Many factors affect fish growth, including diet, fish size, feeding time, stocking density, and feeding frequency. In this experiment, when the fish were fed three times a day, the BWG, WGR, and SGR of fish stocked at a density of 0.55 kg per tank gradually decreased with increasing stocking density and was significantly higher than the other four groups. This suggested that the growth performance of the fish in the 1.10 kg and 1.65 kg per tank stocking densities was better than that of the other groups with the same feeding frequency. In other words, feeding frequency and stocking density influenced the growth performance of this species, which was consistent with previous studies[27, 28]. The poor growth performance observed in the high stocking density was likely due to crowding stress.
Additionally, our findings suggested that feeding three times per day instead of two may have affected satiety levels, thereby resulting in food waste. Several studies have reached different conclusions regarding the effect of stocking density on the growth performance of fish depending on the species. For instance, Rowland et al. (2006)  reported that stocking density had no significant effects on silver perch. In contrast, high stocking density decreased the final weights of Brook charr according to Vijayan et al. (1990), and Boeuf et al. (1989) reported that lower densities had positive effects on the growth of Atlantic salmon during smolting. According to the quadratic regression model based on the BWG of the hybrid groupers studied herein, when fed twice a day, the function was y=-0.4872163x4+6.391186x 3-28.19146x2+45.84394x-6.26（R2=1） and we predicted that the optimal stocking density was 2.004 kg/m3, whereas when the fish were fed three times a day, the function was y=-0.6366255x4+9.53982 x 3-50.46522x2+107.8068x-55.9（R2=1） and the optimal stocking density was 1.343 kg/m3.
Blood can objectively reflect the nutritional metabolism of farmed animals and is an important physiological, pathological, and toxicological indicator. AST and ALT are common indicators of liver cell damage. Specifically, when liver cells are damaged, AST and ALT enzymes will infiltrate into the blood, causing a significant increase in the activity of these enzymes in the blood. Previous studies have linked the occurrence of liver damage with elevated AST and ALT levels [33, 34, 35, 36]. In this study, AST levels increased significantly with stocking density, except at a 2.2 kg/m3. Moreover, ALT decreased significantly with increasing stocking density, particularly at 4.4 kg m− 3 and 5.5 kg m− 3 in both feeding frequencies. Our findings suggest that although increases in AST were indicative of some degree of liver damage, lower ALT levels may explain the 100% survival ratio, as it is an important indicator of fish health .
SOD can eliminate excess free radicals in organisms  and modulate the balance between the formation and elimination of free radicals. Therefore, this parameter is an important physiological indicator of antioxidant capacity and non-specific immune function. LZY and AKP are also important indicators of non-specific immunity in the body. AKP is very important for various metabolic processes including phosphorylation and dephosphorylation, as well as nutrient absorption, and its activity can be used as an indicator of fish immune function.
Previous studies have demonstrated that high stocking densities were associated with decreases in SOD activity in some fish [39, 40] and shrimps [41, 42]. LZY is known to disrupt the structure of the bacterial cell wall, and therefore LZY levels can directly reflect lytic capacity, as well as the impact of pathogens and other environmental factors on fish health. In Senegalese sole, high stocking densities reduced plasma LZY levels. A similar pattern was found in juvenile turbot. In our study, high stocking densities had negative effects on SOD, AKP, and LZY concentrations, which may explain why high stocking densities often lead to diseases. Nonetheless, other studies have demonstrated that high stocking densities could increase AKP activity[44, 45].
TG and CHO are key indicators of blood lipid levels. According to medical research, elevated TG and CHO levels are linked to fatty liver. TP includes GLB and ALB, which are involved in protein metabolism Albumin content generally accounts for more than 50% of total plasma protein content and is known to maintain osmotic pressure balance between blood vessels and tissues, mediate substance binding and transport, protect blood cells and organs, inactivating inflammation, and also acts as an antioxidant and damage repair agent, all of which are closely related to the body’s immune function.
In this study, the CHO levels of groupers gradually decreased with increasing stocking density regardless of feeding frequency, meaning that higher stocking densities resulted in more liver cell damage, which would negatively affect fish digestion and fat translation. Moreover, TP and ALB showed a gradual increase, whereas GLB and GLU decreased gradually with increasing stocking density, indicating that the vital activities represented by the above indicators were gradually weakened as the stocking density increased.
Previous studies have shown that the detection and analysis of digestive enzyme activities can directly reflect the physiological status of fish digestion [46, 47, 48]. In the present study, increases in stocking density had a positive effect on gastric, intestinal, and hepatic pepsin and lipase activities regardless of feeding frequency. However, these results were not consistent with a previous study in which the lipase, trypsin, and amylase activities of Oreochromis niloticus fingerlings were all depressed in high-density conditions (600 organisms m− 3) . This indicated that the fish exhibited better digestive physiology at an appropriate stocking density, which may also explain why the fish had higher growth rates in the corresponding density range. However, the high stocking density in our study may have elicited crowding stress, thereby requiring additional energy expenditure to resist the stress, which in turn required an increased digestive performance. We therefore speculated that increased digestive enzyme activity may be a protective mechanism in high-density conditions; however, this hypothesis must be further explored in future studies.
In this study, feeding frequency and stocking density were found to significantly influence gastric, intestinal, and hepatic tissue histology. When the fish were fed three times a day, even low stocking densities (e.g., 1.1 kg m− 3) resulted in pathological signs or symptoms, which was also observed in fish that were fed twice a day and stocked at 2.2 kg m− 3 and 3.3 kg m− 3 densities. These results were consistent with those of Refaey et al. (2018), who reported that a high stocking density (300 fish m− 3) resulted in intestine and muscle histological changes.