Abstract: The aim of the present research was to provide information on the activities of Alkaline Phosphatase (ALP) in various organs of Red Tilapia cultured under mid-hill condition of Meghalaya in captive condition, where within a year the water temperature ranges from 15-22°C. From the present study, it has been observed that, under mid-hill conditions of Meghalaya, the ALP activity was more in kidney (3983.6609±24.6838 mg L-1) and the decreasing order of activity was recorded in liver (3761.9639±18.6786 mg L-1), intestine (3420.1118±443.3330 mg L-1) and muscle tissue (1969.1100±22.5985 mg L-1). The higher distribution of ALP in kidney and other organs implies that the fish can adapt to cold temperature of mid-hill altitude and can be taken up as a candidate species for mass scale culture in mid-hill condition to meet the nutritional requirements of the people.
INTRODUCTION
The Meghalaya represents unique topographical conditions (Table 1). As a result, all the major water bodies of the state pose low temperature throughout the year. The maximum (22°C) and minimum (12°C) water temperature coupled with other physico-chemical parameters recorded in the year 2005 is represented in Table 2. In the present study, Red Tilapia was chosen as test organism because it is an emerging and attractive species for aquaculture sector, especially in North Eastern Hill Region of India where fish production deficit is above 48%. Beside, it grows quickly, is large when it reproduces, has a low feeding trophic level and is cheap to produce.
The alkaline phosphatase (EC 3.1.3.1) are widely distributed in nature and are characterised by a high pH optima and broad substrate specificity (Mc Comb et al., 1979). Non-bacterial alkaline phosphatases are membrane bound, zinc-containing glycoproteins. The enzyme catalyzes the transfer of phosphate group to water (hydrolysis) or alcohols (Tran phosphorylation) using a wide variety of phosphomonoesters. The physiological role of alkaline phosphatase remains uncertain except for a role in bone mineralization (Harris, 1989) and a possible involvement in various transport processes, such as intestinal phosphate and calcium transfer (Dupuis et al., 1991; Harris, 1989) and placental immunoglobulin mineralization (Makiya and Stigbrand, 1992).
The Red Tilapia used in the present study was produced in the Philippines through process of selective breeding and genetic engineering by the combination of three different species of tilapia viz. Oreochromis niloticus, Oreochromis urolepis hornorum and Oreochromis mossambicus (Majhi et al., 2005). In general, alkaline phosphatase from fish and poikilotherms have not been studied much, but such species like Red Tilapia from cold environments offers an opportunity for kinetic studies of the enzyme in various parts of fish body. Never the less, some comparative studies on impure preparations have been performed with alkaline phosphatase from rainbow trout, eel, carps and catfish (Sorimachi et al., 1983; Yora and Sakagishi, 1986) and the thermal properties of crude enzyme fractions from three deep-water fish have been studied (Gelman et al., 1992; 1989). Recently, the purification and properties of alkaline phosphatase from shrimp were also reported (Lee and Chuang, 1991; Olsen et al., 1991).
Under this backdrop, the present paper describes the distribution of alkaline phosphatase in the liver, kidney, muscle and intestine tissue of Red Tilapia grown in captive condition under mid-hills micro situation and its significance in counteracting stress due to cold environment.
| Table 1: | Characteristics of Mid-Hill situation of Meghalaya |
Source: Bujarbaruah et al. (1996). International Conference
on Organic Food, Shillong, Meghalaya |
|
| Table 2: | Water quality parameters of Red Tilapia culture pond |
| Data show the mean value with standard deviation | |
MATERIALS AND METHODS
Experimental animals: A red tilapia of 1+ age with a weight of 200 g was randomly selected and taken for the study. The fish was collected from ICAR Complex fish farm, Meghalaya. The maintenance of fish and feeding conditions were those of the fish farm. The present study was conducted during November-December 2005 at Division of Fisheries, ICAR Research Complex for NEH Region, Meghalaya, India.
After 48 h without food, the fish was killed and various organs like liver, kidney, intestine and muscle were removed. The samples were immediately placed in deep freeze at 0°C until analysed. Table 3 presents the parameters and biometric indices of the animal used in the assay.
Treatment of the sample: To the 0.3 g each of various organs removed from the fish, 5.7 mL of 0.25 M sucrose was added and homogenized in an electric homogeniser at 0°C. The homogenates were centrifuged at 5,000 rpm for 5 min in a refrigerated centrifuge (REMI C24 Model). After centrifugation, the supernatant was collected and analysed within 8 h.
| Table 3: | Biometrics parameters |
| DSI: Weight of sample tissue (g)/Fish weight (g) x 100 | |
Enzymatic determination: The alkaline phosphatase activity in various organs of Red Tilapia was estimated at 25°C by using Merck Enzyme Kit (Ecoline (R) 1117675.0001) and the methodology described by Bergmeyer (1972). The test concentration was Diethanolamine HCl buffer (pH 9.8), Magnesium chloride and 4-nitrophenylphosphate as substrate. Twenty microliter of supernatant sample was taken and 1000 μL of reaction solution was added in to it. The sample and reaction solution was thoroughly mixed and kept for 1 min. Then after, the absorbance of supernatants was measured spectrophotometrically (Thermo Genesys 10) at 405 nm during a 1-3 min reaction time. The calibration of blank was done by keeping distil water in the blank. The samples were assayed in triplicates and blanks in duplicate. The principle of assay is defined as, the rate of increase in 4-nitrophenolate is determined photometrically and is directly proportional to the alkaline phosphatase activity in the sample material and is expressed as Enzyme activity (mg L–1) = (ΔA/Min)x2754 (Factor).
Statistical analysis: The results are expressed as Mean ± Standard Deviation (SD). The differences among the organs for alkaline phosphatase activity were tested for significance using t-test (MSTAT-C package).
RESULTS AND DISCUSSION
Overall, the enzyme activity in kidney was high (3983.6609±24.6838 mg L–1) followed by liver (3761.9639±18.6786 mg L–1), intestine (3420.1118±443.3330 mg L–1) and muscle tissue (1969.1100±22.5985 mg L–1), (Table 4 and Fig. 1-4).
| Table 4: | Alkaline phosphates activities in studied organs of Red Tilapia |
Different superscript(a, b, c) indicates significant
difference at 5% level. The superscripts(1,2,3,4) indicate studied
organs and are used in presenting t-values between the organs |
|
| Fig. 1: | Alkaline phosphatase in liver sample of Red Tilapia (Absorbance at 405 nm) |
| Fig. 2: | Alkaline phosphatase in kidney sample of Red Tilapia (Absorbance at 405 nm) |
| Fig. 3: | Alkaline phosphatase in intestine sample of Red Tilapia (Absorbance at 405 nm) |
| Fig. 4: | Alkaline phosphatase in muscle sample of Red Tilapia (Absorbance at 405 nm) |
The present study on activities of alkaline phosphatase in various organs of Red Tilapia revealed that, the activity of the alkaline phosphatase was more in kidney. The increase in alkaline phosphatase activity in kidney may be due to the fact that, the phosphatase are very important for regulation of various metabolic processes that occurs by phosphorylation and dephosphorylation with kinase, especially in temperate condition to meet the energy requirement during stress condition, which is also reported by Sparks and Brautigan (1986). Furthermore, Molina et al. (2005) have also reported that, the alkaline phosphatase activity increases significantly in liver of tilapia exposed to physiological stress and the changes are more pronounced in kidney. The physiological stress to Red Tilapia cultured under mid-hill situation of Meghalaya is due to cold water temperature. The Red Tilapia is basically a sub-tropical fish and perform better in a temperature range of 25-28°C, thus adaptation to cold temperature might have put the fish under stress by virtue of which the ALP activities in different tissues have increased to compensate the physiological stress. The alkaline phosphatase is basically a membrane bound enzyme and any perturbation in the membrane properties caused by the interaction with environment could alterate ALP activities (Molina et al., 2005). The functions of alkaline phosphatase are numerous and are widely distributed in the nature. In higher animals, ALP activities is involved in bone formation and in membrane transport activities. In blue crab Callinectes sapidus, ALP modulates the osmoregulatory response (Lovett et al., 1994).
In addition to above, the tissue pH values of most red-flesh fish and white-flesh fish are 5.6-5.8 and 6.2-6.3, respectively (Asgeirsson et al., 1995). In the present study, white-flesh fish was taken for the investigation. Therefore, the largely distribution of ALP in all the tested organs may be due to alkaline properties of fish flesh tissue. Kuda et al. (2002) reported that, the ALP activities in the organs might also be due to leaching from internal organs, apart from stress factor. Furthermore, he revealed that, heavy microbial load in the aquatic system having ALP activity can also lead to accumulation of ALP in different organs. The fish sample used in the present study was collected from a well-maintained culture pond having optimum organic load. Thus, probability of ALP accumulation due to heavy microbial load is rejected. Thus, the ALP distribution in major organs of Red Tilapia is only to compensate the physiological stress arises due to cold water temperature. Karuppasamy (2002) also reports the similar finding, who has studied the ALP activities changes due to environmental stress in fish.
Overall, the ALP are helpful in numerous activities in fish tissues including osmoregulation, membrane transport and bone formation etc. Thus, distribution of ALP in all the studies organs indicate that, the Red Tilapia can be well domesticated in mid-hills conditions for mass-scale production and can be incorporated as a candidate species in integrated fish farming system.
ACKNOWLEDGMENTS
The authors are thankful to the Director of the Institute for providing necessary infrastructure to carryout the present research and to Dr. K. Vinod, Scientist SS (Fishery Division), ICAR Complex For NEH Region, Barapani for helping in analysis of samples.