Development of Cost Effective Nutritionally Balanced Food for Freshwater Ornamental Fish Black Molly (Poecilia latipinna)

The majority (>90%) of freshwater ornamental fish are captive bred, compared to only about 25 of a total of 8000, in the case of marine fish and the ornamental fish industry relies heavily on the export and import of introduced species [1]. In India too, like other countries, Guppy’s, Swordtails, Platys and Molly’s, due to their color, sturdiness, stability, ability to withstand considerable environmental variations, relatively easy maintenance etc., are most popular as aquarium fishes in ornamental fisheries.


Introduction
The majority (>90%) of freshwater ornamental fish are captive bred, compared to only about 25 of a total of 8000, in the case of marine fish and the ornamental fish industry relies heavily on the export and import of introduced species [1]. In India too, like other countries, Guppy's, Swordtails, Platys and Molly's, due to their color, sturdiness, stability, ability to withstand considerable environmental variations, relatively easy maintenance etc., are most popular as aquarium fishes in ornamental fisheries.
Production of aquaculture species, in semi intensive pond culture system, demands the use of artificial feed, as supplementary source of nutrition. Using of such artificial diet for fishes can be traced as early as 1927 [2]. Most commonly used animal protein source in the diet is Fish meal, whose level could range between 10-50% of the operational costs [3]. Increasing cost and short supply of Fish meal has escalated the cost of Fish feed [4][5][6]. Thus, people have started concentrating on low cost Fish feed, by using plant protein sources [7]. Soybean products seem to be more promising, as a substitute, as they are almost half the price of Fish meal [8]. Though there is a tendency to use alternative plant and animal proteins, as a low cost substitute for Fish meal, such alternatives are known to have lower nutritional value, than Fish meal resulting in lower growth rates or a reduced performance of the cultured animals. In addition to the above, many of these protein sources containing substances that, might cause slight to severe effects on nutritional status of an animal. Insoluble fibers, Enzyme inhibitors, Saponins, Lectins, Tannis, Phytic acid and Gossypol are some of the important antinutrients, acting in the Gastro intestinal tract [9]. Despite the above, the search for alternative low cost substitutes is on throughout the world [10][11][12][13].
Ingested food nitrogen in fishes is either excreted as nitrogenous waste product or stored in growth products. The end product of protein catabolism in fishes is ammonia, which is generally considered as toxic, if allowed to accumulate. In many fishes, protein retention may be improved, by partly replacing dietary proteins by lipids. Such protein sparing effects have been demonstrated in Salmon [14,15], Trout [16], Carp [17], hybrid Striped Bass [18], Yellowtail [19], Red Sea Beam [20]. Improving digestibility of diet formulation and optimizing feeding regimens can improve feed utilization efficiency in farmed fishes [21]. It is also known that, fishes are able to regulate their daily food intake, based on their nutrient and energy requirements [22].
Fish meal is known for its high essential Amino acid a Fatty acids contents, high digestibility, low Carbohydrates, etc., Lipid is known to be one of the important nutrient next to protein, which plays a major role in optimum utilization of dietary Protein for growth. Lipids are almost completely digestible by fish and seem to be favored over Carbohydrates as an energy source [23]. Fishes are also known for utilizing protein preferentially over lipid or Carbohydrate as an energy source. Therefore, it is important from nutritional, environmental and economic point of view to improve protein utilization for tissue synthesis, rather than energy purposes.
In view of the above, locally available `Waste material' like Groundnut oil cake, Fish meal, Wheat bran, Snail, marine Fish waste, freshwater Fish waste, Chicken waste, Earthworms, Squids, Mussel, Chicken liver, Prawn meal, etc., were analyzed to see their efficacy as alternative to Fish meal in one of the most popular aquarium fish species viz., Black Molly (Poecilia latipinna).

Materials and Method
One of the most popular Aquarium Fish viz., Black Molly (Poecilia latipinna) was utilized in the present experiments. Uniform sized, healthy fish fingerlings were procured from Freshwater ornamental Fish hatchery of ICAR research complex, Old Goa, India and were treated with 0.05% Potassium Permanganate solution, for two minutes, to make them free from external parasites and pathogens present if any. Before initiating the feeding trials, they were kept in 1.0 ton capacity cement tanks for conditioning/acclimatize to laboratory condition. During this period, they were fed twice daily, with practical diet other than experimental diet.
All the necessary fish feed ingredients such as Snail, Freshwater Fish waste, Marine Fish waste, Chicken waste, Earthworm, Squid, Mussel, Chicken liver, Prawns, Fish meal, Groundnut oil cake, Wheat bran, Vitamin and Minerals, Oil were obtained from local market. The ingredients were dried at 105°C to make them moisture free. Each ingredient was powdered separately using domestic grinder. The powdered material was then sieved through a mesh size of 0.5mm dia. Later ingredients were analyzed, to determine their chemical composition (Table 1). Later, nine different feeds were formulated, based on the nutrient requirement (40% protein and 6% lipid, as determined earlier) ( Table 2). The percent composition and proximate composition of the experimental diet is provided in Table 3. A weight of individual components of the diet was done, by using Sertorius Single Pan Balance and the mixture was prepared by thoroughly mixing the required components in a plastic tray. To these mixtures, required quantity of lukewarm, distilled water was added to prepare the dough, which was then put into hand palletizer and the 2 mm diameter pallets were drawn. These pallets were sun dried for 3-4 days, till they contain 7-8% moisture level. Then, they were stored in air tight containers to be used later. Three each replicate of 10 fishes each, for each of the treatment, was placed in fish tank of 100 L capacity, in which 50 L of water of the quality as mentioned in Table 4 was used. All the batches were fed twice daily, with the experimental diet. The body weight gain after a month of treatment has been mentioned in Table 5. The data obtained was subjected for statistical analyses like ANOVA and the comparison among the groups, was done by Duncan multiple range test at P>0.05. Table 1 provides the data on Composition (% of dry matter) of the food ingredients used for formulating diet for Molly. It can be seen that among the ingredients Fish meal has highest dry matter (93.12%) in comparison with lowest dry matter (15.12%) in Squids. Crude protein was observed to be highest in chicken liver (66.12%) in comparison with 10.94% of crude protein in wheat bran. Further, maximum and minimum of ether extract was recorded in Chicken waste (28.54%) and Wheat bran (2.24%) respectively. Analyses for total Ash, showed that the maximum ash was in Prawn meal (17.96%), compared to a minimum of 7.68% of ash in wheat bran. Table 2 Exhibits the ingredients composition (% of dry matter) of the diet fed to Molly fishes. In all the experimental feeds fish meal and Vitamin and minerals was kept constant (10% and 5.0% respectively) while other ingredients were fed to the experimental batched in varied concentrations.

Results
Proximate composition (% dry matter) of the experimental diet, which provides data on dry matters, crude protein, ether extract and ash, has been provided in Table 3. It can be seen that, dry matter ranged from 92.4 (in Snail) to 94.2% (in Squid meal and Prawn meal). Crude protein was maximum in Marine Fish waste meal (41.90%) and was minimum in Mussel meal (39.96%) though, the difference was statistically insignificant. Ether extract showed maximum in Chicken liver (13.07%) and a minimum of 7.23% in Snail meal. Similarly, Ash content was highest in Prawn meal (13.54%) in comparison with lowest Ash contents in Chicken waste (9.68%).    Sampath [25] found that, females of X. helleri with frequent meals exhibited higher gonad weight, and Gonado-somatic index. In 1964, Hester reported that, scarcity of food will result in reduced fertility, in Guppy Fishes. He further reported that, female Fish needs adequate Protein, Fat, Vitamins and Minerals for egg development and spawning, as yolk is composed of Phospholipo proteins, an amalgam of Minerals (Phosphorus), a Protein and Lipid. It is also known that, Protein is also required for forming follicle in Embryo.

Ingredient Dry matter Crude protein Ether extract Total ash
In the present studies, the replacement of high cost fish meal, with the animal by product meal (ABM) and agro-based products, are used to formulate practical diet, for Black Molly, to observe the growth and dietary performance. The study indicate that, the growth was better, in the fish fed with diet containing Squid meal, Prawn meal, Chicken liver and Mussel meal. Thus, it can be concluded that, such substitution could be used to replace the costly commercial Fish meal [26,27].    Physico-chemical parameters of the water, in which experimental animals were maintained is provided in Table 4. The data reveals that, the water used for maintain the Fishes was very much normal and values obtained were acceptable for conducting such experiments.

Discussion
In aquaculture, diet cost accounts to over 50% of the operating cost, in intensive aquaculture, depending upon many factors, such as protein level, source, type of ingredient, and manufacturer's practices etc., the animal byproducts (ABM) meal, such as the one like Snails, Freshwater Fish waste, Marine Fish waste, Squid, Poultry industry waste can be used as substitute. Added advantage is that, these do not cause any deleterious effects on the fishes. Such attempts have been made earlier too [24], who used Earthworms, Poultry Egg Shell dust, Plant Rhizome, Wheat flour etc., as a substitute, for commercial Fish meal. The study revealed better that growth, length, gonad development, fertility in the Fishes fed with specially prepared feed, followed by dried Earthworm diet. Chong and Hashim observed higher man body length, in red sword tail Xiphophorus helleri, when they were fed several times. They reported that, more energy food is required, before spawning, for gonadal development and production of eggs. While, James and