SAND CRAB ( Emerita sp. ) MEAL AS A NOVEL FEED INGREDIENT FOR KOI CARP ( Cyprinus carpio )

Sand crab ( Emerita sp.) is a marine biodiversity, but it has not been used as a fish feed ingredient. This study aimed to evaluate the nutritional value of sand crabs and to understand its effect when used as feed ingredient on the performance of Koi carp. The study was conducted in two steps, which is evaluation of the nutritional value of sand crab and its effect on fish performance. The proximate composition, amino acids

In Kebumen and Cilacap, the sand crab is one of the most valuable commodities for the economy of the coastal communities.Generally, sand crabs are consumed as snacks by visitors and locals communities in coastal tourist areas (Bhagawati et al., 2016).The nutritional value of sand crab as a food is reported to be relatively high, both in protein, omega-6, and omega-3 (Mursyidin, 2007), depending on the processing mechanism.The best processing of sand crab is steaming, which provide 25.00 g, 11.04 g, 71.86 g, and 2.11 g per 100 g sand crab for amino acids, minerals, fatty acids and cholesterol, respectively (Santoso et al., 2015).However, some researchers reported that sand crab is unfit for human consumption due to its heavy metal content, one of which is mercury (Pérez, 1999) and Pb, which ex-It is therefore necessary to find an alternative to the use of sand crabs that does not endanger human health, so the resource can continue to contribute to the economy of coastal communities.Sand crab can be used in many types of products.Alternative uses of sand crab include as a source of chitosan (Wittriansyah et al., 2018), feed for quail male (Pratiwi, 2016), feed for laying quail (Astriana, 2013), and feed for ducks (Batoro, 2008).However, all these studies still have a risk of heavy metal residues in feed.The best alternative is to use sand crab as a product that is not consumed by humans, for example is to use sand crab as feed for ornamental fish.However, it is necessary to evaluate the nutritional value of sand crab before considering it in feed formulations.Therefore, the aim of this study was to evaluate nutritional value of sand crabs and to know it effect on the performance of Koi carp (Cyprinus carpio).

Sand crab sample preparation
The sand crab (Emerita sp.) was obtained from Jetis beach, Cilacap district, Central Java, Indonesia.Live sand crabs caught by fishermen were rinsed with clean water to remove dirt in the form of sand adhering to the growing parts.The clean sand crab was then fully cooked in a pan and then cooled to room temperature.The cooked sand crab was stored in a freezer at -4°C for 24 hours.A total of 1000 g of frozen sand crab was dried in a freeze dyer (CRIST-ALPHA-LD PLUS-101541, Martin Crist, Osterode am Harz, Germany) at temperature of -40 °C for 20 min and then freeze-dried in a vacuum in two steps: primary drying (-50 °C at 0.040 bar) for 24 h and final drying (-55 °C at 0.021 bar) for a further 24 h.Dried sand crab samples were ground to a powder, and then stored in airtight plastic at a temperature < 25 °C until used.

Analysis of the sand crab nutrient contents
Sand crab samples were analyzed for their proximate composition, i.e., moisture (M), crude protein (CP), ash, crude fat (CF), crude fibre (CF), calcium (Ca), and phosphorus (P) according to AOAC (2005).Analyses of amino acid and fatty acid profiles in the samples were performed using high performance liq-uid chromatography and gas chromatography (Shimadzu Corp., Kyoto, Japan), respectively, according to AOAC (2005).The total carotenoid (TC) concentration in sand crabs was determined using a UV-Vis spectrophotometer.A total of 0.5 g sand crab meal sample were homogenized in 5 ml acetone 98% (Merk, Darmstadt, Germany) containing anhydrous sodium sulfate using a homogenizer (Ultra-turrax IKA-T18 basic, Wilmington, NC, USA).The sample was stored in the refrigerator for 24 h at 4°C and then extracted with acetone two or three times until no color was obtained.The solutions were added 5 ml h-hexane and homogenized using a magnetic stirrer.The total carotenoid concentration was determined using spectrophotometer (Genesys-105 UV-Vis, USA) in n-hexane solutions using extinction coefficients (E1%, 1cm) 2500 for carotenoids at 350, 380, 420, 450, 475, 480, 500, and 663 nm.The total carotenoid (TC) concentration was calculated from the maximum absorbance according to the formulae:

Step two: feeding trial of koi carp (Cyprinus carpio)
A randomized experimental design was used to test sand crabs in ornamental fish food.There were four treatments and three replications.The treatments tested were the level of sand crab meal in the feed formulation: feed without sand crab (T1), feed with sand crab 5% (T2), feed with sand crab 10% (T3), and feed with sand crab 15% (T4).This dose is based on Hertrampf & Pieded-Pascual (2000) suggesting that crab meal can be used at 5-10% in aquaculture diets.Four diets of similar protein content (crude protein 37.00%) were formulated for this study.The dietary composition of experimental diets for juvenile koi with different levels of sand crab is shown in Table 1.
All feed ingredients in the form of flour are ground in a blender to a uniform size and passed through a standard US sieve mash #80.The raw materials are then weighed according to the formula, the raw materials that are in small quantities (wheat flour, fish oil, premix and sand crab) are mixed until they are homogeneous, then they are mixed with the raw materials that are in large quantities in the feed formula (soybean meal, wheat pollard, fish meal) until they are homogeneous.The powder feed mixture is mixed with 12-15% water before being formed into pellets using a meat mincer with a 1 mm die diameter.Subsequently, the pellets are dried by hot air spraying at 37°C for 24 hours.The dry pellets were Indonesian Aquaculture Journal, 18 (2), 2023, 155-167 broken down into crumbles using a coffee grinder.
Only crumble that passed the US standard mash #60 and retained the #80 mash standard was used in this study.
The juvenile koi carp used in the experiment were obtained from the same broodstock and the same batch.A total of 288 fish juvenile fish with a mean initial body weight of 0.55±0.15gand an initial length of 3.09±0.23cmwere used in the experiment.The fish were placed in twelve aquaria (40×30×30 cm 3 ) filled with 18 L of water.Twenty juveniles were stocked in each aquarium.Fish were acclimated for one week and fed a control artificial diet (without sand crab), twice a day up to 3% of the biomass.The fish were then fed the experimental diet according to the treatment.The fish were fed twice a day, morning and evening, for 42 days.The remainder feed was siphoned after 1 hour of feeding to maintain good water quality in aquarium.
Fish were sampled once a week to determine body weight gain (WG), length gain (LG), specific growth rate (SGR), and feed efficiency (FE).In addition, water quality was measured at the beginning and the end of the study to check for similar environmental factors among treatments.

Data analysis
Nutritional data of sand crab were presented as mean ± standard deviation, and then compared with the nutritional composition of crab meal, shrimp head meal, krill meal, and soybean meal from previous studies.Koi carp performance data were analyzed by oneway ANOVA to know the effect of different sand crab meal levels in diet.A Tukey's post-hoc test analysis was used where the analysis indicated significant difference among treatments.Statistical analysis was performed using Minitab software version 16.

Nutritional value of sand crab
The results of the laboratory analysis are shown in Table 2.The protein content of sand crab in this study was 37.89%.This is similar to the results of previous studies, the protein content of crab meal, but lower than the protein in shrimp head meal, krill meal and soybean meal.The mean sand crab has a very high potential as a source of protein in aquafeed.In general, the protein source used in freshwater fish feeds is fishmeal, which accounts for 5-25% (Tacon & Mentian, 2008) and can currently be replaced by soybean meal (Hodar et al.,2020).Due to its protein content, sand crab has the potential as an alternative to reduce the use of one of the two ingredients, as protein sources in feed cannot be eliminated, but can be replaced by other ingredients.As we know that aquatic animals require protein for tissue protein building, enzyme synthesis, hormones, growth, tissue repair, maintenance, and optimal health status (Jia et al., 2022;NRC, 2011;Paul et al., 2019;Teles & Couto, 2019).A detailed comparison of the protein content of the sand crab (37.89%) with the previous studies, as well as comparisons with other ingredients, shows that the sand crab can be considered to be equivalent to other ingredients.According to Mursyidin et al. (2002), the protein content of sand crab ranges from 30-40%, which is also consistent with the report of Kardaya et al. (2011) that the protein content of sand crab is 32.5%.Variations in the protein content of sand crab can occur in fresh, boiled, steamed and grilled conditions of 38.52, 38.40, 37.20 and 35.13% respectively (Santoso et al., 2015).The protein content of sand crab is also similar to that of crab processing waste, which is 31.0-37.6%,but lower than that of shrimp meal, which is 43.3%, krill meal 52-67.3% and soybean meal 42.5-48.6%(Chen et al., 2009;Indonesian Aquaculture Journal, 18 (2), 2023, 155-167 Hertrampf & Piedad-Pascual, 2000;Kuo et al., 1976;Vijayalingam & Rajesh, 2020).
The ash content of sand crab varied when compared to previous studies.The ash content of sand crab in the current study was 23.86%, which was similar to the report of Kardaya et al. (2011), which was 26.4%, but lower than the report of Haq et al. (2018), which was 40.03%.The ash content of sand crab may decrease with different processing methods.According to Santoso et al. (2015), the ash content of fresh, boiled, steamed and grilled sand crab was 35.13, 35.63, 34.63, 32.50, and 31.59%,respectively.Despite the variation in ash content, the Ca and P mineral contents of 8.77 and 0.82% in this study are similar to those reported by Kardaya et al. (2011) and Haq et al. (2018), and close to the amounts of Ca, Mg, Fe, and Cu of 10-11% (Santoso et al., 2015).Ca and P are known to be essential for the development and maintenance of the skeletal system and for many other physiological functions in aquatic organisms (Davis & Gatlin, 1996;Davis & Lawerence, 1997;Prabhu et al., 2016;Lall & Kaushik, 2021).Given that sand crab habitats are known to be sandy intertidal areas (Wardiatno et al., 2014) and sand is an inorganic compound, it is possible that less clean sand was contaminated during sample preparation, thus increasing the amount of ash but not Ca and P, which occurred in previous studies.Another possibility is due to species differences or the phenomenon of molting in sand crabs.
The fat content of sand crabs varies widely, but this component is very important as a source of energy and for reproduction (Hu et al., 2009;Tocher, 2003).The fat content of sand crab in this study was 24.56%, similar to the report of Mursyidin et al. (2002) with a range of 17.22-21.56%in Emerita analoga and Emerita talpoida species.This value is higher than that reported by Kardaya et al., (2011), which was 10.2%, and in Emerita emeritus, which was reported as 8.76, 2.95, 4.32 and 5.65% for fresh, boiled, steamed and roasted conditions, respectively (Santoso et al., 2015).
The difference in research findings on fat content of sand crabs compared to previous studies may be due to several factors.The first factor is the difference in the species studied, as Balzona et al. (2017) stated that the fat content and profile of shrimp and crabs is influenced by the species type.The second factor is the different processing methods.The processing of fishery products affects the quality and quantity of nutrients (Abraha et al., 2018;Akonor et al., 2016;Hairol et al., 2022;Wang et al., 2011;Wu & Mao, 2008), especially the drying step (Chukwu & Shaba, 2009).The third factor is when the crab is caught.This is related to the size, age and stage of the crab itself.Just 1 month before hectic breeding activity (February and August), the percentage of fat in the sand crab body was observed to increase and then decrease sharply during and after spawning (Nagabhushanam & Kulkarni, 1977).
Other parameters presented in Table 2 are fibre content and total carotenoids.The fibre content of 6.0% in sand crab was similar to that reported by Kardarya et al. (2011), which was 4.9% and lower than other crustacean feed ingredients.While carotenoids are an essential nutrient for any biological function in crustaceans, which can be seen when sand crab is heated or boiled, it turns a reddish colour like other crustaceans (Helliwell, 2010;Manikandan et al., 2020;Mohamad-Zuki et al., 2022;Pan et al., 2020).The results of the present studies also showed that the concentration of carotenoids in sand crabs was as high as 1696 µg/g, much higher than the last reported with a range of 40-50 µg/g (Gilchirst & Lee, 1972).This different migh caused by many factors such as an age, developmental stages, metabolism, sex, molting, environmental conditions, carotenoid concentration in feed, and diseases that affect carotenoid deposition in the same aquatic animal species (Contancio, 2011;Meyer & Latscha, 1997;Sukarman et al., 2023).If compared with other fish feed ingredients from crustacean by-product, the sand crab has better carotenoid concentration than crab meal of Figure 1.Colors of fresh Sand Crabs (left) and Sand crabs that have been processed using the freeze-dried method (right).
Sand crab (Emerita sp.) meal as a novel feed.. (Sukarman) 800 µg/g (Kuo et al., 1976), shrimp head meal of 7-72 µg/g, and krill meal 130-330 µg/g (Hertrampf & Piedad-Pascual, 2000).Based on the data of carotenoid content, sand crab is more valuable as an feed ingredient for ornamental fish because fish cannot produce these compounds and must be supplied from their feed (Choubert, 2010;Simpson et al., 1981).Therefore, ornamental fish need to be fed with high carotenoids content such as sand crabs.
A more in-depth study of the nutritional content of the sand crab is related to the amino acids and the fatty acids profile.The results of the present studies showed that the total essential amino acid (TEAA) content of sand crab (12.84%) was higher than that of crab meal (4.72%), but lower than that of shrimp meal, krill meal and soybean meal, as shown in Table 3.This indicates that the protein present in sand crab is not only composed of essential amino acids, but also nonessential amino acids, and may also be nitrogenbonded by chitin compounds.As reported by Wittriansyah et al. (2018), sand crab contains 18.87% chitin, and chitin is known as a nitrogenous polysaccharide found in many crustaceans (Boriae et al., 2020;Kumari et al., 2015;Mohan et al., 2021).Chitin has also been found in crab meal at 17-24% (Broke et al., 2023), shrimp meal at 12.6-17.0%(Hertrampf & Piedad-Pascual, 2000), and krill meal at 2-10% chitin (Landymore et al., 2019).The nitrogen in chitin cannot be utilised by fish, but the presence of chitin in the diet does not affect the growth of common carp Cyprinus carpio (Ringø et al., 2012).Therefore, the authors focus only on the availability of essential amino acids, specifically lysine and methionine.
Lysine and methionine are the main limiting amino acids for the fish growth (Giri et al., 2006;NRC, 2011;Mukhtar et al., 2017;Li et al., 2021).Previous research on lysine requirements in fish diets has varied widely depending on the species.NRC (2011) has summarised the estimated lysine requirements for fish, which range from 1.2-2.5% of the diet, or 4.0-5.8% of the dietary protein.Meanwhile, the methionine requirement for fish ranges from 0.7-1.2% in feed or 1.73-3.3% of feed protein.More specifically, the lysine requirement of Cyprinus carpio is 5.9% of dietary protein (Zhou et al., 2008), while its methionine requirement is 0.86% of diet or 2.13% of dietary protein (Schwarz et al., 1998).The present study showed that sand crabs contained only 0.99 and 0.35% lysine and methionine respectively and are unlikely to be the main source of lysine and methionine in feeds for Cyprinus carpio.However, up to 30% may potentially be used in feeds such as crustaceans or other crustacean by-products (Hertrampf & Piedad-Pascual, 2000;Yi et al., 2015;Hairol et al., 2022).Note: 1 result of Present study 2 adopted from Hertrampf & Piedad-Pascual (2000): original data as percentage of protein content, converted to percentage of samples 3 adopted from NRC (2011) Indonesian Aquaculture Journal, 18 (2), 2023, 155-167 The last nutritional parameter analyzed in this study was the content of essential fatty acids, as shown in Figure 2. Based on the results of the study, the omega-3 components in this form of EPA and DHA of dry sand crab were 1.86% and 0.78%, corresponding to 7.79% and 3.26% of the total fat, respectively.The results are in line with previous research reports, which found that the EPA and DHA content of sand crab was 6.41-8.43%and 1.34-6.57% of total fat, respectively (Mursyidin, 2007).In general, omega-3 fatty acids in the form of EPA and DHA are readily available to marine life, as they are consumed by shrimps and sea fish, as well as by micro-organisms that are rich in these compounds (Adarme-Vega et al., 2014).Similarly, it is thought that sand crab obtains its omega-3s from the same source.At the same time, the other omega-3 components in the form of APA are low in concentration.Alpha-linolenic acid (APA) is usually easily obtained from nuts such as flaxseed (Basch et al., 2007).While omega-6 fatty acids from research results amounted to 0.52% of the total ingredients or equivalent to 2.17% of total fat, much smaller than in species Emerita talpoida and Emerita analoga contained 11.80% and 12.94% omega-6 (Mursyidin, 2007).However, the omega-9 content of sand crabs is relatively high at 1.99% in sand crabs or 8.34% of total fat.The three types of fatty acids in omega-3, omega-6, and omega-9, as well as potential human health supplements, significantly reduce the risk of a heart attack.In addition, when used as feed, these three compounds play an essential role in fish reproduction and seed growth (Hertrampf & Piedad-Pascual, 2000;NRC, 2011).

Feeding trial different level sand crab in feed to juvenile of koi carp (Cyprinus carpio)
The study on ornamental Koi carp revealed that incorporating sand crabs up to 15% in their diet resulted in a significant increase in both body weight and length, compared to other treatments (p<0.05).However, different levels of sand crab meal in diets did not significantly affect the specific growth of these fish during 42 days of feeding experiment.The highest to lowest increase in body weight of Koi carp ranged from 0.44 ± 0.09 g to 0.26 ± 0.08 g (T4 to T1 treatments, respectively).The highest to lowest increase in body length was observed in T4 to T1 treatments, respectively, with consecutive values of 0.93 ± 0.11 cm to 0.66 ± 0.19 cm.The specific growth rate (SGR) of fish did not differ significantly between treatments (P>0.05) and ranged from 0.95-1.45%per day.Figure 3 presents the overall performance data of Koi carp fed with sand crab.
Based on Figure 3, it can be reported that 15% sand crab in the diet (treatment T4) produced the best performance in Koi carp.Until the results of this study were published, there had been no previous studies using sand crab as a fish feed ingredient.However, the use of 40% sand crab in quail feed was reported to produce the best growth (Pratiwi, 2016).Meanwhile, 30% of sand crab meals produced eggs with the highest linoleic acid levels (Astriana, 2013).Experiments have also been carried out on mice to test the efficacy of sand crab.The most effective dose for promoting growth in mice was found to be a 25% concentration in the diet (Kardaya et al., 2011).Similar to this report, another marine crab Chaceon affinis is reported to be able to replace other protein sources in catfish feed (Keremah, 2013), and added up to 15% in shrimp feed can improve growth, feed conversion and protein efficiency of L. vannnamei juveniles (Goytortúa-Bores et al., 2006).In others Sand crab (Emerita sp.) meal as a novel feed.. (Sukarman) report, inclusion of marine crab meal at 10-20% of the diet has been reported to improve growth performance, promote yellow skin colour and delay lipid oxidation in muscle of red porgy Pagrus pagrus (García-Romero et al., 2014a, García-Romero et al., 2014b).
The positive effect of sand crab on the growth of ornamental Koi carp cannot be separated from its high nutrient content, especially fat and calcium.In this study, the protein content was made equal between the treatments, while the other nutrients followed the composition of the raw materials.Adding sand crab to the koi feed at levels 5, 10 and 15% increases the calcium content from 1.77 (control) to 2.16, 2.67 and 3.49% respectively.Also increase the fat content in the feed from 4.00 (control) to 5.03, 5.98 and 6.84%, respectively (Table 1).This means that the fat and calcium content in the treatment was 15% higher than in the other treatments, indicating that the difference in growth between treatments was not due protein content, but rather to other nutrients such as fat and calcium.This follows the theory that calcium and phosphorus affect the growth of juvenile fish (Liu et al., 2021) and essential fats meet the energy needs of fish (NRC, 2011), thus indirectly increasing fish growth.It also improves feed efficiency (Figure 4).
The feed efficiency for each treatment was 40.62, 46.78, 51.24 and 63.5% for T1, T2, T3 and T4, respectively.This is in line with previous reports for the koi carp Cyprinus carpio, which was ranged from 48.59-73.24%(Winarti et al., 2017), and when beta carotene was added in diet, feed efficiency range from 51.35-61.42%(Purba et al., 2020).In general, the diets containing 10 and 15% sand crab could be well utilized by koi carp, as the efficiency value was more than 50% Figure 3.The growth performance of Koi carp was evaluated over a 42-day period by feeding them with varying doses of sand crab in diets.T1: feed without sand crab, T2: feed with sand crab 5%, T3: feed with sand crab 10%, T4: feed with sand crab 15%.Indonesian Aquaculture Journal, 18 (2), 2023, 155-167 (Purba et al., 2020), but the best value was obtained at a dose of 15%, which was significantly different from the others (p<0.05).Based on these results, the use of sand crab up to 15% in the diet resulted in the best performance of juvenile Koi carp, and there is still an opportunity to increase its use.

CONCLUSION
The protein content of sand crab is quite high at 37.88%, but the essential amino acid (EAA) content is lower compared to shrimp meal, shrimp head meal, krill meal and soybean meal.While the fat content is higher than the four ingredients, the omega-3 and omega-9 content is 2.83% and 1.99% respectively.Sand crab has a high carotenoid content of up to 1696 µg/ g, making it a promising source of aquafeed pigments.The inclusion of sand crabs at a level of 15% in the diet of koi fish resulted in the highest values of LG, WG and FE, which were 0.93 ± 0.05 cm, 0.48 ± 0.06 g and 63.50%.These findings lead that sand crab has good nutritional content, and which makes it suitable for Koi carp feed at concentrations of up to 15%.

Figure 4 .
Figure 4. Koi Fish Feed Efficiency using different sand crab levels.

Table 2 .
Proximate composition of the sand crabs compared to others ingredient (in dry matter basis)

Table 3 .
Composition of Essential Amino Acids of Sand Crabs compared to Other Protein Sources (% of sample)