The Effect of Replacement Fish Meal With Earthworm Meal and Maggot Meal in Feed on Protein Digestion, Growth and Survival Rate of Catfish (Pangasius hypophthalmus)

Abstract

The purpose of this study was to examine the replacement of fish meal with earthworm meal and maggot meal in feed on protein digestibility, growth and survival rate of catfish (Pangasius  hypophthalmus).The experimental fish used were catfish (P. hypophthalmus) with an average weight of 8.42 ± 0.16  g. This study used experimental method with a Completely Randomized Design (CRD), 5 treatments, and 3 replicates. The treatments in this study were replacement for fish meal with earthworm meal and maggot meal and with different ratios, namely treatment A (ratio 0:0), treatment B (ratio 2:2), treatment C (ratio 2:4), treatment D (ratio 2.6) and treatment E (ratio 2:8). The variables observed included digestibility for protein (ADCp), feed utilization efficiency (EFU), protein efficiency ratio (PER), feed conversion ratio (FCR), relative growth rate (RGR) and survival rate (SR). The results showed that the replacement of fish meal with earthworm meal and maggot meal as a source of animal protein in feed had a significant effect (P<0.05) on ADCp, EFU, PER, FCR, RGR but had no significant effect (P>0.05) on the survival rate of catfish. The best ratio of fish meal replacement with earthworm meal and maggot meal in catfish (P. hypophthalmus) feed was a ratio of 2:2 (treatment B) the feed was able to produced EFU of 70.78 ± 0.21%, PER of 1.87 ± 0.01, RGR of 4.72 ± 0.16 and FCR of 1.27 ± 0.02. 

Keywords :Earthworms, Maggots, Replacement, Feed, Ratio

1.  INTRODUCTION
Catfish (Pangasius hypophthalmus) is an important freshwater aquaculture species in Indonesia which ranks third in growth rate after tilapia and carp [1][2]. This species is highly favored because of its ease of cultivation, resistance to disease, and tolerance to environmental fluctuations [3]. According to [4], catfish production from fisheries cultivation in Indonesia reached 1.845.324 tons in 2022. This catfish is widely consumed because of its delicious taste, high nutritional value, and contains large amounts of protein, minerals, and fat in its meat. The success of catfish (P.Hypophthalmia) cultivation intensively determined by the existence of artificial feed. Feed costs are the most important variable in intensive cultivation, which is around 70% of the total production costs for each cultivation cycle [5]. This is because protein is the most expensive component of fish feed compared to other nutritional components. Protein functions as a source of essential amino acids (EAA) for tissue repair and fish growth [6].
Fish feed uses fish meal as a source of animal protein up to 50% so that fish meal is the main and most expensive protein ingredient [7]. Fish meal is a source of animal protein feed because it is a source of protein and essential amino acids, essential fatty acids, cholesterol, vitamins, mineral attractants that are very much needed for fish growth [8]. However, due to the increasing demand for livestock and poultry feed, there is a shortage of fish meal for fish feed [9]. Thus, there is a need for a substitute for fish meal to reduce production costs, which can use non-conventional protein sources. Non-conventional protein sources are feed ingredients that have the potential to be used as a mixture of fish feed because of their high availability in various local areas, contain nutrients needed by fish and are less competitive in use with humans, have common anti-nutrient content and must be processed first before they can be used as feed ingredients, for example earthworm meal and maggot meal [10].
Earthworm meal has a considerably high nutrient content, is easily digested, and contains high protein that is relatively similar to that in fish meal. According to [11] earthworm meal (Lumbricus rubellus) contained 63.0% crude protein, 5.9% crude lipid, 8.9% ash, 0.43% Na, 0.53% Ca, 0.62% K, 0.94%, and 1476 kJ/100 g energy for metabolism. In term of the composition of essential amino acids, earthworm meal contained arginine of 2.83 g kg-1, histidine of 1.47 g kg-1, isoleucine of 2.04 g kg-1, leucine of 4.11 g kg-1, lysine of 6.35 g kg-1, phenylalanine of 6.26 g kg-1, tryptophan of 4.43 g kg-1, and valine of 4.43 g kg-1.
Maggot in this study was the black soldier fly (Hermetia illucens) larva from Order Diptera known as decomposer. Maggots use fruits and vegetables as feed source and habitat; therefore, maggots of H. illucens contain a fairly high protein amount and amino acids similar to those in fish meal. According to [12], maggot (H. illucens) contains a high protein amount of 42.1%, lipid of 24.8%, ash of 10.3%, moisture of 7.9%, crude fiber of 7%, calcium of 5%, phosphorus of 1.5%, and NFE of 1.4%. [13] investigated that amino acids in maggot were quite similar to those in fish meal, included arginine of 2.29%, histidine of 1.50%, isoleucine of 1.87%, lysine of 2.71%, methionine of 0.66%, phenylalanine of 1.63%, threonine of 1.70%, tryptophan of 0.555, valine of 2.56%, and leucine of 3.23%. Furthermore, earthworm meal and maggot meal are easy to obtain since they are available in sufficient quantity in nature and relatively cheap, thus potentially used as fish meal replacement in fish feed.
Several studies on substituting fish meal with earthworm meal and maggot meal in artificial feed were reported by [13] on Clarias gariepinus, [14] [15] on Oreochromis niloticus, [10] on Penaeus merguiensis. However, until now, information on substituting fish meal with earthworm meal and maggot meal in feed for catfish has not been found. The purpose of this study was to examine the substitution of fish meal with earthworm meal and maggot meal in feed on protein digestibility, growth and survivalrate of catfish ( P. hypophthalmus).

2.  MATERIALS AND METODS

Experimental animals used in this study were catfish (P. hypophthalmus) with an average weight of 8.42±0.16 g used were obtained from the Muntilan Fish Seed and Cultivation Center, Central Java, as many as 300 tails. Selection of test animals including uniform size, active swimming, completeness of body organs and physical health was carried out during the preparation of test fish before the adaptation process. The adaptation process to the environment and feed was carried out for 7 days [16].
This study used an experimental method with a completely randomized design (CRD) consisting of 5 treatments and 3 replications. The treatment in this study was the replacement of fish meal with earthworm meal and maggot meal and with different ratios, namely treatment A (ratio 0:0), treatment B (ratio 2:2), treatment C (ratio 2:4), treatment D (ratio 2.6) and treatment E (ratio 2:8) referring to [15]. The composition of the raw materials used in the feed was fish meal, earthworm meal, maggot meal, soybean meal, corn meal, wheat flour, fish oil, palm oil, vitamin mix , Cr2O3 and CMC (Carboxyle Methyl Cellulose). The test feed formulation and proximate analysis are presented in Table 1. The earthworm meal used is commercial earthworm meal from Villa Cacing Arjasari Farm with a protein content of 45.86%, while the maggot meal used is commercial maggot flour with the trademark Probsf which comes from CV. Organic Products bsf with 56.08% protein.
Table 1. Formulation and Results of Proximate Analysis of Test Feed Used in   Study
Ingredients
	Feed composition (%/100 g feed)	
	A 	B 	C	D	E	
Fish meal	40.00	  0.00	   0.00	  0.00	  0.00	
Earthworm meal	  0.00	20.00	     13.33	10.00	  8.00	
Maggot meal	  0.00	20.00	  26.67	30.00	32.00	
Soybean meal	30.00	30.00	  30.00	30.00	30.00	
Corn meal	  8.30	  8.30	    8.30	  8.30	  8.30	
Wheat flour	  8.70	  8.70	    8.70	  8.70	  8.70	
Fish oil	  2.00	  2.00	    2.00	  2.00	  2.00	
Corn oil	  2.00	  2.00	    2.00	  2.00	  2.00	
Vit-Min mix	  6.00	  6.00	    6.00	 6.00	  6.00	
CMC	  2.00	  2.00	    2.00	 2.00	  2.00	
Cr2O3	1.00	    1.00		    1.00		   1.00		   1.00		
Total (%)	   100	    100	  100	   100	   100	
Proximate analysis						
Protein (%)	 40.81	 40.84	  40.87	40.83	 40.87	
NFE (%)	19.83	 20.82	  20.30	20.57	  20.71	
Fat (%)	  8.74	   8.94	    8.71	  8.70	   8.50	
Energy. (kcal)	 296.29	  296.16	296.16	   296.58	296.92	
E/P ratio (kcal)	   8.12	    8.20	      8.25	     8.26	     8.29	
Note : 
A = experimental diet with animal protein source of earthworm meal :maggot meal = 0:0
B =experimental diet with animal protein source of earthworm meal : maggot meal = 2:2
C = experimental diet with animal protein source of earthworm meal : maggot meal = 2:4
D = experimental diet with animal protein source of earthworm meal : maggot meal = 2:6
E = experimental diet with animal protein source of earthworm meal:maggot meal = 2:8

The research container used was a plastic container measuring 60 x 40 x 25 cm.3 with a water height of 20 cm and each research container is equipped with a recirculation system. Siphoning of feces and leftover feed is done 2 hours after feeding. Siphoning is done to clean the media so that it remains clean and suitable for the suervival rate of the test fish.
The study began with weighing the test fish to determine the initial weight. The fish were then spread into the experimental unit with a density of 1 fish/ liter. The test fish were kept for 56 days, feeding using the method ad satiation (fish until full) and the frequency of feeding is 3 times a day at 08.00, 13.00, and 17.00 WIB. Observation of the increase in weight of the test fish  conducted every 7 days during the study. The water quality parameters observed included pH (Jenway 3510), DO (Jenway 970), and temperature (Water quality Checker) conducted twice a day at 08.00 and 16.00 WIB, while Ammonia (HANNA: HI. 8633) was conducted at the beginning, middle and end of the study.
The observed data variables consist of feed utilization efficiency (EFU), feed conversion ratio (FCR), protein efficiency ratio (PER), relative growth rate (RGR), survival rate (SR) refers to [8], protein digestibility refers to [17], each calculated based on the following formula :
EFU (%)    = Final weight – Initial Weight/the weight of feed consumed	 x 100 
FCR)          = Feed intake (g)/body weight gain (g).
PER            = 100 × (Final weight – Initial weight)/The amount of diet consumed x Protein content of diet
RGR  (%)   =100 × (final weight - initial weight)/(times of experiment x initial weight)
ADCP         = 100 x [(%Cr2O3 in feed x %protein in feces)/(%Cr2O3 in feces x %protein in feed)]
SR (%)       = 100 (final count/initial count)

	  The observed data variables ware analyzed using variance analysis (ANOVA) to determine the effect of treatment. If the ANOVA results have a significant effect (p<0.05) or a very significant effect (p<0.01), Duncan's multiple area test is carried out to determine the difference in mean values ​​between treatments to determine the best treatment [18]. Water quality parameter data was analyzed descriptively.

3.  RESULTS
The results of observations of feed utilization efficiency (EFU), feed conversion ratio (FCR), protein efficiency ratio (PER), relative growth rate (RGR), protein digestibility (ADCp), and survival rate (SR) of banana shrimp during the study are presented in Table 2.
Table 2. Average values of feed utilization efficiency (EFU), feed conversion ratio (FCR), protein efficiency ratio (PER), relative growth rate (RGR), protein digestibility (ADCp), survival rate (SR) of catfish (P. hypophthalmus) during the research
Variebels	Treatmen	
	A	B	C	D	E	
Initial body weight (g)	2.52±0.12a	2.55±0.10a	2.52±0.12a	2.54±0.14a	2.54±0.14a	
Final body weight (g)	12.68±0.14e	24.46±0.12a	20.19±0.13b	18.37±0.12c	16.28±0.12d	
EFU (%)	50.55±0.32e	70.78±0.21a	63.21±0.27b	60.36±0.32c	58.79±0.21d	
FCR	1.82±0.03e	1.27±0.02a	1.42±0.03b	1.60±0.03c	1.32±0.02d	
PER	1.25±0.02e	1.87±0.01a	1.43±0,02b	1.36±0.01c	1.57±0.02d	
RGR (%/day)	2.13±0.15e	4.72±0.16a	3.02±0.13b	2.69±0.12c	3.64±0.12d	
ADCp (%)	53.27±0.14e	81.26±021a	72.10±0.23b	66.49±0.27c	62.24±0.32d	
SR (%)	100a	100a	100a	100a	100a	
Note : Different superscript values  in the columns indicate significant differences significant (P<0.05)

Table 2 shows that replacement of fish meal with earthworm meal and maggot meal in feed significantly increased ADCp, EFU, FCR, PER and RGR, but was not significant for SR of catfish (P. hypophthalmus). The ADCp, EFU, FCR, PER and RGR values of catfish fed with fish meal replacement with earthworm meal and maggot meal were higher than without fish meal substitution with earthworm meal and maggot meal. The replacement of fish meal with earthworm meal and maggot meal with a ratio of 2:2 is the best ratio for catfish (P. hypophthalmus) because it produces the highest ADCp, EFU, FCR, PER and RGR values compared to other treatments.
The results of measuring water quality parameters in the rearing media are presented in Table 3. Table 3 shows that the water quality parameters during rearing were still within the reference range, made suitable for cultivation of catfish (P. hypophthalmus).
Table 3. Water quality parameter values  during catfish maintenance (P. hypophthalmia)
Parametres Water Quality	Treatment	Reference	
	A	B	C	D	E		
Temperature (oC)	27-30	27-30	27-30	27-30	27-30	27-33*	
DO (mg/l)	5.2-5.8	5.4-5.9	5.6-5.8	5.6-5.8	5.6-5.8	< 5.0*	
pH	7.0-8.0	7.0-8.0	7.0-8.0	7.0-8.0	7.0-8.0	7.0-8.5*	
Amonia (mg/l)	0.04-0.05	0.04-0.05	0.04-0.05	0.04-0.05	0.04-0.05	   < 1,0*	
Note : *[19]

4.  DISCUSSION 
Table 2 shows that catfish (P. hypophthalmus) fed test feeds B, C, D, and E had higher ADCp, EPP, PER, RGR values and lower FCR compared to catfish fed test feed A. This shows that the animal protein source used in test feeds B, C, D, and E consisting of a combination of earthworm flour and maggot flour is rich in  will EAA which is needed by catfish (P. hypophthalmus). Earthworms and maggots are good sources of animal protein because they are rich in EAA [20]. In addition, the cultivation of earthworms and maggots has a short production cycle, making it a good unconventional protein source to replace fish meal as a source of animal protein in fish feed [10][13].
The ADCp value determines the PER and FCR values, there is a linear relationship between ADCp and PER and an inverse relationship between ADCp and FCR [10]. Therefore, the higher the ADCp, the higher the PER and the lower the FCR. The results of this study indicate that catfish (P. hypophthalmus) which was fed B (a ratio of earthworm flour and maggot flour of 2:2 obtained the highest ADCp value, followed by the highest PER and the lowest FCR compared to other treatments. Similar research results were reported by [10] in Penaeus merguiensis.
	Feed with high nutrient content and digestibility can increase feed utilization efficiency. Nutrient absorption by the body is influenced by various things such as protein efficiency ratio and feed utilization efficiency. Good feed utilization efficiency indicates that the nutrients from the feed can be utilized well by shrimp or fish for growth. [20] stated that the greater the feed efficiency value, the better the fish utilize the feed consumed so that the greater the weight of the meat produced. Feed utilization efficiency is closely related to the digestibility of fish to the feed consumed [14]. Replacing fish meal with earthworm meal and maggot meal can have an effect on feed utilization efficiency. From the research results, the best data was obtained in treatment B (ratio 2:2) of 70.78 ± 0.21%. Feed utilization efficiency is directly proportional to the increase in body weight, so the higher the feed efficiency value means the more efficient the fish utilize the feed consumed. The feed utilization efficiency value for each treatment in this study was classified as good because all treatments had values  exceeding 50% in accordance with the opinion of [21] stating that feed efficiency good feed utilization is more than 50% or even approaching 100%.
The feed conversion ratio is a measure that states the amount of feed needed to produce meat. The greater the value of the feed conversion ratio, the more feed is needed to produce meat [8]. Replacement fish meal with earthworm meal and maggot meal can affect the feed conversion ratio. The results of the study obtained the best data, namely treatment B (ratio 2: 2) of 1.27 ± 0.02. The low FCR value indicates that the addition of a number of feeds can result in an increase in the weight of catfish with a greater proportion. The feed conversion ratio is an indicator to determine feed efficiency, the smaller the feed conversion produced indicates the more efficient use of the feed [22].
Catfish (P. hypophthalmus) fed with fish meal replacement with earthworm meal and maggot meal with a ratio of 2:2 had the best RGR value (4.72±0.16%/day) compared to treatments A (2.13±0.15%/day), C (3.64±0.12%/day), D (3.02±0.13%/day) and E (2.69±0.12%/day). This is suspected that feed with fish meal substitution with earthworm meal and maggot meal with a ratio of 2:2 has a complete EAA profile according to the EAA profile of catfish for tissue repair and growth [23]. Similar research results were reported by [13][14][10].	 
The results of the analysis of variance showed that replacement fish meal with earthworm meal and maggot meal had no significant effect on the survival of catfish (P. hypophthalmus). The results of this study indicate that test feed containing a combination of earthworm meal and maggot meal in different ratios as a source of animal protein does not have a negative impact on the health of catfish (P. hypophthalmus). Similar research results were reported by [10] in Penaeus merguiensis.

5. CONCLUSION
Replacement of fish meal with earthworm meal and maggot meal in catfish (P. hypophthalmus) feed has an effect on the efficiency of protein digestibility (ADCp), utilization feed (EFU), feed conversion ratio (FCR), protein efficiency ratio (PER), and relative growth rate (RGR), but did not have a significant effect on survival rate (SR). The best ratio of fish meal replacement with earthworm meal and maggot meal in catfish (P. hypophthalmus) feed was a ratio of 2:2 (treatment B) the feed was able to produced EFU of 70.78 ± 0.21%, PER of 1.87 ± 0.01, RGR of 4.72 ± 0.16 and FCR of 1.27 ± 0.02. 

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