Effect of Ulva lactuca and Sargassum cinereum Supplemented Diets on Growth Performance of Koi Carp (Cyprinus carpio Linnaeus, 1758) Fry in Laboratory Conditions

There is a potential to formulate cost effective aquafeeds using seaweeds in the littoral zone of the coastal belt of Sri Lanka. This study was designed to evaluate the effect of seaweed supplemented diets on the growth of Koi carp. The experimental set up consisted of a control (C) and two experimental treatments (T1 and T2). The control diet (without seaweed) and the two test diets, of which 30% of the weight of control diet was replaced by Ulva lactuca and Sargassum cinereum were fed to Koi carps in C, T1 and T2 respectively. Proximate analyses were carried out for both ingredients, and the diets were prepared following standard methods. Koi carp in fry stage belonging to the same cohort (weight 2.20±0.08 g, length 3.89±0.04 cm) were assigned to C, T1 and T2 in fiber tanks filled with 150L of water (n=20 per tank) with four replicates each. The fish were fed 5% of their body weight twice a day (09.00h and 15.00h) for 14 weeks. The length and weight measurements were recorded once in two weeks. Fish performance was compared using Body Weight Gain (BWG), Daily Growth Rate (DGR), Relative Growth Rate (RGR), Feed Conversion Ratio (FCR), Feed Efficiency (FE), Fulton’s Condition Factor and survival rates. At the end 14th week of the experiment, mean values of the weight, length, BWG, DGR showed a significant difference (p 0.05) among treatments. There was a 100% survival rate in all groups. Aquafeeds supplemented with seaweeds showed promising results in terms of increasing growth rates of young Koi carps.


INTRODUCTION
The ornamental carp Cyprinus carpio Linnaeus,1758 commonly known as Koi, is very popular in the freshwater ornamental fish industry. The marketable size of the Koi carps is considered as over 4 g which can be obtained during 3-5 months of growing period (Jha and Barat, 2005;Yeşilayer et al., 2011). The type of diets that used for feeding the fish plays an important role in the aquaculture industry (FAO, 2017). Nutritional requirements of ornamental fish are important in maintaining colouration, breeding, health, and growth (Sales and Janssens, 2003).
Different food types including herbs have been tested for better growth and immunity of ornamental fish industry (Madhusudan et al., 2011;Radhika and Mohaideen, 2017). Production of costeffective nutritionally balanced diets of fish is the main factor affecting intensive aquaculture due to its influence on growth, health and production cost (Yeşilayer et al., 2011). It has been reported that different seaweed types have been utilized in feed preparation in the ornamental fish industry in other countries due to their growth promoting and antimicrobial activity (Vallinayagam et al., 2009;Raghunathan et al., 2014;Abdel-Warith et al., 2016;Rizzo et al., 2017). Seaweeds or marine algae are one of the abundant natural resources in marine ecosystems classified as red algae (Rhodophyta), brown algae (Phaeophyta) and green algae (Chlorophyta), depending on their nutrient composition and chemical composition. They are rich in proteins, vitamins, minerals, iodine, bromine and bioactive substances (Christobel, 2008;Domettila et al., 2013;Parmar et al., 2016). Utilization of seaweed as supplementary material for preparation of diets in ornamental fish industry is not popular in Sri Lanka (Coppejans et al., 2009;Tacon and Metian, 2013). Therefore, the objective of the current study was to determine the effect of two types of seaweeds; Ulva lactuca and Sargassum cinereum supplemented diets on growth performance of fry stage Koi carp (Cyprinus carpio Linnaeus, 1758).

METHODOLOGY Ethical Approval
Ethical clearance for the study was obtained from the Institute of Biology (IOB), Sri Lanka (Permit No: ERC IOBSL 181 11 18).

Formulation of Test Diets
Seaweeds were collected from the Kamburugamuwa beach (5⁰ 56' 22.64996" N, 80⁰ 29' 51.19994" E) located on the southern province, Matara, Sri Lanka. The collection was thoroughly washed with tap water to remove extraneous materials. Then the samples were sun-dried, finemilled and sieved (mesh size = 500 µm) in order to remove the fibrous portion. Control diet was prepared using the ingredients shown in Table 1. Ingredients were weighed using electronic balance (Mettler PE 3600) and mixed using Kenwood mixer. Gelatin was used as a binding agent in different formulations. Based on the previous findings (Zhou et al., 2011;Anh et al., 2018;Perez-Velazquez et al., 2019), 30% of the weight of the control diet was replaced by the Ulva lactuca and Sargassum cinereum powder to prepare test diets T1 and T2 ( Table 2). The protein level of 40% was maintained for formulation both the test diets and the control diet using the Pearson square method (Wagner and Stanton, 2012). The pellets (2 mm diameter) were prepared by pressing the mixture and dried in a drying cabinet (40°C) until moisture dropped around 10%. The dried pellets were manually broken into small pieces to facilitate to consumption and were packed in sealed polythene bags before storing in a refrigerator at 4 °C.

Proximate Analysis of Dietary Ingredients and Diets
Proximate analyses of experimental diets and ingredients were performed for dry matter, ash, crude protein, lipid using standard methods described by AOAC (2000) and Ergün et al. (2010).

Experimental Design and Setup
The research was carried out at the animal house of the Department of Zoology, University of Ruhuna under the controlled laboratory conditions. The experimental setup consisted of 12 circular fiber tanks of 216 L capacity (79.5 cm in depth, 43.5 cm in width) with a centrally located outflow. Each fiber tank was filled with previously aerated water up to 30 cm level to get 150 L capacity of water. The experimental set up consisted of two treatments (T1 and T2) and a control group (C) in four replicates arranged in Completely Randomized Design. A random number table was used for random assigning of tanks.

Fish Tanks and Sampling
Koi carp fry belonging to the same cohort were purchased from Ornamental Fish Breeding and Training Center (NAQDA), Rambadagalla. Fish were acclimatized while feeding a commercial diet (Prima aquafeed -Crude Protein 36%) for 2 weeks. After the acclimatization period, each tank was stocked with 20 fry. Before assigning fry into tanks initial length and weight of each fish were taken. The average weight and length of the fry were 2.20±0.08 g and 3.89±0.04 cm, respectively. Then fish in untreated control tanks (C), Ulva treatment (T1) and Sargassum treatment (T2) were fed with prepared diets approximately 5% of their body weight daily twice a day (two rations at 09.00h and 15.00h) for 14 weeks. Their weight and length measurements were recorded once in two weeks using electronic balance (Mettler PE 3600) and measuring board. Continuous aeration was maintained throughout the experimental period. Tanks were siphoned to remove fecal matter of fish at the end of each day during the experimental period.

Statistical Analysis
The data were analyzed using Minitab 17.0 statistical package and Microsoft Excel 2016 for Windows 8.1. The One-way ANOVA and Tukey multiple range test were used to compare the differences between treatments and control at the significance level of p=0.05.

RESULTS AND DISCUSSION
The average weight and length of the Koi carp fry were 2.20±0.08 g and 3.89±0.04 cm, respectively. The results of the proximate analyses of dietary ingredients and experimental diets are presented in the Table 3 and Table 4, respectively.  Values are means ± SEM (n=3). Different letters in same row indicate significant differences within groups (p<0.05).
At the end of the 14 weeks of experimental period, mean weight, mean length, mean weight gain and daily growth rate showed a significant difference (p<0.05) among treatments (Figure 1).

Estimation of Growth Performance of Koi Carps in Three Treatment Groups
Tukey test revealed that FCR of T2 and T1 were significantly different (p<0.05) from C ( Figure 2a) and FE% of T2 was significantly different (p<0.05) from C (Figure 2b). At the beginning of the experiment, the mean Fulton's condition factor in the Control group was slightly higher than that in Ulva and Sargassum treatments. During the experimental period of 14 weeks, mean values in the control and two treatments have shown random fluctuations. At 2 nd and 3 rd weeks, Sargassum treatment has obtained the highest mean Fulton's Condition Factor values. A similar pattern of fluctuations can be observed in the 8 th , 10 th , 12 th , 14 th weeks reflecting random variations in survival, reproduction, maturity and health of fish (Figure 3).

Figure 3: Fluctuation of mean Fulton's condition factor values of control and two treatment tanks during the 14 weeks of experimental period
The summary of the results of some selected growth parameters and survival rate of Koi carp in control and two treatment groups for the 14 weeks period are given in Table 5. The mean values of Temperature, pH, Conductivity, Dissolved Oxygen (DO), Nitrate and Phosphate were normally distributed. Therefore, the one-way ANOVA revealed that there was no significant difference (p>0.05) among C, T1, and T2 treatments during the 14 weeks period (Table 6).
In the proximate analysis of dietary ingredients, dry matter content ranged from 78% -88%. When the experimental diets were considered, the dry matter content varied from 90% -92.72%. Samuelson and Oterhals (2016) suggested that the moisture content is important to pellet hardness, a b  physical pellet quality, specific mechanical energy, bulk density and oil adsorption capacity of fish feed. However, Halver and Hardy (2002), pointed out that the high moisture contents in fish diets lead to microbial growth on feed and tanks. Bhuiyan et al. (2018) showed that moisture content of fish feed ingredients varied from 5% -13.28%. Therefore, ingredient moisture should be lowered in future studies in order to achieve optimal moisture contents in fish feed formulations.
According to the experimental results, it was revealed that at the end of the experiment at 14 th week, the Koi carps in the seaweed supplemented treatments (T1 and T2) have achieved a significantly high (p<0.05) mean weight, mean length compared to the Control group (Table 5). Between two experimental diets, Sargassum treatment has achieved higher growth parameters during the experimental period and performed significantly better at the 14 th week of the experiment in terms of mean weight, mean length. It was also found that at the 14 th week the DGR and BWG of fish fed with Sargassum diet was significantly different (p<0.05) from the Control group. Throughout the experimental period of 14 weeks Sargassum treatment (T2) has maintained the highest DGR and BWG (Figure 1). Koi carps in the Sargassum treatment also have shown a higher SGR and RGR than the Control group during the period. This could be due to the high palatability of Sargassum supplemented diet.
Ulva treatment has also shown the second highest overall growth performance, but Sargassum treatment has indicated the highest overall growth performance during the experimental period (14 weeks). Thirty percent of Ulva powder was incorporated with the standard diet in diet formulation. It seems that high levels of Ulva incorporation in the diet leads to reduce the growth performance of Koi carps as they have shown retardation of growth performance compared to Sargassum treatment.
A similar study was carried out by Abdel-Warith et al. (2016) by incorporating green macroalgae Ulva lactuca as a feed supplement in diets on growth performance, feed utilization and body composition of the African catfish (Clarias gariepinus). They suggested that fish fed with a diet containing 20% and 30% Ulva lactuca meal had poorer growth performance and feed utilization than those fed with diet containing 10% Ulva lactuca meal. It may be due to the decrease in palatability of the diets with a high amount of Ulva lactuca. Presence of food particles in the Ulva treatment tanks observed in the present study also supports the fact that fish preferred Sargassum diet over Ulva diet. According to these observations, it could be suggested that, if a lower percentage of Ulva lactuca had been incorporated into the basal diet, it would have performed even better than the Sargassum treatment. According to the suggestions of Azaza et al. (2008), there is a possibility for diminished growth at higher levels of Ulva meal (30%) due to the high fiber content which could affect on digestibility of protein and dry matter. It is possible that fiber structures reduce the accessibility of intestinal enzymes to food nutrients such as starch, protein, and lipids and thus act as physical barriers between nutrients and digestive enzymes in the intestine (Potty, 1996), thereby making the enzymes less active. Zamannejad et al. (2016) carried out a study on the effect of supplementation of Sargassum ilicifolium on growth, survival and body composition of rainbow trout (Oncorhynchus mykiss) and revealed that 5% and 7% replacement of Sargassum ilicifolium in the basal diet made significant effects on feed conversion ratio, specific growth rate, daily growth rate, weight gain, condition factor and survival rate values. Zamannejad et al. (2016) clearly showed that dietary incorporation of Sargassum ilicifolium meal up to 7.5 % of basal diet was sufficient to meet the essential requirements for the growth of Rainbow trout. Makkar et al. (2016) suggested that Sargassum spp. could be introduced at up to 30% in the farm diets without depressing intake, growth performance and diet digestibility. Based on the results obtained from the present study it can be suggested that for Koi carp juveniles, 30% replacement of Sargassum cinereum of the basal diet was successful.
Koi carps in the Sargassum treatment have recorded the highest feed efficiency and the lowest feed conversion ratio (Table 5 and Figure 2). Given the high cost involved in feeding, the feed conversion ratio which is the amount of feed it takes to grow a kilogram of fish, is an important factor for the producer (Craig et al., 2017). Therefore, if a feed has a low feed conversion ratio, it takes less feed to produce one kilogram of fish than a feed with a higher feed conversion ratio. Thus, low feed conversion ratio, indicates that the quality of feed is high (Tuominen, 2003). The acceptability, palatability, and digestibility of feeds vary depending on the ingredients and the quality of the feed. Fish farmers pay special attention to feeding activities to determine feed acceptance, calculate feed conversion and efficiency rates, monitor feed costs, and track feed demand throughout the year (Craig et al., 2017). Therefore, Sargassum supplementation in fish feed could be an effective option in formulating nutritionally balanced diets for Koi carps.

Supplementation of diets of Koi carps with
Sargassum and Ulva showed beneficial effects on their growth indices where Sargassum supplemented diet performed better than that of Ulva supplemented diet. Considering the high palatability and low feed conversion ratio indicating the high feed quality shown it can be concluded that the Sargassum supplemented diet is more beneficial than the Ulva supplemented diet for juvenile Koi carp. The results showed the potential use of seaweeds as a dietary supplement or in developing low-cost diets in aquaculture programmes. Further studies are suggested to incorporate other substances (such as digestive enzymes) when formulating seaweed supplemented diets to increase the effectiveness of the diets.