conclusion, there were no significant differences in weight gain, feed utilization, body composition, protein and energy retention between 2 and 4 meals restricted. However, nitrogen excretion peak values were significantly higher in 2 meals (90%) than 4 meals (90%), indicating that the 4 meals (90%) feeding regime facilitated better water quality and thereby a better environment for the fish.