Evaluation of black soldier fly larva on haematology, intestinal morphology and intestinal histomorphology in commercial broiler chicken

Introduction

The poultry industry plays a major role in supplying affordable animal protein to the growing population. In commercial broiler production, feed accounts for the major portion of production cost, creating the need for sustainable and economical alternative feed ingredients. Conventional protein sources such as soybean meal and fish meal are becoming increasingly expensive and limited in availability. In recent years, the larvae of the Black Soldier Fly have gained attention as an alternative protein source in poultry nutrition due to their high protein content, good nutrient profile, and sustainable production system. Black soldier fly larva meal (BSFLM) also contains bioactive compounds that may improve gut health and nutrient utilization in broiler chickens. Intestinal development and histomorphology are important indicators of digestive efficiency and nutrient absorption in broilers. Parameters such as intestinal length, intestinal weight, villus height, and crypt depth help evaluate the functional status of the intestine. Haematological parameters are also useful in assessing the physiological and health status of birds. Although BSFLM has shown potential as a poultry feed ingredient, limited information is available regarding its effects on intestinal characteristics and haematological parameters in commercial broilers. Therefore, the present study was undertaken to evaluate the effect of black soldier fly larva meal on the production performance of commercial broiler chickens, with special emphasis on intestinal length and weight, intestinal histomorphology, and haematology.

Materials and Methods

The study was carried out as per guidelines and approval of Institutional Animal Ethical Committee (IAEC) with IAEC number 24/SA/IAEC/2024 (24-12-2024). A six-week duration biological experiment was conducted to evaluate the effect of dried BSF larvae on the Haematological, Intestinal morphology and Intestinal histomorphology in commercial broilers. The experimental period divided into three phases namely pre starter (0-7 days), starter (8-21 days), and finisher (22-42) days. A feeding trial was carried out with 160 day-old straight-run commercial broiler chicks (Ven Cobb 430 Y) obtained from Venkateshwara Hatcheries private limited. All the chicks were allotted to four treatment groups viz., T 1 , T 2 , T 3 and T 4 with four replicates in each treatment with 10 chicks in each replicate as given in Table 1. Feeding standards as per BIS (2024) [1] Specification Collection of samples and analysis Haematological Parameters i. Collection of blood: Three milliliters of blood was collected from two males and two females from each treatment in EDTA vacutainer vials for blood analysis.

ii. Total count: Total Erythrocyte Count (TEC) and Total Leucocyte Count (TLC) were estimated by Neubauer counting chamber by using Nambiar's diluting fluid (Bancroft and Marilyn, 2008) [2] .

Intestinal morphology

A total of 12 birds comprising of three birds per treatment were randomly selected for measuring the Small intestinal length and weight. The collected intestines were flushed with Phosphate buffer saline (PBS) to remove all the feed contents. The total intestinal length was measured using a measuring tape in centimeter scale and weighed in grams.

Intestinal histomorphology

Small intestine (middle part of jejunum, duodenum, ileum) samples were taken from three birds per treatment after the experimental period of 42 days and were flushed with phosphate buffer saline and preserved in 10% neutral buffer formalin for processing. Later, it was dehydrated cleaned and embedded in paraffin wax. Serial histology sections of 4 to 5 µm thickness were cut on a microtome and mounted on slides. The slides were stained with Hematoxylin and Eosin stain as described by Bancroft and Marilyn (2008) [2] using image analysis software (Mag vision). Villus height and crypt depth were measured in micrometer (µm) per sample and mean values were used in statistical analysis. The villus height (VH) was measured from the tip of the villus to the villus crypt junction; Crypt depth (CD) was defined as the depth of the invagination between adjacent villi, and epithelial thickness (ET) considered as the distance from the epithelial surface to the basement membrane of the epithelial cell. VH:CD was determined as the ratio of VH to CD.

Statistical analysis

The data pertaining to various parameters were analysed statistically using Analysis of Variance (ANOVA) by software (IBM SPSS version 25.0 for Windows) as per Snedecor and Cochran (1990) [3] . Significant mean differences were determined at 5% probability level (P<0.05) to identify the fixed effect of treatment using Duncan's Multiple Range Test (DMRT) as modified by Kramer (1956) [4] .

Results

Haematological parameters

Effect of feeding different levels of Black Soldier Fly Larva in commercial broilers on haematological parameters (Mean ± SE) is presented in Table 2.

The haematological parameters evaluated, namely packed cell volume (PCV), haemoglobin concentration, total erythrocyte count (TEC), and total leucocyte count (TLC), did not differ significantly (P>0.05) among the treatment groups. The PCV was highest in T 1 followed closely by T 4 while T 2 and T 3 recorded lower values; Similarly, haemoglobin concentration was highest in T 1 closely followed by T 4 whereas T 2 and T 3 showed slightly lower values, with no significant variation among treatments. Total erythrocyte count was numerically highest in T 3 followed by T 4 , T 2 and lowest in T 1 . Total leucocyte count was highest in T 1 whereas T 2 , T 3 and T 4 recorded comparatively lower values. Despite numerical variation, the differences were statistically non-significant.

Intestinal morphology

Effect of feeding different levels of Black Soldier Fly Larva in commercial broilers on Small Intestinal morphology parameters (Mean ± SE) is presented in Table 3.

i. Intestinal length

No significant difference was observed among the treatment groups T 1 , T 2 , T 3 and T 4 and all groups were statistically comparable. Numerically, T 4 (10%) recorded the highest intestinal length followed by T 2 and T 1 while the lowest value was recorded in T 3 .

ii Intestinal weight No significant difference was observed among the treatment groups T 1 , T 2 , T 3 and T 4 and all groups were statistically comparable. Numerically, T 2 recorded the highest intestinal weight followed by T 1 and T 3 while the lowest value was recorded in T 4 .

Intestinal Histomorphology

Effect of feeding different levels of Black Soldier Fly Larva in commercial broilers on Intestinal Histomorphology parameters (Mean ± SE) is presented in Table 4.

i. Duodenum

Villi height, Crypt depth and Villus Height Crypt Depth ratio of duodenum did not differ significantly (P>0.05) among the control and treatment groups.

ii. Jejunum

Villus Height Crypt Depth ratio of jejunum differed significantly (P<0.05) among the control and treatment groups. T 2 recorded the highest villus height to crypt depth ratio, which was statistically comparable with T 1 and T 4 . However, Villi height and Crypt depth did not differ significantly (P>0.05) among the control and treatment groups.

iii. Ileum Villi height, Crypt depth and Villus Height Crypt Depth ratio of ileum did not differ significantly (P>0.05) among the control and treatment groups.

Discussion haematological parameters

The findings of the present study are in agreement with those reported by Belleza et al. (2021) [5] , who observed that supplementation of live BSF larvae in broiler diets did not influence hematological and serum parameters. Similarly, Dabbou et al. (2018) [6] reported that inclusion of defatted BSF larvae meal up to 15% in broiler diets did not significantly affect blood and serum parameters. Contrastingly, Mat K et al. (2021) [7] observed significant effects for several hematological parameters when broilers were fed diets containing defatted BSF larvae meal, although leucocytes, granulocytes and haematocrit remained unaffected.

Intestinal morphology

In accordance with present study, Elangovan et al. (2020) [8] observed no significant differences in the length or weight of different intestinal segments such as the duodenum, jejunum, ileum and caecum between broilers fed diets containing BSF prepupae meal and the control group. Comparable findings were also reported by Belleza et al. (2021) [5] , who noted that the inclusion of live BSF larvae in broiler diets did not influence intestinal weight. However, some studies have reported variations in intestinal characteristics with the inclusion of BSF larvae meal. Attivi et al. (2020) [9] reported that intestinal length increased significantly in broilers fed higher levels of BSF larvae meal (50%, 75% and 100%) compared to lower inclusion levels.

Intestinal histomorphology

The findings of the present study are consistent with the results reported by Dabbou et al. (2018) [6] , who observed that inclusion of defatted BSF larvae meal up to 15% did not significantly influence most intestinal morphometric indices in broiler chickens. Similarly, Moula et al. (2017) [10] reported no significant difference in intestinal histomorphology between control birds and those fed diets containing BSF larvae. Contrastingly, Changqing He et al. (2021) [11] reported significantly higher villus height in the duodenum of birds fed moderate levels of BSF larvae meal, although crypt depth and VH:CD ratio were not significantly affected.

Conclusion

Based on the present study, inclusion of Black Soldier Fly Larva up to 10% had brought no negative impacts on haematological parameters, Intestinal morphology and Intestinal histomorphology to that of control which indicates Black Soldier Fly Larva can be included as one of the potential protein sources in broiler chicken diet.

Acknowledgement

AcknowledgementThis research was funded by Department of Poultry Science, Madras Veterinary College, Tamil Nadu Veterinary and Animal Science University, Chennai -600 007, Tamil Nadu, India.

References

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