Effects of Hermetia illucens (HI) larvae meal as a partial to complete substituttion soybean meal on hematogical and blood biochemical parameters in broiler chickens

Introduction

The demand for animal origin protein sources has tremendously increased during last few decades due to growing global human population and limited amount of agricultural land (Barragan-Fonseca et al. 2017) [3] . Poultry has the potential to fulfill the demand of protein source because of its high feed conversion efficiency (Ndotono et al. 2022) [22] . Poultry production in India has taken a quantum leap in the last few decades and emerged from conventional farming practices to intensive production system. However, feed contributes to about 60-70% of production costs and dietary protein is an impotant and costly nutrient required for optimum broiler production. Soybean meal (plant origin) are most commonly used ingredients as protein source which is becoming costlier for poultry farmers to afford due to shortage of land for cultivation, human consumption and global cost fluctuation (Ndotono et al. 2022) [22] . Insects seems to be a prominent candidate to meet the global challenge of finding new alternative protein sources as it having high protein content, lower space requirement for raising and emission of green house gases (GHGs), high feed conversion efficiency, converting low value organic byproducts into high value proteins (Van Huis and Oonincx, 2017) [30] . In addition, insects can be reared on wide range on organic waste like Fruits and vegetable waste and oranic manure (Marono et al. 2017) [19] . Various insects have been tested as animal feeds, amongst the most promising species being the black soldier fly (Hermetia illucens), yellow mealworm (Tenebrio molitor) and common house fly (Musca domestica) (Ndotono et al. 2022) [22] . Black soldier fly (Hermetia illucens) larvae meal inclusion in broilers (Dabbou et al. 2018; Schiavone et al. 2019) [11,27] , ducks (Gariglio et al. 2019) [15] , quails (Cullere et al. 2016) [10] and partridges (Secci et al. 2018) [28] ration has been studied earlier by researchers but limited up to partial replacement. As insect meal normally contains chitin as an antinutritional factor, studies regarding complete substitution of HILM with soybean meal in broiler health status are limited. Considering above points, present experiment was carried out to study the effects of substituting conventional protein sources with Hermetia illucens larvae meal (HILM) on blood-biochemical parameters in broiler chicken.

Material and method Ethical approval

Present experiment was approved by Institutional Animal Ethics Committee (IAEC) on having approval no. 137/VCN/ANN/2023 dt.26/12/2023.

Experimental diet, design and housing

A total of one hundred and sixty (n=160) day old broiler chick of Vencobb-400 strain day-old broiler chicks (Vencobb-400 strain) were reared from day 01 to day 42 and assigned to five dietary treatments with 32 birds having 4 replicates (8 chicks/replica) in each group. The maizesoybean based diet was used as control without inclusion of partially defatted HI larvae meal while HI25, HI50, HI75 and HI100 were incorporated with 25, 50, 75 and 100% of HI larvae meal as a partial replacement of soybean meal in the basal diet, respectively. HI larvae meal was reared on fruits and vegetable wastes and substituted at different levels with protein source (soybean meal) of basal diet. Isonitrogenous and iso-caloric diets were formulated for 3 phases of pre-starter (0-7 day), starter (7-21 day) and finisher (21-42 day) as per BIS (2007) (Table 1). The entire replica under each group was having facility of individual feeding and watering. Ad libitum feed was offered in weighed quantity once in a day at morning 08.00 hrs. Ad libitum drinking water was also provided to experimental birds throuhout study period. Feed offfered, left over and faceal samples were collected and analysed as per procedure of AOAC (2005) [2] . All the birds were reared on deep litter housing system having rice husk as a bedding material. Formalin and postassium (KMnO4) solutions were used to fumigate poultry shed two days prior to arrival of chicks. Each experimental pen was separated using a wire net for partitioning. Each pen was provided with a 60-watt bulb for brooding and brooding temperature was mainteind at 34 °C up to fisrt 7 days of age. From 2nd week onwards all the birds were kept under a normal environmetal temperature. All the experimental birds were vaccinated for infectious bursal disease and new castle disease.

Chemical composition of feedstuffs

The analyzed chemical composition of HI larvae meal in current study had 94.35% DM, 48.87% CP, 11.88% EE, 17.16% Ash, 5.2% calcium, 0.84% phosphorus and 6.90% chitin. 3000IU; Vitamin E (DL-a-tocopheryl acetate)-60 IU; Vitamin K (menadione sodium bisulfite)-1.02 mg; Riboflavin-2.0mg; Pantothenate-8.0mg; Niacin-6mg; Folic acid-0.5 mg; Biotin-0.10 mg; Thiamine-1mg; Vitamin B12-20 mg; Mn-120 mg; Zn-80 mg; Fe-52 mg; Cu-15 mg; I-1.5 mg; Se-0.4m

Hematology and blood biochemical parameters

Blood was collected at the end of experiment (42 day) from total eight birds per group having 2 birs per replcate. Blood was collected from wing vein in early morning with strilized needles in two different vials for hematological and biochemical analysis. One tube ccontaining EDTA was used for estimation of hematological assay. Approximately 02 ml of whole blood in EDTA vial was used for hematological parameters such as hemoglobin (Hb), hematocrit value/ packed cell volume (PCV) and total erythrocyte count (TEC) and total leucocyte count (TLC) was done by manually in department of pathology. Seocnd tube without anticoagulant was used and allowed the blood to clot for 1 hour at room temperature and then centrifuged at 2000 rpm for 15 minutes to obtain serum (Remi R-8M bench top centrifuge). Clear, non-hemolysed serum collected in tubes was stored at -40°C for various biochemical analysis. Blood biochemical parameters like total protein, glucose, total cholesterol, triglycerides and uric acid were analysed from serum using commercially available analycial kits in spectrophotometer (Model No. 4001/1 Gensys 20), Thermo Fisher Scientific, USA).

Statistical analysis

All the data generated from hematological and biochemical parameters were analysed used using IBM SPSS version (20.0) software. One way -Anova (Analysis of variance) was used and orthogonal polynomial contrast haivg linear and quadratic effects were tested. Significance was declared at 5% level (p<0.05) and differecne amongst means were tested using post-hoc Tukey's HSD test.

Results

Hematological and blood-biochemical parameters

The concentration of Hb, RBCs, WBCs, MCV, PCV, MCH and MCHC lymphocytes and monocytes did not show any significant (p>0.05) changes amongst the treatment groups. (Table 2). Serum glucose (mg/dL), creatinine (mg/dL), uric acid (mg/dL) and troglycerides (mg/dL) remained nonsignificant (p>0.05) between treatment groups (Table 2). Further, serum total protein (g/dL), albumin (g/dL) and globulin (g/dL) quadratically increase in HILM treatment groups with highest values in HI25 and lowest in HI75 groups. Albumin to globulin ratio showed quadratic (P<0.05) effect with HI larvae meal inclusion with highest value in HI100 and lowest in CON. Serum total cholesterol (mg/dl) increased linearly (p<0.001) with increasing levels of HILM in the diet. Serum ALT (U/L) found quadratic increase in treatment groups with highest in HI25 followed by HI75, HI50, HI100 and lowest CON. AST (U/L) increased linearly (p<0.001) with increasing levels of HILM in the broiler diet. Total antioxidant capacity (umol/L), superoxide dismutase (ng/mL) and glutathione peroxidase (umol/L) increased linearly with increased level of HI larvae meal inclusion in broilers ration.

Discussion

All the hematological and blood biochmeical parameters obtained in pressent study were in normal range and results revealed that HI larvae meal inclusion did not affected overall health status of experimental birds (Dabbou et al. 2018; Kim et al. 2022) [11,18] . The Hb, RBCs, WBCs, MCV, PCV, MCH and MCHC, lymphocytes and monocytes values did not show any significant (p>0.05) changes amongst the treatment groups. Hemoglobin (g/dl) level remained similar (p>0.05) amongst treatment group. Normally, Hb level is dependant on numbers of erythrocytes and oxygen levels in the blood (Irawan et al., 2020) [16] . Higher value of hemoglobin indicates a better nutrient supply in the body system of the broiler chickens (Alabi et al., 2013) [1] . No change in overall hematological parameters was found by many researchers like, Kim et al. (2021) [17] ; Dabbou et al. (2018) [11] Bellezza et al. (2021) [5] , Bongiorno et al. (2022) [6] , Tykałowski et al. (2023) [29] [11,19,18] . Serum total protein (g/dl), albumin (g/dl), globulin (g/dl) quadratically increase in HILM treatment groups with highest values in HI25 and lowest in HI75 groups. Similar findings were observed by Chen et al. (2022) [8] who found quadratic increase in total protein (g/dL), albumin (g/dL), globulin (g/dL) in HILM substituted groups at higher levels. In contrast, Marono et al.

(2017) [19] , Schiavone et al. (2018) [26] , Dabbou et al. (2021) [12] , Kim et al. (2021) [17] , Bongiorno et al. (2022) [6] , Mat et al. (2022) [20] , and Tyakalowaski et al. found non-significant serum total protein between treatment groups. Further, according to Gariglio et al. (2019) [15] similar total protein levels between groups indicative of a similar dietary protein bioavailability in the birds. Serum total protein, albumin and globulins are important measures and useful to assess body condition of poultry (Piotrowska et al., 2011) [24] as plasma protein plays a key role in body homeostasis maintenance. Albumin serves as the important source of amino acids for protein synthesis (Filipovic et al., 2007) [14] . Present findings regarding globulin provided an overall increase of circulating immunoglobulins and showed stronger disease resistance response in larvae-feeding birds up to 25% HILM inclusion (Marono et al., 2017) [19] . It was also reflected with lower mortality in HILM substituted group (n=1) as compared to CON (n=1). However, Gariglio et al. (2019) [15] found negative effects of higher level of HILM inclusion in Muscovy ducks as it may compromise auto immunity of the birds due over production of globulin. Serum uric acid (mg/dl) and serum creatinine (mg/dl) remained statistically non-significant between treatment groups. Supporting to our findings, Marono et al. (2017) [19] , Dabbou et al. (2021) [12] , Kim et al. (2021) [17] , Bellezza et al. (2021) [5] , Mat et al.

(2022) [20] and Bongiorno et al. (2022) [6] found similar uric acid and creatinine levels in HILM substituted birds. In contrast, Tyakalowski et al. (2023) found increased uric acid levels with increasing level of HI larvae meal. Uric acid is major nitrogenous end product which represents protein catabolism in broilers (Tyakalowski et al., 2023). Creatinine is a byproduct of phosphocreatine breakdown in skeletal muscle and an important indicator of protein metabolism in birds (Piotrowska et al., 2011) [24] . Further, serum creatinine levels are associated with muscle mass, age, physical activity and diet (Rajman et al., 2006) [25] . Non-significant uric acid and creatinine are indicative that insect meal at higher inclusion levels does not affect kidney functions. Serum total cholesterol (mg/dl) increased linearly (p<0.01) with increasing levels of HILM in the diet while triglycerides remained unaffected (p>0.05) between treatment groups. Present study was supported by Mbhele et al., (2019) [21] , Dabbou et al. (2018) [11] , Dabbou et al.

(2021) [12] , Bellezza et al. (2021) [5] and Mat et al. (2022) [20] .

In present study, the diet was isocaloric in nature and HILM used was partially defatted so, the dietary lipids might have contributed to circulating lipid. In contrast, Gariglio et al.

(2019) [15] and Kim et al. (2021) [17] found linear decrease in total cholesterol and triglycerides level with increasing level of HI larvae meal inclusion in broilers due to chelating effect of chitin in the HI larvae meal (El-Gobary et al., 2016) [13] . Commercial chitin has also reported to decrease the lipid absorption by binding to lipids which lowers plasma cholesterol (Bovera et al., 2018 and Marono et al., 2017) [7,19] . Chitosan, a chitin derivative, has a high anion-exchange capacity and consequently has a bile acid binding capacity which may disturb the enterohepatic circulation and enhance fecal triglycerides (Bedford and Gong, 2018) [4] . Serum ALT (U/L) found quadratic increase in treatment groups with highest in HI25 followed by HI75, HI50, HI100 and lowest CON. AST (U/L) increased linearly (p<0.001) with increasing levels of HILM in the broiler diet. Similar findings were observed by Chen et al. (2022) [8] significant increase in AST activity with partial to complete substitution of HI larvae oil in broilers. In contrast, [9,8,12] . Total antioxidant capacity increased linearly (p<0.002) with increased level of HI larvae inclusion in broilers. Dabbou et al. (2018) [11] also supported present findings and found that total antioxidant capacity increased with HILM in treatment groups. Kim et al. (2021) [17] also found that black soldier fly oil had improved the total antioxidant capacity of broiler chickens by increasing plasma TAC levels. Superoxide dismutase also found linear increase (p<0.021) with increasing level of HI larvae meal in diet. The present findings are supported by Chu et al.

(2020) [9] and Chen et al. (2022) [8] when full-fatted HI larvae and HI larval oil was included in the broiler diet, respectively. Glutathione peroxidase found linear (p<0.031) increase with increasing level of HI larvae meal in the broiler diet. Similar findings were supported by Dabbou et al. (2018) [11] and Chu et al. (2020) [9] , when partially defatted and full fatted larvae meal in the broilers. Black soldier fly contains a proportion of medium chain fatty acids like lauric acid (not measured here) and antioxidant peptide providing important antioxidant ability and antibacterial activity (Chu et al. 2020) [9] . Lauric acid also helps to inhance intestinal immune ability due to its antioxidant and prebiotic properties (Park et [23,31,8] . Further, free radical scavenging effect of a chitin plays a vital role in providing antioxidant capacity by preserving cellular structural integrity and biological functions (Gariglio et al. 2019) [15] . BSF insect meal provides dietary tocopherols, which further contributes to increase glutathione peroxidase activity in insect meal groups (Secci et al. 2018) [28] .

Conclusion

HI larvae meal as a partial to complete substitution of soybean meal in broiler chicken diet did not affect overall hametological and blood-biochemical parameters with improvement in overall antioxidant status of broiler birds. Further, research with higher substitution rate at different agroclimatic conditions will provide better insight about insect meal feeding in tropical conditions in poultry industry.

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