Evaluation of growth performance,feed efficiency and nutrient digestibility of red hybrid tilapia fed dietary inclusion of black soldier fly larvae(Hermetia illucens)

Hsniyti Muin,Norhidyh Mohd Tufek,b,＊

a Aqua-Nutri Biotech Laboratory,Institute of Biological Sciences,Universiti Malaya,Kuala Lumpur,50603,Malaysia

b Ladang Mini,Institute of Biological Sciences,Faculty of Science,Universiti Malaya,Kuala Lumpur,50603,Malaysia

Keywords: Insect meal Black soldier fly larvae Red hybrid tilapia Digestibility Hermetia illucens

ABSTRACT Insects,particularly black soldier fly larvae (BSFM),Hermetia illucens,have been regarded as promising protein and lipid sources in animal feed,including fish.Digestibility is the key factor that affects nutrient utilization,feeding physiology and efficiency.In the present work,the effect of BSFM on the digestibility of red hybrid tilapia (RHT) compared to fish meal (FM) was ultimately unveiled.A 56-day growth trial was performed with 60 RHT (～30 g) randomly distributed into six tanks and fed two experimental diets containing FM as a reference diet and BSFM as a test diet.In addition,faeces were collected to determine their chromium concentration and proximate composition.The apparent digestibility coefficient (ADC) of dry matter,crude protein and energy in the BSFM diet were observed to be significantly higher than those in the FM diet.Besides,the growth performance of fish fed with the BSFM diet exhibited significantly higher (P <0.05) weight gain,specific growth rate,protein efficiency ratio and lower feed conversion ratio than those in the FM group.This study shows that the BSFM feed was well digested by tilapia and improved its growth performance.

1.Introduction

Fishmeal and fish oil (FM and FO) are major aquafeed ingredients due to their excellent nutritional value manufactured from wild-caught fish.However,the market of FM and FO is being heavily exploited as these commodities are being traded internationally at low prices without meeting the minimum standard and overfishing.As a result of the imbalance supply of FM and FO in the past decades,a deliberate attempt to introduce alternative sustainable ingredients has gained wider attention to reduce the dependency on FM and FO.Although the production of FM and FO was reported to be stable recently,it is still unable to fulfil the huge demand of the aquaculture industry (FAO,2016).

Within the animal feed industry,several species have been extensively studied,including black soldier fly,mealworm,silkworm,and crickets,to replace the expensive and unsustainable FM and FO (Gasco et al.,2016;Henry et al.,2015;Roncarati et al.,2015;Taufek et al.,2017,2021).Due to minimal environmental impact,low greenhouse emission,the proper level of the indispensable amino acid (essential)and fatty acids,and cost-effective farming process,these insects are regarded as great candidates for aquafeed ingredients.

Preliminary results have shown that BSFM is a promising protein and lipid source for various fishes (Belghit et al.,2018;Devic et al.,2018;Henry et al.,2015;Iaconisi et al.,2017;Roncarati et al.,2015;Sudha et al.,2022;Zarantoniello et al.,2019).Compared to other insects,BSFM can be easily reared in limited space with numerous types of the organic waste stream and converted into high-quality biomass within a short time (Van Huis et al.,2015).However,there is a certain limitation when supplying insect meal to the fish,particularly chitin’s presence,limiting their digestibility and protein utilization (Belforti et al.,2015;Fontes et al.,2019).

Red hybrid tilapia is one of the commercial fishes consumed by the Asian population,particularly in Malaysia.Conventional tilapia farming that relies on imported FM and FO as a food source is unsustainable and costly,especially for the small-scale farmers in these regions.Based on our previous study,BSFM could replace 50% of FM in the tilapia diet without adverse effects on their growth,feed efficiency and health(Muin et al.,2017).Therefore,this study aims to assess the growth and apparent digestibility in RHT fed with BSFM compared to FM.

2.Material and method

2.1.Experimental diet

The BSFM used within this study was cultivated in the laboratory by utilising soybean curd residue as the food source.They were harvested after 14 days of the feeding period before the prepupal stage and sacrificed before being oven-dried at 50◦C for 24 h.Consequently,the dried larvae were grounded into homogenous powder using the feed mill machine and stored at 4◦C until further use.

Fish feed ingredients including fishmeal soybean meal,corn meal,rice bran meal,dicalcium phosphate (DCP),vitamins and minerals premixes were purchased from a local feed supplier.First,the dry ingredients were grounded to a fine powder using a hammer mill (Disk mill FFC 454).Next,chromic oxide (Cr2O3) (Sigma) was added to the diet at a 1% concentration as an inert marker.The BSFM,FM and other ingredients were then subjected to proximate composition and amino acid analysis to assist in the diet formulation.

Two diets were formulated based on 30% crude protein (isonitrogenous) content.The reference diet (with FM as the primary protein source) and test diet (with BSFM as the primary protein source)were formulated (Table 1) using WinFeed 2.8 software.All the raw ingredients were homogenously mixed before being pelleted with a 1 mm diameter using a mini pellet machine (KCM-Y123M-4) and oven-dried to constant weight for 24 h at 60◦C.The feed was then refrigerated at 4◦C for further use.

Table 1 Ingredients of formulated diets (g/kg) of diet and its nutrient composition.

2.2.Experimental design

A total of six plastic (4′x 3′x 1’) tanks filled with dechlorinated 120 L water equipped with a top filter pump and oxygen supply at 25 L min-1flow rate were used during the feeding trial.The experiment was conducted at the Institute of Biological Sciences,Faculty of Science,Universiti Malaya,over 56 days.

Juvenile tilapia weighed 30.0 ± 1.0 g obtained from the Fisheries Research Institute,Jelebu were acclimatised for 2 weeks and fed commercial pellets twice per day.Before the feeding trial,the fish were weighed and randomly distributed into six tanks,each with 10 fish per tank with triplicate treatments.The fish were fed twice daily at 0900 and 1500,and the feed was given at the amount of 3% body weight (BW)daily during the feeding trial.Feed intake (FI) was recorded by observing the uneaten feed after 15 min of feeding.The feed percentage was adjusted according to BW fortnightly.Any mortality was observed and recorded every day.The water was changed at a 50% rate every two days to maintain water quality.Dissolved oxygen (DO),pH and temperature were monitored every day according to the Standard Methods for the examination of water and wastewater (APHA 1992).The water temperature was maintained at pH 6.0 and 28.13 ±0.15◦C,DO above 4.0 mg L-1,ammonia <1.0 mg L-1,and nitrate <1.7 mg L-1.

The faecal materials in each tank were siphoned 2 h after the feeding session and oven-dried for 24 h at 50◦C before being ground into powder.Daily samples were pooled together from each tank and stored at -20◦C for further analysis.The faecal samples were analysed to estimate the nutrient proximate composition and chromium concentration.

2.3.Proximate and chemical analyses

All dry ingredients,including experimental diets and faecal materials,were analysed for moisture,dry matter,protein,lipid and fibre content using the standard proximate analysis method (AOAC,2003)(Table 2).Crude protein content was determined via the Kjeldahl technique (FOSS Tecator Digestor Auto -Sweden).The crude lipid content was determined by the Soxtec System (petroleum ether extracted).Dry matter was calculated after the weight loss due to the 24-h drying process at 105◦C.Ash was determined after being oven-dried at 600◦C until its weight became constant.Crude fibre analysis was carried out using a fibre cap procedure.Chromium concentration in faecal materials and experimental feed were determined following the Furukawa and Tsukahara (1966).The ADC of dry matter,protein,lipid,ash and fibre in the diets were determined based on the study by Köprücü and Özdemir (2005).Chitin was estimated using the aciddetergent fibre (ADF) and protein linked to ADF (ADIP) according to Marono et al.(2015).Nitrogen-free extract (NFE) was calculated as;NFE: 100% -(crude protein % +crude fat % +crude fibre % +crude ash%).Meanwhile,the gross energy was calculated using the following factors according to Schulz et al.(2005): crude protein=23.9 kJ/g,crude lipids=39.8 kJ/g,and NFE=17.6 kJ/g.

Table 2 Nutrient compositions and amino acids of FM and BSFM.

The amino acid (AA) content of the raw fish meal (FM) and BSFM was determined using the high-performance liquid chromatography(HPLC) with PICOTAG™ method,and the contents were analysed by comparing the peak retention times to a known standard according to Heinrikson and Meredith (1984).

2.4.Analysis of the experimental data

At the end of the study,all fish from each treatment were weighed to determine the following indices:

The apparent digestibility coefficients (ADC) of crude protein,lipid,dry matter and energy in the fish were calculated as (Rahman et al.,2016):

The ADC of protein,lipid and energy:

The ADC of dry matter:

2.5.Statistical analysis

Statistical analysis was performed using the SPSS version 20.0 statistical package.All data for growth performance,feed efficiency and ADC were subjected to an independentt-test,and the differences were regarded as significantly different between the treatment groups (P<0.05).Confidence intervals were calculated as mean ± SEM (standard error mean) with triplicates in each treatment.

3.Results and discussion

3.1.Nutrient content of BSFM,FM and experimental diets

The nutrient composition illustrated in Table 1 shows that both diets are isonitrogenous (30% protein).In regard to nutrient composition,lipid composition in the test diet was higher (12.78%) compared to the reference diet (9.44%) due to the high lipid content in the BSFM(31.08%).The chitin content in the BSFM diet (1%) was estimated according to the raw BSFM composition (3.32%) after ADF analysis.In general,all essential amino acids (EAA) are present in both diets and sufficient for tilapia requirement except for arginine,which is the only EAA observed to have a lower level than the ideal amount required for tilapia.

The proximate composition in this study shows that BSFM contains 43.17% protein and 31.08% lipid (Table 2).This data was similar to previous studies that reported that the prepupae of black soldier fly contained approximately 40% protein and 30% fat (Muin et al.,2017;Newton et al.,2005).Generally,the nutrient composition of BSFM depends on the quality and quantity of food ingested (Barroso et al.,2014;Newton et al.,2005),the stage of development,and the processing method of the larvae (Aniebo &Owen,2010).In terms of amino acids,the values for BSFM were lower than FM.This is due to different protein content in both FM and BSFM,where FM has the highest protein compared to BSFM.Although AA values in BSFM were less than FM,Henry et al.(2015) confirm that the AA profiles of Diptera are considered close to FM.

3.2.Growth performance and feed utilization

The growth performance and feed utilization of the tilapia fed with the experimental diets are shown in Table 3.All fish involved in the experiment survived until the end of the feeding trial.Looking at the growth parameters,fish supplemented with the BSFM diet exhibited significantly higher weight gain,SGR,and PER than those in the FM group.On the other hand,FCR was significantly lower in those fed with BSFM than the FM group,which highlighted the efficiency of BSFM to improve the growth performance of RHT.

Table 3 Growth performance and feed utilization of tilapia fed with experimental diets.

Numerous trials have been conducted where FM was replaced with BSFM at different inclusion levels show a positive effect on growth and feed utilization in different fish species (Abdel-Latif et al.,2021;Abdel-Tawwab et al.,2020;Alfiko et al.,2021;Belghit et al.,2019;Bondari &Sheppard,1987;Bruni et al.,2020;Karapanagiotidis et al.,2014;Li et al.,2017;Magalhães et al.,2017;Rawski et al.,2020;Renna et al.,2017;Taufek et al.,2021;Yamamoto et al.,2022).However,Kroeckel et al.(2012) reported reducing the growth of juvenile turbot with the increasing amount of BSFM in their diet.The negative effect was recorded due to the low palatability of the diet.A similar finding was reported by Muin et al.(2017),where weight gain of red tilapia decreased as the inclusion of BSFM increased further.The result indicates that red tilapia fish could optimally accept the BSFM up to 50%inclusion level.On the other hand,a study on juvenile hybrid grouper shows that BSFM could be incorporated at least 30% into their diet without any adverse effect on growth performance(Mohamad-Zulkifli et al.,2019).

3.3.Digestibility trial

The ADC provides information on how much of the feed was digested and absorbed by the fish.Hence,the digestibility of the feed ingredients is the basis for assessing their bioavailability and sustainability to be included in fish diets (Fontes et al.,2019).In addition,the ADC of the feedstuff will help precisely formulate cost-effective feeds that satisfy fish requirements and maximize feed utilization and profit (Li et al.,2013).However,high outcome variability was observed in fish digestibility when consuming insect meals.In the current study,the ADC of FM and BSFM diets summarised in Table 4 demonstrated significantly higher ADC components (protein,ash and dry matter) in BSFM except for lipid.In this study,the BSFM diets resulted in significantly higher ADC protein and similar outcome was also reported by Tippayadaraet al.(2021).They found higher ADC protein was observed in Nile tilapia (Oreochromis niloticus) fed BSFM compared to FM diets.Overall,the results were in line with previous studies on rainbow trout (Dumas et al.,2018;Renna et al.,2017),and Atlantic salmon (Fisher et al.,2020;Lock et al.,2016),where BSFM significantly increases the ADC nutrients compared to those given only FM.Although there was no significant difference in ADC lipid for both diets,BSFM fed fish shows numerically higher ADC lipid than FM,suggesting that the lipid in BSFM is easily absorbed and digested by tilapia.However,high lipid content in the BSFM diet might influence the poor ADC lipid of RHT in the present study compared to other fish species,including hybrid grouper and rainbow trout as described by Mohamad-Zulkifli et al.(2019) and Renna et al.(2017).

Table 4 Apparent Digestibility Coefficient (ADC %) of crude protein,crude lipid,dry matter and gross energy of tilapia fed with the experimental diets.

With regard to the digestibility of BSFM,Zhou et al.(2018) reported that the full replacement of the FM diet with the BSFM in the Jian carp diet does not affect their growth and digestibility.Furthermore,Mohamad-Zulkifli et al.(2019) also observed highly digestible BSFM when oven-dried when fed to hybrid grouper.However,studies by Belghit et al.(2018) reported that replacing the dietary FM and soy protein concentrate with BSFM significantly reduces the ADC of protein,lipid and all amino acids in Atlantic salmon even though they are still highly digestible.In addition,several fishes such as turbot (Kroeckel et al.,2012),meagre (Guerreiro et al.,2021),sea bream (Takakuwa et al.,2022) and rainbow trout (Belforti et al.,2015) have shown unsatisfactory ADC after being fed with insect meal.Fontes et al.(2019) reported the decrease of ADC due to the presence of higher chitin in insect meals.

Chitin is hypothesised to reduce the access to chitinase and proteinases,affecting intestines’ protein and lipid absorption,thus preventing the absorption of proteins and lipids by the intestine (Lock et al.,2018).Furthermore,according to Taufek et al.(2016),chitin might influence the fish feed intake and nutrient digestibility,resulting in growth suppression.Chitin content in raw BSFM in this study was within the range of 2.86%–5.50%,as reported by Marono et al.(2015),who examined six different samples of dried BSFL collected from different laboratories.However,the value obtained was lower than the one reported by Finke (2013) (5.41%) and Kroeckel et al.(2012),who reported 96 g kg-1dry matter of theHermetia illucenslarvae.

The potential of chitin digestibility varies between fishes.In certain species,chitin is deemed to have immunostimulant property (Lock et al.,2018)that is useful to prevent fishes from contracting the disease and bacterial infection (Esteban et al.,2000;Gopalakannan &Arul,2006;Sakai et al.,1992).This study’s BSFM diet with 1% chitin does not affect tilapia growth.Similar studies were observed in Kono et al.(1987),in which the growth of red sea bream,Japanese eel and yellowtail were not affected after being fed with chitin-supplemented diets.Mari et al.(2014) also proposed the inclusion of 1% chitin and chitosan that could enhance carp’s innate immune response and disease resistance.The chitin supplementation enhanced the growth and feed utilization,improved the resistance to infection by Aeromonas hydrophila,and improved certain indicators of innate immunity of tilapia (Abd El-Naby et al.,2019;Abu-Elala et al.,2015;Qin et al.,2014;S Elserafy et al.,2021).They also recommend using chitin as an aquafeed additive due to its efficient immunostimulant and bio-remediating agent in aquaculture.

4.Conclusion

The results obtained in this study indicate that BSFM is well accepted by tilapia with higher nutrient digestibility than the fishmeal.Furthermore,the results were supported by the increase in growth of the fish supplied with BSFM,suggesting that the inclusion of insect meal in tilapia feed will increase the palatability,nutrient availability and digestibility to fulfill the nutrition requirement.However,further research is required to reduce the lipid composition in BSFM by partially defatting the larvae before processing,hence resulting in a high crude protein level.

Funding

This research was sponsored by the Ministry of Higher Education Malaysia for Fundamental Research Grant Scheme (FRGS) (FRGS/1/2019/WAB01/UM/02/3) and [FP073-2019A] awarded to Dr Norhidayah Mohd Taufek.

Availability of data and material

The authors confirm that the data are available upon request.

Authors’ contributions

Hasniyati Muin: Conceptualization,Methodology,Visualization,Investigation,Writing– original draft,preparation Norhidayah Mohd Taufek: Supervision,Project administration,Writing– review &editing.

Ethics approval

Ethical clearance in handling the animal was obtained from Institutional Animal Care and Use Committee,Universiti Malaya (Ethics Reference Number: S/03102019/10,052,019-01/R).

Declaration of competing interest

The authors declare that there are no conflicts of interest associated with this publication.

Acknowledgement

We want to thank the Ministry of Higher Education Malaysia for the Fundamental Research Grant Scheme (FRGS/1/2019/WAB01/UM/02/3) and FP073-2019A awarded to Dr Norhidayah Mohd Taufek.We also want to thank Mr Hanan Mohd Yusof and his team in Freshwater Fisheries Research Institute,Glami-lemi,Jelebu,Malaysia,for their technical assistance.Finally,our appreciation goes to the Institute of Biological Science for providing the necessary laboratory facilities for this research.