Effect of replacement of fishmeal by black soldier fly larvae meal in diets on growth performance, carcass traits and meat chemical composition of Thai frog (Rana rugosa Temminck and Schelegel, 1838)

Vo Duc Nghia, Pham Thi Phuong Lan and Nguyen Duy Quynh Tram

Hue University of Agriculture and Forestry, Hue University, Vietnam
ndqtram@hueuni.edu.vn

Abstract

This experiment was conducted to evaluate the effect of replacement of fishmeal by black soldier fly larvae (BSFL) meal in the diets on growth performance, carcass traits and chemical composition of meat of the Thai frog Rana rugosa kept in small scale farm. Total 3,600 frogs of 21 g were randomly allocated to five (5) dietary treatments and three (3) replicates. Dietary treatments named NT0, NT10, NT20, NT30 and NT40, in which, fishmeal as control (NT0) and 10, 20, 30 and 40% of fishmeal were replaced by BSFL meal. The results showed that the replacement of fishmeal by BSFL meal (or inclusion of BSFL) in diets for the Thai frogs improved the survival rate (75.6-86.8%), feed conversion ratio, increased live weight (194-240.7 g), and daily weight gain (3.11-3.66 g) and frog’s yield (5.9-8.41 kg/m²); and did not affect carcass traits but increased the contents of crude protein, and ether extract in legs meat at levels of 5.6% and 7.5% as DM diet. In recommendation, BSFL meal can be good feed for frogs and the inclusion level of above 5.6% as DM diet is appropriate in practical small farms.

Keywords: frog farming, insect protein, larval meal, Vietnam

Introduction

Frog meat has a high protein content, complete and balanced amino acid profile, low lipid content and high proportion of unsaturated fatty acids (Noll and Lindau 1987), and low cholesterol (Casali et al 2005). The Thai frog (Rana rugosa) Temminck and Schlegel, 1838 is a species of aquaculture with high economic value and potential for the domestic market in Vietnam.

Since 2003, the Thai frog has introduced in existing farming systems in the country and resulted in improving the farmers’ income and expending frog production in whole country. In frog production, the feed cost consisted of 65 - 80% of total cost. In fact, the farmers have completely used commercial feeds including feeds for catfish, snakehead fish, etc. for frog production, so the feed cost was high and the nutritive value was not guaranteed (Binh and Thao 2016).

On the other hand, the aquaculture industry in recent years is focusing on researching protein-rich ingredients to replace fishmeal in order to reduce feed costs and increase sustainability for aquaculture production (Rana et al 2015; Cammack and Tomberlin 2017). Interest in non-traditional sources of protein from insects as a feed ingredient for aquatic animals is being studied (Veldkamp et al 2018; Lan et al 2022^a). In which, black soldier fly larvae (Hermetia illucens) (BSFL) with rich protein and some essential amino acids such as lysine and methionine, lipid and essential fatty acids such as linoleic acid and alpha-linolenic acid, vitamin and mineral composition are considered as an important alternative animal feed source (Cammack and Tomberlin 2017; Lan et al 2022^b). Similarly, Ngoan et al (2021) reported that BSFL have a high crude protein (37-63% as DM) and crude fat (7-39%) and these values vary widely depending on substractes for feeding the larvae.

Black soldier fly larvae were used in diets for red tilapia (Oreochromissp.), snakehead fish (Channa micropeltes), Asian seabass (Lates calcarifer Bloch, 1790) and showed no adverse effects on growth and meat quality (Hoa and Dung 2016; Katya et al 2017; Khanh and Lan 2019; Lan et al 2022^a,c). However, there recently have been no publications on using BSFL as feed for frogs.

The study aimed therefore to determine the effects of fishmeal replacement with BSFL meal in the diet on growth performance and some carcass traits and meat chemical composition of the Thai frogs kept in small scale farms.

Materials and methods

This two-months experiment was conducted at La Chu Research Farm in Hue City during May-July 2023 and was approved by the Animal Ethics Committee of Hue University No. HUVN0026.

Animal

A total 3,600 frogs of 21 g were used in this study. They were kept in 15 net closed cages with 2 x 3 x2 m measurement, and 240 animals in each cage. The frog cages were fixed in the pond and water in the pond was replaced 30% within one week to maintain an appropriate environmental condition for frogs. Frogs were fed 4 times a day (7.00; 11.00; 15.00 and 19.00h) to satisfy their needs.

Diet preparation

Larvae meal: BSFL were fed by tofu by-products and collected at day 9th after rearing. Larvae meal preparation was followed Kroeckel et al (2012) and Lan et al (2022^c). Larvae were washed with water several times to remove all impurities on the body, boiled at 60°C for 30 minutes, dried at 60⁰C for 48 hrs and milled into full fat BSFL meal.

All ingredients were carefully mixed according to their ratio in five diets. Then the mixtures were extruded through a 2-6 mm diameter die plate using an extruder (Binh Minh Corp., Vietnam).

Experimental design

The experiment was arranged in a completely randomized design with 5 treatments using different diets for frogs and 3 replicates (5 x 3). In each replicate, 240 frogs were kept in the net cage. The experimental treatments included: NT0 - control diet without BSFL meal; NT10, NT20, NT30 and NT40 as replacing 10%, 20%, 30% and 40% of fishmeal with BSFL meal.

Photo 1. Frog at the beginning	Photo 2. The net cage system and monitoring water quality

The experiment was lasted for 60 days, exclusive 2-week preparation time.

The proportions of ingredients and the nutritive value of the diets are presented in Table 1.

Measurements

Water quality parameters were measured including water temperature (°C), pH, dissolved oxygen (DO) and N-NH₃, they were measured periodically (Table 2).

The mean values of Temperature, pH, DO, and Ammonia were within range 28.5 - 31.5 ^oC, 7.15 -7.94, 4.11 - 4.72 mg/l, and 0.01 -0.09 mg/l, respectively.

Growth performance

To determine the growth performance, ten (10) frogs in each cage were randomly collected and were anesthetized with Aqui-S® at a concentration of 10 mL/m ³ before weighing. Live weights of frogs were recorded at the beginning and every 15 days until 60 days.

Daily weight gain (DWG, g/day) was then calculated by = (W ₀ – W_i)/N, in which W ₀ and W_i were live weight at initial day and day i, and N was number day of stocking.

Feed conversion ratio (FCR) was the ratio between feed intake in DM and weight gain.

Survival rate

Survival rate (SR, %) was determined by ratio between survival number at final and at initial experiment.

Yield

Yield (kg/m²) was calculated by total live weight of frogs collected at terminated experiment per square meter of water surface.

Meat quality

At the end of the experiment, three (3) frogs in each cage were randomly collected and anesthetized with Aqui-S® at a concentration of 25 mL/m ³ before slaughter. Carcass traits were taken at the Laboratory of Fisheries Faculty, University of Agriculture and Forestry, Hue University. The carcass traits were measured according to Ayres et al (2015), specifically as follows:

- Weight at slaughter (g): Weighed the frog after 24 hours of starvation;

- Carcass weight (g): Weighed the frog after removing the skin, cutting off the head, cutting off the feet and removing the organs;

- Legs weight (g): Weighed two legs;

- Carcass dressing and leg percentage then were calculated as the proportion of slaughter weight.

Chemical analysis

All feed samples and frog meat were chemically analyzed for dry matter (DM), crude protein (CP), ether extract (EE), crude fibre (CF), and total ash according to the procedures of AOAC (1990) at the Laboratory of Biotechnology of the National Institute of Animal Sciences, Ha Noi.

The gross energy (GE) was calculated according to Ewan (1989):

GE (kcal/kg) = 4143 + 56 x EE + 15 x CP - 44 x ash

Data analysis

Data were presented in the form of the mean (M) and standard error of the mean (SEM). The data were statistically processed by analysis of variance (ANOVA) by General Linear Model in Minitab v.16.2 (2010). The difference between the mean values was determined by the Tukey method at a confidence level of 95%.

Results and discussion

Growth performance, feed conversion ratio, yield and survival

The results on weight gain, feed conversion ratio, yield and survival of frogs in five treatments were presented in Table 3.

Except for the initial weight of frogs, final weight, daily weight gain, FCR and yield were significantly different among treatments (p<0.05). With increasing BSFL meal in diets the final weight, daily weight gain and yield were clearly increased, and improved FCR. The final weights of frogs were the highest in NT30 and NT40 and lowest in NT0 (control diet) and NT10 (p<0.05). Value of the final weight ranged 185.2-240.7 g/animal after 60 days of feeding. Similarly, daily weight gains and yield were also the highest in NT30 and NT40 and lowest in NT0 and NT10 (p<0.05). After 60 days feeding, the yield of frogs ranged 5.2-8.41 kg/m ² of water surface. The results on final weight and gain in this study were in agreement with the previous report. Binh and Thao (2016) reported that the final weight of frogs fed 27-35% CP diets ranged 246.2 – 253.7 g after 50 days feeding. However, the result of this study was different with from the reported by Hung et al (2011) that after 60 days feeding the final weight ranged 110 - 120 g.

Figure 1. The effect of Black soldier fly larvae on DWG

The FCR were lower in NT30 and NT40 than in NT0, NT10 and NT20 (p<0.05). The value of FCR ranged 1.32 - 2.01 in this study was in agreement with the previous publication of Putsate and Veerote (1995), who reported that FCR of the Thai frogs fed commercial 30-35% CP diets ranged 1.5 - 2.0. However, the result of recent study was somehow lower than the many studies in Vietnam, in which the authors reported the FCR of the Thai frogs fed commercial feeds ranged 1.2 - 1.5 (Hung 2004; Thai 2010).

Figure 2. The effect of Black soldier fly larvae on FCR

The yield of the Thai frogs was the highest in NT30 and NT40 (7.7-8.41 kg/m²) and the lowest in NT0 (5.2 kg/m²). The yield in NT10 and NT20 was similar (5.9-6.31 kg/m²). The yield in this study was in agreement with previous publications (Tho and Thi, 2005 but was lower the results of Hoa (2014), who reported that the yield of the Thai frogs kept in liner ponds ranged 17.8 - 25.5 kg/m².

The survival rate of frogs ranged from 70.0 to 86.8% and increased with increasing BSFL meal levels. In NT40 and NT30 (85-86.8%), the survival rates were the highest and was the lowest in NT0 (70%). The survival rate between the NT20 (75.6%) and NT10 (83.3%) had no statistically significant difference (p>0.05). Improving the survival rate of frogs probably resulted from increasing essential fatty acids in diets from BSFL meal. In recent previous publications, essential fatty acid concentrations in BSFL meal were higher than in fishmeal. Cho and Kim (2011) reported that linoleic acid (LA) content ranged 1.1-1.3% and alpha-linolenic acid (ALA) 0.3-0.9% as total fat in fishmeal. Meantime, Lan et al (2022^b) reported that BSFL fed by tofu by-products contained very high LA (omega-6) ranged 27.57-29.7% and ALA (omega-3) ranged 1.89-2.04% as total fat. The results of survival rate of frogs fed BSFL meal ranged 76.5-86.8% in this study were higher than that of 71.1 - 74.1% reported by Tho and Thi (2005), who fed frogs by commercial feeds. Hung (2004) indicated that the survival rate of Thai frogs ranged 60-80%. In addition, Thai (2010) showed the survival rate of the Thai frogs ranged 40-60%.

Generally, the inclusion of BSFL meal in diets for the Thai frogs improved the survival rate, growth performance and yield. As mentioned in the introduction, the use of BSFL for feeding frogs has been not published yet and this study may be preliminarly exploited. However, the effect of BSFL inclusion in diets for fish was documented and BSFL is considered as a good protein-rich feed source for feeding animals including aquatic animals (Hoa and Dung, 2016; Xiaopeng Xiao et al 2018; Yajun Hu et al 2020; Lan et al 2022^a).

Carcass traits and chemical composition of meat

Some carcass characteristics and chemical composition of the Thai frog’s meat were presented in Table 4. The live weight at slaughter ranged from 193.2 g to 242.5 g and differed among treatments (p<0.05). The live weight was higher in NT30 and NT40 (233.7-242.5 g) than in the others (193.2-207.4 g). However, carcass dressing and percentages of legs meat and skin were not different (p>0.05). The carcass dressing ranged 46.3% to 48.7%; the percentage of legs meat was 23.3 - 26.5%, and the percentage of skin was 7.2 - 8.6%. In addition, the carcass dressing and legs meat in treatment NT30 and NT40 seemed to be higher than that of the other treatments but not statistically significant.

Aryres et al (2015) announced that frogs of above 201 g gave the best yield and meat quality, and the carcass dressing ranged 48.97 - 49.96%, legs were 25.94 - 26.75% and skin were 9.42 - 9.98% in frogs reached 151 - 250 g. The results of our study showed that the carcass dressing and the percentage of legs meat were not significantly different from those previously published.

Except for total ash, crude protein and ether extract contents of legs meat were significantly higher in NT40 and NT30 than in other treatments (p<0.05). In this study, CP content ranged 18.4-21.11% was similar to previous publications. Ozogul et al (2008) reported that the meat of frog Rana esculenta has 19.22% CP. Similarly, Cagiltay et al (2011) showed that CP content of natural frogs in different areas in Turkey ranged 16.58-19.37%.

The ether extract content ranged 0.28-0.38% in this experiment, and was lower than in other reports. Cagiltay et al (2014) showed that the EE content in natural frog meat was 0.74% and framed frog 0.93%. According to Ozogul et al (2008), the EE content of frogs Rana esculenta in Cukurova was 0.68%, and Cagiltay et al (2011), the meat of frog Rana ridibunda in Turkey contained 0.48 – 0.89 g EE per 100 g meat.

Total ash content in this study ranged 1.0 – 1.04% and was similar to the report of Cagiltay et al (2014). The authors found that total ash content in frogs at natural and at farms ranged 1.0-1.37%.

In summary, the inclusion of BSFL meal in diets didn’t affect carcass dressing and percentages of legs meat and skin, but gradually increased CP and EE contents in legs meat at the levels of 5.6 and 7.5% BSFL meal.

Conclusions

The inclusion of BSFL in diets for the Thai frogs:

• Improves the survival rate;
  
  
• Increases live weight and daily gain;
  
  
• Improves feed conversion ratio;
  
  
• Increases yield per square meter of water surface;
  
  
• Did not affect carcass dressing, percentage of legs meat and skin;
  
  
• Increases contents of crude protein and ether extract in legs meat at levels above 5.6% as DM diet.

Acknowledgement

This study was financially supported by Thua Thien Hue Department of Science and Technology under the Grant No. TTH.2021-KC.26.

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