The current results revealed that high cornstarch level has a significant impact on the growth response of GIFT tilapia, which is similar to the results of other studies14,15,16,17. Qiang et al. (2019) showed that the WGR and SGR first increased and then decreased with an increase in the carbohydrate level (0, 60, 120, 180, 240 and 300 g/kg) in larval and juvenile tilapias. In addition, Li et al. (2019) found that the specific growth rate decreased linearly with an increase in dietary carbohydrate (52.7, 89.5, 114.9, 143.7, 177.8, 208.2, and 236.5 g/kg)17. Compared with carnivorous fish, herbivorous and omnivorous fish have a stronger ability to utilise starch. The herbivorous and omnivorous fish can adapt to a certain amount of carbohydrates, showing good growth characteristics, but excessive carbohydrates have a negative impact on fish growth18. These results suggest that carbohydrate in feed can be used to meet the energy requirements of fish19, which may partly explain the protein-sparing action of carbohydrate in diet. However, the results of Li et al. (2015)20 in juvenile giant and those of wang et al. (2016)15 in grouper have revealed that carbohydrate has no protein-sparing action. The reason for the different results of the protein- paring action of carbohydrate needs to be further explored, and may be related to fish species, diet formulation, water quality, and management condition. In this present study, the high cornstarch diet significantly decreased the content of crude protein and increased the content of crude lipid in whole fish body. The results indicated that with an increase of corn starch content in feed, the content of crude protein in feed was relatively decreased, leading to an increase of carbohydrate utilisation. This conclusion is consistent with former findings that the content of crude protein in whole fish body decreases linearly, with an increase of carbohydrate inclusion in the diet, while the content of crude lipid increases quadratically (P < 0.05)17. The carbohydrate metabolism in fish is mainly carried out in liver, including glycolysis, gluconeogenesis, and glycogen synthesis. Several studies have shown that the viscerosomatic index and condition factor increase with an increase of carbohydrate ratio in the feed of juvenile giant croaker20, Wuchang bream21, juvenile Elopichthys bambusa22, and juvenile Gift tilapia23. This showed that fish can convert the excess carbohydrate of feed into body fat, and high carbohydrate intake can enhance fat synthesis and accumulation24. The present results showed that the high cornstarch diet significantly improved the viscerosomatic index and condition factor of GIFT tilapia.
In the current research, the datas of blood index showed that high cornstarch level significantly increased the content of blood glucose, and liver/muscle glycogen (P < 0.05). The blood glucose is a very crucial index reflecting the glucose metabolism, the physiological state of cells and tissues, and the endocrine function of fish. The ability of carbohydrate digestion and metabolism is poor in fish, especially carnivorous fish25. The inhibition of high cornstarch diet on growth and feed utilization confirmed the glucose intolerance of GIFT tilapia. One of the metabolic pathways of blood glucose in fish is to synthesise glycogen in the liver and muscle. Studies have shown that there was a positive correlation between glycogen deposition and carbohydrate content in feed, and high carbohydrate diet can result in the deposition of more liver glycogen7. Although liver glycogen also helps to control the high blood glucose after eating carbohydrate feed, the high content of liver glycogen damages the normal function of the fish liver, resulting in a decline of detoxification ability. Insulin is the only hormone that lowers blood glucose levels. Due to insufficient insulin secretion and regulation, fish are considered to possess a congenital "diabetic constitution". Therefore, excessive carbohydrate may lead to the pathological status of fish, which may inhibit immune function. The present study showed that the blood insulin level was not influenced by different cornstarch levels (P > 0.05). This result is consistent with those of previous studies on different types and levels of carbohydrates in Larimichthys crocea26. It is suggested that the secretion of insulin in fish is not related to the type and level of carbohydrate in feed, and the regulation of blood glucose levels mainly depends on liver glycometabolism-related enzymes and liver glycogen content27.
As we all know, the ability of fish to balance blood glucose level is relatively low, therefore excessive carbohydrates often cause various stress reactions of fish28. To prevent oxidative damage, the organism has developed an antioxidant protection system. The antioxidant capacity of organism includes enzymatic and non-enzymatic antioxidant activities, such as T-AOC, GSH-Px and SOD, which are the first defense mechanism against free radicals in organism. Many studies have found that the external defense ability of fish is always affected by feed nutrients29; nonetheless, little is known about how the characteristics and content of feed components affect the oxidative capacity of fish. In the current study, feeding tilapia with a high cornstarch diet seems to elicit strong stress symptoms. The results of previous studies on antioxidative stress caused by a high starch diet in juvenile black carp16 and Wuchang bream30 are similar to those of this study. These studies showed that the higher the activity of antioxidant enzymes, the better the health status of aquatic animals, and the better the metabolic balance16,21. At the same time, this study also proved that the excessive dietary cornstarch also reduced the liver antioxidant capacity of tilapia. However, previous studies on carnivorous fish have shown that feed carbohydrates do not cause oxidative stress31, 32. On the contrary, some studies have pointed out that carbohydrates used as part of the energy have no adverse impact on oxidative stress, or even contribute to oxidative protection33,34. Because of different nutritional requirements, formula composition, and carbohydrate addition level, the ability to tolerate carbohydrate is different for fish with different food habits, therefore, many problems still need to be further studied and discussed.
Similar to other vertebrates, aquatic animals have specific and non-specific responses to infectious agents. When fish are attacked by pathogenic microorganisms, the non-specific defense mechanism is more crucial than the specific defense mechanism, because the former is least influenced by environmental temperature, while the latter takes longer to accumulate antibodies and specific cell activation35. Therefore, when the external pathogens invade, the non-specific defense system of fish is quickly activated, especially through the production of high reactive oxygen free radical phagocytosis, intracellular killing, and other cellular defense mechanisms, so as to protect fish from pathogens invasion. In recent decades, numerous research results have shown a high correlation between dietary nutrients and body immunity. Better dietary nutrition can improve the immunity of fish, thus helping fish resist external stress and disease36,37. Therefore, this study focuses on the humoral and cellular immune indicators that have been proven to be the first line of defense. In this project, we studied the effects of three different cornstarch levels on the immune response and anti-infection ability of tilapia. The results showed that the high cornstarch diet negatively affected the immune response of tilapia. Lysozyme is an important part of animal immune defense, therefore, it is often used as a fish immune index38,39. Lysozyme can destroy the polysaccharide wall of bacteria to prevent pathological infection of animals40. Zhou et al. (2013) showed that Wuchang bream fed a 310 g/kg carbohydrate diet had significantly increased the lysozyme activity than fish fed a 470 g/kg carbohydrate diet (P < 0.05)21. Likewise, Liu et al. (2012) found that the serum lysozyme activity of fish fed 270 g/kg or 340 g/kg carbohydrate diets was significantly lower than that of fish fed a 140 g/kg carbohydrate diet41. In this study, the LYZ activity in serum, liver and gill of fish fed 36% cornstarch was the lowest, which indicated that the high cornstarch diet could reduce the immune ability of tilapia down to a certain level. Phagocytosis is the basic mechanism of the innate immune system of organisms and the first defense line for eukaryotes to resist pathogen invasion42. In phylogeny, phagocytosis is the most primitive and basic defense mechanism of all organisms. Phagocytosis is an important index to evaluate the immune status of organisms. It can be used to evaluate how external factors such as diet, temperature and pathogenic bacteria affect the immune function of organism. The most common technique for assessing phagocytosis in vitro is to calculate the phagocytic index. In this study, we determined the immune function of leukocytes isolated from the peripheral blood of GIFT tilapia. The results showed that the phagocytic activity and phagocytic index of tilapia fed high cornstarch diet were significantly lower than that of fish fed medium cornstarch diet. The bacterial challenge test is also often used as an ultimate indicator of fish health, especially to evaluate the health status of fish after feeding certain feed nutrients4. The Aeromonas hydrophila is a kind of common gram-negative bacterium. Because it can cause haemorrhagic septicaemia and ulcerative diseases in animals, resulting in high mortality, it is widely used in the study of nutritional immunity of fish43. Previous studies have shown that the mortality is higher in Labeo rohita juveniles fed a high carbohydrate diet3,44. Wu et al. (2016) also found that feeding juvenile black carp with diets containing carbohydrate (194.3 and 288.4 g/kg) after A. hydrophila infection could improve the survival rate16. In the present study, the post-challenge mortality in fish fed high cornstarch diet was higher than fish fed low cornstarch and medium cornstarch diets, however, the difference was nonsignificant (P > 0.05).