According to the latest pieces of literature, the synchronizer of gut melatonin synthesis is the feeding entrained oscillators (Mukherjee and Maitra 2015b; Pal and Maitra 2018; Vera et al. 2007; Choi et al. 2015; Pal et al. 2015; Muñoz-Pérez et al. 2016; Pal et al. 2016; Sanjita Devi et al. 2016; Yasmin et al. 2021), as a change in the daily rhythm of gut melatonin was evidenced in association with a change in the timing of food supply, availability of food (Mukherjee and Maitra 2015b), number(s) of feed per day (Yasmin et al. 2021), quality of food supplied (Mukherjee and Maitra 2018). The present study is the first attempt to assess the role of different dietary nutrients (Table 1), viz. protein- in 3 different amounts: SD, PRD1, and PRD2; L-tryptophan (Trp)- in 5 particular amounts: SD, PRD1, PRD2, TrpRD1, and TrpRD2; carbohydrate- in 2 different quantities: SD and CRD; and oil- in 2 different quantities: SD and ORD, from the perspective of melatonin levels, oxidative stress management and digestive regulation in the gut. Further, the work has been performed with two different growth phases of the carp, viz. early juvenile (EJv) and late juvenile (LJv) carp, in between which, according to our present data, distinct differences were noted in the values of the residual gut content (RGC), feeding intensity (FI), gastro-somatic index (GaSI) and specific growth rate (SGR).
As mentioned, the variation in the feeding habit is prominent between the EJv and LJv fish, according to the existing literature (Chondar 1999; Das and Moitra 1963). In addition, our present study too exhibited higher GaSI and lower RGC, as well as FI, in the EJv carp compared to the LJv carp when supplied with the SD. Further, the gut melatonin profile was found to be synchronized with food availability within the gut (Yasmin et al. 2021). Therefore, it was found basis to answer the question of whether such change in feeding capability had any effect on the melatonin content within the gut or not. In the present study, much lower values in RGC, FI, SGR, SOD, and digestive enzyme activities (particularly protease, lipase, and cellulase levels) were noted in EJv fish compared to the LJv fish when fed with the SD. In contrast, the levels of oxidative stress (MDA), melatonin, GaSI, GRd, and α-amylase were found to be higher during the early stage (EJv) compared to its later stage (LJv). As reported earlier by Mukherjee and Maitra (2015b), the positive correlation between gut melatonin and residual gut content was not documented in the current investigation. The present study recorded a negative relationship among gut melatonin, FI, and RGC (Table 2) when the values were compared with EJv and LJv fish. This observation underlines the additional physiological relevance of gut melatonin during different growth phases of carp when the capacity of feeding is reduced in EJv carp compared to LJv carp, but melatonin content was high in EJv carp compared to LJv carp.
The present study has documented that application of any of the diets results in reduced digestibility of food (as noted in the values of digestive enzymes in EJv compared to LJv carp, particularly in protease and lipase activity) and increased stress and its management mechanism exist within the gut wall in the EJv compared to LJv. However, the FI was low during the EJv stage, at least after the SD-fed diet. Such replicating observation on reduced digestive enzymes and enhanced stress management was noted when the same fish was under starvation (Pal and Maitra 2018). These data indicate that the unavailability of food reduces digestive enzyme activities and activates stress management within an animal's gut; such a mechanism already existed during the early stage of gut development, when optimum levels of digestive enzyme activities have not been achieved. It was further evident from the data that the early stage of the carp exhibits reduced feeding activity and increases the gut melatonin content, as noted from the data of RGC, FI, and gut melatonin when compared with EJV and LJv carp following correlation (Table 2). This environment initiates the stress on the gut wall and activates the first line protection by increasing the antioxidative enzyme activities within the gut cells. The increased gut melatonin aids this stimulated antioxidative properties of the gut and allows the food particles to remain within the gut lumen for a longer duration for better digestion and assimilation (Chen 2011). These higher gut melatonin levels might promote accelerated gut growth compared to somatic growth during the early stage for the development of a fully functional gut in the later stage of life, as SGR exhibited a negative correlation with GaSI and FI in EJv carp but not in LJv carp (Table 3). When the proper gut develops, melatonin levels are reduced, and feeding characteristic increases, accelerating somatic growth. The same observation also has been supported by our data of GaSI as noted in EJv and LJv fish. Further, the first appearance of gonadal structures in the LJv carp also might be a reason for reduced melatonin levels in the gut of LJv carp, as melatonin, in general, is known to play an anti-gonadal effect on the development of gonads (Maitra et al. 2005). However, further research on the gut histoarchitecture of EJv and LJv fish and gonadal parameters in LJv carp must confirm these observations.
Introducing better quality of food in terms of PRDs, TrpRDs, CRD, and ORD in the EJv and LJv carp induced different modes of stimulation in the gut melatonin levels and antioxidative agents and digestive enzymes. In any of the different quality diets, protein is considered an essential nutrient source for better growth and quality health (Wang et al. 2017). Generally, fish obtains essential and non-essential amino acids from the protein responsible for muscle formation and different enzymatic actions to maintain optimum physiology (Yang et al. 2002). In our study, the SGR is significantly higher in PRD1 and PRD2 compared to SD in both age groups. Nevertheless, in EJv and LJv, SGR reduced significantly in PRD2 compared to PRD1. After the supply of a higher rate of protein in the diet compared to the standard range, evidence of retarded or inhibited growth was also noted not only in carnivorous (Kpogue et al. 2018) but also in herbivorous fish (Kaushik et al. 1995; Santos et al. 2020). Considering these available reports, it can be assumed that in the present study, the extra energy supplied via the PRD2 may lead to an additional burden on the fish to eliminate excess nitrogen by excretion, which does not reflect in the growth (Ahmed and Ahmad 2020). However, it was not evident earlier whether the high protein content of the diet (PRD2) with higher calorie (energy) value has any effects on the gut melatonin, activities of antioxidative agents, and digestive enzymes. Hence the study has been designed.
After supplying any of the PRDs and TrpRDs, a significant reduction in the RGC, FI, GaSI, and an increase in gut melatonin levels were recorded in EJv and LJv stages compared to the SD. According to a recent report, amino acid sensing receptors can sense the aromatic amino acid, like Trp (Calo et al. 2021), which were supplied in graded increasing doses starting from the lowest value in SD (0.36 ± 0.03%), CRD (0.36 ± 0.03%), and ORD (0.39 ± 0.03%), then in PRD1 (0.46 ± 0.01%), PRD2 (0.58 ± 0.01%), TrpRD1 (0.96 ± 0.03%) to the highest value in TrpRD2 (1.36 ± 0.03%). This increase in Trp levels in the diets may increase the level of cholecystokinin (CCK) in the gut, as found in rainbow trout (Oncorhynchus mykiss) (Calo et al. 2021), hence, decreasing the hunger level by reaching the early satiety in EJv and LJv carp in a different way. The EJv and LJv fish exhibited different responses on the levels of gut melatonin when modulated by dietary ingredients. In the case of EJv carp, the highest levels of gut melatonin were noted after the supply of PRD2, though SGR and FI were reduced in respect of LJv carp. With increased FI, in LJv carp, gut melatonin was reported highest after a supply of TrpRD2, not after high protein-rich diets. This indicates that different rates of AANAT and ASMT enzyme expression (Pomianowski et al. 2020)/activity (Gern et al. 1984; Nisembaum et al. 2013) or serotonin levels for melatonin synthesis may exist in the EJv and LJv carp gut. If the serotonin synthesis in the TrpRD2-fed group is higher in EJv, that may cause reduced food transmit time (FTT) (Bubenik and Dhanvantari 1989) compared to LJv. The reduced level of RGC and FI in EJv carp supports the finding. Further investigation of the AANAT and ASMT enzyme activity (Gern et al. 1984; Nisembaum et al. 2013) and the measurement of serotonin levels in the different diet fed groups in two particular age groups in the future may solve the conjecture. EJv has a significantly higher amount of gut melatonin in each experimental diet than LJv, except TrpRD2, indicating that in the early stages (EJv), proper absorption of that high amount of Trp has not been developed (Herrero et al. 2007). However, in a later stage of life (LJv) gut may develop more potential to utilize a high amount of Trp, and as a result, TrpRD2 exhibited more melatonin in the gut. Notably, the SGR was highest in both growth stages of carp supplied with PRD1. However, a reduced but still higher level was noted only in the late stage after the supply of PRD2, thereby it indicates that a higher protein-rich diet can be managed by the LJv fish but not by the EJv fish, which may be due to a lack of sufficient digestive enzymes, particularly the protease in EJv fish. The EJv carp exhibited a gradual increase in protease levels, but the LJv carp attained the maximum levels of protease activity after the PRD1 supply, as no further increase was noted in its value under the PRD2 diet. This indicates that EJv carp are developing their gut for better digestion, whereas the LJv carp developed the gut and reached to limit of its maximum capability. We have also found a significant increase in α-amylase levels in both the PRDs in EJv and LJv. Previous reports indicate that crude protein availability in diet may alter the α-amylase gene expression in red tilapia juveniles (Oreochromis sp.) (Santos et al. 2020), possibly because of the higher protein availability for the production of digestive enzymes.
In our study, the level of different antioxidative enzymes (SOD, CAT, GPx, GST, GRd) and a non-enzymatic antioxidative agent (GSH) in the gut have been significantly increased, but the level of MDA, a reliable intracellular stress marker, was found to be reduced in both the PRDs and TrpRDs-fed groups, where melatonin was found to be increased. Melatonin is known to play a vital role in regulating intracellular antioxidative molecules (Chen 2011; Pal et al. 2016; Pal and Maitra 2018). According to our data, the activities of all the studied antioxidative agents, viz. SOD, CAT, GST, GPx, GRd, and GSH have been promoted after increasing the gut melatonin levels following the modulation of food ingredients. But such effects were found different in the gut of EJv carp and LJv carp, though a positive correlation with the gut melatonin and antioxidative agents was noted in both cases. But the upregulation of gut melatonin was more prominent with GRd and GSH, indicating more xenobiotics detoxification activity by the gut (Lushchak 2012). The GRd values were found highest in PRD2 diets in the case of EJv carp but in LJv carp after feeding with TrpRD2, parallel with the melatonin profiles noted in these carp. Further, GSH values were not significantly correlated with MDA, SOD, and GST in EJv carp, which was significantly correlated in LJv carp. This indicates the underdeveloped xenobiotic detoxification mechanism in the early stage of the gut compared to its later stage. This suggests that a high level of melatonin in the gut during the early stage of gut development may play a vital role as a direct free radical scavenger (Tan et al. 2000) compared to its later stage when the full potentiality of the antioxidative defense mechanism has developed. The SOD, CAT, and GPx showed a positive stimulatory relationship with melatonin. Still, in such cases, the efficacy of melatonin in the regulation of the activity of the antioxidative enzymes is limited to a certain level (Moniruzzaman et al. 2018), above which melatonin cannot stimulate their actions, as no further increase in their activity was noted when EJv carp was fed with PRDs and having higher melatonin levels compared to the TrpRDs-fed groups. The melatonin possibly activates the GRd and GSH based on the xenobiotics toxicity via the food and activates different antioxidative enzymes, viz. SOD, CAT, GPx, and GST by inducing translational changes in their production, which leads to a decrease in free radicals (Rodriguez et al. 2004). Under increased nutritional intake via PRDs, TrpRDs, CRD, and ORD compared to SD, the metabolic rate within the gut cells may increase, leading to higher production of free radicals. To reduce this stress, free radicals are dismutated by the increased levels of SOD and form a less reactive molecule, H2O2. Higher levels of H2O2 within the gut may be managed by the increased levels of CAT and GPx (Pal et al. 2016), with a positive correlation with gut melatonin. However, the mechanism of actions of melatonin on the antioxidative functions in fish gut tissues should remain speculative until further study.
TrpRDs supply triggered the synthesis of gut melatonin, but the effects were more prominent when supplied with TrpRD2 in LJv fish. Further, indicating higher responsiveness to gut melatonin in the LJv carp to upregulate the higher activities of antioxidative agents and digestive enzymes. In our study, digestive enzyme activities were significantly increased in both the TrpRDs in EJv and LJv compared to SD. Similar kinds of observations were observed in hybrid catfish (Zhao et al. 2019), Jian carp (Cyprinus carpio var. Jian) (Tang et al. 2013), Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) (Mardones et al. 2018). The Trp-mediated enhanced melatonin levels and digestive enzyme activities might be associated with the growth and development of fish digestive tract, at least in the EJv carp, which are the basis for achieving the optimum digestive functions. SGR was increased in TrpRD1 and TrpRD2-fed EJv group compared to SD-fed carp, but the gut growth (GaSI) was inhibited only after the supply of TrpRD2, but not after TrpRD1. The optimal dietary Trp level could modulate the growth performance and feed efficiency, as reported earlier in several fish species (Ahmed and Khan 2005; Ahmed 2012; Tang et al. 2013; Pianesso et al. 2015; Pewitt et al. 2017; Zaminhan et al. 2017).
Optimal protein (Madsen et al. 2017), Trp (Poston and Rumsey 1983), carbohydrate (Villasante et al. 2019), and oil (Magalhães et al. 2020) play a role in maintaining intestinal structural integrity and intestinal development, possibly by maintaining gut microflora. In our data, the GaSI of TrpRD1 and ORD remain unchanged compared to SD. But in the case of PRDs and CRD, GaSI has been reduced. It may be related to the difference in the abundance of gut microbiota in different diet fed groups. After feeding different laboratory-made diets, studying gut microbiota may unravel new insights supporting this observation. Further, the increase in cellulase activity following the supply of better-quality diets may be due to the microbial contribution of the gut microflora. Existing reports indicate that better quality of nutrients in terms of protein, mainly focusing on the amino acid Trp, may play important roles in maintaining gut microflora and intestinal health as protease activity was found positively correlated with cellulase activity, indicating enhanced gut microbiota contribution to cellulase (De et al. 2012). The Trp, accumulated within the gut following a supply of PRDs or TrpRDs, by the formation of melatonin may increase the richness and diversity of the intestinal microbiota, perhaps partly because Trp promotes the growth of the intestinal villi, thus increasing the nutrients available to the intestinal flora (Gao et al. 2018; Liang et al. 2018). Our present investigation found a positive correlation between gut melatonin and cellulase activity in the EJv carp only. This observation suggests that melatonin may play a significant role in the formation of gut microbiota during its early stage of development when α-amylase activity was more prominent than lipase activity, as α-amylase is negatively correlated with lipase during the early stage of gut development. This indicates that immature gut habituates more to CRD than ORD. The equal capability of carbohydrate and lipid digestion develops later in the matured gut of LJv carp when cellulase activity is accompanied by α-amylase activity. However, higher lipase activity in the LJv carp may reduce the cellulase activity or the gut microflora population (Machate et al. 2020), as a significant negative correlation was noted among the two parameters in LJv carp. On the other hand, in the present study, gut melatonin showed a positive correlation with protease in both studied age groups but not with α-amylase and lipase in any of the studied age groups. While a complete time-bound positive correlation between gut melatonin and different digestive enzymes has been seen previously in juvenile carp, Catla catla (Pal et al. 2016). This contradiction invites us to think that protease activity may be associated with gut melatonin synthesis, and the cellulase activity is basically performed by the gut microflora, which was developing during the early stage of the gut and exhibited a positive correlation; the such correlation was found absent in the matured gut of LJv fish (Table 5). However, α-amylase and lipase are related to food intake as higher levels of α-amylase, and lipase was associated after the supply of CRD and ORD diets. This suggests that different quality of fish feed needs to be supplied during the different growth stages of fish. However, further profiling of gut melatonin and digestive enzymes in carp growth stages, from fingerling to adult, is required to understand the best quality of food appropriate for a particular age group.
The supply of CRD and ORD did not exhibit any significant changes in melatonin levels but showed lower values in RGC and FI. These data indicate that when the quality of fish feed increases employing high CRD (Adamska-Patruno et al. 2018) or ORD (Matzinger et al. 1999), the required amount of energy can be absorbed by the gut from the small amount of fish feed. Either CRD or ORD increases the respective enzymes for better digestion, but higher nutritional quality of the diets may delay the gastric emptying for more extended assimilation and suppress the hunger for a longer duration. Hence satiety reaches in a speedy manner (Sánchez et al. 2004). Our present study noted significantly reduced SGR in the CRD-fed group of LJv fish. A similar observation was reported in Japanese Flounder (Paralichthys olivaceus) that the SGR was lowered and the MDA was higher after having a 24% CRD (Deng et al. 2018), the same percentage of carbohydrate supplied in our study as well. The argument has been placed that the supply of CRD may result in hyperglycemia in fish that may lead to metabolic disorders inhibiting growth (Hemre et al. 2002). It was found that the levels of different antioxidative agents were significantly increased after feeding with CRD of both EJv and LJv carp compared with the SD-fed group. Dietary macronutrients substantially affect the metabolic pathway but can be achieved by several different modes. Hence, those have various roles in regulating oxidative stress of different gut cells of the digestive tract. The beneficial effect of dietary carbohydrates on handling oxidative stress may be attained by increasing the level of different antioxidative agents (Sagone et al. 1983; Alvarez et al. 1999; Lygren and Hemre 2001; Pérez-Jiménez et al. 2017). In our study, the level of α-amylase in the gut was significantly highest in CRD in EJv and LJv carps. Similar observations were noticed in a variety of carps (Phadate and Srikar 1990) and catfish (Clarias batrachus) (Aprajita et al. 2017) earlier after feeding CRD.
In ORD, the oil content has been increased by cod liver oil (Fish oil or FO) and sunflower oil (vegetable oil or VO). FO and VO are rich in long-chain polyunsaturated fatty acids (LC-PUFA), and it is well established that due to their high degree of unsaturation, LC-PUFA is more susceptible to peroxidation than PUFA, thus promoting the formation of more free radicals (Halliwell and Chirico 1993). Thereby, it was evidenced that unaltered MDA levels in the gut of both EJv and LJv carp following the replacement of ORD after SD were noted. Hence, oxidative stress was high in ORD-fed group compared to PRDs and TrpRDs, along with the increased levels of different antioxidative agents to combat the stressful situation. ORD may also regulate oxidative stress by secondary metabolites like eicosanoids, arachidonic acid (Herrera et al. 2019), flavonoids, and phenolic compounds of vegetable oil (Martín et al. 2010; Procházková et al. 2011), which are known to act as stress reducers by upregulating the level of different antioxidative agents. This antioxidant effect may result from these compounds’ direct free-radical scavenging action or their role in activating necessary antioxidative enzymes (Castro et al. 2015). However, such a finding needs to be confirmed in future.
Nonetheless, the present study reports for the first time that gut melatonin levels can vary in the early stage of the gut (as in EJv) from its mature stage (in LJv). This variation is likely due to natural variations in the feeding characteristics of the carp during the different growth phases. Possibly gut melatonin has a tremendous role in developing a functional gut along with its full strength of digestive capability and oxidative stress management. Further, dietary nutrients have a significant role in the maintenance of gut melatonin levels, which can be modulated following the supply of different quality diets based on the various needs of the fish by manipulating the quality of the diets, which can reduce the gut stress, maximizing the digestive function, better nutrition, and better gut health.