Protein quality of four indigenous edible insect species in Nigeria

Oibiokpa Florence Inje,Akanya Helmina Olufunmilayo,Jigam Ali Audu,Abubakar Abubakar Ndaman,Egwim Evans Chidi

Department of Biochemistry,Federal University of Technology,P.M.B 65,Minna,Nigeria

ABSTRACT Food security is a serious concern particularly for developing countries.To overcome hunger and malnutrition there is a need for increased research towards finding alternative and cheaper sources of nutrients.Insects have been reported to be rich in protein and could be alternative sources of protein.This work was therefore designed to determine the protein quality of moth caterpillar,termite,cricket and grasshopper and the effects of diets supplemented with these insects on some biochemical and haematological indices of rats.The amino acid compositions of the insects were determined using standard analytical methods.Five iso-nitrogenous and isocaloric diets were formulated on a 10% protein basis with the insects and casein.A Nitrogen free diet was also formulated as control.Thirty six (36) young weanling albino rats(21-28 days old)were divided into 6 groups and fed with the diets ad libitum for 28 days.Their weekly weight gain and daily feed intake were recorded,urine and faeces were collected for nitrogen determination using Kjedahl method and the data obtained used to calculate the various protein quality indices.After the feeding trial periods,the rats were sacrificed,blood samples collected and organs excised for various analyses.Cricket was found to have the highest amino acid score(0.91),protein efficiency ratio(1.78),net protein ratio(3.04)biological value(93.02%)and protein digestibility corrected for amino acid score (0.73) as compared to other insect proteins analysed.The organ body weight ratios of the liver,spleen,lung and heart of rats placed on the insect supplemented diets were not significantly different(p ＞0.05)from those fed with casein and basal diets.Serum total protein concentrations in rats fed with cricket diet were not significantly different(p ＞0.05)from those fed casein diet while serum LDL cholesterol concentration was lowest in rats fed cricket diet.The finding in this work that the selected insects are nutritious and safe for consumption may alleviate the fear of entomophagy thereby reducing the overdependence on conventional animal proteins.

Keywords:Protein quality Insect species Biochemical parameters Haematological parameters Entomophagy

1.Introduction

The nutritional value of a food material largely depends on the quality of protein present in it[1].Dietary proteins provide amino acids for the synthesis of plasma proteins,which are essential for maintaining osmotic balance,transporting substances through the blood,and maintaining immunity.Proteins also play important roles as enzymes and hormones in the body[2].The protein quality of any food is a measure ofits ability to provide all the amino acids required for growth and maintenance of body functions and the digestibility of the protein [3].The quality of proteins has historically been assessed based on rat nutrition studies(in vivoassays).These assays measure growth or nitrogen balance as indicators of protein utilisation and metabolism.Protein quality assays are designed to reflect the essential amino acid content,bioavailability of the amino acids and protein digestibility of the food or food ingredient being tested.Amino acid composition data is also commonly compared to reference proteins,then corrected for digestibility(in vivoorin vitro)to obtain a protein quality estimate[4].

Edible insects have been reported to be promising sources of dietary proteins because they contain higher amounts of proteins as compared to traditional sources of proteins such as meat,dairy products and seeds [5-7].Edible insects are also superior to conventional sources of animal protein because they have high feedconversion efficiency[8].This is due to the fact that they are poikilothermic and spend much less amounts of food energy and nutrients but produce more animal protein than the warm blooded animals.Insects also reproduce and grow faster than conventional livestock[9].However,it has been suggested that whole insect proteins are oflower quality than those proteins obtained from vertebrates because of the presence of chitin in their exoskeletons.Chitin is a long polymer of N-acetyl glucosamine and is considered to be indigestible as it has a similar structure with cellulose[8].

In Nigeria,over 30 species ofinsects are commonly used as food amongst which are locust,termites,ants,grasshoppers,weevils and beetles[10,11].The nutrient composition of various insects species in Nigeria have been carried out by several researchers[11-16]but very few studies on the digestibility of proteins from these insects have been reported.This work was therefore designed to determine the protein quality of the following insects species;Gryllus assimilis(Cricket),Melanoplus foedus(grasshopper)Macrotermes nigierensis(termite),andCirina forda(moth caterpillar).These insects have been found to have high protein contents ranging from 43.75% in

Macrotermes nigierensisto 75.08%inMelanoplus foedus.They were also found to be good sources of minerals particularly potassium,essential fatty acids,vitamins(A,E,K,B2and B12)and low in antinutrients like phytates,tannins,oxalates,saponins and cyanogenic glycosides[16].

The nutritional value ofinsects is highly variable.Some insect species have been shown to be rich in lysine,threonine and tryptophan while some other insects do not contain these amino acids in sufficient amounts.For example the termiteMacrotermes bellicoccushas been found to be a good source oflysine and tryptophan while some other species of termites are not [17].These variations in the nutritional value ofinsects are due to the fact that there are thousands of species of edible insects.Even amongst the same species their nutritional value is dependent on the stage of metamorphosis,origin ofinsects and their diet [18].Due to these variations,it is necessary to determine the nutritional value and protein quality of various insects from different sources,as attempts are being made to compile such data to provide awareness on the benefits of eating insects[8].

Acceptance ofinsects as a viable source of protein remains one of the largest barriers in their use as sources of protein.Entomophagy (use ofinsect as food) has been associated with disgust and regarded as a primitive behaviour by the western world therefore little attention has been given to the possibility ofinsects in addressing the problem of food insecurity in developing nations[19].Edible insects are generally known to be special delicacies in developing parts of the world such as Asia,Africa and Latin America but lately the effect of globalization and civilization has led to a drastic reduction in their consumption[20].Reduced acceptance ofinsects as food may be due to fear of the possible risk of eating insects.Insects harvested from the wild may be contaminated with pesticides or heavy metals[18].Some insects contain potent pharmacologically active substances that are known toxins while others secrete toxic metabolites or toxins for defense and other purposes [21].Such insects are usually eaten after taking precautionary measures such as heating in tepid water,burning off of hair on caterpillars,and complete removal ofintestine [8].It is therefore important to study the toxicity of various insects to ascertain whether they could pose any risk to health.

Protein energy malnutrition has been a major public health problem in developing countries due to high prices of food particularly those of animal origin.One out of five children under the age of five suffers from protein energy malnutrition and nearly half of all deaths in children under the age of 5 have been attributed to under nutrition[22].There is also an increase in demand for meat,worldwide and meat consumption is expected to double by 2050[23].There is therefore a need for alternative and cheaper sources of animal protein to meet this rising demand.This study is therefore aimed at determining the protein quality of some commonly consumed insects in Nigeria and the effect of dietary supplementation of the selected insects on some biochemical and haematological parameters of albino rats.The findings of this work may encourage the use ofinsects in solving the problem of food insecurity.

2.Materials and methods

2.1.Collection,identification and sample preparation

The following insects;termite(Macrotermes nigeriensis),cricket(Gryllus assimilis),grasshopper (Melanoplus foedus),moth caterpillar (Cirina forda) were identified by an Entomologist in the Department of Biological Sciences,Federal University of Technology,Minna,Niger State,Nigeria.

The crickets were purchased from Tyomu village in Guma local Government of Benue State,Moth Caterpillars were purchased from Akurba Village in Gboko Local Government of Benue State,Termites were obtained from Wakpa Village,Lafia central Local Government of Nassarawa State,while grasshoppers were purchased from Katsina Central Market,Katsina State.The insects were washed,dewinged,sun dried and powdered using an electric blender and stored in plastic containers for analyses.

2.2.Reagents and chemicals

All chemicals and reagents used were of analytical grade manufactured by British Drug House(BDH)Limited,England and Sigma Aldrich Chemical Company Incorporation,Milwaukee,Wisconson.USA.

2.3.Amino acid analysis

The Amino acid profile of the insects was determined using the method described by [23]using Technicon sequential Multi-Sample Amino Acid Analyser(TSM).

2.4.Determination of amino acid score

The amino score for each essential amino acid was calculated based on the suggested pattern of amino acid requirements for preschool children(2-5 years)[24].The amino acid score for each amino acid was calculated using the formula

The lowest ratio is the amino acid score of the protein.The amino acid with the lowest score indicates the first limiting amino acid[25].

2.5.Protein quality determination

The protein quality of the various insects were determined using growth methods,nitrogen balance methods and the protein digestibility corrected for amino acid score(PDCAAS)method.The growth methods used were the protein efficiency ratio (PER) and Net protein ratio while the nitrogen balance methods used were the true digestibility(TD),Net protein utilisation(NPU)and Biological value(BV).

2.5.1.Dietary formulation

Six experimental diets were formulated as described by [26].Each diet contained 10%of the test proteins.The diets were isonitrogenous and isocaloric (Table1).The components of the diets were mixed manually and stored in well labeled plastic containers.

Table1 Composition of basal,reference and insect supplemented diets(g/kg diet).

2.5.2.Experimental design

Thirty six(36)young male weanling albino rats approximately 21-28 days old were used for the study.The animals were divided into 6 groups of 6 rats.Group 1 was fed with N-free basal diet,Group 2 was fed with nutritional casein (reference protein).The remaining groups were fed with diets containing the different insects sample as the sole source of protein.The rats were allowed to adjust to their respective diets for 3 days.The rats were housed in metabolic cages and were offered their respective dietsad libitumfor 28 days.All experimental rats were handled using the guide for care and use oflaboratory animals by the Institute of Laboratory Animal Resources,Commission of Life Sciences,National Research Council,USA[27].

2.5.3.Determination of daily feed intake

A 24 h feeding trial was conducted.The daily feed intake was determined by weighing the remnants of the feed and subtracting from the amount of feed(in grams)given to the rats on a daily basis.

2.5.4.Determination of body weight gain

The rats were weighed using electronic compact scale(Labtech BL10001).The initial weights of rats were taken and their weights monitored on a weekly basis.Body weight gain was determined using the formula

Body weight gain = Final body weight-Initial body weight

2.5.5.Collection of urine and faeces

Urine and Faeces of rats were collected on a daily basis from each group of rats in metabolic cages.The faecal sample for each group of rats was dried to constant weight and ground to fine powder and stored in plastic containers.Nitrogen content of the faeces and urine was determined using Kjedahl method[28].

2.5.6.Calculation of protein efficiency ratio(PER)

The PER was determined by dividing the total weekly gain/loss in body weight by the total protein intake[29].

2.5.7.Calculation of net protein ratio(NPR)

The net protein ratio was determined by summing up the average weight gain of animals fed with test protein and the average weight loss of animals fed with the nitrogen free diet divided by average protein intake[30].

2.5.8.Calculation of true digestibility,net protein utilisation and biological value

The nitrogen intake and nitrogen excreted in urine and faeces of animals fed with the test protein,reference protein and nitrogen free diets were used to calculate the true digestibility,biological value and net protein utilisation of the proteins[31,21,32].

Where I is the nitrogen intake(mg)

F=Faecal nitrogen of rats on test diet(mg)

FO=metabolic faecal nitrogen(from basal diet)

U=nitrogen excreted in urine of rats on test diet(mg)

UO=the endogenous urinary nitrogen from basal diet(mg)

2.5.9.Calculation of protein digestibility corrected for amino acid score(PDCAAS)

The Protein digestibility corrected for amino acid score for each protein was calculated using the formula:

Protein digestibility corrected for amino acid score(PDCAAS)=amino acid score×%true digestibility[4].

2.6.Collection of blood sample

At the end of the 28 days feeding experiment the final weights of the rats were taken.Each rat was anaesthetized with chloroform in a dessicator and sacrificed.Blood samples were collected in EDTA sample bottles for haematological analysis and plain bottles for biochemical analysis.Blood collected for biochemical parameters were spinned using Eppendorf centrifuge 5702 at 503×g for 10 min to obtain the serum.

2.7.Assays for haematological parameters

The following haematological parameters were determined,Packed cell volume(PCV),White blood cell count WBC,Red blood cell count (RBC),Haemoglobin concentration (Hb),platelet count and white blood cell differentials using Mindray Auto Hematology analyser BC-5300 model.

2.8.Assays for biochemical parameters

The serum obtained was analysed for AST,ALT,ALP,Albumin,Total protein,Total cholesterol,LDL cholesterol,HDL cholesterol and Triglyceride concentrations using Cobas C111 Roche Autoanalyser.

2.9.Statistical analysis

All values were expressed as mean±SEM.Statistical analyses was performed using oneway analysis of variance (ANOVA) followed by Duncan’s multiple range test using SPSS program 20.0.p values ＜0.05 was considered to be significant.

3.Results

3.1.Amino acid composition

The result of the amino acid contents of the selected insect species is shown in Table2.The insects analysed were found to contain all the 20 naturally occurring amino acids,they are therefore sources of both essential and non essential amino acids.The most abundant essential amino acid was threonine in grasshopper (10.60±0.05 g/100 g) while the least abundant was methionine in moth caterpillar(0.62±0.00 g/100 g).Glutamic acid was found to be the most abundant non essential amino acid (13.00±0.20 g/100 g) while cystine was the least abundant(0.40±0.00 g/100 g).

Table3 shows the score of each essential amino acid in the selected insect species.The score for lysine,threonine,valine,phenyalanine+Tyrosine and histidine was highest for grasshopper(1.09,3.12,1.90,2.05,2.40 and 2.95 respectively).The sulphur containing amino acid (methionine+cystine) was found to have the highest score in crickets (1.37) and lowest score in termite (0.47)while isoleucine had the highest score in termite(1.54)and lowest in grasshopper(0.54).The score for leucine was highest for termite(1.18)and lowest for moth caterpillar(0.90).The amino acid score of crickets was higher than the other insects analysed,with termite having the lowest amino acid score.

3.2.Feed intake and weight gain

Fig.1A and B shows the feed intake and weekly weight gain of rats fed with the experimental diets (respectively).At week 1 rats fed with termite diet had the lowest feed intake while crickets had the highest feed intake.There was no significant difference(p ＞0.05)in the feed intake of rats fed with basal,grasshopper,termite and moth caterpillar diets at week 2.The feed intake of animals placed on casein decreased while the feed intake of rats on moth caterpillar diet increased at the third week of the experiment as compared to previous weeks(week 1 and 2).There was also no significant difference(p ＞0.05)in the feed intake of animals placed on basal,grasshopper and termite diet on the third week of the experiment.Rats fed with cricket diet had the highest feed intake at the last week (week 4).Rats fed with basal diet had a drastic loss in weight after the first week of the experiment and lost weight continuously till the end of the experiment while rats fed with casein diet had a slight loss in weight after week 1 but picked up in the subsequent weeks.Amongst rats fed with various insect diets,those fed with termite supplemented diets had the highest weight loss.Rats fed with grasshopper and moth caterpillar supplemented diets had an initial loss in weight at week 1 and week 2 but were found to gain slight weight at week 4 of the experiment.The weight gain of rats fed with cricket supplemented diet was significantly higher(p ＜0.05)than those placed on other supplemented diets.

Fig.1.A:Weekly feed intake of rats fed insect supplemented diets.B:Weekly weight gain of rats fed with insect supplemented diets.

3.3.Protein quality

The protein quality of the insects analysed is shown in Table4.Cricket was found to have the highest PER and NPR values(1.78±0.02 and 3.04±0.37 respectively) while termite had the lowest values for PER and NPR(-2.25±0.09 and 1.32±0.20 respectively).Casein had a significantly higher (p ＜0.05) value for true digestibility as compared to other insect proteins.Termite protein had a higher digestibility as compared to cricket,moth and grasshopper proteins.There was no significant difference(p ＞0.05)in the values of net protein utilisation of all the proteins analysed.Casein had the least biological value while cricket had the highest biological value.

3.4.Organ body weight ratio

Table5 shows the organ/body weight ratios of rats fed with insect supplemented diets.The organ body weight ratios of the liver,spleen,lung and heart of rats placed on the insect supplemented diets were not significantly different from rats fed with casein and basal diets at p ＞0.05 while the organ body weight ratio of the kidney of rats fed with termite supplemented diet was significantly higher (p ＜0.05) than the relative organ body weight ratio of the kidney of rats fed with basal and casein diets.Table2 Amino acid composition of some edible insect species(g/100 g protein).

Values are means of triplicate determinations±SEM.Values along rows with different superscript are signifycantly different(p ＜0.05).* Essential amino acids.

Table3 Amino acid score of essential amino acids in some edible insect species.

Table4 Protein quality of selected insects.

Table5 Organ body weight ratio of rats fed with insect supplemented diets(g/100 g).

Table6 Serum biochemical parameters of rats fed insect supplemented diets for 28 days.

3.5.Biochemical parameters

Serum biochemical parameters of rats fed with insect supplemented diets for 28 days are shown in Table6.The serum ALT activity of rats fed with termite diet was statistically higher(p ＜0.05)than those fed with the basal casein and other experimental diets.There was a significant decrease (p ＜0.05) in the serum AST activity of rats fed with grasshopper supplemented diets as compared to rats fed with other experimental diets.The serum ALP activity of animals placed on casein diet was also significantly lower(p ＜0.05)than animals fed with the other experimental diets.

Rats fed with casein supplemented diet had the highest serum concentration of albumin (48.15±1.37 g/L) while those fed with the basal diet had the lowest concentration of serum albumin(28.21±1.68 g/L).There was no significant difference (p ＞0.05) in the serum albumin concentrations of animals fed with cricket and moth caterpillar diets.There was also no significant difference(p ＞0.05) in the serum albumin concentrations of rats fed with grasshopper and termite diets.The total protein concentration in the serum of rats fed with moth caterpillar and cricket diet was found to be comparable with the that of rats placed on the casein diet while the concentration of total protein in the serum of rats fed with basal,grasshopper and termite diets were significantly lower(p ＜0.05)than those placed on casein supplemented diet(Table6).

The concentration of total protein in serum of rats fed casein diet was found to be statistically similar (p ＞0.05) to those of rats fed moth caterpillar and cricket supplemented diets but statistically higher(p ＜0.05)than rats placed on other insect diets.

Dietary inclusion of the insects analysed was found to have no significant effect on the total cholesterol and HDL cholesterol concentrations in the serum of rats as compared to those placed on casein diet but a significantly lower (p ＜0.05) level of LDL cholesterol was observed in rats fed with cricket supplemented diet as compared to rats fed with casein and other insect supplemented diets.

3.6.Haematological parameters

Results for haematological parameters of rats fed insect supplemented diets for 28 days is shown in Table7.The packed cell volume (PCV) and haemoglobin levels of rats fed with the insect supplemented diets were comparable to those of rats fed with basal and casein diet while RBC count of rats fed with grasshopper was statistically lower(p ＜0.05)than the those of rats fed with casein,termite,and moth caterpillar diet.The platelet counts of rats fed with insect supplemented diets were not significantly different(p ＞0.05) from those of rats placed on the basal and casein diets while the white blood cell count of rats fed diets supplemented with grasshopper and moth caterpillar were found to be significantly higher (p ＜0.05) than those of rats fed with other experimental diets.The neutrophil count of rats fed with basal,grasshopper and termite supplemented diets were significantly higher(p ＜0.05)than rats fed casein,moth caterpillar and cricket diets.The lymphocyte counts of rats fed with casein,basal and insect proteins were not significantly different at p ＞0.05.The monocyte count was highest in rats placed on cricket diet but lowest in rats fed with the basal diet.The eosinophil count of rats on termite diet was found to be significantly higher(p ＜0.05)than those of rats fed with other experimental diets.There was a significant difference(p ＜0.05)in the basophil count of rats placed on the basal and cricket diet while there was no significant difference (p ＞0.05) in basophil count of rats placed on casein,termite and moth caterpillar diet.

4.Discussion

The insects analysed were found to be good sources of several essential amino acids like lysine threonine,leucine,isoleucine,valine,phenylalanine and tyrosine.The concentration of these amino acids in the insects was found to meet the FAO/WHO suggested pattern as requirement of these amino acids for preschool children and adults.Grasshopper was found to be the best source oflysine as compared to other insects.Ahmad et al.,[33]reported lower values as lysine content of grasshopper(6.39 mg/100 g protein) and termite (7.36 mg/100 g protein).Variations in the lysine content may therefore be as a result of differences in the species ofinsects,their habitat and their diet.Lysine is known to be generally low in all cereal proteins which are staple diets in developing countries.It is also the limiting amino acid in most diets[34].The high values for lysine in insects suggest that they may be used for dietary supplementation.

The significantly higher concentration (p ＜0.05) sulphur containing amino acids (methionine and cystine) in cricket as compared to other insects analysed suggest that they may be better sources of these amino acids.The sulphur containing amino acids are important sources of sulphur in the body.Methionine in the form S-adenosyl methionine is required for transmethylation reactions[35]while cysteine is also an important detoxicant of specific substances and an important component of gluthathione (an antioxidant in cells).Methionine is essential for protein synthesis,synthesis of antioxidants and lipotropic compounds like taurine,glutathione,choline,carnitine and S-adenosyl methionine[36].

The selected insects were also found to be good sources of semi essential amino acid,histidine and arginine.These amino acids are referred to as growth promoting factors since they are not synthesised in sufficient amounts during growth,hence they are essential in growing children,pregnancy and lactating women[37].Inclusion of the selected insects in diets of children,pregnant and lactating mothers may therefore meet their daily requirements for these amino acids.

The finding that methionine and cystine were the limiting amino acid in termite(0.47)and moth caterpillar(0.51)while isoleucine was the limiting amino acid grasshopper with a score of 0.54 is inagreement with the report of Ande [38]who reported thatCirina forda(Moth caterpillar),had an amino acid score of 56.70%.Methionine and cystine have been reported to be the limiting amino acids in most insects [39].van Huis et al.,[8]also reported tryptophan and lysine to be the limiting amino acids in most edible insects,however limiting amino acids vary according to species ofinsect[40].Although the limiting amino acid in cricket was threonine,it had a high score of 0.91.Based on the amino acid score of the insect proteins,cricket may therefore have a higher protein quality compared to moth caterpillar,grasshopper and termite.

Table7 Haematological parameters of rats fed insect supplemented diets for 28 days.

The high feed intake of rats fed with cricket supplemented diet as compared to diets supplemented with other insects indicated that cricket may have been more palatable to the rats compared to casein,termite,grasshopper and moth caterpillar diets.The low feed intake of rats fed with moth caterpillar,grasshopper and termite diets is in agreement with findings of Ande[38],who reported that rats fedCirina fordadiet had lower feed intake as compared to rats fed skimmed milk diet (control diet).Low feed intake has been attributed to deficiency or imbalance of amino acids,presence of antinutrients in the protein source or unpalatability of diets[26].The selected insects have been analysed to determine their antinutrient composition and were reported to contain very low concentrations of antinutrients like phytates,tannins,saponins,oxalates and cyanogenic glycosides [16]therefore the low feed intake observed in rats may not be due to the presence of antinutrients but deficiency of amino acids in the rat’s diet.

The significantly higher (p ＜0.05) weight gain of rats fed with cricket supplemented diet as compared those placed on other supplemented diets may be as a result of the higher feed intake and therefore higher protein intake of rats fed with cricket diet.High weight gain has been attributed to high feed intake in experimental animals.The weight loss observed in rats placed on termite,grasshopper and moth caterpillar diets may be as a result oflow feed intake and hence low caloric and protein intake of rats.Ande[38]also reported that the poor growth performance of rats fed withCirina fordadiet as compared to rats on skimmed milk diet may have been due to essential amino acid limitation,low feed intake and intolerance to insects by rats.These reasons may also account for the loss in weight in rats fed insect diets in this study.The presence of antinutrients like saponins and tannins are known to affect protein bioavailability as they inhibit the activity of chymotrysin and trypsin[16].Since all the insects analysed were found to be low in antinutrients,the poor growth performance observed in rats placed on termite,moth caterpillar and grasshopper diets may therefore be attributed to deficiency of sulphur containing amino acids and isoleucine.These insects were found to be rich in other essential amino acids particularly lysine,tryptophan and threonine which are usually low in many cereal proteins that are staple diets in most developing countries [8],therefore termites,moth caterpillars and grasshoppers can still be used to complement cereal proteins to boost the protein quality of diets of people living in developing countries.

Protein efficiency ratio(PER)is a reflection of protein digestibility and amino acid bioavailability of a test protein [4].Results obtained for the PER of the insects showed that crickets may have a higher nutritional value as compared to moth caterpillar,termite and grasshopper.The higher PER value for Crickets as compared to casein is similar to reports of Ekpo [26]who also discovered thatRhynchophorus phoenicishad a higher PER than casein.The PER of cricket obtained in this study were higher than PER ofOrcytes rhinoceros(1.15±0.27),Imbrasia belina(0.87±0.38),Macrotermes bellicoccus(1.18±0.28)andRhynchophorus phoenicis(1.31±0.15)[16].This suggests that crickets may be a good source of high quality protein.

The NPR of cricket in this study was higher than NPR values reported by Ekpo[16]but lower than values reported by Ande[38]for the insects they analysed.Several authors have also reported that crickets may also be a source of quality proteins for livestock[39,41].Their findings also support the results obtained in this study.

True digestibility (TD) indicates how well a protein is digested which is a measure of the bioavailability of amino acids present in a protein[26].Casein was found to have a higher true digestibility as compared to the selected insects.The lower true digestibility value of the insects as compared to casein may be as a result of the presence of chitin in the insects.The results for TD obtained in this study fall within the range reported as TD values(76-98%)for insects[40].

Net protein utilisation (NPU) measures how well a protein is digested and utilised[4].The NPU value for casein obtained in this study is similar to the value reported by[42].The values of NPU for the selected insects were not significantly different(p ＞0.05)from that of casein.Ande [38]also reported high NPU values forCirina forda.This finding may suggest that proteins from these insects are utilisable.

Biological value (BV) of a protein defines the percentage of absorbed nitrogen that is retained and is therefore an indication of how well a protein is utilised.Results obtained in this study showed that cricket had the highest BV as compared to casein,grasshopper,moth caterpillar and termite.The biological value for casein obtained in this study is similar to value of 70.8% reported by Mitchell [43]and lower than values reported by Agbede [44](93.4%).The low biological value for casein diet obtained in this study may be as a result ofincreased metabolism.At higher a level of protein intake,there is a tendency for increased metabolic wastage of proteins.The biological value of a protein increases with decreased protein intake as maximal utilisation of good quality proteins occur at lower levels of protein intake[26].

Protein digestibility corrected for amino acid score (PDCAAS)accounts for protein digestibility and amino acid composition of aprotein and therefore is a better indicator of protein quality.The value 73% for protein digestibility corrected for amino acid score(PDCAAS)for cricket is also an indication that cricket are of higher nutritional value as compared to grasshopper,moth caterpillar and termite.A value of 68%has earlier been obtained for edible beetle by[40].

Organ /body weight ratio is a marker of cell constriction and inflammation [45].The Organ/body weight ratios of the liver,spleen,lung and hearts of animals placed on the insect supplemented diets were not statistically different from those of rats fed the casein and basal diet,this therefore suggests that dietary inclusion ofinsects did not cause any adverse damage to these organs.Although the kidney/body weight ratio of rats fed termite diet was significantly higher than rats placed on casein diets this increase may be as a result oflow PER value of termite protein as observed by Ekpo [26]that low PER value results in a higher kidney/body weight ratio.

Serum activities of alanine amino transferase (ALT) and aspartate amino transferase(AST)are elevated whenever the integrity ofliver cells are disrupted leading to release of these enzymes in the blood from damaged cells hence are markers for liver damage[46].Previous studies have also shown that feeding poor quality proteins to rats could lead to a significant increase in serum activities of ALT and AST[26].The result obtained in this study shows that inclusion ofinsects did not cause any alteration in serum activities of ALT and AST of the experimental rats as compared to rats fed with the control diet (Casein and basal diet) suggesting that these insects did not have toxic effects on the rats.However,the significantly higher(p ＜0.05)ALT activity observed in the serum of rats fed termite supplemented diet may not necessarily be as a result ofliver damage but to low protein quality of the termite supplemented diet.

Alkaline phosphatase(ALP)is present in high concentrations in the bone,mucosa of the small intestine,kidney,liver and plasma.Damage in any of these organs may cause a release of ALP into the bloodstream.ALP is also a marker for the plasma membrane and endoplasmic reticulum used to assess the integrity of plasma membrane and endoplasmic reticulum [47].An increase in serum ALP activity is also associated with bone growth [48,49].The increase in ALP activity (Table5) observed in the serum of rats fed cricket diet may not be as a result ofliver damage,since Serum ALT and AST activities were not elevated in the rats.The higher serum ALP activity may therefore be associated to bone growth,since the rats were young and actively growing.

The concentration of total protein in serum is an indicator of the synthetic ability of the liver[50].Concentration of protein in serum is also used to monitor the protein / nutritional status of animals since its synthetic rate is dependent on protein intake.Decreased concentrations of serum total protein are therefore markers ofimpaired liver function or protein energy malnutrition[51].

The significantly lower (p ＜0.05) concentration of serum total protein in the experimental rats when compared to control rats(rats placed on casein diet) may not be as result ofliver damage since the serum activities of ALT and AST were not elevated in the rats fed insect diets.The results obtained were therefore a reflection of the protein intake of the rats,since the rats fed with casein,cricket and moth caterpillar diets had higher feed intakes and hence a higher protein intakes than rats fed with other experimental diets.The higher concentration of serum total protein observed in rats fed casein diet may also be due to a higher bioavailabilty of essential amino acids required for synthesis of proteins as reflected in the true digestibility value for casein (Table4).This result is also reflected in the value of the true digestibility of casein obtained in this study,as casein was found to have a higher true digestibility as compared to the insects analysed.

The significantly lower (p ＜0.05) concentration of serum LDL cholesterol of rats placed on diet supplemented with cricket is of significance as high levels of total cholesterol and LDL cholesterol in blood have been associated with increased risk of coronary heart disease[52].Consumption of some conventional animal proteins such as meat and dairy products have been known to lead to increase in the plasma levels of LDL cholesterol because they are composed primarily of saturated fatty acids[3].Crickets may find relevance as substitute for conventional sources of animal proteins due to its high protein quality and ability to lower serum levels of LDL cholesterol.The marked decrease in the serum levels of triglyceride in rats fed with termite diet (Table6) as compared to rats placed on other diets may be due to the low feed intake.Inadequate feed intake may have led to increased mobilisation of triglycerides for energy production[2].

Haematological parameters are good indicators of the physiological,nutritional and pathological status of animals.They are also valuable in monitoring feed toxicity [53,54].The non significant difference (p ＞0.05) in the packed cell volume,haemoglobin concentration,red blood cell and platelet count of rats fed with insect supplemented insects with those of rats supplemented with casein dietis an indication that dietary inclusion of these insects did not have any cytotoxic effect on the rats.Similar results have been reported by Ekpo [26]that insect supplemented diets had no significant effect on haematological indices of rats.The significantly higher (p ＜0.05) white blood cells in rats fed with moth caterpillar may imply that these rats had increased immunity against infection.Animals with low white blood cells and white blood cell differentials(lymphocytes,eosinophils,basophils,monocytes and neutrophils)are exposed to high risk ofinfection while those with adequate white blood cell counts are known to be capable of generating antibodies and have a high degree of resistance[55].

5.Conclusion

In this study,cricket was found to have the highest protein quality compared to termite,grasshopper and moth caterpillar as reflected in the various protein quality indices determined.The low protein quality of termite,moth caterpillar and grasshopper can be attributed to deficiency of sulphur containing amino acids and isoleucine in these insects but not low digestibility and net protein utilization.These insects were however rich in other essential amino acids like lysine and can be still be used to complement staple cereal foods that are usually deficient in this amino acid.Inclusion of the insects in the diets of rats did not have any adverse effect on serum concentrations of AST,ALT,ALP,total protein,HDL cholesterol,total cholesterol and haematological indices.Cricket diet was however found to lower serum LDL cholesterol concentration of rats.This finding is of relevance because a high serum level of LDL cholesterol is implicated in increased risk of cardiovascular diseases.These insects are therefore nutritious and safe for consumption.

Conflict ofinterest

The authors have declared no conflict ofinterest

Acknowledgments

The authors would like to acknowledge TETFUND for funds and staff of the animal housing unit,University of Jos,Nigeria for technical assistance.