Early life stress can shape behaviour, physiological metabolisms, stress responses, and immune and antioxidant systems of organisms later in life. While either low or high levels of early stress produce negative impacts (for example, poor immune system responses), the right sort and at the right time of early mild stress (EMS) result in positive outcomes. EMS is like preparedness and might help fish deal with stress appropriately. In this project (six trials: six published papers), we tested scheduled EMS for the first time in farm animals and also its interaction with protein, lipid and fish meal levels in diet and environmental factors (photoperiod and temperature). Oscar Astronotus ocellatus (4-9 g), as one of the most traded aquarium fishes, was selected. We ran six trials for nine weeks in the same condition in 100L tanks and fed mostly the same diets to decrease the bias. Fish were fed three times a day to apparent satiation levels. After collecting samples in week 9, the left fish were randomly transferred to the new tanks. Then, fish rested for one week and we applied acute confinement stress (AC) in week 10. More than 40 health parameters were measured.
The output of trial 1 showed consecutive stress and not enough recovery time , which negatively affected the survival rate, stress responsiveness, immune and antioxidant system, and health of oscar. Dietary protein and lipid contents drove changes in growth but not EMS, which was optimum at 51% and 18% (trials 2 and 3). After AC stress, a better survival rate, immune system, stress responsiveness, antioxidant defence and haematology in the 2EMS group resulted in a higher survival rate than others (trials 2 and 3) . Fish meal contents in diets (11-25%) did not affect growth, but the number of EMS (0, 2, and 3 weeks) did. Most of the health parameters were affected by Fish meal contents but not EMS numbers (trial 4). Five scheduled photoperiods did not affect the growth and survival of oscar. After AC stress, fish farmed in 24 light hours had the lowest health parameters, while these results were not observed in EMS-exposed fish (trial 5). Too high (32°C) or low (17°C) temperature decreased growth. The EMS did not positively or negatively affect fish physiology in this study (trial 6) . There was no significant difference in survival rate before and after AC stress among groups (trial 6).
We understood from this project while EMS can greatly help fish tackle stress, the roles of diet components and environmental parameters are key drivers. While the general trend was improved haematology, immune and antioxidant systems, stress responsiveness, and serological enzymes, no parameter showed the same trend across trials. Although fish welfare must be considered, EMS can result in a greater survival rate after fish are exposed to later acute stress in life.