| Livestock Research for Rural Development 34 (1) 2022 | LRRD Search | LRRD Misssion | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
Two experiments were conducted to determine the optimal harvesting time of black soldier fly larvae (BSFL) and effect of replacement of trash fish in seabass’s diet rearing in fresh and brackish water. In the experiment 1, larvae of 5 days old fed by tofu by-products were randomly allocated into 4 treatments and 5 replicates according to harvesting time at 3 (D3), 5 (D5), 7 (D7), and 9 (D9) days after rearing. At day 7, larvae color was changed into dark yellow as adult stage; and day 9, around 5% of total larvae have black color (as pre-pupa). The results showed that larvae biomass and protein productivities were higher in D7 and D9 than in D3 and D5 (p<0.05), and not significant difference between D7 and D9 (p>0.05). Biomass and protein yields at D7 and D9 were 1.42 kg/m2 and 0.18 kg/m2, and 1.49 kg/m2 and 0.2 kg/m2, respectively. Moreover, 1 kg fresh larvae harvested at D7 consumed 3.65 kg fresh tofu by-products, and very much lower at D9 6.48 kg. Therefore, harvesting larve at 7 days after rearing was recommended. In the experiment 2, a total of 360 seabass fingerlings of 4.35 g were randomly allocated into 2 x 2 factorial design treatments (fresh -F and brackish water-B; and trash fish-T and black soldier fly larvae-BSFL). Fish were kept in a 160 L aquarium, with density of 180 fish/m3. The results showed that weight gain and length of fish cultured in brackish water with trash fish were higher than that of fresh water with BSFL. In contrast, the survival rate of fish cultured in fresh water is higher than in brackish water. Fish productivity was not affected by the water environment but it was higher in fish fed trash fish than in the one fed BSFL. Thus, using BSFL to total replacement of trash fish reduced the productivity of seabass in both fresh and brackish water environments.
Key words: biomass yield, insect protein, tofu by product
Seabass (Lates calcarifer Bloch, 1790) is a species that grows well in water with different salinity, it is commonly cultured in brackishwater (Singh 2000; Van et al 2020) and freshwater (Katya et al 2018) in Vietnam and other countries. The popular feeds are trash fish and commercial feed, in which fishmeal accounts for a high proportion resulting in a 70% of total feeding cost (Wilson 2002). Moreover, the lack of trash fish source used for fish become a serious issue these days, hence the substitution protein sources play an important role and black soldier fly larvae (BSFL) are one of the potential sources (Ngoan et al 2021).
The published studies demonstrated that BSFL are able to use feces, food processing byproducts, brewer’s grains, tofu processing byproducts, etc, so it is low production cost (Green and Popa 2012; Webster et al 2016). BSFL was reported as good quality as fishmeal and is safe to use as feeding in aquaculture (Kroeckel et al 2012; Sánchez-Muros et al 2016; Vaun et al 2017). BSFL was illustrated that replacement up to 30-50% of fish meal or soybean meal in diets of nile tilapia (Oreochromis niloticus), grass carp (Ctenopharyngodon idellus), rainbow trout (Oncorhynchus mykiss), seabass (Lates calcarifer), atlantic salmon (Salmo salar) did not effect on performance, physiology indices and meat quality (Ronghua Lu et al 2020; Rana et al 2015; Katya et al 2018). In contrast, many authors believe that the current high cost of BSFL will not be able to compete with fishmeal, the reason is that BFSL culture on poor nutrient substrates and harvesting of larvae at the wrong time affects the quality, chemical composition, as well as productivity (Nguyen et al 2015; Rachmawati 2010).
In Vietnam, there have been limited publications on the BSFL topic. Hoa and Dung (2016) reported that replacing trash fish by BSFL in snakehead fish ( Channa micropeltes) diet did not affect the growth rate and eye muscle quality. Another study also reported the same result when replacement of 20% fishmeal by BSFL on red tilapia (Oreochromis sp.) (Khanh and Lan 2019).
The current study aimed to determine the effect of harvesting times on biomass yield, chemical composition of BSFL; and effect of total replacement of trash fish by BSFL on performance of seabass rearing in fresh and brackish water (10‰).
The experiment was conducted at Center for Research and Training (CRT) of the Faculty of Animal Husbandry and Veterinary Medicine, Hue University of Agriculture and Forestry, Hue city, Vietnam.
Experiment was arranged as completed randomized design (CRD) including 4 treatments and 5 replicates. Four treatments were four haversting times: at day 3 (D3), day 5 (D5), day 7 (D7) and day 9 (D9) after rearing.
BSFL were reared in 40 x 25 x 15 cm (Length x width x height) plastic boxes with the density of 2 head/cm2 (2000 head/box). All boxes were put in a room with the temperature in a range of 26-33 oC and relative humidity of 65-70%. BSFL were offered with fresh tofu by-product (DM: 26.3%; CP: 5.4%; EE: 3.1%; Ash: 1.1% as fresh matter basis). BSFL were fed 100 mg/larvae/day (Diener et al 2009) and was adjusted in the whole the experiment period to ensure supplying enough feed. Feeding was supplied at 8 a.m daily together with water to maintain the substrate’s humidity of 70%. The experiment finished when 5% of BSFL reached to the prepupal period (black color).
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| Photo 1. Experimental design |
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| Black soldier fly larvae at initial | |
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| Black soldier fly larvae after 3 days of rearing (D3) | |
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| Black soldier fly larvae after 5 days of rearing (D5) | |
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| Black soldier fly larvae after 7 days of rearing (D7) | |
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| Black soldier fly larvae after 9 days of rearing (D9) | |
Photo 2.The larvae measured at 0, 3, 5, 7 and 9 days | |
The larvae weight was measured at 0, 3, 5, 7 and 9 days of experiment period. One hundred larvae per replicate were selected randomly and weighed, the procedure was repeated three times. The larvae productivity was calculated as total fresh weight (kg) per one square meter at each measurement time.
Feed conversion ratio (FCR) = Feed intake (kg)/Larvae weight gain (kg)
Protein efficient ratio (PER) = Larvae protein gain (kg)/protein intake (kg)
BSFL and feed samples were analysed for chemical composition as follow: The DM, Ash, and EE content were determined following protocols of AOAC 930.15, AOAC 942, 1990 and AOAC 930.15, 1990, respectively. The crude fibre content was measured following TCVN 4329, 2007 standard. The total nitrogen was measured following protocol of AOAC 930.15, 1990 and crude protein was calculated as 6.25 x N.
Data were presented as mean (M) and standard error of mean (SEM). Data were analysed using analysis of variance (ANOVA) by the GLM (General Linear Model) application of Minitab software version 16 and a post-hoc Tukey test to compare the difference between each treatment.
Yij = µ + αi + eij (1)
Where Yij = dependent variable; μ = the overall mean; α i = the fixed effect of treatment (i = 3, 5, 7, 9 day measurements); eij = the random error.
Seabass juveniles were purchased from Phan Toan fish farm, in Phu Thuan commune, Thua Thien Hue province. The experiment was carried out at the laboratory of the Faculty of Fisheries, Hue University of Agriculture and Forestry, Viet Nam. The experiment was from January to April 2021.
A total of 360 seabass juveniles with weight of 4.35 g were randomly assigned into 4 treatments according to a factorial design (2 x 2) with 3 replicates. Two factors were water salinity (0-Fresh and 10‰-Brackish water) and the feed sources (trash fish-T and black soldier fly larvae-L). The treatments are denoted as follows: Treatment 1 (FT): fish raising in fresh water (0‰) and feeding trash fish; Treatment 2 (FL): fish raising fish in fresh water (0‰) and feeding black soldier fly larvae; Treatment 3 (BT): fish raising in brackish water (10‰) and feeding trash fish; and treatment 4 (BL): fish raising fish in brackish water (10‰) and feeding black soldier fly larvae.
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| Photo 3. Feeding experimental system |
Seabass were acclimatized in 1 m3 composite tanks for 7 days with Lai Thieu industrial pellets (40% CP). After 7 days, the seabass were randomly arranged into 12 aquarium with a capacity of 200 L, containing 160 L of water and a stocking density of 180 fish/m3. The experimental system has separate water filtration system and are aerated all time. The experiment was run for 60 days.
BSFL were reared on a substrate of tofu by-product and harvested at the 7 th day after culture under the same temperature and humidity conditions of the substrate as in experiment 1. After harvesting, BSFL were washed with water several times to remove impurities. Trash fish were bought in the local market. Both of them were frozen at -20 oC for use throughout the experiment. The chemical composition of the feed is shown in Table 1.
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Table 1. Chemical composition of trash fish and larvae (% DM) |
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Feed |
DM |
CP |
EE |
Ash |
CF |
|
|
Trash fish |
20.50 |
71.40 |
12.60 |
7.26 |
3.47 |
|
|
BSFL |
21.35 |
58.43 |
19.04 |
9.24 |
10.72 |
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|
DM: dry matter, CP: crude protein, EE: ether extract, Ash: total ash, CF: crude fiber |
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Seabass were fed twice daily (8:00 and 17:00 h) with 10% of body weight. Regularly check the amount of feed eaten by the fish and siphon off the leftovers (if any) after each feeding. About 50% of the water in the aquarium was changed bi-daily. Every 10 days, vitamin C was added to the feed with a dose of 1 g/kg of feed; After adding vitamin C, the feed mixture was stirred and left for 5 minutes, then fed to the fish. Every 15 days, fish were bathed with KMnO4 at a dose of 5 ppm within 15 minutes to prevent disease.
Environmental factors such as water temperature (°C) and pH were measured with the HI98107/Hanna handheld meter; NH3 measured by HI 700/Hanna; alkalinity measured by the kH test kit of Sera (Germany); dissolved oxygen (DO) by DO test kit of Sera (Germany); and salinity was measured with a refractometer (Atago Model 2491-master's, Japan). All water quality factors were measured periodically every 7 days and measured at twice (7:00 and 14:00 h).
Growth performance: Ten fish in each aquarium were randomly selected to weigh and measure at the beginning and end of the experiment for calculate:
Specific growth rate in weight (SGRW, %) = [(Ln Wf – Ln Wi)/ day] x 100; and Specific growth rate in length (SGRL, %) = [(Ln Lf – Ln Li)/ day] x 100.
In which, Wi: mean of initial fish weight (g); Wf: mean of final fish weight (g); Li: mean of initial fish length (cm); Lf: mean of final fish length (cm).
Total weight gain; and then feed conversion ratio (FCR) = total feed intake (kg)/total weight gain (kg)
At the end of 60 days of the experiment, all fish were collected to determine:
Survival rate (SR, %) = (number of fish harvested/ number of fish stocked) × 100; and
Yield (kg/m3) = Weight of fish harvested in 1 m3 of water.
Data were presented as mean (M) and standard error of mean (SEM). The data were statistically processed by analysis of variance (ANOVA) by the GLM (General Linear Model) application of Minitab 16 software. The difference between the mean values was determined by the Tukey method with 95% confidence
Statistical model: Yijk = + Si + Fj + (SF)ij + eijk
Where: µ is the average value; Si is the level effect of salinity; Fj is the level effect of the feed; (SF)ij is the interaction between salinity and feed; eijk is the random effect; i is the salinity level (0‰ and 10‰); j is the type of food (trash fish and black soldier fly larvae); k is the interaction between salinity and feed.
Life cycle of BSFL develop in different periods (young larvae, adult larvae and pre-pupa) and each period have a typical colour including ivory-white, light yellow and dark yellow (or black), respectively. In the current study, BSFL were ivory-white at D3 and D5 and then changed into dark yellow at D7 and reached to 5% of total BSFL turned into dark yellow or black at D9 after rearing (prepupal stage).
Data in Table 2 show that final weight of larvae increased with the increasing harvesting times (p<0.05). In which, the final weight of larvae in D9 was the highest (0.081 g) and the lowest weight was in D3 (0.025 g). The biomass and protein yields of the larvae increased during growing stage, and highest in pre-pupa in D9 (p<0.05). Meanwhile, FCR was the lowest in D7 and highest in D3 (p<0.05). While, the PER was the higher in D7 than in other treatments (p<0.05).
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Table 2. Effect of harvest time on growth performance and protein efficiency of black soldier fly larvae |
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Treatment |
D3 |
D5 |
D7 |
D9 |
SEM |
p |
|
Final weight, g/larvae |
0.025a |
0.051b |
0.076c |
0.081c |
0.001 |
<0.001 |
|
Fresh yield, kg/m2 |
0.51a |
1.01b |
1.42c |
1.49c |
0.022 |
<0.001 |
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Dry matter yield, kg/m2 |
0.09a |
0.20b |
0.30c |
0.36d |
0.006 |
<0.001 |
|
FCR |
7.98c |
6.11b |
3.65a |
6.48b |
0.245 |
<0.001 |
|
PER |
0.62a |
0.81a |
1.38b |
0.76a |
0.057 |
<0.001 |
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Protein yield, kg/m2 |
0.06a |
0.13b |
0.18c |
0.20d |
0.003 |
<0.001 |
| a, b c, d: Means in the same row without common letter are different at p<0.05 | ||||||
The larvae weight and yield increased as increasing growing stage and reached the highest values at the prepupal stage, then decreased in the pupa stage. These findings are in line with the previous studies (Xiu Liu et al 2017; Abduha et al 2020). The larvae weight at D9 in this study was 0.081 g/larvae which is lower than finding by Kinasih et al (2018) (0.13 g/larvae) with the same feeding but longer rearing time (20 days). However, BSFL weight was reported a lower than current study result at 0.07 g/larvae, which reared by mixed of waste avocado and soybean residue at the ratio 1:1 (Abduha et al 2020) and equal with current study at 0.08 g/larvae when reared by brewery residues (Manyara 2018; Kinasih et al 2018). The BSFL weight in this study was low might be because of the high temperature in summer time. Jeffery et al (2009) reported that if the temperature is lower than 3oC then the larval rearing time is 4 days longer on average, and the larval weight is also smaller. Besides, feeding management such as one meal or different meals per day, also might effect on larvae growth rate.
The feed conversion ratio in this experiment was most effective at day 7 of culture and much higher than on days 3, 5 and 9 but the results of this study were lower than that of Manyara (2018).
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Table 3. Chemical composition of larvae at different harvest stages (% DM) |
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Treatment |
Initial |
D3 |
D5 |
D7 |
D9 |
SEM |
p |
|
|
DM |
14.35a* |
17.89b |
19.74c |
21.09d |
24.04e |
0.176 |
<0.001 |
|
|
CP |
59.86c |
68.13e |
62.87d |
58.68b |
54.30a |
0.148 |
<0.001 |
|
|
EE |
17.35c |
8.32a |
14.64b |
18.77d |
23.95e |
0.079 |
<0.001 |
|
|
CF |
7.04a |
8.58b |
10.40c |
10.70d |
10.98e |
0.032 |
<0.001 |
|
|
Ash |
7.88a |
8.78b |
8.62b |
9.18c |
10.02d |
0.076 |
<0.001 |
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| a, b c, d, e: Means in the same row without common letter are different at p<0.05 | ||||||||
Table 3 shows chemical composition of BSFL is affected by growing stage or harvesting times (p<0.05). DM and EE contents were gradually increased and highest in D9 (p<0.05). Meanwhile, CP content declined and highest in D3 (68.13%) and lowest in D9 (54.3%). CF content also increased by time but not much, e.g. CF of 5-day old larvae (initial) was 7.04% and 10.98% of D9 (14 days old).
The chemical composition of BSFL in this study changed with the increase of rearing time. Xiu Liu et al (2017) reported that BSFL fed commercial layer feed reached the highest CP content after 3 days at 46% and declined to 39.2% after 9 days, however the EE was decreased as the longer time of rearing, in range of 13.4 to 28.4%. The protein and fat content of BSFL fed the mix of waste avocado and soybean residue after 20 days reached the highest values of 47.2% and 28.9%, respectively (Abduha et al 2020; Xiu Liu et al 2017).
The data in Table 4 showed that most of the environmental factors have no statistical difference (p>0.05); Only pH and NH3 in water with different salinity have statistical difference (p<0.05). However, all environmental factors in cashew culture were within the appropriate range for the growth and development of seabass (Rimmer and Rusell 1998). Seabass is a species that adapts well to environmental conditions, especially tolerant of salinity ranges from 0 to 56‰ (FAO 2017). Other studies have shown that seabass could be cultured in fresh water, brackish and salt water environments, but the optimum range of salinity is 10 to 33‰ (Ercan et al 2015; Katya et al 2018).
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Table 4. Changes in environmental factors during the experiment |
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Item |
Brackish water |
Fresh water |
SEM |
p-value |
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|
T |
L |
T |
L |
W |
F |
WxF |
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Temperature, oC |
26.13 |
26.15 |
26.13 |
26.12 |
0.016 |
0.422 |
0.760 |
0.371 |
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Dissolved oxygen, mg L-1 |
4.01 |
4.04 |
4.02 |
4.04 |
0.013 |
0.712 |
0.092 |
0.760 |
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|
pH |
Morning |
7.62b |
7.58b |
7.32a* |
7.34a |
0.024 |
<0.0001 |
0.867 |
0.259 |
|
|
Afternoon |
7.75b |
7.76b |
7.49a |
7.50a |
0.030 |
<0.001 |
0.645 |
0.947 |
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|
NH3,
|
Morning |
0.10bc |
0.11c |
0.06a |
0.08ab |
0.006 |
<0.001 |
0.167 |
0.731 |
|
|
Afternoon |
0.14 |
0.14 |
0.11 |
0.13 |
0.009 |
0.035 |
0.203 |
0.613 |
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| a, b, c: Means in the same row without common letter are different at p <0.05 T: trash fish; L: black soldier fly larvae; W: water source; F: feed type; WxF: interaction | ||||||||||
The survival rate and growth performance of the fish in the treatments are shown in Table 5. The data show the influence of water environment and feed in some parameters. The weight, length and specific growth rate in weight at the end of the experiment were influenced by the water salinity and feed types. However, the specific growth rate (SGRL) in length was not affected by the interaction between the water salinity and the feed. Weight in treatments (FL and BL) using BSFL was statistically significant (p<0.05) compared to using trash fish (FT and BT). The SGRW of fish in brackish water was higher than in fresh water (p<0.05) and fish fed with trash fish was higher than that of larvae (p<0.05). Similarly, SGRL was faster in brackish water than in fresh water, and trash fish culture was higher than larvae. Meanwhile, the survival rate of fish in brackish water was lower than in fresh water (p<0.05) with no interaction between water salinity and feed type (p>0.05). In contrast, growth performance was not affected by the water salinity but it was higher in fish fed trash fish compared with fed BSFL (p<0.05). Similarly, FCR was lower in trash fish (BT and FT) than in BSFL (BL and FL).
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Table 5. Survival rate, growth performance and yield of seabass fingerling |
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|
Item |
Brackish water |
Fresh water |
SEM |
p-value |
||||||
|
T |
L |
T |
L |
W |
F |
WxF |
||||
|
Survival, % |
84.44c |
91.11b |
95.55ab |
97.77a |
1.111 |
<0.001 |
0.004 |
0.080 |
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|
Initial weight, g |
4.35 |
4.36 |
4.35 |
4.35 |
0.003 |
0.960 |
0.889 |
0.992 |
||
|
Final weight, g |
43.27a |
32.28b |
39.30c |
28.85d |
0.009 |
<0.001 |
<0.001 |
<0.001 |
||
|
SGRW, % |
3.827a |
3.339c |
3.668b |
3.152d |
0.0014 |
<0.001 |
<0.001 |
<0.001 |
||
|
FCR |
4.11d |
6.24b |
4.167c |
6.34a |
0.0025 |
<0.001 |
<0.001 |
<0.001 |
||
|
Initial length, cm |
6.681 |
6.677 |
6.677 |
6.668 |
0.0038 |
0.801 |
0.676 |
0.560 |
||
|
Final length, cm |
15.743a |
13.697c |
14.637b |
12.80d |
0.0286 |
<0.001 |
<0.001 |
0.006 |
||
|
SGRL, % |
1.43a |
1.20c |
1.31b |
1.08d |
0.004 |
<0.001 |
<0.001 |
0.364 |
||
|
Yield, kg/m3 |
5.48a |
4.41b |
5.63a |
4.23b |
0.0605 |
0.826 |
<0.001 |
0.025 |
||
| abcd : Means in the same row without common letter are different at p<0.05 T: trash fish; L: black soldier larvae; W: water source; F: feed type; WxF: interaction | ||||||||||
In this experiment, the growth performance of seabass fed trash fish was higher than fed BSFL and reared in the salinity of 10‰ higher than that in brackish water. The growth performance results in this study were similar to those studied on snakehead fish (Chana micropeiles) reared with different feeds, and similar results when studied on seabass (Lates calcarifer) with different salinity (Hoa and Dung 2016; Hassan et al2020). The results of this study showed a higher growth rate than the results of Hai (2013) when raising seabass in fresh water and fed trash fish. However, raising seabass in a salinity of 5‰, the SGRW and
SGRL were higher (4.84% compared with 3.83% and 1.95% compared with 1.43%) of the other authors (Khanh et al 2010; Thoai 2000). The results of SGRw are similar to the results of Sen Sorphea et al (2019) when feeding seabass commercial feed with 58% CP. The survival rate of fish cultured in this experiment was 84.44% and higher than 40% - 56.7% of other reports (Khanh et al 2010; Thoai 2000).
In the world, there have been many studies to replace fish meal (FM) with BSFL in diets for aquatic animals. According to Aniebo et al (2009), replacing 25% of FM with BSFL in the diet of African catfish (Clarias gariepinus) could help to reduce production costs. The study showed that when replacing FM with BSFL meal, it did not affect the growth, FCR and meat quality of fish with a proportion less than 50% in diets for seabass (Lates calcarifer) cultured in fresh water and tilapia (Oreochromis niloticus), however in yellow catfish (Pelteobagrus fulvidraco) it is 20 to 25% (Katya et al 2018; Rana et al 2015; Hu et al 2017; Xiaopeng Xiao et al 2018). Bruni et al (2019) reported that when replacing 100% FM by BSFL, it affected the quality of fish fillet of Atlantic salmon (Salmo salar) and encouraged rearing of BSFL on a substrate supplemented with brown algae to improve fish meat quality.
On the other hand, Ronghua Lu et al (2000) indicated that replacement of less than 50% soybean meal (SBM) with BSFL in the diet of grass carp (Ctenopharyngodon idellus) did not affect the yield and quality of fish meat. Kroeckel et al (2012) pointed out that replacing soybean oil with oil of BSFL in the diet of carp (Cyprinus carpio var. Jian) did not affect the growth rate but reduced the amount of fat in fish meat.
The authors acknowledge the partial finance support of the Strong Research Group Program of Hue University and the partial support of the funding for science and technology research from the Hue University, code DHH2021-02-149.
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