## 2 Materials and Methods  
### Ambient Oxygen Concentration Forecasting Model Design  
Waterless Transportation Process for Flatfish  
The work flow of live sea fish delivery with cooling condition can be roughly divided into three parts: the pre-transportation processing stage, the packaging processing and monitoring stage, and the waterless live transportation and recovery stage. The more specific procedure of delivery is demonstrated as follows.  
* _The pre-transportation processing stage_: in this stage live fish are caught from the aquaculture pond, and the healthy fish are picked and moved to the aquatic plants for pre-transport processing. The fish to be transported must be healthy and in good condition. Weakened individuals should be eliminated from the consignment.
* _The packaging processing and monitoring stage_: the hibernated fish are caught from the pond, and are carefully placed on a tray. After weighing, they are piled up in a fresh-keeping box that is equipped with observation sensors for each box. Then, the box is filled with pure oxygen and sealed, inducing a hypopnea state in live fish. The fresh-keeping box is transferred to the chill car and waterless delivery is carried out with a relatively low inside temperature from 0 to 5 \({}^{\circ}\)C.
* _The waterless live transportation and recovery stage_: in this stage, fish are delivered in waterless conditions with low temperature and pure oxygen at the beginning of transportation. The aquatic products are maintained in dormancy at the lowest state of metabolism. At the destination of delivery, fish will be aroused gradually in the docking ponds and are expected to regain consciousness and return to their live state.  
The workflow of waterless transportation for live flatfish is shown in Figure 1. Over the whole period, the quality of fish transported is a key decisive criterion. The fish to be transported must be healthy and in good condition. Weak fish are killed at a much higher rate than fish in good condition when the transport time is longer.  
The most important single factor in transporting fish is providing an adequate level of oxygen [19; 20]. However, an abundance of oxygen within a container does not necessarily indicate  
Figure 1: The process of flatfish waterless transportation.  
that the fish are in good condition. The ability of fish to use oxygen depends on their tolerance to stress, ambient temperature, and metabolic products such as concentrations of carbon dioxide. In a live flatfish delivery system, the fish will require more than the minimum amount since they are not in a fully resting state. Furthermore, if they are excited at loading, or there are vibrations or disturbances during transport, they may consume more amount of oxygen than we expected.  
The amount of oxygen a fish consumes does not only depend on the amount of oxygen available. In closed systems, the high levels of atmospheric oxygen required to maintain the live condition are not well sensed or predicted. During long-distance delivery when the container with fish are undergoing abnormal movement, the fish may consume more oxygen at a certain level of stress response. This research applies mainly to solve such problems because an uncertain oxygen atmosphere is not suitable for maintaining a reasonable survival rate. Next, we will introduce the on-line oxygen forecasting methods.