Feeds and Feeding Management Biological Evaluation of Existing Feed Formulations for the Grow-out Culture of the Mud Crab, Scylla serrata Mae R. Catacutan Aquaculture Department Southeast Asian Fisheries Development Center Tigbauan 5021, Iloilo mrc@seafdec.org.ph Abstract This study was conducted to identify a formulated pelleted feed for the grow-out culture of the mud crab. A commercial crab feed (CCF) was evaluated together with fish by-catch (FBC) and three test diets (formulated and pelleted) of similar dietary energy (1723 MJ/Kg diet) based on published works. The test diets contained 48% (D-1), 43% (D-2), and 38% (D-3) crude protein (CP), respectively. All the five dietary treatments were fed to crablets (5 to 6 g body weight) that were individually stocked in 60-L tanks. Each dietary treatment was fed to six crabs. Crustaceans grow by molting which is not simultaneous, hence, each crab was monitored daily. After the third molt (M3) survival rates in D-1, D-2 and D-3 ranged from 33%-66% compared with only 16% in FBC, and 0% in CCF where no crab survived to molt for the second time (M2). Nutrients in FBC may not be sufficient to sustain growth and survival, while water stable CCF may lack a feed attractant. Diets D-1, D-2, and D-3 were evaluated in another feeding experiment to identify the basal diet for the crab. All crabs molted until M3 and no significant differences were noted on growth performance between treatments. However, D-1 (48% CP) was identified as the basal diet due to better numerical values observed in terms of feed conversion ratios and specific growth rates. Keywords: Scylla serrata, feed formulation, grow-out culture Introduction There is increasing interest in the culture of the mud crab, Scylla serrata, as an alternative to shrimp farming. The source of crab seeds is from the wild and is heavily exploited due to an increasing market demand for this species. The market potential of the mud crab is well recognized by the Philippine government and it has stepped up funding researches on mud crab production and building hatcheries in strategic locations in the country. With this development, and in anticipation of an increasing production from the grow-out culture, studies on formulated feed for this species is very relevant. Philippines: In the forefront of the mud crab industry development 69 The traditional feed for crab is trash fish or fish-by-catch (FBC) which has many disadvantages such as unreliable supply, freshness, a need for storage equipment, and it can be a potential carrier of disease agents. Formulated diets have been tried on juvenile mud crab (Chin et al., 1992; Kuntiyo, 1992). A study on nutritional requirement has been conducted by Sheen and Wu (1999). Digestibility studies have also been done for common aquaculture feed ingredients for the mud crab (Troung et al., 2008; Catacutan, 2003). Formulated pelleted feed also enhances growth and survival of crabs in pens where natural food is present (Primavera et al., 2010). An efficient formulated feed is one of the components in the grow-out culture of crabs that is considered vital for a sustainable production. This research aims to identify a diet that could promote growth and survival of the mud crab and can be utilized to substitute partially the traditional feed. diet, similar size crabs were selected and stocked individually in 30 and 18 units of 60 li tanks for Experiment 1 and 2, respectively. Tanks were provided with aeration and flowing seawater. Test diets and preparation The five dietary treatments for growth in Experiment 1 were commercial crab feed (CCF), FBC, and three test diets formulated based on published works to contain similar dietary energy levels of 1723 MJ/Kg. The test diets were formulated to contain crude protein (CP) at 48% (D-1), 43% (D-2) and 38% (D-3) with protein to energy ratio (P/E in mg protein/KJ) of 27.9, 25.0 and 22.1, respectively. Proximate analysis (expressed in dry matter) showed that CP levels were 50.1, 46.9 and 43.3%, respectively (Table 1).The CCF has a CP level of 47.8%. For Experiment 2, only D-1, D-2 and D-3 were evaluated and diets were slightly modified but were formulated to have the same nutrient levels as in Experiment 1. Materials and Methods The efficiency of crab feeds (test feeds and commercial crab feed) was compared with that of traditional feed which is the low value fish or fish-bycatch (FBC) in feeding experiments. Diets were prepared according to Catacutan (2002). After drying, the strands were allowed to cool and then broken down manually to suitable pellet size before storing in labelled plastic containers. The containers were stored in a cold room at about 20°C. Experimental animals Crabs (average body weight of 3 g) from the Crab Hatchery of the Aquaculture Department of the Southeast Asian Fisheries Development Center were stocked in 1.5 ton fiberglass tanks and fed FBC in the morning and pelleted maintenance diet in the afternoon. After 2 weeks, when crabs were weaned to the pelleted formulated Test diets were analyzed for proximate nutrient levels (CP, crude fat or CrFat, crude fiber or CrFb, crude ash or CrA, and Nitrogen Free Extract or NFE) while samples of flesh from wild and pellet-fed crabs, commercial feed and test diets were sent to a laboratory for the analysis of amino acid profile. Proximate analysis of samples followed the methods in AOAC (2000). 70 Philippines: In the forefront of the mud crab industry development Table 1. Feed composition of mud crab formulated test diets with different dietary Crude Protein (CP) levels at similar dietary lipid and energy levels* Ingredients D-1 (48% CP) D-2 (43% CP) D-3 (38%CP) Danish fish meal 370 310 250 Whole fish (Tuna) 120 100 80 Squid liver powder 20 20 20 Acetes 85 85 85 Corn meal 30 90 150 Seaweed 10 10 10 Defatted Soybean Meal 90 90 90 Breadflour 150 150 150 Danish fish oil 20 20 20 Soybean Oil 20 20 20 Lecithin 0.3 0.3 0.3 Vitamin C 0.5 0.5 0.5 Dicalcium phosphate 10 10 10 Carboxymethylcellulose 30 30 30 Mineral mix 20 20 20 Vitamin mix (Tas mix) 10 10 10 Rice bran 14.2 34.2 54.2 1000 1000 1000 Digestible Energy (MJ/kg diet) 1723 1723 1724 Protein/Energy Ratio (mg protein/kJ 27.9 25 22.1 %Proximate nutrient composition in Dry matter Crude protein 50.92 46.89 43.26 Crude fat 4.85 5.01 5.36 Crude fiber 1.7 2.14 2.53 Crude ash 15.18 15.83 16.33 Nitrogen-free extract 27.35 30.13 32.52 *Commercial Crab Feed (CCF) proximate nutrient composition in dry matter: 47.83 % crude protein, 8.32 % crude fat, 1.33 % crude fiber, 11.97 % crude ash and 30.55 % NFE (Nitrogen-Free Extract). The moisture content is 6.5%. Stocking, feeding and termination of experiment Crabs stocked individually in 30 fiberglass tanks with 60 li capacity were fed with a pelleted maintenance diet. Feeding of dietary treatments commenced when a crab first molted in the assigned tank. The following parameters were taken on a newly molted crab before a test diet was given: body weight (BW), carapace Philippines: In the forefront of the mud crab industry development 71 width (CW), and carapace length (CL). Newly molted crabs (MO) were fed daily with their respective dietary treatments. Each test diet was fed to six crabs that were individually stocked in tanks. Feeding was twice a day after cleaning the tanks in the morning (0900 H) and in the afternoon (1600 H). Feeding rate was based on BW of newly molted crabs. The BW of an individual crab was monitored at day 4, 8, 14, and 20. At days 1-8, feeding rate was at 20% BW, and this was adjusted starting day 14 to 10% and day 20 to 10 or 15%. For FBC, the amount of feed given has the factor of 2.5 (BW X feeding rate X 2.5), due to moisture content. Test diets were then given continuously 10 days after the third molt. The following parameters were determined for each crab at the end of the experiment: 1) number of days at intermolt, 2) total number of days until third molt, 3) BW, 4) CW,5) CL, 6) percent weight gain (PWG), and 7) survival rates. The exuviae were collected at each molt for each crab as it was included in the calculation of final PWG. The same procedures were followed for the two feeding experiments. For the statistical analysis, only Oneway ANOVA was used to determine the differences in BW, CW, CL, SGR and FCR on results of Experiment 2. Results and Discussion Results of Experiment 1 (Table 2) showed that crabs fed formulated test diets (D1-D3) molted three times (M3) in tanks with survival rates of 33 to 67%, while crabs fed CCF molted only once (M1) and none survived to molt for the second time. It was noted that CCF pellets were not attractive or sought by the crabs. Attractant in crustacean feed is important because it takes a while for these species to find the pelleted feed compared to fish. Although CCF is a formulated pelleted diet and very stable in the water, it may lack attractant for crabs. The crabs fed FBC or natural food were observed to reach for the food by extending its claws as soon as FBC was placed in tanks. The consumption of FBC enabled some crabs in this treatment to survive after M2 but none of the crabs molted for the third time. The FBC-fed crabs showed a relatively faster growth rate or a shorter molt interval or number of days between molts of 87.5 days (43.5 days + 44 days) between M0 to M2 compared with other treatments. On the other hand, crabs fed D-1, D-2 and D-3, had average molt intervals of 90, 102, and 114 days, respectively. In addition, most crabs in these treatments reached M3 (Table 2). FBC, on the other hand, was not able to sustain crabs until M3 nor enhanced crab survival. It was clear that survival rates improved with formulated pellet diets, especially with D-1, with the highest survival (66.6%) and numbers of survivors that reached M3 (100%). This experiment showed that formulated diets (same dietary energy of 1723 MJ/kg diet) as the sole food for crabs in tanks were able to sustain growth and survival until M3 compared with the CCF and FBC. The level of dietary energy in this present study is the level recommended by Cuzon and Guillaume (1997) for crustaceans. Crabs require a high dietary protein of about 48% as shown in this study. The FBC has insufficient nutrients to sustain growth and survival of crabs, but this can be 72 Philippines: In the forefront of the mud crab industry development enhanced when fed together with adequate nutrients incorporated in the formulated pelleted diet. Levels of dietary carbohydrate generally required by crustacean is from 20 to 30% of the total diet, while higher levels of dietary carbohydrates can cause slow growth and low survival in crustaceans (Xiaodan et al., 2016). The test diets for crab in the current study had 15% breadflour and the other sources of carbohydrate in the formulation were corn meal and soybean meal. The proximate analysis showed that levels of digestible carbohydrate or NFE in test diets and CCF were from 27.35 to 32.52% (Table 1). In Experiment 2, only D-1, D-2 and D-3 were evaluated. Crabs fed these diets survived until M3 in tanks. This confirmed results obtained in Experiment 1 that formulated diets were able to sustain growth and survival in tanks until M3. There were no significant differences in the various parameters when crabs were fed diets containing dietary protein levels of 48%, 43% or 38% at similar dietary energy content (Table 3). However, the average numerical values showed that SGR and FCR were better in crabs fed with D-1 which is similar to the basal protein level used by Sheen and Wu (1999) in his study on dietary lipid for the mud crab. The diet composition of D-1 in Table 4 was identified as the basal diet for the crab. Results of amino acid profiles (Table 5) showed that amino acids such as threonine, lysine and phenylalanine in wild crab flesh are generally higher than those of pellet-fed crabs. In the wild, various types of food are eaten by the crab which are good sources of amino acids. The amino acid contents of both crab samples were higher than in CCF and 3 test diets. The amino acid profile of test diets (D-1, D-2 and D-3) were decreasing with dietary protein level of diet and that amino acid profile of CCF seemed to be in between that of D-1 and D-2. Although this is the case with CCF, the performance of D-2 and D-3 were much better in terms of crab reaching M3 and survival rate. The performance of test diets vis-a-vis with CCF could be due to diet attractability and digestibility of protein sources used. The test diets contained Acetes sp. and corn meal with high Table 2. Survival and molt interval of crablets (initial body weight of 3-6 g) individually stocked in tanks and fed 3 formulated diets, a commercial crab feed (CCF), and natural food or fish by-catch (FBC). Crabs were fed diets after molting (MO). First, second and third molts are indicated as M1, M2 and M3, respectively. Six crabs (replicates) were used for each diet treatment in Experiment 1 Diets % CP M0 - M1 Survival (%) Molt interval (days) M1 - M2 Survival (%) Molt interval (days) M2 - M3 Survival (%) Molt interval (days) Survivors that reached M3 (%) 1 48 100 36 100 54.16 67 58.25 100 2 43 100 39 50 63 33 73 50 3 38 100 65 67 49.2 50 67 16 CCF 33 52.8 0 - - - 0 FBC 100 44.5 50 44 16 - 0 Philippines: In the forefront of the mud crab industry development 73 Table 3. Increase in body weight (BW), carapace width (CW), and carapace length (CL), number of days from start of feeding to termination, specific growth rate (SGR) and feed conversion ratios (FCR) of crabs fed formulated test diets with % crude protein (CP) levels at 48%, 43%, and 38% (Experiment 2) Tank BW CW CL Feed weight No. of days No. (grams) (mm) (mm) (grams) MO-termination SGR FCR Diet 1 43 79.60 27.00 20.00 298.16 102.00 4.29 3.75 44 45 44.51 16.00 15.00 310.79 118.00 3.22 6.98 46 54.14 19.00 18.00 211.18 80.00 5.05 3.90 47 48 34.99 18.00 199.85 118.00 3.01 5.71 Mean SE Diet 2 53.31 9.60 20.00 17.67 2.42 1.45 255.00 28.78 104.50 9.00 3.89 5.09 0.48 0.77 49 50 42.90 31.00 17.00 206.69 51 52 33.90 18.00 16.00 181.85 110.00 113.00 3.42 4.82 3.12 5.36 53 54 Average 33.95 38.35 37.28 23.00 19.00 22.75 14.00 14.00 15.25 279.05 199.57 216.79 132.00 108.00 115.75 2.67 8.22 3.38 5.20 3.15 5.90 SE 2.15 2.95 0.75 21.40 5.51 0.17 0.78 Diet 3 55 19.22 13.00 13.00 377.65 106.00 2.79 56 52.10 18.00 19.00 357.45 112.00 3.53 6.86 57 58 59 60 Average SE 35.62 47.15 48.34 40.49 5.99 17.00 24.00 21.00 18.60 1.86 13.00 19.00 16.00 16.00 1.34 277.19 313.59 244.49 314.07 24.62 119.00 145.00 134.00 123.20 7.18 3.00 7.78 2.66 6.65 3.77 5.06 3.15 6.59 0.21 0.57 Note: One-way ANOVA showed no significant differences in the increase in BW, CW, and CL, SGR and FCR apparent protein digestibility for crab at 94.5% and 96.4%, respectively, and apparent crude fat digestibility of 87.2% and 94.5%, respectively (Catacutan et al., 2003). Furthermore, crude ash which is the mineral component of a feed considered important for molting, was more than 15% in test diets while it was only 11.97% in CCF (Table 1). Summary and Conclusion The basal diet for mud crab was identified in this study and this can be used to supplement the traditional or Table 4. Composition of the basal diet for the mud crab Ingredients Sardines (head and tail) Acetes Defatted soybean meal Squid liver powder Breadflour Danish fish oil Soybean oil Lecithin Vitamin C Dicalcium phosphate Mineral mix Vitamin mix Rice bran Total g (1000g-1) diet 260 160 160 80 100 30 10 0.3 0.3 30 10 5 154.4 1000 74 Philippines: In the forefront of the mud crab industry development Table 5. Amino acid profiles of the mud crab flesh from the wild, formulated pelleted feed fed crab the three test diets and commercial crab feed expressed in % protein of sample Amino Acids Crab Flesh Wild Pellet fed crab crab Diet - 1 Test Diets Diet - 2 Diet - 3 Commercial crab feed Aspartic acid 5.960 5.410 4.085 3.455 3.077 3.39 Threonine 2.739 2.491 1.862 1.563 1.399 1.54 Serine 2.611 2.243 1.991 1.661 1.481 2.379 Glutamic acid 8.233 7.711 6.388 5.634 5.163 6.507 Glycine 4.384 3.641 2.445 2.089 1.923 2.217 Alanine 3.768 3.335 2.674 2.288 2.095 2.125 Cystine 0.996 0.842 0.573 0.512 0.449 0.862 Valine 2.644 2.527 2.022 1.795 1.627 1.903 Methionine 1.496 1.55 1.09 0.878 0.752 0.549 Iso-leucine 2.372 2.195 1.657 1.472 1.329 1.563 Leucine 4.519 4.078 3.302 2.89 2.621 3.169 Tyrosine 2.583 2.377 1.443 1.245 1.112 1.142 Phenylalanine 2.628 2.398 1.879 1.600 1.458 1.897 Histidine 2.224 2.012 1.405 1.16 1.072 1.196 Lysine 4.724 4.188 3.163 2.572 2.347 1.894 Arginine 5.991 5.107 2.594 2.252 2.008 2.442 Tryptophan 0.615 0.608 0.406 0.365 0.317 0.351 Proline 3.412 4.217 2.0740 1.809 1.622 2.739 Taurine 0.79 0.924 0.758 0.631 0.585 0.274 TOTAL 62.689 57.854 41.811 35.871 32.437 38.139 natural food of crabs. 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