The growth performance of snubnose pompano in the present study, in terms of WG%, SGR, ADG and TGC were found higher in control group followed by 100CSM containing 343 mg FG kg −1 diet, which indicated that it can tolerate the FG than most of fishes and thus growth performance was not affected. This is in agreement with Rinchard et al. (2003a) found that complete replacement of FM using CSM does not influence the growth performance in terms of WG% and SGR% in male rainbow trout.
The major limitation associated with the use of CSM is the free gossypol toxicity which could decrease the digestibility of lysine and rendering it less bioavailable when fish ingested diets with high concentration of gossypol (Liu et al. 2009). According to Rinchard et al. (2003b) growth depression in fishes fed with CSM based diets are usually linked with the reduction in the bioavailable lysine contents of the diets. In the present study, the maximum inclusion level of CSM was 34.7% of diet which did not show any negative effects on growth due to supplementation of adequate level of lysine in the diet and availability of more quantity of arginine in the CSM replaced diets when compared with 0CSM diet. This is because the fact that FG present in CSM reacts with the free epsilon-amino groups from lysine and arginine to form bound gossypol which affects the digestibility of these amino acids (Fernandez et al. 1995). In the same way, higher inclusion rate of 24.5% of CSM was reported for on-growing grass carp (Yan et al. 2014) and 36.6% for sub-adult stage of grass carp (Liu et al., 2016) without adverse effects on growth. As the FM replaced diets in the present study were supplemented with lysine and methionine on par with the control diet and availability of higher arginine content as the level of CSM increased in the diet, the growth performance of pompano fed with CSM was not depressed.
Haematological status of fish is a useful indicator for monitoring fish health and physiological responses to any stressors (Ahmed et al. 2020). Free gossypol found in the CSM readily binds with dietary iron to form a gossypol-iron complex, which inhibits its absorption that leads to iron deficiency and affects erythropoiesis (Gadelha et al. 2014). The FG may also cause a reduction in Hct and Hb of fish (Mbahinzireki et al. 2001) and leukocyte counts which affect the immune-competence of the animal (Braga et al. 2012). The hemoglobin, RBC, WBC and hematocrit values in the present study were not significantly influenced by the complete replacement of FM using CSM in snubnose pompano. This result is in line with Rinchard et al. (2003a) who reported similar response in terms of RBC and hematocrit in male rainbow trout fed with graded levels of CSM in the diet. Similarly, Dabrowski et al. (2000) fed adult rainbow trout with the diets containing up to 990 mg FG/kg through dietary CSM for 131 days had lower hematocrit and hemoglobin than fish fed 495 mg/kg or less concentration. The deleterious effects of CSM on hematological response of fishes are species-specific (Liu et al. 2016) and fish can endure relatively higher levels of FG than other monogastric animals (Cheng and Hardy 2002). In the present study, the levels of FG content was lower and also the diets were adequately supplemented with lysine which may bound with FG and effectively minimized the formation of gossypol-iron complex and hence, did not affected the RBC and hematocrit values seriously.
According to Summerfelt and Penne (2007) the nutrient accretion in different fish species is primarily dependent on the level of dietary nutrient composition and other associated biological factors. In the current study, whole-body proximate composition of experimental fishes in terms of total ash, calcium and phosphorous content was influenced by the reduction in the level of inclusion of FM content of the diet and thus the lower values were observed in FM free diet fed fishes (100CSM). This is in agreement with Garcia-Abiado et al. (2004) and Hassaan et al. (2019) reported the reduction of total ash content in tilapia as the level of inclusion of CSM in the diet increased by reducing FM content of the diet. Calcium (Ca) and phosphorous (P) concentration of fish vertebra, scales and whole body tissue have been commonly used as an insightful indicator of dietary Ca or P status in fish nutritional research (Zhang et al. 2006; Hossain and Yoshimatsu 2014). In corroboration with the present result, Mbahinzireki et al. (2001) reported that dietary CSM with varying level of FG on mineral composition especially calcium and phosphorous content of whole body of tilapia found a decreasing trend as the CSM inclusion increased. Similarly, Hassaan et al. (2019) reported the plasma calcium and phosphorous content of comparable response in tilapia. In the present study, other proximate parameters such as crude protein, lipid and NFE were not affected by CSM inclusion in the diet. The results of the current study corroborates the results of Ahmed et al. (2018) and Zhou et al. (2017) in juvenile blunt snout bream fed with complete plant based diet and cottonseed meal, respectively.
The essential amino acid profile of the experimental diets indicating that the levels of limiting amino acids, lysine and methionine were on par with their requirements of lysine (2.4% of diet) and methionine (1.16-1.18% of diet) of snubnose pompano (Ebeneezar et al. 2019; Ebeneezar et al. 2020). In the present study, the experimental diets were supplemented with external sources of lysine and methionine to maintain their levels equally in all the FM replaced diets on par with the requirements of snubnose pompano. The lysine content was slightly reduced to the level of requirements in the higher inclusion of CSM groups (75CSM and 100CSM) which may be due to the reaction of FG with lysine and formation of bound gossypol. This is in accord with the results of Liu et al. (2020) in the experimental diets prepared with graded level of inclusion of low-gossypol CSM in the diet of silver sillago, Sillago sihama. The EAA profile of whole body samples of experimental fishes showing that lysine, methionine and arginine contents were comparatively lower in CSM incorporated diets than the than 0CSM groups. This result is comparable with Choi et al. (2020) in sub-adult olive flounder, Paralichthys olivaceus fed the diet containing FM replaced with either animal proteins or animal and plant proteins revealed that most of the amino acids were not significantly changed among the treatments.
The levels of energetic metabolites such as triglycerides and cholesterol of fish are considered as important indices reflecting the nutritional and physiological status of fish (Xu et al. 2013) and body fat metabolism (Shen et al. 2020). Gossypol content of dietary CSM reduces the cholesterol in the body which may be attributed through reduced intestinal absorption of dietary cholesterol and may reduce the synthesis of LDL in the liver (Wang et al., 2009). In the present study, serum cholesterol, total lipid, LDL and VLDL contents of experimental fishes showing a declining trend as the level of inclusion of CSM increased in the diet which may be due to FG contents of the diets that reduced the intestinal absorption. This is in agreement with Liu et al. (2016) reported similarly lower level of cholesterol and LDL content in the pre-adults of grass carp fed with varying levels of CSM. Correspondingly, Choi et al. (2020) reported that FM replaced with either animal proteins or animal and plant proteins in sub-adult olive flounder effectively reduced the levels of cholesterol when compared with FM based diet.
Serum protein profile in terms of higher albumin and globulin is a valuable indicator of better health status of fish through enhanced innate immune system function (Liang et al. 2018). In the present study, the serum protein profile did not show significant changes among the treatments which indicate that CSM content of experimental diets did not influenced the health status of snubnose pompano. This is in agreement with Shen et al. (2020) found no significant difference in total protein content in golden pompano fed with the diet containing varying levels of FM substitution with cottonseed protein concentrate. Similarly, Ahmed et al. (2018) reported that complete replacement of FM with plant proteins including CSM) and supplemented EAA in juvenile of M. amblycephala did not affect plasma total protein, albumin and triglyceride which in turn did not affect the immune response.
Digestive enzyme activities depend on the age, feeding habits and nutritive profile of the ingredients used for feed formulation (Deguara et al. 2003). In the present study, the protease activity was decreased as the inclusion level of CSM increased in the diet of snubnose pompano. This is in agreement with Liu et al. (2016) who reported likewise decreasing trend of protein digesting enzyme, chymotrypsin activity in grass carp fed with increasing level of CSM. In the present study, the amylase activity was increasing with increased inclusion of CSM in the diet. This is true in silver sillago that showed an increasing amylase activity as the level of inclusion of CSM was increased in the diet (Liu et al. 2020).
Activities of the metabolic enzymes such as AST, ALT and ALP are the indicators of the metabolic processes and function of liver (Prabu et al. 2021). In the current study, the activities of AST, ALT and ALP in the FM replaced with CSM groups were found slightly lower than the fishes fed with control diet without cottonseed meal (0CSM). This is agreement with Bu et al. (2017) who reported similarly lower AST and ALT enzyme activities in juvenile Ussuri catfish fed with FM replaced using CSM in the diet. Wang et al. (2014) reported that the AST and ALP activities showing a fluctuating trend among the treatments and suggested that asynchronous effect of dietary CSM on their activities. Further, Cai et al. (2011) reported that dietary CSM up to 400 g kg−1 diet did not show any difference in plasma levels of ALT and AST of crucian carp. In the present study, the LDH and MDH activities were showing a slightly decreasing trend as the level of inclusion of CSM increased in the diet. This result is in line with Rinchard et al. (2003a) who reported similar trend of LDH activity as the level of CSM increased in the diet of male rainbow trout.
Dietary FG accumulates higher amount in the liver and kidneys of animals (Wang et al. 2014) and in the muscle tissue the accumulation was very meagre that depends on the levels of dietary FG content and the nutritive profile of diet (Li and Robinson 2006). In the present study, the FG content of muscle tissues of snubnose pompano was at the maximum of 1.28 mg kg−1 body mass on as is basis in 100CSM which was insignificant while comparing the permissible limit of FG (450 mg kg−1) in any cotton seed related products (FDA 1974). This result is in accord with the results of Lee et al. (2006) who fed the rainbow trout with 58.8% dietary CSM for 3-years and concentration of FG in the muscle tissue was lower than 1 mg kg−1 of wet tissue sample. Similarly, Wang et al. (2014) reported that juvenile common carp fed with 54% CSM corresponding to 647 mg kg−1 FG for 8 weeks showed the FG concentration in the muscle tissue was 6.07 mg kg−1 on DM basis. Based on these results, it is evident that it is very safe to consume the fish fed with CSM based diets as the accretion was insignificant. Further, the half-lives (𝑡1/2) of (+) and (−) FG in rats was estimated at 25.26 and 10.53 h, respectively (Chen et al. 1987) which indicated that the accumulated FG may be completely depleted from the fish tissues once the animals fed with CSM free diet for a short stint.
The alterations in liver morphological structure and cytology significantly affect the normal liver function as liver is an excellent indicator of water quality, nutritional and physiological status of fishes (Xu et al. 2013). In the present study, the liver sections of experimental fishes did not show any serious gross structural changes even at higher FG content of 100CSM group except for the mild vacuolation of hepatocytes, few hypertrophic hepatocytes and few hepatocytes observed with pyknotic nucleus in 50CSM group. The present observation is in accord with the histological observations of Wang et al. (2014) who noticed that slight decrease in the volume of the hepatocytes with increased numbers in the juveniles of common carp.