Stress Modulated Physiological Responses in Nile Tilapia, Oreochromisniloticus, Treated with Non-Ascorbic Acid Supplemented Feed

Stress caused by the aquaculture environment hampers normal growth and immune response of fish and makes them susceptible to diseases. Ascorbic acid (vitamin C), on the other hand, has been known to improve the immune response. Experiments were conducted to investigate the effects of the absence of vitamin C in fish dietgrown in stressed condition induced by crowding. Fish fingerlings were obtained from a local fish farm and were acclimated in the new environment for two weeks. After the acclimation period, fish were maintained in two different conditions: controlled and stressed (density > 50 g/l). Within each condition, fish were further divided into two diet groups, each with two replicates. The experimental groups were fed vitamin C supplemented (1000 mg/kg) feed and the control groups were fed vitamin C free feed for fifteen days. Six fish from each group were sampled to assess the physiological parameters: plasmacortisol, blood glucose, plasma protein, packed cell volume, spleen somatic index, and condition factor. Fish in the stressed conditions showed significantly higher level (p<0.05) of plasma cortisol, blood glucose indicating stress, both of which were reduced when fed vitamin C supplemented diet. Significantly higher (p<0.05) hematocrit concentration was observed in the vitamin C fed group indicating better physiological adaptation.


Introduction
In chronic stress situation, hypothalamus of fish releases hypothalamic factors which excite pituitary gland to synthesize adrenocorticotropic hormone (ACTH), melanophore stimulating hormone (MSH), and β-endorphin (β-END). These hormones induce inter-renal cells to synthesize cortisol, a steroid hormone with broad spectrum activities enabling the fish adapt to stress [1]. Its primary function is to increase blood sugar through gluconeogenesis and thus increase energy availability. Activities of cortisol also include downregulation of the Interleukin-2 receptor (IL-2R), enhancement of glycogenolysis, and proteolysis (Barton and Iwama, 1991). Cortisol has been linked directly with growth reduction, immunosuppression and declined reproductive capabilities [1].
Ascorbic acid(vitamin C) is an important molecule for normal growth and metabolic functions of fish. It has been widely used as dietary supplement asalmost all fish lack the last enzyme of vitamin C biosynthetic pathway, L-gulonolactone oxidase [2]. Protection of living cells from oxidative damage is provided by ascorbic acid by neutralizing the reactive oxygen species [3]. Collagenization is an important step in development of connective tissue and wound healing. Ascorbic acid is a critical cofactor in formation of collagen molecules through hydroxylation of proline and lysine [4]. Jaunceyet al. [5]demonstrated that lack of ascorbate in fish diet slows wound healing. Ascorbic acid has also been reported to decrease the toxic effects of heavy metals such as nickel, cadmium [6] and mercury [7].
Immune system is boosted by vitamin C in a number of ways -protectionfrom free-radical mediated protein inactivation in macrophages, chemotaxis, stimulation in production of antibody and interferon, signaling of rapid proliferation of immune cells and complement activation [8,9]. Ascorbic acid induces regeneration of vitamin E, a fat-soluble antioxidant, which is present in the lipid of cell membranes and lipoproteins to fight free radical-mediated peroxidation [10].
In stressful conditions of aquaculture depletion of ascorbic acid takes place faster than normal situations [11].Stocking density of more than 40g/l is considered to be stressful for most teleost [12]. The response mechanisms against even sub lethal disturbances in chronic situations result a net detrimental effect on the fish health, and is regarded as maladaptive [13]. High dose of vitamin C supplementation 40 Stress Modulated Physiological Responses in Nile Tilapia, Oreochromisniloticus, Treated with Non-Ascorbic Acid Supplemented Feed has been suggested to mitigate the effects of physiological stress [14]. Kibatchi [15] stated that ascorbate in high concentration inhibit steroid synthesis and therefore might reduce the severity of cortisol mediated stress responses. The objective of this experiment was to determine the physiological effects of absolute ascorbic acid deficiency in tilapia that are stressed by crowding.

Fish Acquisition and Maintenance
Nile tilapia,Oreochromisniloticus fingerlings (mean weight 30 grams) were purchased from Troyer's Farm, IN and transported to the Life Sciences Resource Center at Indiana University-Purdue University, Fort Wayne. The fish were acclimated in 500 liter tanks containing recirculating dechlorinated water for two weeks. Within the system, large reservoirs were used to mix circulating water drained from the fish tank with city water. Keeping the mixed water uncovered under light allowed slow evaporation of chlorine. The water then was passed through filtration chambers containing charcoal, coarse sand and denitrifying bacteria culture. The filtered water was then used to fill the fish tanks.
In the beginning of the experiment, all the fish were fed vitamin C free feed for two weeks. The fish were then distributed in eight tanks among which four tanks had normal fish density and were considered as unstressed controlled group. Four other tanks had crowded fish with a density of 50g/l representing stressed group.
A natural photoperiod of 12 light hours and 12 dark hours was maintained throughout the study. Water quality parameters were monitored and optimal water quality was maintained (dissolved oxygen was at a minimum of 6 mg/l and pH between 6 and 7.5). Dissolved oxygen and pH were measured using YSI portable DO and pH probes.Fish were cared for according to approved animal care protocol and fed twice daily in an amount of 1% of their average body mass. Excess feed was removed from the tanks using filtration systems attached to the tanks.

Feed Preparation
Diets were formulated using MIXIT-WIN 2+ (Ag Software Consultants, San Diego, CA), based on the essential amino acid (EAA) requirements of Nile tilapia. All diets contained 36% crude protein (CP) and 8% lipid ( Table  1). Availability of EAA was assumed to be 80% for all protein-containing ingredients. Ingredients were mixed in a Hobart mixer and pelleted using the chopping end attachment. Diets were air dried to a constant moisture concentration and stored frozen at -20 0 C prior to feeding. Vitamin C was added in the phosphorylated form.

Sampling
Sampling was performed after 14 days of feeding with the experimental feed. The fish were starved for 12 hours before sampling. On the sampling day, 3 fish were randomly taken from each tank with the help of a dip net, making the number of samples to be 6 per group. As soon as they were caught, they were put in different marked buckets containing a lethal amount of 200mg/liter of tricaine methane sulfonate (MS-222) [16] for rapid euthanization. After measuring weight and length, blood was taken from each fish from the caudal vein. The health and stress levels of these fish were then determined by the following measures:condition factor, blood glucose, plasma cortisol, plasma protein, and packed cell volume. Length and weight of each individual fish were measured using a meter scale and an electronic balance respectively. Then the weight and length data were used to calculate the condition factor (K) using the following formula [17]:

Blood glucose
A TRUEtrack ® glucose meter (Nipro Diagnostics Inc., Osaka, Japan) was calibrated by inserting a test strip into the test slot. A drop of blood from the syringe was added on the designated area of the test strip to measure the glucose [17]. This method has been validated for use in glucose analysis for fish [18].

Plasma cortisol concentration
Blood were collected in microfuge tubes containing no anticoagulant and incubated in an upright position at 4 0 C overnight to allow clotting. Carefully the plasma was aspirated at room temperature for quantitative analysis by cortisol EIA Kit (Enzo Life Sciences Int'l Inc., PA) following a slightly modified protocol of the manufacturer.
Instead of using 100µl of sample as suggested by the manufacturer, 10µl was used and a total volume of 100 µl was prepared by diluting with the assay buffer. 1/10 th sample dilution was necessary because of high concentration of cortisol in the plasma. Spectra Count Microplate Reader (Packard Instrument Company, Meriden, USA) was used to measure optical density at 405 nm. The measured optical density of the standard cortisol solution was used to formulate the following equation: The value of percent bound, Y was put into the equation to calculate the value of x, the concentration of cortisol.

Plasma Protein
A handheld protein refractometer (VEEGEE Scientific Inc. Kirkland, WA) was used to measure plasma protein content. Using distilled water on to the surface of the prism by adjusting the focus and closing the day-light the machine was calibrated. Two drops of plasma from hematocrit tube were added on to the surface of the prism, the level of total protein was measured through the eyepiece [16].

Packed Cell Volume
A single glass capillary tube was filled with blood from the syringe and then one end was capped with Critocaps [16]. The tubes were kept standing for 15 minutes at room temperature. After sampling all the 24 fish, the blood-filled tubes were centrifuged in the micro centrifuge machine for 1000g for 5 minutes, resulting separation of the plasma from the blood cells. The hematocrit levels were determined using Micro-Hematocrit Capillary Tube Reader (Monoject Scientific, St. Louis, MO) after the centrifugation [17].

Statistical Analysis
The statistical analysis of the data was performed by one way analysis of variance (ANOVA). A post-ANOVA comparison of multiple means was done by Holm-Sidak method. All the experimental groups were compared with each other. The graphical representation was also produced using SigmaPlot 11.0. The error bars within the graphs are depicted as means ± standard errors of the means (SEM).

Plasma Cortisol Concentration
After two weeks, all the experimental groups had significantly different concentration of cortisol in blood plasma (p<0.05). The control stressed group showed maximum plasma cortisol concentration. The control unstressed had lower level than that of the control stressed group but yet it was significantly higher than the groups with vitamin C in their feed (p<0.05). The unstressed fish receiving vitamin C complemented feed had the lowest plasma cortisol level of all ( Figure 1).

Blood Glucose Concentration
Significant variation in concentration of glucose in blood was observed among all three groups (p<0.05), the control stressed group having the highest glucose level. The control unstressed had no significant difference from the vitamin C stressed group. The control group of unstressed fish fed with vitamin C complemented feed had the lowest blood glucose concentration (p<0.05) (Figure 2).

Plasma Protein
An overall analysis revealed that although not significant, the vitamin C supplemented groups showed lower plasma protein concentration than the groups without any vitamin C. Similarly the total protein amount was lower in the control groups than their stressed group counterparts. When compared individually, the stressed group without receiving any vitamin C had significantly higher (p<0.05) protein content in their plasma than the unstressed group receiving vitamin C supplemented feed (Figure 4).

Packed Cell Volume
Among the different treatment groups, there exists a significant difference (p<0.05) in the percentage of packed cell volume of blood. The two groups -stressed and unstressed without any vitamin C in their feed had almost equal percentage of blood hematocrit level, which was significantly (p<0.05) lower than the stressed unit with vitamin C supplementation. Although the unstressed vitamin C diet group had lower percentage of hematocrit in their blood, it was not statistically significant ( Figure 5).

Condition Factor
There was no significant difference in condition factors among fish groups after two weeks of experimentation ( Figure 6).

Discussion
Experiments have been performed to observe the effect of absence of vitamin C in farmed fish diet. Aanorexia, lethargy, scoliosis, and lordosis were observed in rainbow trout [19,20]. Soliman et al. [21] observed Nile tilapia fed Vitamin C free diet exhibited erratic and convulsive swimming, anorexia, lethargy, caudal fin erosion, skin haemorrhages and mortality. Objective of the current experiment was to analyze the combined effect of absence of vitamin C and crowding stress in Tilapia.
The significant difference in both plasma cortisol concentration and blood glucose level between the stressed and the unstressed groups implies that crowding condition exerts stress on fish. This data supports the finding of Rotllant and Tort [22] when they subjected Red porgy (Pagruspagrus) to 3 weeks of chronic stress by crowding. After an initial spike the cortisol levels return to basal values to avoid tissue damage. Therefore, reviewers like Martinez-Porchaset al. [23] has suggested that cortisol might not be very reliable for chronic stress. Some other stressors like Sea lice challenge in Atlantic salmon [24] and organochlorine injection in rainbow trout [25] also produced elevation in plasma cortisol in chronic experiments. Ortuñoet al. [26] showed that plasma cortisol level in Gilthead seabreams shoots up during intense short-term crowding stress of 100 kg/m 3 for 2 hours. Significant higher cortisol concentration of the experimental group after two weeks of rearing in crowded condition supports the validity of plasma cortisol as a good crowding stress biomarker that spikes up in absence of ascorbate.
During their crowding stress experiment on Red porgy (Pagruspagrus), Rotllant and Tort [22] did not observe any significant difference in plasma glucose level. But,Basruret al. [27] observed rise in both plasma cortisol and glucose level during a repeated crowding stress investigation on Atlantic salmon (Salmosalar). Glucose metabolism is affected by cortisol concentration [13,28], and so a linear relationship is expected between these two chief stress parameters. From our experimental data, a regression analysis reveals a fairly strong correlation (R 2 = 0.802) between glucose and cortisol. So, the primary stress response by production of hormones was responded by concurrent rise of blood glucose, a secondary stress response to adapt with the stressed situations.
From the results of plasma cortisol concentration and blood glucose level it can be proposed that vitamin C helps to reduce the stress response induced by crowding. Vitamin C has been reported to reduce plasma cortisol level in Tilapia, Oreochromisniloticus exposed to cold stress [29]. We have found that vitamin C supplementation in an amount of 1000 mg/kg as suggested by Verlhac and Gabaudan [30], reduces the cortisol concentration in the stressed group even lower than the unstressed group lacking the minimum required amount of dietary vitamin C. The finding that absence of ascorbate is increasing cortisol level somewhat contradicts [31], who concluded that high concentration of ascorbate does not inhibit steroidogenesis. Ortuñoet al. [22] reported that in gilthead seabream, vitamin C happens to reduce glucose level in stressed groups but not cortisol. The drastic upsurge of both glucose and cortisol in the control group might be due to the fact that our control feed was totally devoid of vitamin C while in aforementioned researchers maintained a minimum level of ascorbate in their diet.
In terms of total plasma protein, there is no significant difference between the fish, which received vitamin C from those which did not. Hardieet al. [9] in their experiment on Atlantic salmon and Sealey and Gatlin [32] while experimenting with hybrid striped bass observed no effect of vitamin C on plasma protein content. On the other hand, pirarucu (Arapaima gigas), in net culture, showed elevation of plasma protein [33]. Similar results were obtained by Pimpimolet al. [34],who worked with Mekong giant catfish (Pangasianodongigas). The contradicting results might arise due to variation of species and experimental settings.
High packed cell volume or blood hematocrit level usually indicates hemoconcentration due to gill damage, dehydration of polycythemia induced by stress [13]. But we did not observe any significant difference of hematocrit level between the stressed and nonstressed groups within each diet group. But we marked a significant rise of packed cell volume in the vitamin C supplemented stressed group than others. It has been reported by de Menezeset al. [33] that vitamin C supplementation resulted in increase of Hemoglobin percentage, number of Red blood cells and packed cell volume in pirarucu (Arapaima gigas). Falcon et al. [29] demonstrated that Tilapia submitted to low temperature stress exhibit significantly reduced Hematocrit level when fed with vitamin C free diet. It has been hypothesized that impairment of iron metabolism and distribution due to deficiency of vitamin C might decrease RBC production [35]. As we observed from our experiment, the stressed fish group without vitamin C, despite having significantly higher cortisol and glucose concentration, had significantly lower hematocrit level than their vitamin C fed counterpart. Hence the higher hematocrit level of the group receiving vitamin C might be an indicator of adaptation. Solimanetet al. [21] observed that fish feeding with diet devoid of ascorbic acid exhibited significantly (p < 0·01) higher condition factor. But in our experiment there were no tangible difference in condition factor among the experimental groups. Had this experiment been carried on over a longer period of time, there might have been some observable difference in condition factor.
The overall result of the experiment clearly illustrates a negative effect of vitamin C deficiency in commercially cultured Nile tilapia.