Introduction
Evolution has selected organisms that were able to anticipate fluctuations of their environment and adapt themselves accordingly (DeCoursey, 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.
Materials and methods
Three experimental groups were constituted (ML was fed during the light phase at ZT6, MD was fed during the middle of the dark phase at ZT18, and finally, ALE, the last group had no fixed feeding time). Each per treatment was considered individually and the three corresponding sampling sand isolated in metabolic tanks. In order to measure ammonia excretion rate at a given time, 6 fish were selected per treatment and isolated in metabolic tanks. The ammonia concentration was measured in the metabolic tanks right before adding the fish, and then 4 hours after the introduction of the fish. The differences in ammonia concentration allowed us to calculate ammonia excretion rate in throughout thse 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, gill and liver samples were collected on 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 and rhcg in the gills).
Results and discussion
NB: qPCR results are not yet available, they should be released by the end of April, so the current section will only cover ammonia excretion rate.
Statistical analysis (one-way ANOVA and its post-hoc Tukey test) have showed significant differences in the daily variation of ammonia excretion rate based on the experiment groups. Cosinor confirmed the rhythmic nature of ammonia excretion in ML and MD, but fish that had no fixed feeding time did not display such rhythmicity. Acrophase of ammonia excretion occurred at ZT18 in ML (12 hours after feeding time), while the highest ammonia excretion rate was observed at ZT2 in MD, only 6 hours after feeding. This suggests that feeding time is not the lone factor controlling the dynamics of ammonia excretion.
References:
DeCoursey, P.J., 2004. The behavioral ecology and evolution of biological timing systems.
Dunlap, J.C. and Loros, J.J., 2017. Making time: conservation of biological clocks from fungi to animals. Microbiology spectrum, 5(3), pp.10-1128.