Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 4 (april 2022) : 444-450

Effect of Different Dietary Protein Levels on Physio-metabolic Response during Stunting of Milkfish, Chanos chanos (Forsskal, 1775) Reared under Pond Conditions

Mamidala Shyam Prasad1,2, Muralidhar P. Ande1,*, Karthireddy Syamala1, Narinder Kumar Chadha2, Paramita Banerjee Sawant2, Biji Xavier3, P. Gireesh-Babu4
1ICAR-Central Institute of Fisheries Education, Brackishwater Fish Farm, Kakinada Regional Centre, Kakinada-533 007, Andhra Pradesh, India.
2ICAR-Central Institute of Fisheries Education, Mumbai-400 061, Maharashtra, India.
3ICAR-Central Marine Fisheries Research Institute, Visakhapatnam-530 003, Andhra Pradesh, India.
4ICAR-National Research Centre on Meat, Hyderabad-500 092, Telangana, India.
Cite article:- Prasad Shyam Mamidala, Ande P. Muralidhar, Syamala Karthireddy, Chadha Kumar Narinder, Sawant Banerjee Paramita, Xavier Biji, Gireesh-Babu P. (2022). Effect of Different Dietary Protein Levels on Physio-metabolic Response during Stunting of Milkfish, Chanos chanos (Forsskal, 1775) Reared under Pond Conditions . Indian Journal of Animal Research. 56(4): 444-450. doi: 10.18805/IJAR.B-4813.
Background: Stunting is a process of suppressing growth from unfavourable conditions. The protein supplementation during stunting gives scope to maintain the nutrient reserves of fish and its quality.

Methods: A feeding trial was conducted for eight months to study the effect of three hetero-nitrogenous diets with 25% (control), 30% (T1) and 35% (T2) crude protein (CP) levels on growth and physio-metabolic responses of Chanos chanos fingerlings during stunting. Milk fish fingerlings with a mean body weight of 11.71±0.18 g were stocked in earthen ponds @ 20 no/m2 in each replicate (n=3) was fed @ 2% biomass throughout the experiment. 

Result: Fish fed with T1 diet showed better specific growth rate (0.64±0.01% d-1), weight gain percentage (362.56±14.95) and protease activity (7.53±0.25 U/mg protein). Whereas, lower activity was observed for the enzyme assay, namely superoxide dismutase (45.41±2.50 U/min/mg protein), aspartate aminotransferase (34.01±1.88 U/min/mg protein) and alanine aminotransferase (39.64±0.64 U/min/mg protein). Hence, it may be concluded that the dietary protein inclusion level of 30% CP showed better growth performance and lower physio-metabolic response in milkfish fingerlings during the stunting.
Milkfish is suitable candidate species for intensive brackishwater farming (CIBA, 2018). Despite the progress made in the induced breeding of milkfish, seed demand is fulfilled through the wild collection (CIBA annual report, 2015). Stunting is the production of dwarf and quality individuals from unfavourable environmental conditions like high stocking density and limited food availability, which leads to utilization of stored nutrition such as lipid and protein and thus affecting growth (Ferki et al., 2018; Klinger et al., 1983) to get year-round availability (Sahoo et al., 2021). In milkfish, an eight-month stunting period with 20 no/m2 stocking density has been reported to be sufficient to obtain optimum compensatory growth under grow-out culture conditions when fed with de-oiled rice bran (Lingam et al., 2018).
However, stunting is assumed to deplete the nutritional quality of fish (Yeannes and Almondes, 2003). This in turn affects energy levels, metabolic activity in stunted fingerling due to limited nutrient availability (Ferki et al., 2018). Hence, nutrient supplementation during stunting is essential to maintain the quality of fingerlings by reducing the stress response (Khalil et al., 2015). Protein is one of the crucial components of feed that greatly influences fingerlings’ growth rate and quality (Surjobala et al., 2021). Protein requirement for different life stages of milkfish such as fry, fingerling/adult and broodstock was reported to be 40%, 24-36% and 36%, respectively, under intensive farming (CIBA, 2018). Protein supplementation may compensate for the loss of stored nutrients in the fish body and mitigate the stress during stunting (Ferki et al., 2018). Although protein-rich feeds are reported to improve the quality of fingerlings during stunting, the excessive dietary protein metabolized as an energy source may result in increased nitrogen discharge due to de-amination (Pascual et al., 2003; Babaei et al., 2016).
Hence, it is imperative to determine the optimum dietary protein inclusion levels and their effect during stunting to preserve fingerling quality with minimum stress. Therefore, the present study was carried out to determine the optimum protein inclusion level by examining the digestive enzyme activity, growth and physio-metabolic response.
Experimental setup and diet
The experiment was conducted in 2020 (March to October) in the earthen ponds at BWFF, ICAR-CIFE, Kakinada Regional Centre, Andhra Pradesh. Uniform weight (11.71±0.18 g) of pond reared milkfish fingerlings were stocked in 0.02 ha ponds @ 20 no/m2 (Lingam et al., 2018) in each replicate. The experiment comprised three groups that indicated hetero-nitrogenous diets, including control [C; 25% crude protein (CP)], Treatment group 1 (T1; 30% CP) and Treatment group 2 (T2; 35% CP) (Table 1). The experimental diets were formulated and prepared by following De Silva and Anderson’s standard methods (1995). The experiment was conducted in triplicate by following the completely randomized design.

Table 1: Ingredients and proximate composition of the experimental diets.

Sample collection and preparation
Milkfish fingerlings (n=40) from each replicate were sampled (at 30 days intervals) to record growth data and released back into the respective replicate group. For enzyme assay, liver and intestine tissue samples (n=3) were collected (at 60 days intervals) in chilled 0.25M sucrose and prepared 5% tissue homogenate for analysis (Kumar and Tembre 1998).
Growth parameters
Growth performance of the fish calculated as follows:
Weight gain (WG) = Final weight (FW) - Initial weight (IW)

Biochemical analysis
Intestinal protease activity was determined by the casein digestion method (Drapeau, 1974). Amylase activity was assayed with 2% (w/v) starch solution as substrate (Rick and Stegbauer, 1974) and lipase activity was estimated by the titrimetric method of Cherry and Crandell (1932).
Superoxide dismutase (SOD) was assayed based on the oxidation of epinephrine-adrenochrome transition (Misra and Frodovich, 1972). Catalase (CAT) was assayed according to the method described by Takhara et al., (1960). Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) activity were estimated as described by Wooton (1964).
Statistical analysis
Statistical analysis was performed SPSS version 25.0 using one-way analysis of variance to test the significant difference among the treatments. Comparison of the means was demonstrated by Duncan’s multiple range tests.
Growth performance and survival
Dietary CP level significantly affected the growth performance of milkfish fingerlings during the stunting period (Table 2). Significantly higher WG (Fig 1), WG%, feed intake, protein intake, SGR and PER were also recorded in the T1 group (30% CP). The least WG% observed in the T2 group (35% CP) could be attributed to the limited acceptanceof a high protein diet by fish during starvation. Previous studies on other fish species Siberian sturgeon, Acipenser baerii (Babaei et al.,  2016) suggested that fish do not readily accept the high protein diet due to insufficient non-protein energy diet. During stunting of fingerlings, nutritional imbalance with high protein diets negatively affects growth, leading to stress, growth suppression and less survival (Kiron, 2012). It is speculated that the excess protein in diet undergoes de-amination in the body resulting in the supply of protein-energy for metabolism rather than body growth (Furne et al., 2008). It is also worth mentioning that feeding fish with lower protein levels leads to slower growth and poor survival (Khalil et al., 2015). The results suggest that the 30% crude protein inclusion levels were better suited for stunting compared to other protein fed groups based on the observed parameters i.e., weight gain, SGR, feed intake, WG%,  ANPU, FCR and PER .

Table 2: Growth performance of C. chanos fingerlings during stunting.


Fig 1: Average weight gain (g) of C. chanos fingerlings during stunting.

Digestive enzyme assay
Protease, amylase and lipase activities of stunted milkfish fingerlings were significantly affected (P<0.05) in the treatment groups and confirmed the response of digestive enzymes against dietary protein levels (Kumar et al., 2019).
T1 group showed higher protease activity during the entire experimental period (Fig 2a) with a value of 7.53±0.25 U/mg protein at the end of the experiment. The protease enzyme plays a crucial role in activating and stimulating the reactions related to the mobilization of energy through the metabolic pathways, resulting in variation in growth (Furne et al., 2008). It also indicates that the difference in potential feed utilization results in the efficient uptake of protein for better growth performance (Babaei et al., 2016). Lower protease activity in the high protein supplemented group (35% CP) suggests that the capacity to digest a high amount of proteins is compromised due to feeding deprivation and stress. In this respect, Abolfathi et al., (2012), observed lower protease activity in juvenile Caspian roach, Rutilus rutilus caspicus, with a high protein diet.

Fig 2a: Protease activity (U/mg protein) of stunted C. chanos fingerlings.

Amylase activity showed a decreased trend with an increase in protein level (Fig 2b). The highest amylase activity (4.68±0.10 U/mg protein) was recorded in the control group at the end of the experimental period (240 days). The lower amylase activity observed at a higher dietary protein level (35% CP) could be due to degeneration of pancreatic tissue (Abolfathi et al., 2012) and intestinal mucosal cell damage during food deprivation (Kumar and Tembre, 1998).

In silver barb, Puntius gonionotus (Mohanta et al., 2008) and rohu, Labeo rohita (Mohapatra et al., 2003), it is reported that the liver and alimentary tract are depleted during starvation leading to a possible reduction in digestive efficiency due to decreased digestive enzyme secretion.

Fig 2b: Amylase activity (U/mg protein) of stunted C.chanos fingerlings.

In the control and T1 groups, the lipase activity increased until 120 days and gradually decreased later (Fig 2c). The higher lipase activity observed in the 35% CP group could be attributed to the possible influence of dietary protein on the pancreatic lipase secretions to hydrolyze the triglycerides during digestion. Similar reports were also made Adriatic sturgeon, Acipenser naccerii and rainbow trout, Oncorhynchus mykiss (Furne et al., 2008).

Fig 2c: Lipase activity (U/mg protein) of stunted C.chanos fingerlings.

Anti-oxidant enzyme assay
Anti-oxidants and specialized enzymes like SOD and CAT can neutralize reactive oxygen species [ROS-includes superoxide anion, hydrogen peroxide and hydroxyl radicals] generated through metabolic activity (Rahman, 2007). The highest SOD and CAT activities were observed in T2 and T1 groups, respectively.
The superoxide dismutase (SOD) activity of milkfish fingerlings was significantly affected with varying dietary protein levels (P<0.05) during stunting. The liver displayed fluctuation in the SOD activity across the variables. While the 120 and 240 days sampling points recorded the highest SOD activity, the 60 and 140 days sampling points showed the lowest activity (Fig 3). The higher SOD activity recorded in the T2 group could be due to the increased production of free radicals because ofthe physiological imbalance between the level of anti-oxidants and oxidants during oxidative stress with a high protein diet (Pascual et al., 2003). Research reports on yellow croaker, Pseudosciaena crocea (Zhang et al., 2008) and Siberian sturgeon, A. baerii (Babaei et al., 2016) suggests that high protein diet is unproductive for compensating the oxidative stress during stunting.

Fig 3: SOD activity (U/min/mg protein) in liver tissue of C. chanos fingerlings.

The catalase activity was higher during the entire experimental period. At the end of the experimental period, the T1 group recorded the highest catalase activity in the liver (0.23±0.001 U/min/mg protein). When compared across the sampling intervals, the catalase activity was seen to be more or less stable (Fig 4). The higher CAT activity in the 30% CP group indicates sufficient enzymes to degrade hydrogen peroxide into molecular oxygen and water (Kumar et al., 2019). Similar studies on Siberian sturgeon, A. baerii (Babaei et al., 2016); Adriatic sturgeon, A.naccarii and rainbow trout, O.mykiss (Furne et al., 2009) indicate that prolonged starvation induces a significant increase in CAT activity. SOD activity increased, while CAT activity decreased in the liver of milkfish subjected to partial deprivation in control and T2 groups. This might be due to production levels of ROS during starvation to diminish oxidative stress. Similar findings were observed in gilt-head seabream, Sparus aurata exposed to partial food deprivation (Pascual et al., 2003).

Fig 4: CAT activity (U/min/mg protein) in liver tissue of C. chanos fingerlings.

Metabolic enzyme assay
The dietary protein levels significantly (P<0.05) influenced the AST and ALT activities. A steady increase in AST activity was observed across the time points tested during the stunting (Fig 5). At the end of the experimental period (240 days), the T2 group registered the highest AST activity in the liver (63.37 U/min/mg protein). Similarly, ALT activity was found to be highest in the T2 group (59.98 U/min/mg protein) tissues (Fig 6).

Fig 5: AST activity (U/min/mg protein) in liver tissue of C. chanos fingerlings.


Fig 6: ALT activity (U/min/mg protein) in liver tissue of C. chanos fingerlings.

The liver-specific enzymes, ALT and AST, indicate nutrient utilization of amino acids and greatly influence metabolic functions under stressful conditions for meeting the high energy demand in fish (Jiang et al., 2015; Huang et al., 2018). Increased AST and ALT enzyme activity during stunting suggests that the high dietary protein forms substrates such as glycogenic aminoacids and lactates, resulting in higher gluconeogenesis under stress conditions (Perez-Jimenez et al.,  2012). The AST and ALT activities were recorded minimum in the 30% CP group for the entire experiment. This may be due to sufficient production of glucogenic substrates under the gluconeogenesis cycle as a protective strategy to prevent excess oxidation generated under stressful conditions (Perez-Jimenez et al.,  2012). The higher activity observed in the 35% CP may be attributed to the higher production of lactates to utilize the energy to meet the metabolic functions. Similarly, the ALT and AST activities increased with a high protein diet in Cyprinus carpio (Jiang et al., 2015) and L.rohita (Kumar et al., 2019).
This study concludes that the dietary protein level has a significant role in producing quality milkfish fingerlings during stunting. The 30% dietary protein levels showed better WG, WG%, SGR, PER, higher digestive (protease activity) and metabolic enzyme activities. Fish fed with 30% CP have lower SOD enzyme activity than the 25% and 35% CP groups. Further, low levels of metabolic enzymes (AST and ALT) in the 30% CP group indicate that the fingerlings are healthy and are of good quality. Considering all the above, the present study concludes that a 30% dietary protein level is better suited for C. chanos fingerlings during 8-month stunting under pond conditions.
The authors would like to thank The Director and Vice-Chancellor of ICAR-CIFE, Mumbai, for the constant support to experiment.

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