Effect of hydrolyzed red worm (<i>Eisenia fo&eacute;tida</i>) on production parameters in red tilapia (<i>Oreochromis sp.</i>)

Fabian Gerardo Muñoz García; Nelson Vivas Quila; Luis Fernando Londoño Franco; Crispulo Perea Roman; José Luis Hoyos Concha

doi:10.12688/f1000research.154622.1

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Research Article

Effect of hydrolyzed red worm (Eisenia foétida) on production parameters in red tilapia (Oreochromis sp.)

[version 1; peer review: 1 approved, 2 approved with reservations]

Fabian Gerardo Muñoz García ¹, Nelson Vivas Quila², Luis Fernando Londoño Franco³, Crispulo Perea Roman⁴, José Luis Hoyos Concha⁵

Fabian Gerardo Muñoz García ¹, Nelson Vivas Quila², [...] Luis Fernando Londoño Franco³, Crispulo Perea Roman⁴, José Luis Hoyos Concha⁵

PUBLISHED 15 Jan 2025

Author details Author details

¹ Zoot, Universidad del Cauca, Popayán, Cauca, Colombia
² Zoot, Universidad del Cauca, Popayán, Cauca, Colombia
³ Colombian Polytechnic Jaime Isaza Cadavid, Medellín, Antioquia, Colombia
⁴ Universidad del Cauca, Popayán, Cauca, Colombia
⁵ Universidad del Cauca, Popayán, Cauca, Colombia

Fabian Gerardo Muñoz García
Roles: Data Curation, Formal Analysis, Investigation, Project Administration, Resources, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Nelson Vivas Quila
Roles: Formal Analysis, Investigation, Methodology

Luis Fernando Londoño Franco
Roles: Investigation, Supervision, Validation

Crispulo Perea Roman
Roles: Conceptualization, Formal Analysis, Investigation, Methodology

José Luis Hoyos Concha
Roles: Formal Analysis, Investigation, Methodology

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Agriculture, Food and Nutrition gateway.

Abstract

Background

Conventional fish feed based on fish meal, meat, and soy cake presents procurement difficulties and high costs, affecting the profitability and sustainability of the aquaculture industry.

Objective

To evaluate the effect of hydrolyzed red worm (HRW- Eisenia foétida) in red tilapia (Oreochromis sp.) diet on production parameters.

Methods

The study was conducted at the aquaculture farm of the Politécnico Colombiano Jaime Isaza Cadavid (PCJIC) at 780 m.a.s.l.,with an average temperature of 28 °C. Ninety red tilapia fingerlings, averaging 7,5±0,5 g, were distributed in nine aquariums containing 75 liters of water. Fish underwentweight and size measurements at the beginning and end of the trial. They were fed experimental diets to apparent satiation three times daily. Water quality parameters and productive rates of growth and nutrient utilization were measured. The experimental design included three treatments with three replicates each: T1 (control diet, 0% hydrolysate inclusion), T2 (10% hydrolysate inclusion), and T3 (20% hydrolysate inclusion). ANOVA (p<0,05) was applied to growth and nutrient utilization variables, with mean comparisons using α<0,05 in SPSS version 25.

Results

Significant differences (p<0,04) were found between the control diet T1 (0% inclusion) and T2 (10% inclusion) in favor of weight gain (31,87 g). There were no statistical differences in size increase (p<0,217). As HRWinclusion increased, feed consumption decreased, likely due to higher hydrolyzed protein availability. Feed conversion rates showed significant differences (p<0,001) between T2 and T3 compared to T1, indicating better assimilation of the hydrolyzed protein. T2 and T3 also showed better protein and energy efficiency (p<0,001), demonstrating the hydrolyzed protein’s nutritional quality and assimilation. Diet cost decreased with higher hydrolyzed inclusion (p<0,034).

Conclusion

Inclusion 10% and 20% hydrolyzed red worms significantly improved production parameters and reduced costs, making it a viable alternative for feeding red tilapia for small and medium-scale producers.

Keywords

Energy, hydrolyzed, fish, production, protein

Corresponding author: Fabian Gerardo Muñoz García

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2025 Muñoz García FG 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 work is properly cited.

How to cite: Muñoz García FG, Vivas Quila N, Londoño Franco LF et al. Effect of hydrolyzed red worm (Eisenia foétida) on production parameters in red tilapia (Oreochromis sp.) [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2025, 14:90 (https://doi.org/10.12688/f1000research.154622.1) First published: 15 Jan 2025, 14:90 (https://doi.org/10.12688/f1000research.154622.1) Latest published: 15 Jan 2025, 14:90 (https://doi.org/10.12688/f1000research.154622.1)

Introduction

Currently, the scarcity of raw materials and high costs for fish feeding necessitate the use of new or innovative resources to address this issue,¹ which should also be environmentally friendly and sustainable. In this regard, vermiculture emerges as an alternative in bioconversion processes, such as Hydrolyzed Red Worms (HRW), for inclusion in fish diets.² Additionally, it is known that the red worm (Eisenia foétida) is an important source of nutrients: proteins (62%), fiber (7%), fats (8%), ash (9%), and energy 3.9 kcal.³ Likewise, the hydrolyzed contains free amino acids and low molecular weight peptides, promoting absorption and making them highly digestible due to their nutritional quality.⁴ Consequently, this study aims to obtain an alternative food source by utilizing the nutrients from the red worm to determine a nutritional and productive effect on the development of the early stages of red tilapia (Oreochromis sp.).

Methods

Location: The study was conducted at the experimental and aquaculture production farm of Politécnico Colombiano Jaime Isaza Cadavid - PCJIC, located in the municipality of San Jerónimo-Antioquia-Colombia, at coordinates 6° 26′ 49.88″ North Latitude and -75° 43′ 55.42″ West Longitude, at an altitude of 780 meters above sea level, with an average temperature of 28°C and relative humidity of 50%, classified as Tropical Dry Forest (TDF).

Experimental biological material

Animals: Red tilapia fingerlings (Oreochromis sp.) with an average body mass of 7.5±0.5 g from the PCJIC farm. Additionally, 8 kg of HRW (Eisenia foétida) is provided by the Institución Educativa Noroccidente Popayán farm.

Hydrolyzed preparation: Two kilograms of worms without bedding residues were mixed with 2.5% formic acid (85% m/v) to lower the pH and facilitate the hydrolytic action of endogenous enzymes. The mixture was supplemented with 0.25% sodium benzoate as an antimicrobial and 0.1% butylhydroxytoluene as an antioxidant. Three replicates were performed.^2,5 The hydrolyzed was placed in sealed plastic containers with a 30% headspace and left at room temperature for 10 days (enzymatic hydrolysis) until the product was ready for subsequent use.^1,6,7

Experimental environment

Ninety red tilapia fingerlings were weighed and randomly distributed into nine glass aquariums measuring 36 × 35 × 80 cm, with a capacity of 75 liters. The aquariums were pre-disinfected with 50 ppm sodium hypochlorite and a 100 ppm iodine solution on the ceiling, walls, nets, and floors. There were placed 10 fingerlings per aquarium. The setup was as follows: three aquariums for diet 1 (control with 0% hydrolyzed worm), three for diet 2 (10% hydrolyzed worm), and three for diet 3 (20% hydrolyzed worm). Each treatment was triplicate, with an average body mass of 7.5 ± 0.5 g per fingerling.

The aquariums were equipped with an aeration system consisting of a diffuser stone connected by a hose to an air pump, model-51, 2,5 HP (Sweetwater brand).

The water volume in the aquariums was supplied by the Guaracú stream, which feeds the entire fish station and meets the conditions suitable for fish survival. The water was pre-aerated and filtered using a Hydrofiltro, Mardal brand, model FCM 100, 2 HP (USA). The light-dark photoperiod cycle was 12:12. The water temperature in the aquariums ranged from 24.5 to 27.5 °C, with an average of 26 ± 0.0 °C. The physicochemical parameters of the water were monitored daily with a multiparameter device, Hanna brand, model HI98194,⁸ ensuring they met the requirements for the species. The water temperature was kept constant according to the farm environment.

Experimental feeding

An acclimatization period of 10 days was conducted, during which the experimental diets were supplied to adapt the digestive system to the type of experimental food. During the study phase, the evaluation diets were provided for apparent satiation three times daily (8:00 a.m., 12:00 p.m., and 4:00 p.m.). The food was offered according to the biomass of the animals, considering an average water temperature of 26 °C and voluntary food ingestion as determined by the following equation 1.⁹

(1)

Constant temperature at 26 ° C Food ingestion (\frac{\frac{g}{fish}}{day}) = 0.15 * weight (g)^0.600

Uneaten food and feces were removed daily in the morning (9:00 a.m.) and in the evening (5:00 p.m.) by siphoning, and 60% of water changes were performed in the aquariums.¹

Experimental diets

Analysis of hydrolyzed red worm (Eisenia foétida) was conducted at the Universidad Nacional de Medellín to determine the nutrient content: dry matter (AOAC 934.01; AOAC, 1990), crude protein by Kjeldahl method (AOAC, 1987), ether extract (EE) by gravimetry (AOCS, 1998; AOAC, 1990), calcium (AOCS, 1998; AOAC 985.35; AOAC, 2005), phosphorus (AOAC 995.11; 2012), and the gross energy (GE) was determined precisely using the CAL2k^® bomb calorimeter system, in the institution’s research laboratory.¹⁰ The analysis results are presented in Table 1.

Table 1. Proximate analysis of hydrolyzed red worm (Eisenia foétida).

Variable	Quantity g/100 g
Dry matter digestibility	82±0.6
Crude protein	59±0.2
Ether extract	7±0.6
Digestible energy	4.817±0.0 Cal
Phosphorus	0.8±0.2
Calcium	0.4±0.06

The dry matter digestibility values obtained for the Hydrolyzed Red Worm (HRW) were greater than 82%. This can be attributed to a higher degree of liquefaction, resulting in simple peptides, dipeptides, and tripeptides from the hydrolysis process, thereby increasing the amount of dry matter available for fish feed (see Table 1).

It is important to highlight that the crude protein content was above 59%, due to the quality of the animal-derived raw material and probably the acidic conditions promoting greater lysis of strong peptide bonds like glycine and alanine. These amino acids have properties that ensure soluble protein stability under ideal temperature conditions.¹¹ Similar results were reported by Muñoz et al.,⁶ in their study on the preparation and characterization of hydrolyzed red worm (Eisenia foétida), where they demonstrated the nutritional quality of the hydrolyzed and its protein potential for use in animal feed.

The composition in grams of the ingredients used in the experimental diets is presented in Table 2. Additionally, the inclusion levels of the hydrolyzed red worm are shown, with three replicates conducted for each treatment.^2,12

Table 2. Composition in grams of diets with red worm hydrolyzed red worm (Eisenia foétida).

Diet ingredients	T1 0%	T2 10%	T3 20%
Fish meal	35±0.00	23±0.00	26.5±0.00
Wheat flour	3±0.00	3±0.00	3.9±0.00
Vegetable oil	3±0.00	3±0.00	3±0.00
Dicalcium phosphate	0.4±0.01	0.4±0.00	0.89±0.00
DL-methionine	0.2±0.01	0.2±0.00	0.69±0.00
Premix1	1±0.00	1±0.00	1±0.00
Tryptophan	0.3±0.00	0.3±0.00	0.28±0.0
Corn bran	9±0.00	9±0.00	7±0.00
Soy cake	25±0.00	26.3±0.0	10.5±0.00
Yellow corn flour	5±0.00	6±0.00	9±0.00
Wheat muffin	13.1±0.00	11.1±0.00	10.24±0.00
Cassava flour	3±0.00	3±0.00	4.5±0.00
Bentonite	1±0.00	1.7±0.00	1.5±0.00
Salt	1±0.00	2±0.00	1±0.00
Hydrolyzed red worm (HRW2)	0±0.00	10±0.00	20±0.00
Total	100±0.0	100±0.0	100±0.0

1 Premix=premix of vitamins, minerals, and additives.

2 HRW= Hydrolyzed red worm.

The formulation of the experimental diets was carried out in the Rheology Laboratory of the Universidad del Cauca. The following ingredients were used: hydrolyzed red worm (Eisenia foétida), fish meal, soy cake, yellow corn flour, wheat flour, corn bran, cassava flour, wheat muffin, vegetable oil, premix (vitamins, minerals, and additives), among others. Bentonite was used as a binder. Additionally, three isoproteic diets, T1, T2, and T3, were prepared for the evaluation, with a protein level of 36% ( Table 3) and isoenergetic with a digestible energy of 3,25 cal/g. These protein-energy levels are recommended for red tilapia (Oreochromis sp.).^13,14

Table 3. Composition of diets with red worm hydrolyzed (Eisenia foétida).

Nutrient	T1 0%	T2 10%	T3 20%
Crude Protein	36.7±0.0	36.6±0.0	36.6±0.0
Ether extract	7.5±0.0	7.3±0.0	8.0±0.0
Gross fiber	2±0.0	2±0.0	1.6±0.0
Ash	14.9±0.0	11.3±0.0	11.1±0.0
Digestible energy (Kcal/100g)1	325.5±0.0	325.5±0.0	325.6±0.0
Calcium	2.1±0.0	1.5±0.0	1.9±0.0
Phosphorus	1.2±0.0	1±0.0	1.2±0.0

1 Kcal/100 g = Kilocalories per 100 grams.

The diets were prepared by incorporating the dry ingredients, followed by the addition of hydrolyzed, vegetable oil, and water. The mixtures were then homogenized in a Kitchen Aid mixer with a capacity of 10 kg, packed in polypropylene bags, and stored for 24 hours at 10°C to equilibrate the moisture.¹⁵ The diets were then processed in a Haake Polylab OS twin-screw extruder. The pellets underwent a drying process in a Centricol Ltda equipment, series 0827, at 50°C for 4 hours until reaching 9% moisture. Variables such as compression, durability, water stability, floatability, and water absorption rate were monitored.¹⁶

The resulting pellets were 2 mm in length and 1.3 mm in diameter.¹⁵ Subsequently, the physical and mechanical characteristics of the pellets were evaluated.^13,16

Feeding protocol

The fish were fed to apparent satiation three times daily (8:00 a.m., 12:00 p.m., and 4:00 p.m.) for 30 consecutive days. Daily records of the feed supplied in each treatment were maintained to determine the feed consumption during the evaluation period.^17,18

Diet cost

The cost of the diet was determined based on the value of each ingredient used in the formulation for each treatment (see Table 4). Additionally, the processing cost of the feed was considered to quantify the value of each experimental diet in Colombian pesos, which was then converted to US dollars.^12,13 Table 4 shows each ingredient in kilograms and the corresponding value for each treatment. Furthermore, the total value in pesos and dollars (currency exchange in 2022) is indicated.

Table 4. Value of ingredients in diets with red worm hydrolyzed (Eisenia foétida).

Raw materials	Cost kg RM¹	T1 0%		T2 10%		T3 20%
Raw materials	Cost kg RM¹	Qty/kg²	Value	Qty/kg	Value	Qty/kg	Value
Fish meal	3.250	0.35	1.137.50	0.23	747.50	0.265	861.25
Wheat flour	850	0.03	25.50	0.03	25.50	0.039	33.15
Vegetable oil	3.600	0.03	108.00	0.03	108.00	0.03	108.00
Dicalcium phosphate	2.800	0.004	11.20	0.004	11.20	0.00894	25.03
DL-methionine	14.000	0.002	28.00	0.002	28.00	0.0069	96.60
Premix	20.000	0.01	200.00	0.01	200.00	0.01	200.00
Tryptophan	156.000	0.003	468.00	0.003	468.00	0.0028	436.80
Corn bran	1.000	0.09	90.00	0.09	90.00	0.07	70.00
Soy cake	2.062	0.25	515.50	0.263	542.31	0.105	216.51
Yellow corn flour	1.350	0.05	67.50	0.06	81.00	0.09	121.50
Wheat muffin	437	0.131	57.25	0.111	48.51	0.10236	44.73
Cassava flour	1.200	0.03	36.00	0.03	36.00	0.045	54.00
Bentonite	2.380	0.01	23.80	0.017	40.46	0.015	35.70
Salt	1.000	0.01	10.00	0.02	20.00	0.01	10.00
HRW	1.000	0.00	0.00	0.1	100.00	0.2	200.00
Maquila			$356.00		$356.00		$356.00
Diet cost “pesos”		$3.134.25		$2.902.47		$2.869.27
USD diet cost per year (2022)		US$ 0.84		US$ 0.78		US$ 0.77

1 RM= Row Material.

2 Qty/kg= quantity per kilogram.

Two samplings were conducted, one at the beginning and one at the end of the trial, where the weight of the animals was obtained to evaluate the performance of each treatment. The evaluated variables are presented in detail in Table 5.

Table 5. Variables evaluated in Oreochromis sp., fed with red worm hydrolyzed (Eisenia foétida).

Variable	Rates
Growth	Weight gain
	Size increase
	Thermal growth coefficient
Nutrient utilization	Feed consumption
	Feed conversion
	Protein efficiency rate
	Energy efficiency rate

Growth rates

The evaluated growth rates were: weight gain (WG) (Eq. 2), size increase (SI) (Eq. 3), and thermal growth coefficient ratio (TGC) (Eq. 4), as estimated according to Refs. 12, 19. Biometries were performed at the beginning and end of the study. The obtained data were independently compiled into tables to standardize the observation and analysis of the growth and nutrient utilization parameters.^2,20

(2)

Weight gain = WG (g) = final weight - initial weight

(3)

Size increase = SI (cm) = final size - initial size

Thermal growth coefficient = TGC

(4)

TGC = \frac{100 * (final weight 1 / 3 - initial weight 1 / 3)}{Sum of day temperature in °C}

Nutrient Utilization Variables: these variables were estimated according to equations 5, 6 and 7.^21–23

Feed Conversion Rate (FCR)

(5)

Feed Conversion Rate = FCR = total feed ingestion (g) / weight gain (g)

Protein Efficiency Rate (PER)

(6)

Protein Efficiency Rate : PER (g) = weight gain / protein consumed

Energy Efficiency Ratio (EER)

(7)

Energy Efficiency Ratio : EER (kcal / g) = weight gain / energy consumed

Statistical analysis

A randomized block design (RBD) was applied, evaluating 3 treatments with three replicates each (each aquarium with 10 fish) considered as an experimental unit to determine growth parameters and nutrient utilization. Each variable was reviewed through the application of an analysis of variance (p<0.05). Additionally, Duncan’s test was used for mean comparison with α=0.05 as a significant statistical difference, using SPSS version 23 to statistically compare the results of each treatment during the study.^2,24

Results and Discussion

It is important to mention that during the research, no mortality was observed and the monitored physicochemical parameters of the water were within the suitable limits for red tilapia (Oreochromis sp.) production.^13,25 The mean values of dissolved oxygen were (5.55 ± 0.10 mg L⁻¹), pH (7.25 ± 0.17), carbonate alkalinity (0.50 ± 0.12 mmol L⁻¹), chemical oxygen demand (5.60 ± 0.42 mg L⁻¹), biological oxygen demand (3.98 ± 0.10 mg L⁻¹), ammonium (0.08 ± 0.22 mg L⁻¹), nitrites (0.10 ± 0.00 mg L⁻¹), and nitrates (0.07 ± 0.18 mg L⁻¹), indicating that the inclusion of the hydrolyzed did not affect fish mortality or the productive variables.^1,12

Table 6 presents the evaluated variables of growth rate and nutrient utilization in red tilapia with the calculated variance statistics and Duncan’s test.

Table 6. Growth and use of nutrients in Oreochromis sp., fed with hydrolyzed Eisenia foétida.

Variable	T1	T2	T3	ANOVA P< 0.05
Growth
WG¹ (g)	81.13±13.55^b	113±9.68^a	90.5±16.49^ab	0.047
SI² (cm)	0.3567±0.2743^a	0.8430±0.1305^a	0.60±0.4187^a	0.217
TGC³ (%)	1.71±0.11^a	2.2±0.06^a	1.94±0.36^a	0.830
Nutrient utilization
FCR⁴ (g)	1.47±0.04^a	1.14±0.04^b	1.15±0.06^b	0.001
PER⁵ (g)	1.89±0.06^b	2.43±0.08^a	2.42±0.11^a	0.001
EER⁶ (kcal/g)	2.14±0.06^b	2.75±0.09^a	2.74±0.13^a	0.001
Diet cost (US $ kg⁻¹)⁷	0.86 ± 0.00^a	0.82 ± 0.00^b	0.81 ± 0.00^b	0.034

1 WG= Weight Gain.

2 SI= Size Increase.

3 TGC= Thermal Growth Coefficient.

4 FCR= Feed Conversion Rate.

5 PER= Protein Efficiency Rate.

6 EER= Energy Efficiency Ratio.

7 (US $ kg⁻¹) = Dollars per kilogram.

Different letters in the same row indicate statistical differences (p<0.05).

Weight Gain: According to the variance analysis shown in Table 6, the weight gain in treatments T2 (10% inclusion) and T3 (20% inclusion) did not show significant differences between them. However, there was a statistical difference (p<0.047) between the control diet (T1: 81.13±13.55) and Treatment T2 (113±9.68) (see Figure 1). In summary, T2 presented a weight gain of 31.87 g compared to the control diet (T1). Similar results to this study were reported by Perea et al.,¹⁹who evaluated the inclusion of 0, 10, 20, and 30% fish waste silage in the feeding of Oreochromis sp., obtaining better results for the 10% and 20% inclusions, with weight gains of 80.75 and 89.94 g respectively.

Figure 1. Weight gain of red tilapia (Oreochromis sp.) fed with hydrolyzed red worm (Eisenia foétida).

Similarly, Yucra,²⁰ in his study on feeding trout (Oncorhynchus mykiss) with biological silage from trout viscera, obtained similar weight gain results of 37.36 g and 35.74 g for treatments T2 (27.9% silage) and T1 (commercial), respectively, demonstrating the nutritional quality of the silage. Furthermore, silage can replace fish meal by up to 27.9%, standing out as a low-cost alternative.

Size increase: The ANOVA analysis in Table 6 shows no statistical differences (p<0.217) between treatments. However, the best result for size increase was obtained with treatment T2, which presented a greater growth of 0.486 cm and 0.243 cm compared to T1 and T3, respectively (see Figure 2).

Figure 2. Size increasse of red tilapia (Oreochromis sp.) fed with hydrolyzed red worm (Eisenia foétida).

Consequently, increasing the levels of hydrolyzed red worm inclusion in the diets of red tilapia fingerling improved weight gain and to a lesser extent, growth (size). This can be attributed to factors favoring weight and growth gain, including protein quality, feeding rate, and appropriate water temperature (26 °C).²⁶ Furthermore, the decrease in growth between T2 and T3 can be attributed to the excess protein in the diet with 20% hydrolyzed inclusion, likely due to an extra energetic effect, leading to lower consumption and progressive growth reduction as the extra ingested energy is used for other purposes rather than weight gain and size increase.²³

Similarly, research by Perea et al.,²⁷ on juvenile tilapia sp. with the inclusion of fish silage (10, 20, and 30%, respectively), reported that fingerlings fed with diets containing higher silage inclusion showed greater weight gain and size increase, possibly due to the quantity and quality of amino acids present in the silage, making it a viable alternative in animal feeding.

Thermal Growth Coefficient: Table 6 shows no statistical differences in the thermal growth coefficient between treatments in the study. However, the best values were obtained with treatments T2 (10% inclusion) and T3 (20% inclusion) compared to T1 (0% inclusion). The thermal growth coefficient is an index that relates the effect of water temperature on the productive performance of fish.² In the present study, the temperature (26 ± 0.00 °C) was maintained within the adequate range for red tilapia (Oreochromis sp.) production and was similar for each evaluated treatment.¹⁹ Therefore, the differences in the thermal growth coefficients are directly attributed to the effect of the diets.

Moreover, the study by Mora et al.,²¹ which evaluated the effect of crude protein levels (28, 32, and 36%) in extruded commercial feeds on the growth of Leiarius marmoratus (catfish) fingerlings, demonstrated no significant differences in the thermal growth coefficient (TGC), with values ranging between 0,667 and 0,712. This contrasts with the values of the present study, which supports the quality of the hydrolyzed used in this study.

Another study by Aguilar et al,²⁸ which examined the effect of feed processing (extruded versus pelleted) on the productive performance including the thermal growth coefficient in Oreochromis niloticus (nile tilapia) fingerlings and juveniles, found similar results to our study and reported no significant differences in this parameter during the feeding phases, with values of 0.206±0.0025 and 0.203±0.0031 in the fingerling stage, and 0.103±0.0028 and 0.098±0.0021 in the juvenile stage. These values are lower than those obtained in diets with hydrolyzed red worm (Eisenia foétida) inclusion, highlighting the hydrolyzed’s significant energy and nutritional content for use in animal feeding.

The following results relate to different productive parameters of nutrient utilization rates: feed conversion rate, protein efficiency rate, and energy efficiency ratio with the inclusion of hydrolyzed red worms in the feeding of red tilapia (Oreochromis sp.).

Evaluation of Nutrient Utilization Rate: Table 6 presents the nutrient utilization evaluation, which determined indices such as feed conversion rate (FCR), protein efficiency rate, energy efficiency ratio, and diet cost ratio.

Feed Conversion Rate: As shown in Table 6, the variance analysis (p<0.001) demonstrated significant differences between treatments. Additionally, Duncan’s mean test determined significant differences (p<0.05) between the diet without hydrolyzed inclusion (T1) and the diets containing hydrolyzed T2 (10%) and T3 (20%), with values of 1.47±0.04, 1.14±0.04, and 1.15±0.06, respectively (see Table 6 and Figure 3). However, treatments T2 and T3 were similar and did not show statistical differences between them.

Figure 3. Feed conversión rate with inclusión of hydrolyzed red worm (Eisenia foétida).

It is worth noting that in feed conversion, lower values are better from a productive standpoint.²⁹ The trend in this variable indicates that an inclusion above 10% hydrolyzed significantly improves feed conversion. The best feed conversion rates achieved with 10% and 20% inclusion levels are likely due to the nutritional quality of the hydrolyzed (protein, energy, and amino-lipid profile), which enhances the metabolism of red tilapia ( Figure 3). This confirms the advantages of including the hydrolyzed in fish diets.^1,2 It also supports the acceptance and palatability of the feed by the fish.

Protein Efficiency Rate (PER)

According to Table 6, there were statistical differences (p<0,001) between the treatment T1 without hydrolyzed inclusion (HRW) compared to T2 and T3. T2 (2,43%) showed better protein efficiency, followed by T3 (2,42%) and T1 (1,89%). Furthermore, T2 and T3 demonstrated better performance by 0,53 and 0,54 grams compared to T1, respectively ( Figure 4), favoring improved protein digestion and metabolism, likely due to the quantity and nutritional quality of the hydrolyzed.^1,6 This demonstrates that appropriate inclusion levels can constitute an important protein source in red tilapia production, a fish that seems to efficiently utilize the nutrients present in the hydrolyzed.

Figure 4. Protein efficiency rate with inclusión of hydrolyzed red worm (Eisenia foétida).

The results obtained are possibly due to the greater availability of hydrolyzed energy and protein, resulting from the acidification process that facilitates enzymatic hydrolysis, increasing low molecular weight proteins, releasing amino acids, and raising unsaturated fatty acid content, thus optimizing nutrient digestibility and utilization.^12,23 The higher quantity and quality of nutrients in the fish’s intestinal lumen improve metabolism and absorption, enhancing feed conversion.¹²

The protein efficiency rate results obtained in this study contrast with those cited in a study on Clarias gariepinus (African catfish) by Llanes et al.,³⁰ where they evaluated the partial replacement of fishmeal with meat silage with inclusion levels of 10% and 20%, obtaining values of 2.17 and 1.50, respectively. Additionally, they differ and are lower than those reported by Martinez-Castillo et al.,³¹ who evaluated the zootechnical performance of Piaractus brachypomus (cachama) fingerlings fed diets with different gross energy levels between 3750 and 4440 kcal/kg, obtaining results of 3.97 and 4.80. Similarly, they differ from those presented by Llanes y Parisi.,³² who evaluated hydrolyzed fishery products in Clarias gariepinus (African catfish), reporting values of 3.1 and 3.2 in protein efficiency. Furthermore, they contrast with the results obtained by Murillo et al.,³³ who reported in their study on growth, efficiency, and composition of Oreochromis aureus fed with red worm (Eisenia foétida) that there was no significant difference in weight gain or length. However, there was a significant difference in feed conversion (p<0.05).

Energy Efficiency Ratio

The T1 and T2 treatments showed significant differences (p<0.001) compared to the control diet T1, which was surpassed by T2 and T3 by 0.61 and 0.60 kcal/g, respectively (see Figure 5). This indicates better utilization and performance in the metabolic energy of lipids and proteins. The differences obtained are determined by the different inclusion levels of the hydrolyzed in the diets. This could be due to the quantity and nutritional quality of the hydrolyzed used, demonstrating once again that appropriate inclusion levels can constitute a viable protein and energy source in red tilapia (Oreochromis sp.) production, reiterating the efficient use of the nutrients present in the hydrolyzed.^12,32,34

Figure 5. Energy efficiency ratio with hydrolyzed red worm (Eisenia foétida).

Figure 5 shows that treatments T2 and T3 achieved better energy efficiency ratio (2.75 and 2.74) compared to T1 (2.14) without hydrolyzed (p<0.001).

Similarly, the greater availability of energy could be related to the acidification of the diet generated by the hydrolyzed, which facilitates nutrient digestibility and, consequently, fish growth.³² Additionally, endogenous proteases may have influenced protein solubility, favoring energy absorption and utilization in the fish. The results obtained in this study for the Energy Efficiency Ratio (EER) are superior to those reported by Martinez-Castillo et al.,³¹ who evaluated the zootechnical performance of Piaractus brachypomus (cachama) fry fed with different levels of gross energy, obtaining results between 0.35 and 0.41%. They are also superior to those reported by Botello-Leon et al.,³⁵ who evaluated the substitution of fishmeal with protein cane meal for fattening Oreochromis sp. (red tilapia), finding that the diets presented significant conversion rates with an appropriate protein rate-energy ratio of 0.94 and 0.96 of digestible energy values.

Diet cost

It is important to note that with a higher inclusion of hydrolyzed, the cost of the diet decreases significantly, showing statistical differences (p<0.05) between treatments with and without hydrolyzed inclusion ( Table 6). T3 (20%) presented the lowest cost, followed by T2 and T1. Regarding this, there is a difference between the price of the control diet (T1) and T3 of $0.05 per kg, and with T2 of $0.04 per kg, representing a cost reduction of 5.8% and 4.6%, respectively.

Similar behavior has been reported in studies with fish hydrolyzed in red tilapia feed,³⁶ rainbow trout,³⁷ and even broilers,³⁸ were increasing the inclusion of silage in the diet significantly decreases feed cost.

The results of this study are similar to those reported by Llanes y Parisi,³² where they replaced fishmeal with chemically made silages using sulfuric and formic acids with fish by-products in extruded diets for Clarias gariepinus. They also align with those cited by Vilchez,³⁹ who evaluated three inclusion levels of poultry by-product meal in finishing diets for Piaractus brachypomus, where higher inclusion levels in the evaluated diets significantly reduced the price per kilogram of feed. Similarly, they match the findings of Perea et al.,¹⁹ who conducted an economic evaluation of the use of fish waste silage in the feed of Oreochromis sp.

Conclusions

Hydrolyzed California red worm (Eisenia foétida) is a high-quality nutritional alternative for use in diets for red tilapia at inclusion levels of 10% and 20%, optimizing protein and energy efficiency, weight gain, and feed conversion rate. In addition, the inclusion of the hydrolyzed could provide an interesting economic profit due to the lower cost of the diet for small and medium-scale fish producers.

Ethics statement

Animal Welfare Protocol in the study “Effect of hydrolyzed red worm (Eisenia foétida) on production parameters in red tilapia (Oreochromis sp.)”

At the beginning of the document, you can see the approval: The Ethics Committee for Scientific Research of the University of Cauca, endorses the project according to minutes No. 6.1-1.25/15 of August 5, 2019.⁴⁰

Animal ethics: Every effort was made to mitigate the suffering of the animals. Procedures were also implemented in strict compliance with animal welfare standards. All necessary measures were taken to recreate the natural habitat conditions of the species, in order to minimise pain, stress and suffering of the animals, ensuring their well-being at all times. The measures and conditions adopted are detailed in the Declaration of Commitment to Animal Welfare https://doi.org/10.5281/zenodo.14477924⁴¹

Data availability

Underlying data

The project contains the following underlying data: Statistics “Effect of hydrolyzed red worm (Eisenia foétida) on production parameters in red tilapia (Oreochromis sp.)”.XLSX [Data set]. Zenodo. https://doi.org/10.5281/zenodo.14081025⁴²

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Reporting guidelines

Arrive 2.0: ARRIVE checklist for ‘Effect of hydrolyzed red worm (Eisenia foétida) on production parameters in red tilapia (Oreochromis sp.) ‘https://doi.org/10.5281/zenodo.13139560⁴³

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

We extend our gratitude to Politécnico Colombiano Jaime Isaza Cadavid for its collaboration in the development of this research at its aquaculture farm facilities and to the Acuícola GIA (SAS) research group for its guidance. We also thank the Universidad del Cauca and its research groups NUTRIFACA and ASUBAGROIN for their support and guidance, the Universidad Antonio Nariño for allowing us to use the biochemistry laboratory, and the Institución Educativa Noroccidente Popayán for providing biological material (California red worm).

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Version 1

VERSION 1 PUBLISHED 15 Jan 2025

Author details Author details

Nelson Vivas Quila
Roles: Formal Analysis, Investigation, Methodology

Luis Fernando Londoño Franco
Roles: Investigation, Supervision, Validation

Crispulo Perea Roman
Roles: Conceptualization, Formal Analysis, Investigation, Methodology

José Luis Hoyos Concha
Roles: Formal Analysis, Investigation, Methodology

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (1)

version 1

Published: 15 Jan 2025, 14:90

https://doi.org/10.12688/f1000research.154622.1

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Muñoz García FG, Vivas Quila N, Londoño Franco LF et al. Effect of hydrolyzed red worm (Eisenia foétida) on production parameters in red tilapia (Oreochromis sp.) [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2025, 14:90 (https://doi.org/10.12688/f1000research.154622.1)

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 1

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PUBLISHED 15 Jan 2025

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Reviewer Report 14 Mar 2025

Zulhisyam Abdul Kari, Universiti Malaysia Kelantan, Kafrelsheikh University, Universiti Sains Malaysia, Kelantan, Malaysia

Approved

https://doi.org/10.5256/f1000research.169671.r368929

Here are some minor comments for the article:
General Comments

Clarity in Introduction:
- The introduction is well-structured, but it would benefit from a clearer problem statement. The rationale for using hydrolyzed

Here are some minor comments for the article:
General Comments

Clarity in Introduction:
- The introduction is well-structured, but it would benefit from a clearer problem statement. The rationale for using hydrolyzed red worm (HRW) should be explicitly connected to existing challenges in aquaculture feed.
- Consider incorporating more references on the use of insect or worm-based protein sources in aquaculture to strengthen the background.
- Please also read the latest article published which can help the authors in the introduction part as below: (reference 1 to 9)

My intention was not to self-promote but rather to provide the authors with relevant and recent references that could enhance their study, particularly in the context of aquaculture nutrition. The suggested citations serve as a reference point for the authors to:

Improve the Introduction Section – The cited works illustrate how introductions in similar aquaculture nutrition studies are structured, integrating the latest research trends and background information in a comprehensive manner. This guidance aims to help the authors refine their context-setting and literature synthesis.
Ensure the Use of Up-to-Date References – The field of aquaculture nutrition is rapidly evolving, and my recent work contains key insights that are directly relevant to the study’s focus on red tilapia and alternative protein sources. Citing recent studies, including my own, ensures that the manuscript aligns with the latest scientific developments.
Enhance Scientific Rigor – By referring to well-documented findings in related studies, the authors can strengthen their discussion, reinforce their hypotheses, and make well-supported conclusions. My recommendations were purely scientific, aimed at improving the quality of the manuscript rather than for personal or professional gain.

Methodology Enhancements:
- The justification for selecting 0%, 10%, and 20% HRW inclusion levels should be elaborated. Were these levels based on prior studies, preliminary trials, or nutrient availability considerations?
- In the water quality monitoring section, specifying how often parameters were measured (daily, weekly) would enhance reproducibility.
- The statistical analysis section should clarify whether assumptions (e.g., normality and homogeneity of variance) were tested before applying ANOVA.
Results Interpretation:
- The phrase "no statistical difference (p<0.217)" should be revised to "p > 0.05, indicating no significant difference" for clarity.
- While weight gain showed significant improvement at 10% inclusion, size increase did not. The discussion should explicitly state whether size growth is a limiting factor or if hydrolyzed proteins primarily contribute to mass gain.
- Since no significant difference was found between 10% and 20% HRW inclusion, a cost-benefit analysis might help determine whether 10% is the optimal level.
Discussion and Conclusion Adjustments:
- The conclusion should specify that while weight gain and feed efficiency improved, size growth did not show a significant difference across treatments.
- Highlighting potential long-term effects (e.g., immune response, fillet quality, or digestive enzyme activity) could add depth to future research recommendations.
- The limitations of the study should be acknowledged, such as the relatively short experimental duration (30 days) and the need for trials at different life stages.

Minor Corrections:

Spelling & Formatting
- Eisenia foétida → should be consistently italicized as Eisenia foetida throughout the text.
- "size increasse" (Figure 2 caption) → "size increase"
- "feed conversión" (Figure 3 caption) → "feed conversion"
Figures & Tables:
- Ensure all figures have clear legends and units where applicable (e.g., Figure 5 on Energy Efficiency Ratio).
- The proximate composition of HRW could be compared more explicitly with fish meal in a supplementary table.

By implementing these minor suggestions, the article can improve clarity, precision, and scientific rigor while maintaining its strong contribution to aquaculture research.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Abdul Kari Z, Kabir M, Mat K, Rusli N, et al.: The possibility of replacing fish meal with fermented soy pulp on the growth performance, blood biochemistry, liver, and intestinal morphology of African catfish (Clarias gariepinus). Aquaculture Reports. 2021; 21. Publisher Full Text
2. Dawood MAO, El Basuini MF, Yilmaz S, Abdel-Latif HMR, et al.: Exploring the Roles of Dietary Herbal Essential Oils in Aquaculture: A Review.Animals (Basel). 2022; 12 (7). PubMed Abstract | Publisher Full Text
3. Mathew RT, Alkhamis YA, Alngada RS, Whed RA, et al.: Dose response effects of dietary clove and peppermint oils on the growth performance, physio-metabolic response, feed utilization, immunity, and organ histology in African catfish (clarias gariepinus).Vet Res Commun. 2025; 49 (2): 101 PubMed Abstract | Publisher Full Text
4. Rashid N, Abdul Kari Z, Kallem P, Sukri S, et al.: The potential of hydrolyzed chicken feather meal as a partial replacement for fish meal and its effects on the growth and health status of African catfish (Clarias gariepinus) fingerlings. Aquaculture International. 2025; 33 (3). Publisher Full Text
5. Ahmed R, Jastaniah S, Alaidaroos B, Shafi M, et al.: Effects of dietary Spirulina platensis supplementation on growth performance, whole body composition, antioxidant activity, histological alterations, and resistance to Vibrio parahaemolyticus in Pacific white shrimp, Litopenaeus vannamei. Aquaculture Reports. 2025; 40. Publisher Full Text
6. Bazina WK, Tawfik WA, Abd Elghany NA, Saadony S, et al.: Effects of nano-selenium and/or vitamin E supplementation on growth performance, antioxidant status, histopathology and resistance to Aspergillus flavus in Nile tilapia (Oreochromis niloticus).BMC Vet Res. 2025; 21 (1): 50 PubMed Abstract | Publisher Full Text
7. Alafari H, Albaqami N, Abd El‑Aziz Y, Reyad Y, et al.: Correction to: The effects of nano‑selenium and/or vitamin C on the growth performance, blood health, organ histology, molecular alterations, and disease resistance of Nile tilapia (Oreochromis niloticus) against Saprolegnia ferax. Aquaculture International. 2025; 33 (1). Publisher Full Text
8. Acharya D, Kari Z, Wei L, Ghosh K: Nutrient Profiling of Some Terrestrial Plant Leaves for Their Prospective Use as Nonconventional Ingredients in Carp Diets. Aquaculture Research. 2025; 2025 (1). Publisher Full Text
9. Radhakrishnan D, AkbarAli I, Velayudhannair K, Kari Z, et al.: Exploring the role of plant oils in aquaculture practices: an overview. Aquaculture International. 2024; 32 (6): 7719-7745 Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Aquaculture nutrition

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response 21 Mar 2025

Fabian Gerardo Muñoz García, Zoot, Universidad del Cauca, Popayán, Colombia

21 Mar 2025

Author Response

We sincerely thank the reviewer for his valuable input, which significantly contributed to the improvement of this article. His highly relevant comments were carefully considered and incorporated into the manuscript ... Continue reading We sincerely thank the reviewer for his valuable input, which significantly contributed to the improvement of this article. His highly relevant comments were carefully considered and incorporated into the manuscript update.
We sincerely thank the reviewer for his valuable input, which significantly contributed to the improvement of this article. His highly relevant comments were carefully considered and incorporated into the manuscript update.
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 21 Mar 2025

Fabian Gerardo Muñoz García, Zoot, Universidad del Cauca, Popayán, Colombia

21 Mar 2025

Author Response

We sincerely thank the reviewer for his valuable input, which significantly contributed to the improvement of this article. His highly relevant comments were carefully considered and incorporated into the manuscript ... Continue reading We sincerely thank the reviewer for his valuable input, which significantly contributed to the improvement of this article. His highly relevant comments were carefully considered and incorporated into the manuscript update.
We sincerely thank the reviewer for his valuable input, which significantly contributed to the improvement of this article. His highly relevant comments were carefully considered and incorporated into the manuscript update.
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 11 Mar 2025

Fredrick Syanya, Cochin University of Science and Technology, Kochi, India

Approved with Reservations

https://doi.org/10.5256/f1000research.169671.r370242

The research is interesting since it's using red worms (Eisenia foetida) with a CP of 62% as a source of protein, which can be a perfect replacement for fish meal that has been traditionally used as the main source of protein for fish feed formulation.

However, Expound on your introduction, as extensive work has been done in this area by incorporating more literature.

Let your problem statement come out clearly in the introduction.

The methodology is sound; however, it could be improved on some parameters.

What informed the diet percentage determination? Elaborate in your work. 0% hydrolysed worm, 10% hydrolysed worm, and 20% hydrolysed worm.

Instead of presenting no statistical differences (p<0.217), you would rather say p>0.05 for a 95% chance of occurrence (page 8).

Elaborate more in your conclusion and give clear recommendations on the percentage dosage that is appropriate for red tilapia fingerlings.

Highlight the limitations of this study.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Aquaculture

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 04 Apr 2025

Fabian Gerardo Muñoz García, Zoot, Universidad del Cauca, Popayán, Colombia

04 Apr 2025

Author Response

We sincerely thank the reviewer for his valuable contribution, which significantly contributes to the improvement of this article. His relevant comments were carefully considered, and the most relevant ones were ... Continue reading We sincerely thank the reviewer for his valuable contribution, which significantly contributes to the improvement of this article. His relevant comments were carefully considered, and the most relevant ones were incorporated into the manuscript update.
We sincerely thank the reviewer for his valuable contribution, which significantly contributes to the improvement of this article. His relevant comments were carefully considered, and the most relevant ones were incorporated into the manuscript update.
Competing Interests: No competing interests were disclosed. Close
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Author Response 04 Apr 2025

Fabian Gerardo Muñoz García, Zoot, Universidad del Cauca, Popayán, Colombia

04 Apr 2025

Author Response

We sincerely thank the reviewer for his valuable contribution, which significantly contributes to the improvement of this article. His relevant comments were carefully considered, and the most relevant ones were ... Continue reading We sincerely thank the reviewer for his valuable contribution, which significantly contributes to the improvement of this article. His relevant comments were carefully considered, and the most relevant ones were incorporated into the manuscript update.
We sincerely thank the reviewer for his valuable contribution, which significantly contributes to the improvement of this article. His relevant comments were carefully considered, and the most relevant ones were incorporated into the manuscript update.
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 14 Feb 2025

Farah Izana Abdullah, International Institute of Aquaculture and Aquatic Sciences, Universiti Putra Malaysia, Port Dickson, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.169671.r363739

1. Summary of the Article

The study investigates the use of hydrolyzed red worm (HRW) as a protein source in red tilapia (Oreochromis sp.) diets. The experiment involved 90 red tilapia fingerlings distributed across nine aquariums, with ... Continue reading

T1 (Control Diet, 0% HRW)
T2 (10% HRW inclusion)
T3 (20% HRW inclusion)

The key parameters evaluated included weight gain, size increase, feed conversion ratio (FCR), protein efficiency rate (PER), energy efficiency ratio (EER), and diet cost.
Key Findings:

Weight gain: T2 (10%) showed significantly higher weight gain compared to T1 (p=0.047). No significant difference was observed between T2 and T3.
Size increase: No statistically significant difference was found among treatments (p=0.217).
FCR, PER, and EER: Significant improvements were observed in T2 and T3 compared to T1 (p=0.001).
Diet cost: Higher HRW inclusion reduced the overall feed cost (p=0.034).
No mortality observed: Water quality remained within acceptable limits.

The authors conclude that HRW is a viable and cost-effective protein source for red tilapia.

2. Assessment of Data Availability and Reproducibility

Are all the source data underlying the results available to ensure full reproducibility?

Yes, but verification is needed.

The authors provide access to a dataset hosted on Zenodo (DOI: 10.5281/zenodo.14081025).
The article states compliance with ARRIVE 2.0 guidelines, ensuring transparency in animal research.

Recommendations for Improvement:

Verify that the dataset contains all raw data, including:
- Individual fish measurements (initial and final weight, size)
- Daily feed intake per tank
- Water quality parameters over time
- Statistical analysis scripts (if applicable)
Ensure metadata is provided for full reproducibility.

3. Evaluation of the Scientific Soundness of the Conclusions

Are the conclusions drawn adequately supported by the results?

Partly. The study presents strong evidence supporting the benefits of HRW inclusion, but certain aspects require further clarification or additional data to strengthen the conclusions.

Strengths:

✔ Statistically significant improvements in key parameters: The weight gain, FCR, PER, and EER results support the claim that HRW enhances growth and feed efficiency. ✔ Economic benefit: The reduced diet cost further justifies HRW as an alternative protein source. ✔ No negative health effects: The lack of mortality and stable water quality parameters indicate that HRW does not introduce harmful effects on tilapia health.

Weaknesses and Required Revisions:

1. Lack of Statistically Significant Improvement in Size Increase

The study finds no significant improvement in size increase (p=0.217), yet the conclusion broadly states HRW "improves production parameters."
Required Revision: Clearly specify that while weight gain improved, length growth did not show a statistically significant difference.

2. Lack of Clarity on Optimal Inclusion Level

No significant difference was observed between T2 (10%) and T3 (20%) in growth performance, yet both are recommended.
Required Revision: The authors should discuss whether 10% is the optimal level since increasing to 20% did not yield additional benefits.
If possible, a cost-benefit analysis should be included to determine the most economically viable HRW level.

3. Short Experimental Duration (30 Days)

Long-term effects on fish growth, health, or reproductive performance were not evaluated.
Recommended Improvement: Acknowledge this limitation and suggest future research on extended feeding trials.

4. Need for More Discussion on Protein and Energy Utilization

The article attributes improved feed efficiency to better protein digestibility but does not detail the amino acid profile of HRW.
Recommended Improvement: If amino acid analysis is available, include a discussion on how HRW’s amino acid profile compares to conventional fish meal.

4. Final Recommendations

To improve the scientific rigor of the paper and ensure clarity in conclusions, the following revisions are mandatory for scientific soundness:

Clarify that size increase was not significantly improved and discuss possible reasons.
Reassess the recommendation of 20% HRW inclusion, given that 10% yielded similar results.
Acknowledge the short duration of the study and the need for long-term assessments.
Ensure the dataset is fully detailed and contains all necessary raw data for reproducibility.

Additionally, the following improvements are strongly recommended to enhance clarity:

Provide more discussion on HRW’s amino acid composition.
If possible, conduct a cost-benefit analysis to determine the most practical HRW inclusion level.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: My expertise includes protein hydrolysate, particularly its nutritional characteristics, bioactive properties, extraction methods, and applications in aquaculture, functional foods, and pharmaceuticals.

CITE

Report a concern

Author Response 19 Mar 2025

Fabian Gerardo Muñoz García, Zoot, Universidad del Cauca, Popayán, Colombia

19 Mar 2025

Author Response

I would like to thank the reviewer for his comments, which are intended to strengthen the scientific quality of this article. Some of the proposed adjustments will be made.

... Continue reading I would like to thank the reviewer for his comments, which are intended to strengthen the scientific quality of this article. Some of the proposed adjustments will be made.

However, I consider it necessary to refute some of the points raised, as follows:

In response to point 2, regarding the evaluation of data availability and reproducibility, it is confirmed that the raw measurements for each experimental unit and for all evaluated parameters are available in the first sheets of the data set hosted in Zenodo (DOI: 10.5281/zenodo.14081025).

In reference to point 3. Evaluation of the scientific solidity of the conclusions.
The article concludes that California red worm (Eisenia foetida) hydrolyzate represents a high-quality nutritional alternative for red tilapia diets, specifically at inclusion levels of 10% and 20%. It is observed that these levels optimize protein and energy efficiency, improve weight gain and feed conversion rate. Additionally, it is suggested that the inclusion of the hydrolyzate could generate economic benefits due to the reduction in the cost of the diet.

As evidenced in Table 6, the treatments with 10% and 20% hydrolyzate presented a statistically superior performance compared to the treatment without hydrolyzate.

In response to the observation that suggests: 'Revision required: The authors should discuss whether 10% is the optimal level, since increasing to 20% did not produce additional benefits', it should be noted that the statistical results showed no significant differences in most of the parameters analyzed. Furthermore, the numerical differences observed were minimal, which precludes the claim that the 10% inclusion level is optimal. To determine with certainty the optimal inclusion level, an additional study with more specific inclusion levels would need to be conducted, that allow a more detailed and precise evaluation."

4. Need for more debate on protein and energy utilization
Recommended improvement: If an amino acid analysis is available, include a discussion of how the amino acid profile of HRW compares to conventional fishmeal.
Regarding the suggested improvement, it is important to note that the amino acid analysis was not considered as part of the objectives of this research, so it was not carried out.
I would like to thank the reviewer for his comments, which are intended to strengthen the scientific quality of this article. Some of the proposed adjustments will be made.

However, I consider it necessary to refute some of the points raised, as follows:

In response to point 2, regarding the evaluation of data availability and reproducibility, it is confirmed that the raw measurements for each experimental unit and for all evaluated parameters are available in the first sheets of the data set hosted in Zenodo (DOI: 10.5281/zenodo.14081025).

In reference to point 3. Evaluation of the scientific solidity of the conclusions.
The article concludes that California red worm (Eisenia foetida) hydrolyzate represents a high-quality nutritional alternative for red tilapia diets, specifically at inclusion levels of 10% and 20%. It is observed that these levels optimize protein and energy efficiency, improve weight gain and feed conversion rate. Additionally, it is suggested that the inclusion of the hydrolyzate could generate economic benefits due to the reduction in the cost of the diet.

As evidenced in Table 6, the treatments with 10% and 20% hydrolyzate presented a statistically superior performance compared to the treatment without hydrolyzate.

In response to the observation that suggests: 'Revision required: The authors should discuss whether 10% is the optimal level, since increasing to 20% did not produce additional benefits', it should be noted that the statistical results showed no significant differences in most of the parameters analyzed. Furthermore, the numerical differences observed were minimal, which precludes the claim that the 10% inclusion level is optimal. To determine with certainty the optimal inclusion level, an additional study with more specific inclusion levels would need to be conducted, that allow a more detailed and precise evaluation."

4. Need for more debate on protein and energy utilization
Recommended improvement: If an amino acid analysis is available, include a discussion of how the amino acid profile of HRW compares to conventional fishmeal.
Regarding the suggested improvement, it is important to note that the amino acid analysis was not considered as part of the objectives of this research, so it was not carried out.
Competing Interests: Se presentan consideraciones respecto a lo propuesto por el evaluador Close
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COMMENTS ON THIS REPORT

Author Response 19 Mar 2025

Fabian Gerardo Muñoz García, Zoot, Universidad del Cauca, Popayán, Colombia

19 Mar 2025

Author Response

I would like to thank the reviewer for his comments, which are intended to strengthen the scientific quality of this article. Some of the proposed adjustments will be made.

... Continue reading I would like to thank the reviewer for his comments, which are intended to strengthen the scientific quality of this article. Some of the proposed adjustments will be made.

However, I consider it necessary to refute some of the points raised, as follows:

In response to point 2, regarding the evaluation of data availability and reproducibility, it is confirmed that the raw measurements for each experimental unit and for all evaluated parameters are available in the first sheets of the data set hosted in Zenodo (DOI: 10.5281/zenodo.14081025).

In reference to point 3. Evaluation of the scientific solidity of the conclusions.
The article concludes that California red worm (Eisenia foetida) hydrolyzate represents a high-quality nutritional alternative for red tilapia diets, specifically at inclusion levels of 10% and 20%. It is observed that these levels optimize protein and energy efficiency, improve weight gain and feed conversion rate. Additionally, it is suggested that the inclusion of the hydrolyzate could generate economic benefits due to the reduction in the cost of the diet.

As evidenced in Table 6, the treatments with 10% and 20% hydrolyzate presented a statistically superior performance compared to the treatment without hydrolyzate.

In response to the observation that suggests: 'Revision required: The authors should discuss whether 10% is the optimal level, since increasing to 20% did not produce additional benefits', it should be noted that the statistical results showed no significant differences in most of the parameters analyzed. Furthermore, the numerical differences observed were minimal, which precludes the claim that the 10% inclusion level is optimal. To determine with certainty the optimal inclusion level, an additional study with more specific inclusion levels would need to be conducted, that allow a more detailed and precise evaluation."

4. Need for more debate on protein and energy utilization
Recommended improvement: If an amino acid analysis is available, include a discussion of how the amino acid profile of HRW compares to conventional fishmeal.
Regarding the suggested improvement, it is important to note that the amino acid analysis was not considered as part of the objectives of this research, so it was not carried out.
I would like to thank the reviewer for his comments, which are intended to strengthen the scientific quality of this article. Some of the proposed adjustments will be made.

However, I consider it necessary to refute some of the points raised, as follows:

In response to point 2, regarding the evaluation of data availability and reproducibility, it is confirmed that the raw measurements for each experimental unit and for all evaluated parameters are available in the first sheets of the data set hosted in Zenodo (DOI: 10.5281/zenodo.14081025).

In reference to point 3. Evaluation of the scientific solidity of the conclusions.
The article concludes that California red worm (Eisenia foetida) hydrolyzate represents a high-quality nutritional alternative for red tilapia diets, specifically at inclusion levels of 10% and 20%. It is observed that these levels optimize protein and energy efficiency, improve weight gain and feed conversion rate. Additionally, it is suggested that the inclusion of the hydrolyzate could generate economic benefits due to the reduction in the cost of the diet.

As evidenced in Table 6, the treatments with 10% and 20% hydrolyzate presented a statistically superior performance compared to the treatment without hydrolyzate.

In response to the observation that suggests: 'Revision required: The authors should discuss whether 10% is the optimal level, since increasing to 20% did not produce additional benefits', it should be noted that the statistical results showed no significant differences in most of the parameters analyzed. Furthermore, the numerical differences observed were minimal, which precludes the claim that the 10% inclusion level is optimal. To determine with certainty the optimal inclusion level, an additional study with more specific inclusion levels would need to be conducted, that allow a more detailed and precise evaluation."

4. Need for more debate on protein and energy utilization
Recommended improvement: If an amino acid analysis is available, include a discussion of how the amino acid profile of HRW compares to conventional fishmeal.
Regarding the suggested improvement, it is important to note that the amino acid analysis was not considered as part of the objectives of this research, so it was not carried out.
Competing Interests: Se presentan consideraciones respecto a lo propuesto por el evaluador Close
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Farah Izana Abdullah, International Institute of Aquaculture and Aquatic Sciences, Universiti Putra Malaysia, Port Dickson, Malaysia
Fredrick Syanya, Cochin University of Science and Technology, Kochi, India
Zulhisyam Abdul Kari, Universiti Malaysia Kelantan, Kafrelsheikh University, Universiti Sains Malaysia, Kelantan, Malaysia

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14 Mar 2025 | for Version 1

Zulhisyam Abdul Kari, Universiti Malaysia Kelantan, Kafrelsheikh University, Universiti Sains Malaysia, Kelantan, Malaysia

6 Views Cite this report Responses(1)

Approved

Here are some minor comments for the article:
General Comments

Clarity in Introduction:
- The introduction is well-structured, but it would benefit from a clearer problem statement. The rationale for using hydrolyzed red worm (HRW) should be explicitly connected to existing challenges in aquaculture feed.
- Consider incorporating more references on the use of insect or worm-based protein sources in aquaculture to strengthen the background.
- Please also read the latest article published which can help the authors in the introduction part as below: (reference 1 to 9)

Improve the Introduction Section – The cited works illustrate how introductions in similar aquaculture nutrition studies are structured, integrating the latest research trends and background information in a comprehensive manner. This guidance aims to help the authors refine their context-setting and literature synthesis.
Ensure the Use of Up-to-Date References – The field of aquaculture nutrition is rapidly evolving, and my recent work contains key insights that are directly relevant to the study’s focus on red tilapia and alternative protein sources. Citing recent studies, including my own, ensures that the manuscript aligns with the latest scientific developments.
Enhance Scientific Rigor – By referring to well-documented findings in related studies, the authors can strengthen their discussion, reinforce their hypotheses, and make well-supported conclusions. My recommendations were purely scientific, aimed at improving the quality of the manuscript rather than for personal or professional gain.

Methodology Enhancements:
- The justification for selecting 0%, 10%, and 20% HRW inclusion levels should be elaborated. Were these levels based on prior studies, preliminary trials, or nutrient availability considerations?
- In the water quality monitoring section, specifying how often parameters were measured (daily, weekly) would enhance reproducibility.
- The statistical analysis section should clarify whether assumptions (e.g., normality and homogeneity of variance) were tested before applying ANOVA.
Results Interpretation:
- The phrase "no statistical difference (p<0.217)" should be revised to "p > 0.05, indicating no significant difference" for clarity.
- While weight gain showed significant improvement at 10% inclusion, size increase did not. The discussion should explicitly state whether size growth is a limiting factor or if hydrolyzed proteins primarily contribute to mass gain.
- Since no significant difference was found between 10% and 20% HRW inclusion, a cost-benefit analysis might help determine whether 10% is the optimal level.
Discussion and Conclusion Adjustments:
- The conclusion should specify that while weight gain and feed efficiency improved, size growth did not show a significant difference across treatments.
- Highlighting potential long-term effects (e.g., immune response, fillet quality, or digestive enzyme activity) could add depth to future research recommendations.
- The limitations of the study should be acknowledged, such as the relatively short experimental duration (30 days) and the need for trials at different life stages.

Minor Corrections:

Spelling & Formatting
- Eisenia foétida → should be consistently italicized as Eisenia foetida throughout the text.
- "size increasse" (Figure 2 caption) → "size increase"
- "feed conversión" (Figure 3 caption) → "feed conversion"
Figures & Tables:
- Ensure all figures have clear legends and units where applicable (e.g., Figure 5 on Energy Efficiency Ratio).
- The proximate composition of HRW could be compared more explicitly with fish meal in a supplementary table.

By implementing these minor suggestions, the article can improve clarity, precision, and scientific rigor while maintaining its strong contribution to aquaculture research.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

References

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Aquaculture nutrition

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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11 Mar 2025 | for Version 1

Fredrick Syanya, Cochin University of Science and Technology, Kochi, India

5 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Aquaculture

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5 Views

14 Feb 2025 | for Version 1

Farah Izana Abdullah, International Institute of Aquaculture and Aquatic Sciences, Universiti Putra Malaysia, Port Dickson, Malaysia

5 Views Cite this report Responses(1)

Approved With Reservations

T1 (Control Diet, 0% HRW)
T2 (10% HRW inclusion)
T3 (20% HRW inclusion)

The key parameters evaluated included weight gain, size increase, feed conversion ratio (FCR), protein efficiency rate (PER), energy efficiency ratio (EER), and diet cost.
Key Findings:

Weight gain: T2 (10%) showed significantly higher weight gain compared to T1 (p=0.047). No significant difference was observed between T2 and T3.
Size increase: No statistically significant difference was found among treatments (p=0.217).
FCR, PER, and EER: Significant improvements were observed in T2 and T3 compared to T1 (p=0.001).
Diet cost: Higher HRW inclusion reduced the overall feed cost (p=0.034).
No mortality observed: Water quality remained within acceptable limits.

The authors provide access to a dataset hosted on Zenodo (DOI: 10.5281/zenodo.14081025).
The article states compliance with ARRIVE 2.0 guidelines, ensuring transparency in animal research.

Recommendations for Improvement:

Verify that the dataset contains all raw data, including:
- Individual fish measurements (initial and final weight, size)
- Daily feed intake per tank
- Water quality parameters over time
- Statistical analysis scripts (if applicable)
Ensure metadata is provided for full reproducibility.

The study finds no significant improvement in size increase (p=0.217), yet the conclusion broadly states HRW "improves production parameters."
Required Revision: Clearly specify that while weight gain improved, length growth did not show a statistically significant difference.

2. Lack of Clarity on Optimal Inclusion Level

No significant difference was observed between T2 (10%) and T3 (20%) in growth performance, yet both are recommended.
Required Revision: The authors should discuss whether 10% is the optimal level since increasing to 20% did not yield additional benefits.
If possible, a cost-benefit analysis should be included to determine the most economically viable HRW level.

3. Short Experimental Duration (30 Days)

Long-term effects on fish growth, health, or reproductive performance were not evaluated.
Recommended Improvement: Acknowledge this limitation and suggest future research on extended feeding trials.

4. Need for More Discussion on Protein and Energy Utilization

The article attributes improved feed efficiency to better protein digestibility but does not detail the amino acid profile of HRW.
Recommended Improvement: If amino acid analysis is available, include a discussion on how HRW’s amino acid profile compares to conventional fish meal.

4. Final Recommendations

To improve the scientific rigor of the paper and ensure clarity in conclusions, the following revisions are mandatory for scientific soundness:

Clarify that size increase was not significantly improved and discuss possible reasons.
Reassess the recommendation of 20% HRW inclusion, given that 10% yielded similar results.
Acknowledge the short duration of the study and the need for long-term assessments.
Ensure the dataset is fully detailed and contains all necessary raw data for reproducibility.

Additionally, the following improvements are strongly recommended to enhance clarity:

Provide more discussion on HRW’s amino acid composition.
If possible, conduct a cost-benefit analysis to determine the most practical HRW inclusion level.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

My expertise includes protein hydrolysate, particularly its nutritional characteristics, bioactive properties, extraction methods, and applications in aquaculture, functional foods, and pharmaceuticals.

Respond to this report

Responses (1)

Author Response

19 Mar 2025

Fabian Gerardo Muñoz García, Zoot, Universidad del Cauca, Popayán, Colombia

I would like to thank the reviewer for his comments, which are intended to strengthen the scientific quality of this article. Some of the proposed adjustments will be made.

However, I consider it necessary to refute some of the points raised, as follows:

In response to point 2, regarding the evaluation of data availability and reproducibility, it is confirmed that the raw measurements for each experimental unit and for all evaluated parameters are available in the first sheets of the data set hosted in Zenodo (DOI: 10.5281/zenodo.14081025).

In reference to point 3. Evaluation of the scientific solidity of the conclusions.
The article concludes that California red worm (Eisenia foetida) hydrolyzate represents a high-quality nutritional alternative for red tilapia diets, specifically at inclusion levels of 10% and 20%. It is observed that these levels optimize protein and energy efficiency, improve weight gain and feed conversion rate. Additionally, it is suggested that the inclusion of the hydrolyzate could generate economic benefits due to the reduction in the cost of the diet.

As evidenced in Table 6, the treatments with 10% and 20% hydrolyzate presented a statistically superior performance compared to the treatment without hydrolyzate.

In response to the observation that suggests: 'Revision required: The authors should discuss whether 10% is the optimal level, since increasing to 20% did not produce additional benefits', it should be noted that the statistical results showed no significant differences in most of the parameters analyzed. Furthermore, the numerical differences observed were minimal, which precludes the claim that the 10% inclusion level is optimal. To determine with certainty the optimal inclusion level, an additional study with more specific inclusion levels would need to be conducted, that allow a more detailed and precise evaluation."

4. Need for more debate on protein and energy utilization
Recommended improvement: If an amino acid analysis is available, include a discussion of how the amino acid profile of HRW compares to conventional fishmeal.
Regarding the suggested improvement, it is important to note that the amino acid analysis was not considered as part of the objectives of this research, so it was not carried out.

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Competing Interests

Se presentan consideraciones respecto a lo propuesto por el evaluador

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Effect of hydrolyzed red worm (Eisenia foétida) on production parameters in red tilapia (Oreochromis sp.)

Abstract

Background

Objective

Methods

Results

Conclusion

Keywords

Introduction

Methods

Experimental biological material

Experimental environment

Experimental feeding

(1)

Experimental diets

Table 1. Proximate analysis of hydrolyzed red worm (Eisenia foétida).

Table 2. Composition in grams of diets with red worm hydrolyzed red worm (Eisenia foétida).

Table 3. Composition of diets with red worm hydrolyzed (Eisenia foétida).

Feeding protocol

Diet cost

Table 4. Value of ingredients in diets with red worm hydrolyzed (Eisenia foétida).

Table 5. Variables evaluated in Oreochromis sp., fed with red worm hydrolyzed (Eisenia foétida).

Growth rates

(2)

(3)

(4)

(5)

(6)

(7)

Statistical analysis

Results and Discussion

Table 6. Growth and use of nutrients in Oreochromis sp., fed with hydrolyzed Eisenia foétida.

Figure 1. Weight gain of red tilapia (Oreochromis sp.) fed with hydrolyzed red worm (Eisenia foétida).

Figure 2. Size increasse of red tilapia (Oreochromis sp.) fed with hydrolyzed red worm (Eisenia foétida).

Figure 3. Feed conversión rate with inclusión of hydrolyzed red worm (Eisenia foétida).

Figure 4. Protein efficiency rate with inclusión of hydrolyzed red worm (Eisenia foétida).

Figure 5. Energy efficiency ratio with hydrolyzed red worm (Eisenia foétida).

Conclusions

Ethics statement

Data availability

Underlying data

Reporting guidelines

Acknowledgements

References

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