# Rearing Conditions and Automated Feed Distribution Systems for Zebrafish (_Danio rerio_)
## 4 Feeding Requirements
### Feeding Management  
The lack of standardization of zebrafish feeding protocols not only concerns the great consideration for implementing factors affecting the daily nutritional requirements [27, 56, 79], but also includes feeding management practices, which are equally important and should be designed by taking into consideration the nutrient and physical properties of the diet. Many studies are used to report both feed amount and daily/weekly feeding regime (feed ratio and frequency), but each study follows its own personal scheme, and the direct effects of the various feed management criteria on specific outcomes have seldom been investigated [31]. Feeding ratio is the amount of diet administered per group or per group of individuals, which is usually referred to as grams per individual or percentage of body weight [31]. Feeding frequency, on the other hand, is defined as the number of times feeding is provided (ratio per individual per unit time) [31]. Both practices are often determined by the availability and the economic resources of the operators [31], thus significantly affecting zebrafish nutrition, especially when using formulated diets [65; 80; 81]. Therefore, it is not surprising to find a wide variety of feeding frequencies, ranging from once per day to several times per day or even during the night. Moreover, the feeding frequency should also be adjusted for different sizes and ages of zebrafish with the aim of reducing the suspension time in the water before ingestion [31]. Furthermore, the common practice in laboratories is to feed zebrafish _ad libitum_ (i.e., the animals are offered as much food as they want), which can be followed by leaching from uneaten food and mixing of feeds with fecal material in the bottom of the tanks, thus reducing much of the ability to quantify feed intake--an essential practice in determining daily nutrient requirements--as well as water quality and, thus, fish welfare [31]. In addition to feeding frequency and ratio, feeding time (the time of day or night when the diet is provided) is also greatly affected by the operators [31]. For example, feeds can be administered any time of the day or night, depending on the operators' availability. Since feeding time can affect zebrafish behavior and feed intake [82], specific time(s) of feeding should be standardized and reported. Fish, in fact, do not usually feed continuously in the wild, but consume meals at certain times of the day or night, exhibiting distinct daily feeding rhythms [83], and although no preferential feeding times have been reported, food-anticipatory activities when fed on a fixed schedule have been shown [84]. Moreover, if multiple ratios per day are provided, the amount of each food ratio should be determined in order to optimize feed ingestion at specific times [31], since ingestion of a ratio may vary depending on the time per day (or night).  
To fully assess the potentiality of zebrafish as a model and its increasing use in research laboratories, facilities, and biotech companies (which invariably implies highly controlled fish rearing conditions), the need to combine novel standard diets which satisfies all nutrient requirements with a controlled and reproducible administration setup, far and independent from variables introduced by the operators, is increasingly urgent. As a starting point, an initial reference growth curve is already established [17].