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Seaweed Ulva reticulata a Potential Feed Supplement for Growth, Colouration and Disease Resistance in Fresh Water Ornamental Gold Fish, Carassius auratus
ISSN: 2155-9546
Journal of Aquaculture Research & Development

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Seaweed Ulva reticulata a Potential Feed Supplement for Growth, Colouration and Disease Resistance in Fresh Water Ornamental Gold Fish, Carassius auratus

Rama Nisha P1,2*, Elezabeth Mary A1, Uthayasiva M2, and Arularasan S2

1Department of Zoology, St. Mary’s College (Autonomous), Thoothukudi–628 001, Tamil Nadu, India

2CAS in Marine Biology, Faculty of Marine Science, Annamalai University, Parangipettai-608502, Tamil Nadu, India

*Corresponding Author:
Rama Nisha P, Ph.D.
Research Scholar, CAS in Marine Biology
Faculty of Marine Science, Annamalai University
Parangipettai-608502, Tamil Nadu, India
Tel: 91-9952666281
E-mail: [email protected]

Received Date: June 14, 2014; Accepted Date: July 26, 2014; Published Date: August 04, 2014

Citation: Rama Nisha P, Elezabeth Mary A, Uthayasiva M, Arularasan S (2014) Seaweed Ulva reticulata a Potential Feed Supplement for Growth, Colouration and Disease Resistance in Fresh Water Ornamental Gold Fish, Carassius auratus. J Aquac Res Development 5:254 doi: 10.4172/2155-9546.1000254

Copyright: © 2014 Rama Nisha P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Gold fish (Carassius auratus) being an omnivorous fish was chosen as the candidate fish. Fingerling of C.
auratus 2.34 gm after acclimated to laboratory condition for a period of 15 days were used in various studies. Five feeding types with 35% dietary protein level and with varying levels of algal meal were prepared and used. The fish were divided in to 5 groups. Group I was fed on Ulva reticulata free diet. Fish belonging to groups II, III, IV and V were fed on diets with 2, 4, 6 and 8% of Ulva reticulata supplementation respectively for 40 days. At the end of the experimental period growth, proximate composition, haematological parameters and carotenoid content were analyzed. It can be conclusively deduced from this study that Ulva reticulata had positively improved growth performance, haematological parameters and coloration in gold fish C. auratus. Reasons could be the high nutrient and mineral profile and carotene and chlorophyll a, b content of Ulva reticulata. This study suggests that U. reticulata can be included as an ingredient of gold fish diet up to 8%. Owing to the presence of disease resistance imparting antibacterial substances in the algae gold fish remained active and healthy and were free from bacterial infection, during the period of investigation. Antibacterial activity of U. reticulata on the tested fish pathogens indicates the scope of using extracts of U. reticulata an immuno- prophylactic for health management in the culture of Gold fish. Based on the results of this study, it was concluded that short term feeding had no adverse effect on gold fish but long term effect needs future research. Future work should focus on digestibility coefficients of different nutrient classes in U. reticulata prior to inclusion in formulated diets.

Keywords

Goldfish; Seaweed; Ulva reticulate; Carassius auratus; Supplement feed

Introduction

Ornamental fishes can be defined as attractive, colorful fishes of peaceful nature that are kept as pets in confined spaces of an aquarium or a garden pool as visually exciting objects [1]. However, some fish species loved by aquarists are quite ugly, in such cases the peculiar appearance is a source of attraction for the aquarium lovers and naturalists [2]. Inspiring popularity of aquarium keeping in households in many parts of the world, ornamental fish has become an important part in international trade. The world trade of ornamental fish is valued at about US $ 9.0 billion [3].

In Indian domestic trade in ornamental fish is conservatively estimated at Rs.10 crores and it is growing at the rate of 20 percent annually and the present domestic demand is higher than the supply [4]. Carassius auratus is a traditional ornamental gold fish which the body and fin shape; size and colour are features that affect its market price [5,6]. Attractive coloration is one of the most important quality criteria dictating their market value [7]. Carotenoids are the primary source of pigmentation on the skin of fishes. Like other animals, fish are unable to perform de novo synthesis of carotenoid [8]. In natural environment the fishes meet their carotenoid requirements by ingesting aquatic plants or through their food chain [9].

Skin colour determination in the gold fish involves cellular factors like existence of cells called choromatophores which store specific pigment black or brown (melanophores), yellow or orange (xanthophores), red (Erythrophores), iridescent, blue, silver or gold (iridophores) and white (leucophores) [10]. However, pigmentation efficiency is associated with different factors such as pigment source, fish size, feeding time, diet composition, sexual maturity and genetics of organisms [5,11]. Haematological parameters have been recognized as valuable tools for monitoring fish health. The analysis of blood indices has proven to be a valuable approach for analyzing the health status of farmed animal as these indices provide reliable information on metabolic disorders, deficiencies and chronic stress status before they are present in a clinical setting [12].

The immunity stimulating capacity of algae Spirulina and Cladophora have well been established [13,14]. Algae meals are an alternative plant feed stuffs that are increasingly being used in aqua feeds because of their nutritional quality, lower cost and availability [15,16]. Fish meal representing the protein source, is the main ingredient in fish feed. Up to 1990, about 10% of the global fish meal production was used for aquaculture; however this has tripled over the last decade [17]. The addition of small amounts of algae meal to fish diets resulted in considerable effects on growth, feed utilization [18,19]. Ulva species have become important macro algae, as it contains proteins, pigments, vitamins, minerals and unknown growth factors for several cultivable food fishes which have been investigated as a dietary ingredient including black sea bream, Acanthopigrus schelegli [18], snake head, Channa striatus [20], red sea bream, Pagrus major [21] and striped mullet, Mugil cephalus [22,23] for a wide range of cultivable food fishes. Low level dietary incorporation of Ulva meal has resulted in improved growth, feed utilization, physiological activity, disease resistance, carcass quality and reduced stress response.

The gold fish is an omnivorous fish and can utilize feed including plant materials effectively. Seaweeds are recognized as a potential source of bioactive natural products [24]. They are considered as a source of secondary metabolites characterized by a spectrum of biological activity. Seaweeds are rich and varied source of biocidal and pharmaceutical agents. Recent findings evidenced that seaweeds contained antibacterial [25], antiviral [26,27], antifungal, Cytotoxic [28] and larvicidal potential [29]. In aquaculture the bacterial pathogens create with fish diseases is worldwide problem. The secondary metabolite directly acts as antibiotics in aqua culture. Hence, the interest in macro algae as a potential and promising source of pharmaceutical agents has increased during the last years [30,31]. Extracts from seaweeds have let to the discovery of the variety of secondary metabolites with antimicrobial activities against fish pathogens.

The present study was carried out to examine the efficiency of seaweed Ulva reticulata as a feed ingredient for gold fish Carassius auratus. Evaluate growth and coloration of Carassius auratus fed on diets with various level of Ulva reticulata meals as feed additives so as to determine the optimal level of dietary supplementation. Estimate the proximate chemical composition from fish Carassius auratus. Access the haematological parameters of Carassius auratus fed feed with the varying level of Ulva meal. Determine the antimicrobial efficacy of Ulva reticulata on some selected bacterial fish pathogens viz., Pseudomonas aeruginosa and Aeromonas hydrophila.

Materials and Methods

Sample collection

Fresh water ornamental fishes were collected from ornamental fish trades, Chennai and they were accumulated with the laboratory condition. Raw samples of seaweed were collected from Hare Island, Thoothukudi, Tamilnadu.

Experimental species

The gold fish is one of the earliest fish to be domesticated, and the most commonly known aquarium fish. Among ornamental fish, gold fish is considered to be the most popular and attractive pet fish due to its many variations such as colour, finagles, tail, shape, size and body and has great demand both in domestic as well as international market. The gold fish is an omnivorous fish and can utilize feed including plant materials effectively. The ornamental fish Carassius auratus (Figure 1a) was chosen as the candidate fish for the present study. It belongs to Order-Cypriniformes, Family-Cyprinidae, Genus-Carassius and Species-C. auratus.

aquaculture-research-development-experimental-fish

Figure 1a: Experimental fish-Carassius auratus.

Fish collection and maintenance

Full orange colour gold fish of more or less uniform size (2.75 ± 0.09 cm in length and 2.89 ± 0.02 cm in weight) were procured from a private fish farm Thirunavukarasu aquarium, near Korampallam, Thoothukudi, Tamil Nadu, India. They were transported to the laboratory in an oxygenated bag and stocked in a trough. The water was changed on alternative days and the fish were fed with a balanced fish diet with 35% protein. The excess feed and fecal materials were siphoned out once a day. The temperature of the water in the fish trough was kept between 28-29°C. Other physiochemical parameters of water were also analyzed systematically at 7 days interval to maintain optimum level of dissolved oxygen 6.8-7.2 mg/l. pH 7.7-8.5 and ammonia 0.08-0.12 mg/l throughout the experiment.

Preparation of seaweed meal

The seaweed Ulva reticulata was collected from the coastal areas of Tuticorin. This species was chosen because of its abundance, distribution and proximity to areas and seaweed also chosen because it is known as one of the species that give good colour. The collected samples were thoroughly washed with sea water, dried at 40°C for 48 to 72 hrs and then ground to powder.

Diet formulation

In the present experiments 35% protein was used as basal diet for Ulva meal supplementation. Fresh feed ingredients were procured in dry form. Using the ingredients such as dried fish meal, ground nut oil cake, rice bran, wheat flour, sardine oil, vitamin and mineral mixtures the feed was formulated following the square method of food compounding [32]. The ingredients were mixed in five different proportions in order to get five diets with five levels of algae Ulva reticulata (0, 2, 4, 6 and 8%) in the diet. The ingredient were mixed and homogenized to wet dough by adding sufficient water. The dough was cooked under steam in a pressure cooker, cooled and to this feed vitamin mix, cod liver oil and Ulva meal were added and mixed then pelletized through hand pelletizer until between 1 and 4 mm and dried in shade to reduce the moisture content of the feed below 10% and stored in air tight container and used for experiment. Composition of the feed is given in Table 1.

Ingredient (g) Diet (Ulva Content)
F1
0
F2
2%
F3
4%
F4
6%
F5
8%
Fish meal 40 40 40 40 40
Ground nut oil cake 36 36 36 36 36
Tapioca flour 14 13 12 11 10
Wheat flour 8 7 6 5 4
Sardine oil 1 1 1 1 1
Vitamin & Mineral mix 1 1 1 1 1
Ulva meal 0 2 4 6 8
Proximate composition (%)
Moisture 7.27 ± 0.13 7.51 ± 0.05 7.74 ± 0.08 7.86 ± 0.08 7.89 ± 0.02
Crude protein 34.13 ± 0.17 34.84 ± 0.3 34.92 ± 0.14 34.92 ± 0.14 35.19 ± 0.7
Crude fat 6.11 ± 0.06 5.83 ± 0.09 5.69 ± 0.17 5.12 ± 0.23 4.78 ± 0.08
Ash 9.26 ± 0.06 10.94 ± 0.09 11.54 ± 0.10 11.69 ± 0.08 14.02 ± 0.06
Nitrogen Free Extract 43.22 ± 0.27 41.45 ± 0.14 40.35 ± 0.23 39.82 ± 0.34 39.96 ± 0.35

Table 1: Formulation and composition of the experimental diets and their proximate composition (% dry matter basis).

Proximate composition analysis of feed

Proximate analysis is usually the first step in the chemical evolution of a feed ingredient, where the materials is subjected to a series of relatively simple chemical tests so as to determine the content of moisture crude protein, crude fiber, lipid, ash and digestible carbohydrate.

Estimation of protein from formulated feed: The protein content of the feed was estimated following the method of Lowry [33]. 100 mg of tissue was taken and homogenized with 5 ml of 10% trichloroacetic acid in a tissue homogenizer. The homogenates were centrifuged for 15 minutes at 3000 rpm. The supernatant was discarded. The precipitate was dissolved thoroughly in 5 ml of 0.1N sodium hydroxide solution and kept in a water bath at 60-70°C for 10 minutes. From this 0.5 ml of solution was pipetted out and poured in a clean dry test tube.

To this 4 ml of carbonate copper solution was added. It was mixed well by thorough lateral shaking and kept in the room temperature for 15 minutes. To this 0.4 ml of folin phenol reagent was added. The test tubes were shaken well for uniform mixing and kept in the room temperature for another 30 minutes. The blue colour appeared was read at 640 nm against a reagent blank. The standard curve was obtained by using bovine serum albumin.

Estimation of lipid from formulated feed: Lipid was estimated following the method of Bragdon [34]. A known quantity of the tissue was homogenized well with 5 ml of chloroform and the solution was centrifuged at 3000 rpm for 15 min. The supernatant was evaporated to dryness by keeping it in an oven. Then 3 ml of 2% potassium dichromate in conc. Sulphuric acid was added followed by 3 ml of distilled water. The greenish brown colour appeared was read at 640 nm against a reagent blank in a spectrophotometer. The standard curve was obtained by using cholesterol and the lipid was expressed in mg/g.

Determination of Ash: Ash content was estimated by the method of Lovell [35]. A weighed amount of dry sample was heated to higher temperature (550-600°C). All the organic constituents are burnt leaving only the inorganic constituents in the form of ash. The percentage of ash content was estimated as per the calculation given below.

On moisture free basic/Per cent by weight=100(W2-W)/W1-W

Where,

W2=Weight in g of dish with ash

W=Weight in g of empty dish

W1=Weight in g of the dish with the dried material taken for the determination of ash.

Experimental design

Active and healthy fish (2.79 ± 0.15 g) were chosen from the acclimation tank and starved for 24hrs prior to the commencement of the experiment. The fish were divided into 5 groups of 6 individuals each and reared in circular plastic troughs containing 20 L of water (Figure 1b).

aquaculture-research-development-experimental-set-up

Figure 1b: Experimental set-up.

Group 1 served as control and fed with Ulva free diet (F1). Test animals belonging to 2nd, 3rd, 4th and 5th groups were fed on diet (F2, F3, F4 and F5) with varying Ulva levels viz., 2, 4, 6 and 8% respectively. Fish were fed on allotted experimental diet at 5% of their total body weight per day. Feeding was done daily at 0800hrs. The unfed was removed after 2 h of feeding.

Triplicates were maintained for each group. The experiment has run for 40 days. The following experimental parameters were maintained throughout the experiment; temperature 26 ± 2°C; pH 7.7, dissolved oxygen 8 mg/l; photoperiod 12LL:12DD.

Growth parameters: At the beginning of the experiment, the total wet weight of the fish in each group was determined by weighing in an electronic balance. All fish in each group were weighed at the end of the experiment.

Absolute growth rate (g)=Final weight–Initial weight

Specific growth rate (%)=[(In final weight–In initial weight)/ Rearing period (days)]×100

Weight gain (mg/g)=[(Final weight–Initial weight)/Initial 0weight]×100

Carotenoid estimation

Pigment extraction in fish tissue: Analysis of total carotenoids in the fish tissue was carried out prior to and after the termination of the experiments following [36]. One gram of entire gold fish body tissue (without head and alimentary canal) was taken in a 10 ml screw capped clear glass vials and 2.5 g of anhydrous sodium sulphate was added.

The sample was gently meshed with a glass rod against the side of the vial and then 5 ml of chloroform was added and left overnight at 0°C. When the chloroform formed a clear 1-2 cm layer above the caked residue, the optical density was read at 380, 450, 470 and 500 nm, in a spectrophotometer taking 0.3 ml aliquots of chloroform diluted to 3 ml with absolute ethanol. A blank prepared in a similar manner was used for comparison. The wavelength, at maximum absorption, was used for the calculation.

The total carotenoid content was calculated as μg per wet weight of tissue as follows:

Total carotenoid content=[Absorption at maximum wave length/(0.25×sample weight (g))]×10 (Where, 10=Dilution factor; 0.25=Extinction coefficient)

Proximate composition analysis

Analysis of proximate composition of muscle tissue of fish from all groups was made both at the beginning and at the end of the experiment.

Estimation of protein: The protein content of the feed was estimated following the method Raymont [37]. About 20 mg of dried material was taken and homogenized with 1.0 ml of distilled water. 4.0 ml of Biuret reagent was added into two installments of 2.0 ml each and the tissue grinder was cleaned before being transferred to centrifuged tube. After 30 minute and this sample were centrifuged for 10 minutes and the supernatant fluid was transferred to another tube. Then the optical density of supernatant tube in spectrophotometry was taken using 540 ml against blank reading. Bovine serum albumin was used as standard protein. Optical density is equal to 18.44 mg of protein. The percentage of protein was calculated using the formula.

Percentage of protein=18.44×O.D of the sample X 100/Weight of tissue

Estimation of carbohydrate: The Carbohydrate content of the feed was estimated following the method Seifter [38]. A known weight of tissue 10 mg was homogenized with 2 ml of 10% TCA and 8 ml distilled water. The homogenate was centrifuged at 3000 rpm for 10 minutes. The supernatant was collected. One ml of the supernatant was taken in a clean test tube. To this 5 ml of anthrone reagent was kept in boiling water bath for 10 minutes. The tube was then cooled at room temperature. The developed colour was read at 620 nm against a reagent blank in a spectrophotometer. Standard curve was obtained by using glucose and the carbohydrate was expressed in mg/g. The values were then converted into percentage.

Percentage of Carbohydrate=2.16×O.D of the sample X 100/ Weight of tissue

Estimation of lipid: The Lipid content of the feed was estimated following the method Bragdon [34]. The same procedure was followed as above.

Hematological studies

Blood sampling: Blood sample was collected from fish of each group. The fishes were collected and gently wiped with a dry cloth to remove water. Caudal peduncle was cut with a sharp blade and the blood was collected in a watch glass containing EDTA, an anticoagulant (6% Ethylene Diamine Tetra Acetic Acid). The blood was mixed well with the EDTA solution by using a needle and this sample was used for determining the RBC, total WBC and haemoglobin content.

RBC count: For RBC count, a method devised by Yokayama [39] and later modified by Christensen [40] was followed. The standard RBC diluting pipette and a 1:200 dilution was used for the RBC count. Hayem’s solution was used for dilution.

Blood was drawn in the pipette up to the 0.5 mark. The tip of the pipette was wiped with a filter paper to adjust the volume exactly to 0.5 marks. The pipette was immediately filled to the 101 mark with the diluting fluid. Partial rotation of the pipette while it was being filled assured the complete mixing its ends grasped between the thumbs and diluting fluid and prevented clotting. With its ends grasped between the thumb and second finger the pipette was then shaken for 5 minutes, which ensured the through mixing of the blood with diluting fluid.

The improved Neubauer counting chamber was used for counting the cells. A glass cover slip was placed over the ‘H’ groove on the chamber. Then the blood was allowed to enter the chamber by simply touched to the edge between the cover slip and the chamber, where the blood spread under the cover slip by capillary action. The counting area is divided into 25 squares of 16 small squares. Only 5 squares were taken to count RBC marked as E1-E5. Two minutes time was giving for the corpuscles to settle down, the total number of cells in the five squares were counted, calculated and expressed as RBC per mm3 of blood.

Haemoglobin content: The Sahli Hellige method was followed for haemoglobin determination. Sahlis pipette was filled slightly above the 20 mm mark the pipette was wiped with a filter paper or cotton to remove excess blood and the volume was adjusted to exactly 20 mm3 by blotting the tip. The blood was expelled into a calibrated (transmission) test tube containing 2 ml of 0.1N HCL. The pipette was rinsed several times in the acid solution. The sample was allowed to stand for 15 minutes.

The principle behind the method is the conversion of haemoglobin to acid haematin. The acid haematin was then diluted with distilled water till the colour matched with colour of the standard in haemoglobinometer. The height of the column at which the match obtained gives the value of haemoglobin in g%.

Determination of antibacterial activity of ulva reticulata against bacterial pathogens

The antibacterial activity of extract of seaweed Ulva reticulata was investigated against select bacterial fish pathogens such as Gram +ve (Aeromonous hydrophila) and Gram -ve (Pseudomonas aeruginosa).

Preparation of seaweed extracts: The collected Ulva reticulata samples were cleaned and the necrotic parts were removed. The seaweeds were washed with tap water to remove any associated debris and shade dried at room temperature (28 ± 2°C) for 5-8 days or until they are brittle easily by hand. After completely drying, the seaweed materials (1.0 kg) were ground to a fine powder using electrical blender. Twenty gram of powdered seaweed was extracted successively with 200 ml of solvents (Methanol, and Ethyl acetate) in Soxhelet extractor until the extract was clear. The extracts were evaporated to dryness and reduced pressure using rotary vacuum evaporator and the resulting pasty form extracts were stored in a refrigerator at 4°C for future use.

Preparation of algal disc for antibacterial activity: 5 mm diameter discs were prepared using sterile Whatmann No.1 filter paper. The solvent such as (Methanol, and Ethyl acetate) extracts of seaweed Ulva reticulata were mixed with 1ml of Dimethyl sulfoxide (DMSO). The discs were impregnated with 20 μl of different solvent extracts of Ulva reticulata at three different concentrations ranging 1 mg/ml, 10 mg/ml and 100 mg/ml to check their antibacterial activity. The streptomycin (5 mg/ml) was used as positive control.

Bacterial inoculum preparation: Bacterial inoculums was prepared by inoculating a loopful of test organisms (Pseudomonas aeruginosa and Aeromonas hydrophila) in 5 ml of Nutrient broth and incubated at 37°C for 3-5 hours till a moderate turbidity was developed. The turbidity was matched with 0.5 Mc Farl and standards and then used for the determination of antibacterial activity.

Disc diffusion method: The antibacterial activities of Ulva reticulata extract were determined by Disc Diffusion method proposed by Bauer [41]. Petri plates were prepared by pouring 20 ml of Nutrient agar and allowed to solidify for the use in susceptibility test against bacteria. Plates were dried and 0.1 ml of standardized inoculum suspension was poured and uniformly spread. The excess inoculum was drained and the plates were allowed to drying for five minutes. After drying the discs with extract were placed on the surface of the plate with sterile forceps and gently pressed to ensure contact with the agar surface. The streptomycin (5 mg/disc) was used as positive control. The plates were incubated at 37°C for 24 hours. The zone of inhibition was observed and measured in millimeters.

Statistical analysis

The statistical analysis of the data was performed as per the method described by Snedecore and Cochran [42]. Mean, Standard Deviation and Analysis of variance was analysed.

Analysis of variance (ANOVA): Variance between different means of a single variable was tested following one way calculation of variance described by Zar [43]. Sum of X for all the values was squared and a correction factor ‘C’ was obtained.

Equation

Equation

Where, X2 represents the sum of squared values.

Equation

Where, N- number of observations in each group.

Error sum of square=Total SS- Group SS

Considering the degrees of freedom for each source of variance, mean square was calculated.

Total SS=Number of values in the Table–1

Group SS=Number of groups–1

Error SS=Df of total SS–df group SS

Significance level at the corresponding df was read from table D.11 of Zar [40].

Results

Growth

All diets were readily accepted by fish, indicating no issues relating to palatability of algal supplemented diets. No mortalities were observed during the experimental period. Data indicates that dietary Ulva meal, significantly improved fish growth when compared with the non Ulva meal supplemented group. (Table 2). The mean weight of the gold fish on the first day of stocking in the control feed (F1) was 2.34 gm. The initial mean weight of the fish fed on diets F2, F3, F4 and F5 were 2.52, 2.79, 2.49 and 2.44 gm respectively.

Feed AGR
gm
SGR
%
WG
Mg/g %
Control 3.01 ± 0.22 7.53 ± 0.55 128.47 ± 9.45
2% 4.06 ± 0.72 11.54 ± 1.82 183.28 ± 29.01
4% 4.66 ± 0.42 11.66 ± 1.06 167.05 ± 15.11
6% 5.29 ± 0.14 13.24 ± 0.35 214.75 ± 5.72
8% 5.68 ± 0.26 14.09 ± .65 231.24 ± 10.71

Table 2: Dietary supplementation of Ulva reticulata on growth parameters of Carassius auratus. Values are mean ± SD of 3 replicates.

The result indicates that mean weight gain of the fishes fed with diets F2, F3, F4 and F5 were found to be 4.62, 4.66, 5.29 and 5.63 gm. The absolute, specific growth rate and weight gain of the fishes also showed a similar trend of an increase with increase in concentration of Ulva meal in diet. The maximum values of absolute, specific growth rate and weight gain were observed in fish fed on 8% Ulva meal supplemented diet viz., 5.68 ± 0.26 gm, 14.09 + 0.65% and 231.24 ± 10.71 mg/g% respectively. Analysis of variance for the data on absolute growth rate, specific growth rate and weight gain indicated that the changes were significant at 1% level (Table 3).

Absolute Growth Rate
Source of variation SS DF MS F F Crit
Between group Within group 12.22 2.44 4 10 3.06 1 3.06 3.48
TOTAL 14.66 14      
Specific Growth Rate
Source of variation SS DF MS F F Crit
Between group Within group 76.38 9.59 4 10 19.1 0.96 19.9 3.48
TOTAL 85.97 14      
Weight Gain
Source of variation SS DF MS F F Crit
Between group Within group 20299.85 1780.02 4 10 5074.96 178 28.51 3.48
TOTAL 22079.87 14      

Table 3: Summary of analysis of variance for the data on the feeding parameters of Carassius auratus in relation to dietary Ulva supplementation.

Body composition

Whole body composition of the fish at the beginning and at the end of the experiment is summarized in Table 4. The crude protein content of fish fed on algal diets increased proportionally with the concentration of algal meal compared to the initial measurement. Maximum tissue protein of 35.04% was realized in group fed on 8% algal supplemented diet (Figure 1a). In the present study compared with non-Ulva supplemented groups, fish fed Ulva meal resulted in a significant reduction of body lipid. The lipid level tented to decrease with increasing level of Ulva meal. In group fed on 8% algal diet exhibition a decrease in lipid content from the initial value of 4.2% to 3.4%.

Feed Protein Carbohydrate Lipid
Initial Final Initial Final Initial Final
F1
F2
F3
F4
F5
8.30%
8.28%
8.52%
8.36%
8.96%
17.52%
24.89%
31.35%
32.37%
35.04%
3.75%
3.54%
3.10%
3.46%
3.24%
10.15%
9.07%
8.21%
6.48%
5.83%
4.6%
4.12%
4.16%
4.0%
4.2%
3.6%
3.6%
3.5%
3.4%
3.4%

Table 4: Proximate Composition of muscle tissue of gold fish fed with Ulva meal.

Coloration

The result clearly showed that the maximum carotenoid content was present in the fish fed with F5 diet (8 g/100 g) dry weight feed. For instance, the total carotenoid content in the 8 per cent fed group was found (Figure 3) to be 0.60 ± 0.07 μg/g wet weight and for the control group it was found to be 0.42 ± 0.07 μg/g wet weight. Similarly, the carotenoid content in F2, F3, F4, F5 diet groups increased significantly and analysis of variance showed that the carotene content between treatments differed significantly. The carotenoid content among the variation was found to be significant (0.001) (Tables 5 and 6).

aquaculture-research-development-antibacterial-activity

Figure 2: Antibacterial activity of U.reticulataextract against two fish pathogens.

aquaculture-research-development-pigment-profile

Figure 3: Pigment profile of C. auratus fed with Ulva reticulata supplemented feed (F1- Control, F2-2%, F3-4%, F4-6%, F5-8%).

aquaculture-research-development-proximate-composition

Figure 4: Proximate composition.

F1 F2 F3 F4 F5  
0.42 ± 0.070 0.51 ± 0.2 0.56 ± 0.11 0.57 ± 0.17 0.60 ± 0.07
One way ANOVA for carotenoid content of Carassiusauratus feed with different experimental diets 
Source of variation SS  DF  MS  F Crit
Between group 97.51 4 24.38    
Within group 298.6 10 29.86    
TOTAL 396.11 14      

Table 5: Carotenoid content (μg/g wet weight) of Carassius auratus fed with different experimental diets.

Pathogens Solvent
Methanol Ethyl Acetate
Pseudomonas aeruginosa Aeromonashydrophila 10 mm
-
7 mmv
-

Table 6: Antibacterial activity of seaweed extracts of Ulva reticulata against two bacterial fish pathogens.

Antibacterial activity

The results of antibacterial activities of the seaweed are summarized in Table 7. In our observation highest inhibition zone 10 mm was recorded in methanol extract (100 mg/ml) and ethyl acetate extract 7 mm of Ulva reticulata against Pseudomonous aerogenosa. The Aeromonas hydrophila did not show exhibitory activity against tested seaweed extracts. The positive control streptomycin was effective against all the tested bacterial pathogen and it exhibited nearly 20 mm of zone of inhibition in all the pathogens. During the entire period of investigation the fish remained active and healthy. There was no incident of any bacterial infection.

Discussion

Natural resources as possible feed ingredient or additives need to be investigated for the development of inexpensive feeds in the world. Macro and micro algae, seaweed and other alternative aquatic plants have been researched and used for decades in aquaculture. Ulva meal is one of the most studied and promising source in this sense. In this study, we primarily laid out the possibility of inclusion of Ulva meal in gold fish Carassius auratus without affecting their health status negatively.

In the present study, growth performance of fish tended to increase with an increase in Ulva meal concentration. The growth rates recorded in the present investigation were in accordance with the finding of Mensi et al., who fed Orechromis niloticus fry on 30% dietary protein containing 3% and 18% Ulva meal for 45 days [44]. Betoikut Gorey, reported an enhancement in growth rate of Oreochromis niloticus fed 5-10% Ulva supplemented diet [45]. Whereas, Kissil observed no effect of Ulva inclusion on fish growth in the gift head sea bream, Sparus aurata [46].

Growth performance in rainbow trout Oncorhynchus mykiss was reported to be reduced when fed on U. lactuca and Enteromorpha linza meal. Onder Yildirim, attributed the decline in growth performance of Oncorhynchus mykiss fed on U. lactuca and Enteromorpha diets when compared to group fed the non- algal diet to significantly higher fiber content [19]. Ibrahim, demonstrated increase in growth performance and higher body protein gain concentration for common carp Cyprinus carpio fed on dietary Ulva rigida meal supplementation in higher concentration (upto 15%) [47]. Bindhu and Shoba, reported very good response in terms of growth and feed utilization in Ctenopharyngodon idella fed with algal diet containing Ulva fasciata, Spyridia insignis and Sargassam wightii [48].

Ray and Das, successfully incorporated composted Salvinia cuculata until 20% dietary rate without compromising the growth performance in Labeo rohita [49]. Bairaji, concluded that feeding rohu (Labeo rohita) fingerlings with diet incorporating upto 30% fermented duckweed (Lemna polyrhiza) leaf gave better growth response, feed efficiency, higher protein and lipid gain compared to 10% raw duckweed inclusion level contrastingly catla (Catla catla) fingerlings fed with increasing amount of dietary Spirogyra sp meal [50], belonging to the class Chloraphyceae showed worse growth performance even at the lowest dietary inclusion level [51]. Studies of Chitra showed that fishmeal mixed with the marine algae powder Sargassum wightii gave better growth and weight increments of Oreochromis mossambicus [52].

Jafri and Farrog Anwar, stated that protein from plant sources showed higher digestibility than animal protein [53]. Supplementation of 5% Ulva meal for Nile tilapia was used in their study with no negative effect on growth performance, feed utilization and body composition. However; negative consequences resulted from using levels between 10% and 20% [54,55]. In contrast, Onder Yildirim, reported a decline in feed utilization and growth performance of rainbow trout fed diets with Ulva lactuca and Enteromorpha linza when compared with fish fed (10% level) a control diet [19]. A carnivorous fish like trout would accept rather animal origin ingredient than plant food stuffs. The growth retardation was attributed to the antinutritional characteristics of seaweeds.

In the present investigation, in gold fish being omnivorous the algal meal elicited a growth promoting effect. This may be due to their high water stability and lower disintegration, which are limiting factors in aquaculture as carp are slow feeders. Olin observed that the incorporation of the marine algae in formulated diets increases the water stability of pellets because of the nature of the phytochemical and stabilizers present in them [56]. Ulva fed fish displayed a marginal improvement in carcass protein. Our reports are in conformity with the finding of Sebshattin, Erugan who reported a similar observation in body composition of juvenile Nile tilapia (Oreochromis niloticus) fed on Ulva meal [57]. Reports of Guroy and Azaza also revealed an enhancement in carcass composition of Nile tilapia fed on Ulva meal [54,55]. Onder Yildirim reported a significant increase in the crude protein content of Oncorhynchus mykiss fed on U.lactuca and Enteromorpho linza [19].

Inclusion of algae meal had reduced lipid content of gold fish. Our reports are in conformity with Onder Yildirim who reported a similar reduction in lipid content of Oncorhynchus mykiss fed diet with U.lactuca and Enteromorpho linza [19]. The effect of dietary algal meal on carcass lipid can be contradictors, where as some reports suggest a decrease [56,57]. The effective substance provided by algae is not known but protein, fiber, mineral and trace elements need to be considered. The observed benefits may be explained by the high vitamin C content of Ulva meal [58,59] which affect lipid metabolism and may result in alternative of body composition and nutrient deposition in fish [60,61].

An observation of considerable interest during this study was increase in the carotenoid content of the test fish fed on Ulva supplemented diets. Ulva sps contain various pigments such as beta carotene, chlorophyll a, b and xanthophylls [62] which may possibly cause the enhancement of carotenoid pigments. Ulva, a natural pigment source may enhance the potential of seaweed inclusion in fish feed and may perhaps replace and reduce artificial colourants currently used by the industry [63]. Seaweed meal was found to be an effective colour enhancer at a cheaper price. Anna Soler-vila et al reported dark orange pigmentation in Oncorhynchus mykiss fed on red algae Porphyra compared to the control fish [64]. Ezhil found maximum carotenoid content in red sword tail Xiphophorus helleri fed on 15 gms of marigold pellet meal (6 g/100 g) [65].

A proportionate increase in carotenoid content in the fin, skin and muscle with dietary supplemention of Spirulina in Xiphophorus helleri was reported by James [13]. Similarly, Relul Guroy, demonstrated an enhancement in yellow cichlid fed the diet containing Spirulina meal [66]. The dietary supplementation of algae was proved to enhance colour in fish. Enhancements of colour in fish fed algal diets are well documented [48]. The RBC counts were higher for the fish fed on 8% algal diets which was similar to the finding of Terry that striped tilapia fed with algae had a red blood cell count of 1.91-2.83×106 cell/ml [67]. Feeding Spirulina plantensis in processed form enhanced specific and non-specific immunity and resistance against Edward ictaluis infection in channel cat fish, Ictaluras punctatus [68].

James et al reported an increase in the monocytes, neutrophils and lymphocytes with increase in dietary level of Spirulina with 8% level fed group the maximum [13]. Similarly, fingerlings of Clarias gariepinus fed 5% Spirulina diet recorded higher values for red and white blood cell counts and the lysozymes activity [69]. Increased in WBC indicate that Ulva in the diet may benefit the immune system in C. auratus as well. U. reticulata contains carotenoids, which affect the health of fish, specifically improving health and increasing the ability to fight off infections through the reduction of stress level [70]. Antimicrobial activity depends on both algal species and the solvents used for their extraction. Similar to our finding, Periasamy Mansuya recorded the maximum activity (45 mm) from 200 mg aqueous extract of Ulva reticulata against Salmonella typii and minimum 9 mm by Ulva lactuca [71]. Streptococcus pyogenes at 50 mg level whereas, the methonalic extract showed the maximum activity (40 mm).

Seeenivasan, performed antibacterial activity studies in vitro with 3 extracts namely acetone, methanol and ethanol [72]. They observed that Ulva fasciate in selective media produced good results against E.coli. Cox et al screened the microbial activity of 6 species of edible seaweeds on food pathogenic bacteria such as Listeria monocytogenus, Salmonella abony, Enterococcus faecalis and Pseudomonas aerugenosa [31]. They found that dried methanol extracts of red and green seaweeds had significantly lower antimicrobial activity than brown seaweed.

Studies of Felix and Rajeev showed better inhibitory activity of Graiularia verrucosa against both Vibrio alginolyticus and V. parahaemolyticus [73]. Kolinjinathan and Stella reported zone of inhibition of Ulva reticulata and U. lactuca extract against gram positive and gram negative bacteria in the range of 7 mm to 16 mm [74]. the methanol extract showed highest mean zone of inhibition (15 ± 0.6 mm) against the gram positive cocci Streptococcus pyogenes followed by Staphylococcus aureus (13 ± 0.3 mm), Streptococcus epidermis (12 ± 0.6 mm), Bacillus subtilis (11 ± 0.6 mm) and Bacillus cereus (10 ± 0.55 mm). For gram negative bacteria, maximum zone of inhibition was recorded in methanol extract of Ulva reticulata against Klebsiella pneumonia (13+0.6 mm) followed by Escherichea coli (12+0.8 mm), Enterobacter aerogenes (10+0.03 mm), Vibrio cholarae (9+0.6 mm) and Salmonella typhi (9+0.3 mm). The zone of inhibition obtained from the hexane extracts of seaweed Ulva reticulata against bacterial pathogens was comparatively very less when compared to the other solvent extracts.

Alang, isolated eight compounds from Ulva lactuca for their antimicrobial activities and concluded that different extracts and fractions show moderate to significant microbial activity to the tested bacterial S. aureus, S. epidermidis, P. aeruginosa and E. coli [75]. Highest zone of inhibition of 10mm in methanol extracts of green algae Pithophora oedogonia against gram negative bacteria, Bacillus subtilis and Staphylococcus aureus was observed by Anand Prakesh Singh and Chaudhary [76]. Radika, suggested that brown seaweed had high antibacterial activity against Klebsiella [77]. Antimicrobial activity of U. lactuca was correlated to its active compounds such as carbohydrates, steroids and glycosides [75].

Karthigai Devi, evaluated for antibacterial activity of commonly occurring green algae Codium adherens, Ulva reticulata and Halimeda tuna [78]. The ethanol extract was found to be more effective than other. Vallinayagam, screened the antibacterial activities of two important seaweeds namely Ulva lactuca and Gracilaria edulis were screened against human bacterial pathogens Staphylococcus aureus, Vibrio cholerae, Shigella dysentriae, Shigella boydii, Salmonella paratyphi, Pseudomonas aeruginosa and Klebsiella pneumonia [79]. The maximum activity was recorded from the extract of Gracilaria edulis against Staphylococcus aureus and minimum by Ulva lactuca against Pseudomonas aeruginosa.

Conclusion

The present study of anti-bacterial activity of seaweeds Ulva reticulata extracts showed promising antimicrobial activity against fish pathogens, suggesting their potential for the development of antipathogenic agents in aquaculture. The enhancement in growth rate, colouration and hematological parameters observed in the test fish could be due to the disease resistance imparting antibacterial substances present in the Ulva reticulata.

Acknowledgements

Authors are thankful to the authorities of Department of Zoology, St. Mary’s College (Autonomous), Thoothukudi–628 001 and CAS in Marine Biology, Annamalai University, Parangipettai for provided necessary facilities.

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