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 .
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.
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.
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).
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.
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).
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).
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).
