EFFECT OF FEEDING TIME ON THE INTRADAY VARIATION OF AMMONIA EXCRETION RATE IN NILE TILAPIA Oreochromis niloticis
Evolution has selected organisms that were able to anticipate fluctuations of their environment and adapt themselves accordingly (D unlap et al. , 2004). As a result, organisms have developed specific biological clocks that act as natural timing devices, regulating their metabolism according to the cyclic changes in conditions (Dunlap and Loros, 2017). These biological clocks respond to a broad range of parameters known as zeitgebers. Photoperiod and feeding time are some of the foremost zeitgebers controlling farmed-fish metabolism. The present study aims to investigate the variation of intraday rhythms of ammonia excretion in the commonly farmed Nile tilapia (Oreochromis niloticus) exposed to 3 different feeding protocols (ML group: fed during the middle of the light phase; MD group: fed during the middle of the dark phase; aleatory group: no fixed feeding time).
Materials and methods
A total of 189 fish were used in this study (8.87 ± 1.23 cm total length, and 9.39 ± 2.23 g total weight) e qually distributed among the 3 different experimental groups. The 3 groups were exposed to the same 12:12 LD (light:dark) photoperiod. ML group was fed at ZT6, 6 hours after the beginning of the light phase, MD group fed at ZT18, 6 hours after the beginning of the dark pha se, and the aleatory group were fed at a different time everyday).
After 3 weeks of treatment, the sampling took place. In order to measure ammonia excretion rate at a given time, 9 fish were selected per treatment and isolated in metabolic tanks. The ammonia concentration was measured in the metabolic tanks right before adding the fish, as well as 4 hours after the introduction of the fish. The differences in ammonia concentration allowed us to calculate ammonia excretion rates throughout these 4 hours. After 4 hours, the fish were sacrificed. The same methodology was consecutively repeated 7 times in total, allowing us to measure ammonia excretion rate throughout a period of 28 hours.
Additionally, in order to track ammonia excretion down to the molecular level, gills and liver samples were collected from the sacrificed fish. qPCR analyses were performed to measure the intraday variation of the expression of key genes involved in ammonia metabolism in fish (glsn and gludmit in the liver; ca , rhag , rhbg an d rhcg in the gills).
Results and discussion
NB: qPCR results are not yet available, they should be released by the end of the month, so the current section will only cover ammonia excretion rate.
The data collected proved the rhythmic nature of ammonia metabolism in Nile tilapias.
Statistical analysis (one-way ANOVA and its post-hoc Tukey test) have showed significant
differences in the daily variation of ammonia excretion rate. Fish in the MD group had their highest ammonia excretion rate at ZT 20 (2 hours after their feeding time), ML group displayed its highest ammonia excretion rate at ZT 16 (10 hours after feeding time) and the aleatory group had its peak ammonia excretion rate at ZT 16 as wel l (17 hours after the last feeding time). Interestingly, this group is the only one that displayed a second minor peak of ammonia excretion at ZT 8. The experimental groups displayed different rhythmicity but the magnitude of their excretion rates were also significantly different (Student test, p<0.05), with the Aleatory group having the highest mean excretion rate (43.73g/kg/h) followed by MD (21.51g/kg/h), and ML (12.40g/kg/h).
These preliminary results showed the strong endogenous nature of ammonia excretion in this species. Even though ML and MD had their feeding times shifted by 12 hours (ML was fed at ZT 6, MD was fed at ZT 18), their resulting peaks of ammonia excretion were only shifted by 4 hours. The aleatory group received its last meal at ZT 2 3 the day before the sampling began and displayed a peak of ammonia excretion at ZT16 the day of sampling. This group distinguishes itself from the others by having a second minor peak at ZT 8. When considering mean ammonia excretion rates, the aleatory group has the highest value, more than twice the value observed for MD and almost 4 times the mean ammonia excretion rate in ML. This difference may be the result of higher stress level in fish exposed to this aleatory feeding protocol, which would trigger a higher protein catabolism rate and consequently increase the amount of ammonia excreted.
When available, qPCR results will allow to follow daily variations of selected gene expression, and provide us an insight on the molecular mechanisms involved and their relation with photoperiod and feeding time. It is possible that some of these genes are sensible to light while others depend on feeding time, this would explain the strong endogenous nature of the re sults.
The main objective of this project is to gain knowledge on ammonia metabolism in order to control nitrogen cycles in aquaponic systems.
Dunlap, J.C. and Loros, J.J., 2017. Making time: conservation of biological clocks from fungi to animals. Microbiology spectrum, 5(3), pp.5-3.
Dunlap, J.C., Loros, J.J. and DeCoursey, P.J., 2004. Chronobiology: biological timekeeping. Sinauer Associates.