Ph.

D Zoology Synopsis IMBB/2022/-------

Impact of insect meal on growth performance, nutrient digestibility and

proximate composition of Labeo rohita

Submitted By

Name: NIDA ISMAT

Registration No.: DZOO02203004

Degree Program: Ph. D Zoology

Date of Submission: 10/02/2023

Supervisor I: DR. SIKANDAR HAYAT _______________________

Supervisor II: DR. MAJID HUSSAIN _______________________

Coordinator: DR. SIKANDAR HAYAT _______________________

Pro-Rector IMBB: PROF. DR. MUHAMMAD ASHRAF _______________________

Institute of Molecular Biology and

Biotechnology

The University of Lahore

2023
Ph. D Zoology Synopsis IMBB/2022/-------

UNDERTAKING

I, NIDA ISMATREG#DZOO02203004under the supervision of DR. SIKANDAR HAYAT,

declare that it is sole responsibility of me and my supervisor to execute and complete the
research work titled “Impact of insect meal on growth performance, nutrient digestibility
and proximate composition of Labeo rohita” in the given time. We will be responsible if we
fail to complete it in time.

_____________________ ___________________

NIDA ISMAT DR. SIKANDAR HAYAT

Assistant Professor
Ph. D Zoology Synopsis IMBB/2022/-------

Abstract

For rapidly growing human population maximum food production is necessary.

Aquaculture is an emerging field to compensate this food requirement of whole world living
human population. Fish is good source of protein and well accepted food item. It is major
product of aquaculture system. For maximum fish production, proper fish food is necessary. This
study is aimed at preparation of fish food from insect larvae meal and to examine its effects on
growth performance, nutrient digestibility and proximate composition of specific fish species.
Fish, Labeo rohita is selected in this study purpose because of its high demand, high survival rate
and adaptation to different environmental conditions.
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1. Introduction

The population pressure on earth is increasing day-by-day. Human population is main cause
of urbanization and according to UN projections, population is expected to reach 9.3 billion in
2050 (Division, 2010). It is estimated that food requirements will increase about sixty percent
globally in 2050 (Alexandratos & Bruinsma, 2012). Due to population pressure, most countries
in the world are facing problems like shortage of proteins and food supplies (Cochrane et al.,
2009). To support this large number of human population maximum production of food is
necessary. Aquaculture industry is important for production of aquatic animals like fish,
mollusks and crustaceans and its growth rate recorded annually is 5.8% (FAO, 2012). It is also
regarded as fastest food producing sector in world (Newman, 2000). A huge concentration of
proteins, omega 3 poly unsaturated fatty acids is present in aquatic food (Tacon & Metian, 2013).
Aquatic food is beneficial for human health and also reduces the risk of coronary heart diseases
(Rudkowska et al., 2010). Food from aquaculture sources supplies vitamins like B12 and D3,
amino acids and minerals such as iron, zinc and selenium (Lund, 2013).

Fish food is the vertebral column of aquaculture industry as fish production is dependent on
it (Haghbayan & Shamsaie Mehrgan, 2015). It is exhibited that composition of fish food has
strong effects on quality and chemical properties of fish flesh (Ostbye et al., 2018). Fishmeal is
main food item in aquaculture industry and rich in proteins, vitamins, minerals and lipids
(Lindberg et al., 2016). It is used as protein source in fish production system and crude protein
value estimated in fishmeal is 60-72% (Cline & Richert, 2000). It contains maximum
concentration of vitamins, fatty acids and essential amino acids (NRC, 2011). Fish meal contains
huge concentration of unsaturated fatty acids like eicosapentaenoic acid (EPA) (20:5n-3) and
docosahexaenoic acid (DHA) (22:6n-3) mostly beneficial against inflammatory, autoimmune and
heart diseases (Simopoulos, 2012) Fish oil is extra item obtained during fishmeal preparations
from fish scrapes and is rich in proteins (Turchini et al., 2010). It is also used along with
fishmeal for aqua-cultural production (Hardy & Masumoto, 1990).

As competition for fish meal and fish oil is increasing day by day by both humans and other
food manufacturers organizations, this competition raised their cost value of so high making it
less beneficial for aquaculture industry (Tschirner & Simon, 2015). As aquaculture industry will
progress more due to increasing demands of food, fishmeal and fish oil will not be able to
Ph. D Zoology Synopsis IMBB/2022/-------

support this industry in next few years (Gasco et al., 2018). So, there is need to minimize the
dependence of feed industry upon these expensive and finite commodities of wavering supply
and cost and search for new feed preparation resources that should be cheap and easily available
(Bondari & Sheppard, 1981).

Insects are important in preparations of cheap fish diet for aquaculture industry (Van Huis et
al., 2013). High concentration of proteins is found in insects (Bosch et al., 2014). Carnivorous
and omnivorous fish species mostly use insects as natural source of food (Henry et al. 2015).
Among insects black soldier fly (BSF) and mealworm possess a superlative importance in
aquaculture industry (Meneguz et al., 2018). BSF converts organic waste into high energy
proteins and fats (Ewald et al., 2020). The protein content of BSF larvae is 35-46% (Meneguz et
al., 2018). The value of lipid content present in BSF is 19-37% (Makkar et al., 2014). Ash value
in BSF larvae is nearly 11-28% (St‐Hilaire et al., 2007). The concentration level of saturated
fatty acids present in BSF larvae lies in range of 58-72% (Surendra et al., 2016). Likewise, the
crude protein value of mealworm (Tenebrio molitor) is about 47- 60. % (NRC, 2012). Average
value of fat present in mealworm is 30.8% and ash value is 4.2% (NRC, 2012). The crude fiber
value lies in range of 4.19-22.35% (NRC, 2012).

Fish is a good source of proteins for humans and is mostly used as food item in every part of
world (Béné et al., 2016). It provides about sixteen percent animal proteins to human’s
population (Food & States, 2013). Due to nutritive qualities fish meat plays key role in human
nutrition (Sary et al., 2022). Thirty percent of food eaten by fish is converted into proteins
(Hasan & Halwart, 2000). Rahu (Labeo rohita) is an omnivorous species and tolerates wide
environmental conditions and temperature fluctuations (Menaga and Fitzsimmons, 2017). It is
well accepted bony fish as food item in most parts of world (Abdel et al., 2000). Optimum
temperature for growth of Rahu is 28-32o C (Guo et al., 2011). Its meat is tasty and white in color
(Oliveira Filho et al., 2010). It retains essential zootechnical characteristics and good acceptance
in different country market (Albergaria et al., 2022). Growth rate of rahu is so fast and converts
plant proteins into high quality proteins in fish flesh (El-Sayed, 2006). Environmental stress
resistance of rahu like population pressure, water pollution and pH is high than other fish species
(Abdel-Tawwab et al., 2014).
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2. Literature review

Antony et al. (2022) nourished goldfish fingerlings on BSF larvae meal. The rearing period
was sixty days. The objective of experiment was to find the effects of BSF based fish meal on
growth rate of under trial fish fingerlings. Six types of test diets were formulated and ratio of
BSF larvae meal in each test diet was 0% (T0), 20% (T20), 40% (T40), 60% (T60), 80 % (T80)
and 100 % (T100) respectively. During the trial period, fingerlings were provided with two times
prepared test diets daily. Test diet containing 60% BSF meal expressed maximum growth rate
and high acceptance by fingerlings. Test diets containing more than 60% BSF larvae meal
resulted in reduced growth rate and less feed acceptance by goldfish fingerlings. Form results, it
was concluded that 60 % based BSF larvae meal test diet is better for goldfish fingerlings
growth. Impacts of insect meal on fish growth have been studied by Hua, (2021). He reported
that different quantity levels of insect meal in place of fish meal did not retard growth activities
of fish. However, he expressed that concentration of insect meal should be less than 29%, higher
concentration impose negative effects on growth of fish or any other aquatic animal. A similar
study was conducted by Su et al., (2017) who used mealworm in fish food replacing with fish
meal and provided it to yellow catfish juveniles. The inclusion level of mealworm in test diets
was 0%, 25%, 50% and 75% respectively. He expressed that test diet containing 25%
concentration of mealworm is beneficial for growth activities with no negative effects.

Belforti et al., (2015) conducted a study to determine the effects of insect meal on nutrient
digestibility in Rainbow trout. He used mealworm larvae in preparation of test diet and made
three types of test diets. The ratio of mealworm in test diets was 0%, 25% and 50%. He reported
that feeding rate and its conversion ratio of diet containing 0% accumulation of mealworm was
higher than other test diets. Rainbow trout juveniles group reared at 50% test diet expressed
lowest protein digestibility than other two groups. However results related to digestibility of
organic matter, dry matter and lipids was similar from all three treatments. Increased levels of
protein and decreased level of lipids were recorded from fillets of 25% and 50% test group’s
respectively as compared to control group (0%). Concentration level of fatty acids in trout dorsal
muscle was 0%>25%>50% respectively. He suggested that mealworm should be included in
diets of Rainbow trout juveniles.
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Mastoraki et al., (2020) expressed the effects of insect based fish meal on proximate
composition of European sea bass. He used three types of test diets from larvae of insects namely
Tenebrio molitor (TM), Musca domestica (DM) and Hermetiaillucens (HI). The concentration of
insect’s meal was 30% in all test diets replacing fish meal. His experimental period was twelve
weeks. Sea bass juveniles reared at MD, HI and control group expressed same results of whole
body composition and nutrient retention. However nutrient efficiencies of TM group was higher
from other treatment groups. Fatty acid (n-3) was higher in control and HI group whereas n-6
was higher in TM and DM test diet groups. The amino acid profile of control group and test
groups was similar. Cholesterol level of blood plasma was lower in TM and DM groups. Similar
results related to plasma triglycerides, phospholipids, liver amino acid catabolizing enzymes
activities (ALT, AST and GDH) were recorded from control and test groups. Form his results,
he demonstrated that 30% insect meal can be successfully replaced with fishmeal with no
negative effects on proximate composition of European sea bass.
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3. Hypothesis
4. Aims and Objectives
The objectives of current study are:

 Preparation of cost effective diet for Gift tilapia fingerlings.

 Effects of insect meal based diet on growth performance, nutrient digestibility and
proximate composition of Gift tilapia fingerlings.


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5. Methodology
5.1. Study design:

Experimental fish Rahu Labeo rohita fingerlings will be obtain from “Fish nursery unit
Head Balloki”. Fingerlings will be transported in the aerated bags. Before the start of
feeding trials fish will be dipped in NaCl (5g/L) solution for few minutes to make them free
of any present ectoparasites. The fingerlings will be acclimatized in laboratory conditions
for a period of 2 weeks in glass aquaria. During this period, the fish will be fed basal diet
once a day. Physicochemical parameters will be evaluated on daily basis. Replacement of
water will be done after 2 days.

Two trials will be set separately. 15 no of aquaria will be used for each trail. 05 feeding
setups will be handle under each feeding trial. And 03 replicates for each feeding setup will
be carried out. Total no of days for each trial will be 90. 15 no of fingerlings will be stocked
in each water tank. Growth performance will be check fortnightly.

Test diets will be prepared by using following ingredients and on the same formula.

5.2. Test diet preparations:

Black Soldier Fly Larvae Feed

Ingredient 0% 25% 50% 75% 100%

Fishmeal 20 15 10 5 0

Soyabean Meal 22 22.5 23 23.5 24

Wheat Flour 10 8.5 7 6 5

Sunflower Meal 8 8 8 8 8

Canola Meal 10 10 10 10 10

Corn Gluten 30% 15 16 17 17.5 18

Vegetable Oil 3 3 3 3 3

Rice Polish 10 10 10 10 10

Black Soldier fly Larvae 0 5 10 15 20

Ph. D Zoology Synopsis IMBB/2022/-------

Vitamin Premix 1 1 1 1 1

Mineral Mixture 1 1 1 1 1

100 100 100 100 100

CP% 32.255 32.33 32.235 32.138 32.040

Mealworm Feed

Ingredient 0% 25% 50% 75% 100%

Fishmeal 20 15 10 5 0

Soyabean Meal 22 22 22 22 22

Wheat Flour 10 9 8 7.5 7.5

Sunflower Meal 8 8 8 8 8

Canola Meal 10 10 10 10 10

Corn Gluten 30% 15 16 17 17.5 17.5

Vegetable Oil 3 3 3 3 3

Rice Polish 10 10 10 10 10

Mealworm 0 5 10 15 20

Vitamin Premix 1 1 1 1 1

Mineral Mixture 1 1 1 1 1

100 100 100 100 100

CP% 32.255 32.33 32.405 32.393 32.293

Ph. D Zoology Synopsis IMBB/2022/-------

5.3. Growth Activities Measurement:

The growth of all fingerlings involved in experiment will be determined after every two
weeks. Standard length, fork length, total length, body weight, fee conversion ratio (FCR),
specific growth rate (SGR) shall be measured. Calculations shall be performed on the basis of
following formulas.

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

Condition Factor (K) = body weight (g)/body length3 (cm) x 100.

Feed Conversion Ratio (FCR) = feed intake (g)/weight gain (g).

Specific Growth Rate (SGR) = [(ln final mean weight (g) – ln initial mean weight (g)/days fed) x
100].

Protein Efficiency Ratio (PER) = weight gain (g) / total protein fed

5.4. Nutrient Digestibility:

 Chemical Analysis of Feed and Faeces

Moisture content of test diets and faeces will be calculated after oven drying of homogenised
samples at 105oC for 12 h. Micro Kjeldahl Apparatus (InKjel M behr Labor Technik GmbH D-
40599 Dusseldorf) will be used to determine the crude protein (CP) content (N × 6.25). Soxhlet
system (Soxhlet Extraction Heating Mantels, 250 ml 53868601) will be used to analyze the crude
fat (EE). Crude fiber content will be estimated by digestion with 1.25% H 2 SO4 and 1.25%
NaOH. The difference in weight after calcination indicates the quantity of fiber present. Ash
contents will be determined by ignition at 650 oC for 12 h in electric furnace (Naberthern B170)
to constant weight.

Total carbohydrates (N-free extract) will be calculated by using standard formula:

Total carbohydrates (%) =100-(EE % + CP % + Ash % + CF %).
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Oxygen bomb calorimeter will be used to estimate the gross energy (GE) value of
samples. Chromic oxide contents in the diets and feces will be estimated after oxidation with
molybdate reagent using a UV-VIS 2001 Spectrophotometer at 370 nm (Divakaran et al., 2002).

Apparent nutrient digestibility coefficient (ADC) of test diets will be calculated using the
standard formula (NRC, 1993):
ADC (%) = 100 - 100 × (% Marker in feces × % Nutrient in diet / % Marker in diet × %
Nutrient in feces)
 Digestive enzymes activity analysis

After the completion of experiment, from each aquarium three fish (n=3 triplicate per
treatment) will be selected randomly for collection of intestinal samples. Chilled phosphate
buffer (pH 7.8) will be used to homogenize the samples. Next the samples will be centrifuged at
4500 rpm for five minutes. The collected supernatant will be stored at -20°C until the enzyme
assay activity will start. Quantifying of amylase activity will be fulfilled through 3.5 dinitro
salicylic acid colorimetric technique that is according to Clark method (1964). Cherry and
Crandel (1932) method will be utilized to expose the activity of lipase by measuring the fatty
acid release due to enzyme hydrolysis of olive oil. Lowry, Rosebrough and Farr technique will
be used to measure protease activity. The enzyme activity will be examined on the basis of
changes in absorbance using a spectrophotometer (Lamba 25UV Win Lab V 6.0). One unit of
enzyme activity will be shown as 1µg of maltose, fatty acid and tyrosine released per minute.

5.5. Hematological study:

Blood samples will be collected from the caudal vein using heparinized syringe and will be
analyzed for hematological indices. Micro-hematocrit technique will be used to determine
hematocrit/packed cell volume (PCV) value as per Brown (1980). Red blood cells (RBC) and
white blood cells (WBC) counts will be performed using a Neubauer counting chamber (Blaxhall
and Daisley, 1973). Hemoglobin (Hb) concentration will be measured as described by
Wedemeyer and Yastuke (1977). Mean corpuscular hemoglobin concentration (MCHC); mean
corpuscular hemoglobin (MCH) and mean cell volume (MCV) will be calculated through
formulae:

MCHC = Hb /PCV × 100

Ph. D Zoology Synopsis IMBB/2022/-------

MCV = PCV/RBC × 10

MCH = Hb /RBC × 10

5.6. Proximate composition analysis:

Three fish (n=3 triplicate per treatment) fishes from each experimental tank will be collected
at the end of feeding trial to expose the whole-body composition. For proximate composition
analysis moisture, crude protein, crude lipid and ash contents of experimental diets as well as
whole body will be measured by following standard protocols (AOAC, 2010). The amino acid
composition of control and treatment diets will be determined through ultra-pressure liquid
chromatography (UPLC; Model—Waters ACQUITY-UPLC, Waters), following the method
described by Ishida et al. (1981).

5.7. Statistical Analysis:

All the experimental data that will be obtained after completion of feeding trial will be
expressed as mean values ± standard deviation (SD). Each and every data will be tested for
normality (Shapiro–Wilk test) and homogeneity of variance (Levene’s test) and will be
transformed if the data not expressed normal distribution. One-way ANOVA will be applied to
test the significant differences of data, wherein the Tukey's test will be used to express
significant differences between experimental groups. Differences will be demonstrated as
significant if p < 0.05. The software SPSS 20.0 for windows (SPSS) will be utilized to
statistically analyze the whole data.
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