Methionine Modulated Bioavailability of Inorganic Zinc (ZnSO4. 7H2O) in Common Carp (Cyprinus carpio L.) Through Diets Containing Tricalcium Phosphate

The present work has been conducted to study the efficacy of methionine for the intake of dietary inorganic Zinc (ZnSO4. 7H2O) in the fingerlings of common carp (Cyprinus carpio L.). The experiment was performed in triplicate for which young ones of common carp of average 3.39±0.68g weight and 6.02±0.25 cm length were stocked in the indoor glass aquaria (60× 30 ×30cm) @11 fish/ aquarium. Five diets including control (D1) and four experimental diets (D2 to D5) were formulated. In treatment diets D2 and D3 ZnSO4.7H2O was added @88.4 and 176.8mg/kg, while in D4 and D5 ZnSO4.7H2O was added @88.4 and 176.8mg/kg along with 1% DL Methionine to observe its role in uptake of zinc. Crude protein content in the experimental diets ranged between 36.68 39.14%, while zinc concentrations in diets (D1 to D5) were recorded 31.80, 57.40, 61.60, 56.60 and 62.80 mg kg, respectively. Highest growth w.r.t. net weight gain was recorded (4.01g) and SGR (0.92%), feed conversion ratio (2.42) and protein efficiency ratio (1.08) was also recorded highest in fish fed with diet D5. Moisture content (%) in fish flesh ranged between 77.20 – 78.90, protein 14.80 – 16.70, lipid 1.75 – 2.73, ash 1.82 – 2.61 and carbohydrate 1.13 – 1.92%, among the fish fed with diet D1 to D5. Zinc concentration was recorded significantly high in muscle (36.90 mg kg ), liver (60.40 mg kg) and bone (109.56 mg kg). The present study indicates that Zn uptake in different tissues has been significantly improved due to addition of methionine in the formulated diets for young ones of common carp. Keywords— Common carp, Cyprinus carpio, Methionine, Tricalcium phosphate, Zinc uptake.


INTRODUCTION
Minerals serve as an essential requirement for the variety of functions, as both intra and extra cellular co mponents. Zinc is an essential micro mineral which is directly or indirectly involved in a wide variety of physiological processes including growth, development, reproduction and immune function (Watanabe et al., 1997). In fish, its deficiency leads to poor growth, high mortality, erosion of fins and skin, lo w content in bone (Takeuchi et al., 2002). Co mmon carp (Cyprinus carpio L.) is an important fresh water carp, cultured across the world as food fish. Success of fish culture depends on rearing of quality seed, fed with nutritionally balanced diet and good management practices . For quality seed production with high survival rate, young ones must be fed with supplementary d iets containing all essential nutrients in balanced quantity. Fish meal is always considered as one of the best protein sources containing all essential amino acids, hence are widely used in fish feed. Co mmon carp requires high levels of dietary protein during early stage of life cycle, for this addition of supplementary protein of animal orig in in the fo rmulated diets is a co mmon practice. Rendered (recycled) by-products from an imal waste serve as cheap source of quality protein (El Seyed 1998), but their applicat ion is limited due to low digestibly and restricted bioavailability of nutrients as it tends to bind with organic co mpounds and forms insoluble comp lexes , due to presence of certain limit ing factors (Cho et al., 1982;Gill, 2000). Diets containing animal p rotein especially fish meal as protein source contains Tricalciu m phosphate (TCP), wh ich acts as inhibitory factor for Zn uptake in fish (Davis et al., 1993). Organic co mpounds or chelated forms are important source of trace minerals, because they protect trace elements fro m forming insoluble co mplexes (such as phytate and TCP) in the digestive tract and facilitate transport across the intestinal mucosa (Ashmead1993). During production process of fish meal, calcareous compound present in fish converts into calciu m phosphates and their derivatives (Butler and Gross, 2017), even though fish scales also form a variety of calciu m phosphate salts (Belouafa et al., 2017). Methionine is such sulfur containing amino acids essentially required in fish diets, especially those containing high levels of plant protein source (Mai et al., 2006). Being a wide cultivable fish species in India as well as in the world, common carp is still not well studied fish species with respect to response against the inhibitory effect of TCP, more over to find out the possibilities to mitigate the negative effect of TCP through amino acid modulation. Keeping in v iew the above facts, the present work had been conducted to study the efficacy of methionine to enhance uptake of Zinc as ZnSO4. 7H2O in the fingerlings of common carp (Cyprinus carpio L.).

II. MATERIALS AND METHODS
The experiment was conducted at the Department of Aquaculture, College of Fisheries, Gu ru Angad Dev Veterinary and Anima l Sciences University, Ludhiana. The experiment was conducted in triplicate through rearing the youngones of common carp of average 3.39±0.68g weight and 6.02±0.25 cm length in the indoor glass aquaria (60× 30 ×30cm) @11 fish/ aquariu m. Five diets including one control (D1) and four experimental diets (D2 to D5) were formulated and prepared by mixing different ingredients such as casein, dextrin, gelat in, fish meal, sodiu m alginate, soybean meal, oil, carbo xy methyl cellulose, Zn free mineral, vitamin mix, zinc sulphate (ZnSO4.7H2O), TCP and DL-methionine as per ratio given in table 1. Fishes were fed with prepared experimental diets in crushed crumb le form in two split doses @ 2% (each time) of fish body weight (BW) for 90 days. Physico-chemical parameter of water was observed for temperature, pH, d issolved oxygen, total alkalinity and total hardness as per standard methods given in APHA (2005)  Base: q.s. (g 100g -1 ) ***Added as over and above 100%

STATISTICAL ANALYSIS
Data recorded for Physico-chemical parameters of water, Body weight and growth parameters of fish, biochemical composition of flesh and change in zinc concentration (mg kg -1 ) in d ifferent tissues were analysed by two-tailed bivariate Pearson's correlation coefficient for average values, standard deviation and correlation coefficient using one way ANOVA by SPSS 16.00 software.

III.
RESULTS AND DISCUSSION Fish, being an aquatic vertebrate is direct ly influenced by water quality parameters for their survival and growth. During the experimental period, physico-chemical parameters of water like temperature, p H and dissolved oxygen (DO) varied within the desirable range for fis h culture as suggested by Boyd (1990) and Bhatnagar and Devi (2013) for warm water fish species. However, total alkalinity and hardness were slightly higher than optimu m range but no negative effect in terms of behavioral changes recorded in fish. Details of the water quality recorded during the study period are given in table 2.  (Table 3). A mong experimental diets, highest crude protein content recorded in D5, fo llowed by D4, it may be possibly due to addition of methionine, which is itself a n itrogen containing biomolecule i.e. an essential amino acid. In prepared diets, higher concentration of zinc recorded as compared to rate of addition, which may be due to Zn content already available in different feed ingredients in different ratio, naturally. Garling   The growth in fish stocked during experimental period was assessed as increment in terms of net weight gain (NW G), specific gro wth rate (SGR) and efficiency of feed was assessed as feed conversion ratio (FCR) and protein efficiency ratio (PER) for each treatment (Table 4).
In fishes fed with different experimental d iets, highest weight gain recorded in fish fed with D5 diet, however in D3 diet fed fish lowest growth rate was recorded, wh ich may be possibly due to negative effect of tricalciu m phosphate and no element/ carrier was added to cater the negative effect of TCP, however in treat ment D2 and D4 in between h ighest and lowest growth rate recorded, wh ich may be either due to less concentration of TCP or negative effect was mitigated due to addition of methionine. Similarly SGR, FCR and PER values also improved in fish fed with D5 diet, it may be due to addition of methionine. The values recorded in present study were well close to the observations recorded by different workers. Sultana et al.(2001) reported the SGR in between 2.53 -3.24, FCR 1.22 -1.78 and PER 1.68 -2.48 in co mmon carp fry fed with 33.34% crude protein @ 5% body weight (BW) fo r 45 days. Kiaalvandi et al.(2011) reported the FCR in between 4.76 -6.25 and PER 0.38 -0.47 in co mmon carp juveniles (8.6 g) fed with 26 -28% crude protein @ 5% BW daily while Jader and Su levany (2012) recorded the SGR 0.71 -0.87, FCR 2.27 -3.01 and PER 0.79 -1.05 in juveniles of common carp when fed with 25-35% crude protein. in fish flesh fed with diet D5, wh ich indicates protein is not only digested but also absorbed and assimilated well in the fish flesh.

IV.
CONCLUSION In the present study, high NWG, SGR, FCR, PER and soluble protein in fish fed with diet D5 indicates feed containing even though 1% methionine helps in improving growth parameters, better feed utilization, absorption and assimilation of nutrients particularly protein, in the body of fish. Similarly, Zinc uptake was also improved in fish fed with d iet D5, which g ives an indication that methionine, helps to improve zinc absorption and accumulation in body even though from its inorganic source i.e. ZnSO4. 7H2O and mitigates the negative effect of TCP up to certain extent.