
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00695-3
10.1016/j.psj.2024.104116
104116
MANAGEMENT AND PRODUCTION
Nano-emulsified black soldier fly oil concerning performance traits, health, and immunity of broilers
Dewanti Ratih ratihdewanti@staff.uns.ac.id
⁎†1
Martien Ronny ‡
Ariyadi Bambang §
Zuprizal †
⁎ Department of Animal Science, Faculty of Animal Science, Universitas Sebelas Maret, Surakarta, Indonesia
† Department of Animal Nutrition and Feed Science, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia
‡ Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia
§ Department of Animal Production, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia
1 Corresponding author: ratihdewanti@staff.uns.ac.id
29 7 2024
10 2024
29 7 2024
103 10 1041165 6 2024
24 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
In the antibiotic-free era, traditional antibiotics have been suggested as alternatives to antibiotic-based growth promoters. Among the various methods, self-nano-emulsifying drug delivery systems (SNEDDS) are increasingly utilized to improve the bioavailability of oils containing essential substances. In this study, we evaluated the effects of black soldier fly oil (BSFO) SNEDDS in chicken drinking water on growth performance, small intestine histomorphology, and poultry health status. We divided 225 male Indian River strain broiler chickens into five treatment groups, each consisting of 5 replicates. The chickens were reared from to 0 to 35 d of age in a controlled environmental housing system. The BSFO SNEDDS was administered via drinking water. Treatments included ordinary drinking water (P1), bacitracin (P2), and 10 mL/L (P3), 20 mL/L (P4), and 30 mL/L (P5) BSFO SNEDDS. The observed parameters included growth performance, carcass yield, blood hematology, intestinal histomorphology, digestive microflora, and immunoglobulin (Ig) levels. Data were analyzed using analysis of variance (ANOVA) and Duncan's test. The results indicated that administering BSFO SNEDDS via drinking water improved feed conversion (P < 0.05), enhanced the performance index (P < 0.05), increased carcass percentage (P < 0.001), and increased the weight of the carcass parts. Additionally, it increased villus height (P < 0.01), lowered jejunal pH (P < 0.001), reduced pathogenic bacteria in the jejunum, and decreased the leukocyte count. BSFO SNEDDS in drinking water is expected to be used as a traditional antibiotic with the potential to replace synthetic antibiotic growth promoters in broiler chickens.

Key words

black soldier fly oil
broiler
growth performance
intestinal histomorphology
SNEDDS
==== Body
pmcINTRODUCTION

Synthetic antibiotics are widely used as growth promoters in commercial feed mixtures by feed mills. However, the use of antibiotic growth promoters that are not in accordance with recommended doses and their continuous administration can cause antibacterial resistance, residues in livestock products (Alagawany et al., 2021), weakened disease resistance, and environmental pollution (Salim et al., 2018). Thus, the use of antibiotics as growth promoters in poultry feed is restricted in some countries (Smagieł et al., 2023). Feed antibiotics have been banned in the European Union since January 2006 (Regulation EC/1831/2003). The banning of antibiotics can result in economic losses. The ban on the use of antibiotics has encouraged the development of alternatives to synthetic antibiotics (Cheng et al., 2014), such as traditional antibiotics used as feed additives. Various feed additives, including phytobiotics, probiotics, prebiotics, synbiotics, enzymes, and organic acids have been used as antibacterial agents. Although plant-derived organic acids have been extensively studied, animal-derived organic acids remain under researched. Insects are a potential alternative to food and feed owing to the increasing demand for renewable and sustainable products (Chou et al., 2020). Insects have the advantages of being reproduced quickly, easy to cultivate in limited spaces, nontoxic, and not competing with human needs. One potential source of animal organic acids from insects is obtained from black soldier fly oil (BSFO). BSFO contains the bioactive ingredient lauric acid (C:12) as the main fatty acid which is a medium chain fatty acid (MCFA) that has the potential to act as an antibacterial and immunity booster. BSFO contains 44.55% lauric acid, which has the highest fatty acid content amongst fatty acids in BSFO (Dewanti et al., 2024).

To date, the utilization of BSFO has been limited to substituting soybean and fish oils in animal feed mixtures (Schiavone et al., 2017; Rawski et al., 2020; Kim et al., 2021). However, the addition of BSFO biomass to the feed is less effective. On the other hand, administering it through drinking water is practical, economical, and allows for precise dosing. Unfortunately, BSFO has low solubility in water-based solvents. Therefore, the enhancement of BSFO formulation using nanotechnology in the form of self-nano emulsifying drug delivery system (SNEDDS) is needed to overcome substances that are difficult to mix and insoluble by increasing their solubility through a mixture of oils, surfactants, and cosurfactants (São Pedro et al., 2013; Prajapati et al., 2023a) Besides, the utilization of SNEDDS has the potential to accelerate and broaden drug absorption with low water solubility properties (Prajapati et al., 2023b).

The use of traditional antibiotics in the form of BSFO SNEDDS is expected to reduce the number of pathogenic bacteria in poultry bodies because it contains lauric acid, a bioactive compound with bacteriolytic properties that damages bacterial cell membranes through a physicochemical process. Previous research shows that SNEDDS from plant sources, such as SNEDDS lemongrass essential oil, can improve poultry performance and reduce pathogenic bacteria in the poultry digestive tract (Ujilestari et al., 2019) Meanwhile, SNEDDS cinnamon essential oil can improve broiler feed conversion (Baskara et al., 2020). Furthermore, it has been reported that the bioavailability of docosahexaenoic acid (DHA) from fish biomass was more than three times higher following SNEDDS injection using a surfactant compared to that of an aqueous solution (Alhakamy et al., 2020). Moreover, SNEDDS are increasingly being utilized to enhance the bioavailability of volatile lipophilic components in essential oils to be administered in broiler diets (Abd El-Hack et al., 2022). The presence of pathogenic bacteria in the intestinal lumen significantly affects poultry performance. Broiler performance can be assessed by improving intestinal villus morphology and microbial balance. A well-balanced microbial community can promote mucosal well-being and sustain the intestinal barrier function (Ren et al., 2023). Toxic emissions from pathogenic microorganisms compromise the physical integrity of the cell wall of the small intestine, which is essential for absorption. The BSFO SNEDDS also improved nutrient absorption in the gut. Therefore, in this study we aim to evaluate the in vivo effects of different doses of BSFO SNEDDS in drinking water on the performance traits, health, and immunity of broilers.

MATERIALS AND METHODS

Broiler chickens used in this study were registered with the Ethical Clearance Commission of the Faculty of Veterinary Medicine at Gadjah Mada University (No. 019/EC-FKH/Eks). /2023.

Preparation of Black Soldier Fly Oil Self-Nano Emulsifying Drug Delivery System

Black soldier fly oil (BSFO) was obtained from Mikayste Farm (Bekasi, Indonesia), while polyethylene glycol (PEG 400) (Sigma-Aldrich, Germany) and Tween 80 were purchased from PT. Brataco (Bekasi, Indonesia). The SNEEDS formulation was prepared by mixing BSFO (11.906%) with PEG 400 (11.647%) using a magnetic stirrer at 37°C and 250 rpm for 10 min. Then Tween 80 (76.447%) was added and mixed for 20 min, followed by an ultrasonicator (Aczet CUB-10L, Aczet Pvt. Ltd, India) at 40°C for 10 min (Dewanti et al., 2024).

The fatty acid compositions of BSFO and BSFO-SNEDDS were analyzed using gas chromatography (GC-2010 Plus, Shimadzu, Japan). The fatty acid compositions of BSFO and BSFO SNEDDS are presented in Table 1.Table 1 Fatty acid profiles of black soldier fly oil (BSFO) and self-nanoemulsifying drug delivery system of black soldier fly oil (BSFO SNEDDS).

Table 1Fatty acid profiles	Concentration (%)	
BSFO	BSFO SNEDDS	
Methyl decanoate	0.94	0.26	
Methyl laurate	44.55	13.79	
Methyl myristate	9.81	2.98	
Methyl palmitate	17.43	9.37	
Methyl heptadecanoate	<0.1	0.79	
Methyl stearate	1.66	<0.1	
Methyl arachidate	<0.1	2.88	
Methyl heneicosanoate	<0.1	2.94	
Myristoleic acid methyl ester	0.27	<0.1	
Methyl palmitoleate	2.50	0.13	
Methyl trans-9elaidate	<0.1	1.72	
Methyl cis-9-oleate	15.35	<0.1	
Methyl linolelaidate	<0.1	60.85	
Methyl linoleate	6.74	0.22	
Gamma-Linolenic acid methyl ester	<0.1	0.20	
Methyl cis-11-eicosenoate	0.52	0.17	
Methyl linolenate	0.22	3.46	
Methyl cis-11, 14-eicosadienoate	<0.1	0.26	
Other fatty acids not listed are in the range of <0.1%.

Animals and Housing

This study used 225 male day-old chick (DOC) of the Indian River strain from Aviagen produced by PT Widodo Makmur Unggas in Yogyakarta, Indonesia. Day-old chick were vaccinated with triple vaccines comprised of Newcastle disease (ND I), Gumboro, and ND 2, conducted by the company in the hatchery. Broilers were housed in controlled-environmental housing system with brooding temperature of 0 to 3-days-old chicks (30°C) and were decreased by 2°C every 3 d until reaching 20°C as needed. The chicks were placed in pens with litter bases measuring 1 × 1.7 m (9 chicks per box), each equipped with a manual feeding and drinking system. Drinking water was obtained from groundwater without chlorine. A ventilation system with an exhaust fan and a cooling pad with a relative humidity of 60 to 70% was used. The lighting program consisted of 23 h of light and 1 h of darkness (from 0 to 7 d of age). After 7 d of age, the dark period was approximately 5 h (4–6 h). Biosecurity was applied using Rodalon disinfectant (PT Pyridam Farma Tbk., Indonesia) and fumigation using Formac (PT IMA, Indonesia). In addition, the waste management system was implemented at the end of the study by collecting dried litter into sacks and utilizing it as an agricultural fertilizer. Broiler rearing was conducted according to the Indian River Broiler Management Handbook (Aviagen, 2018).

Experimental Treatments and Design

This study used a completely randomized design. The study used five treatments; each treatment was repeated 5 times, and each replicate consisted of 9 chickens. The treatments included regular drinking water as the negative control (P1), drinking water + 25 mg/L bacitracin as the positive control (P2), drinking water + BSFO SNEDDS 10 mL/L (P3), drinking water + BSFO SNEDDS 20 mL/L (P4), and drinking water + BSFO SNEDDS 30 mL/L (P5). Feed and water were provided ad libitum. Drinking water was provided from the age of 8 to 35 d. A sterile sorbitol energy supplement (Multifarma Satwa Maju, Tangerang, Indonesia) was administered upon DOC's arrival of DOC (10 mL/L). Drinking water was provided by mixing BSFO SNEDDS with drinking water and stirring until homogeneous. The period from d 0 to 7 served as the brooding and adaptation phases. The starter phase (1–7 d) used commercial feed (PT Japfa Comfeed, Indonesia) and d 8 to 35 utilized a specific feed formulation. The feed formulations are listed in Table 2.Table 2 Percentage and nutrient content of feed.

Table 2Feed ingredients	Percentage (%)	
Yellow corn	58.50	
Soybean meal	27.50	
Meat-bone meal	7.00	
Rice bran	2.50	
Crude palm oil	2.60	
Limestone	0.80	
L-Lysine	0.20	
DL-Methionine	0.20	
Premix	0.25	
NaCl	0.25	
Toxin binder	0.20	
Total	100	
Nutrient content		
 ME (Kcal/kg)	3013.05	
 Crude protein (%)	22.08	
 Crude fat (%)	6.12	
 Crude fiber (%)	5.28	
 Calcium (%)	1.21	
 Available phosphor (%)	0.72	
 Methionine	0.50	
 Lysin	1.26	

Growth Performance and Carcass Composition

At 35 d of age, weighing using electronic scale (electronic scale quattro Macs 3 kg x 0.1 g, Taiwan) was conducted to obtain the final body weight (BW), body weight gain (BWG), feed intake (FI), water intake (WI) and feed conversion ratio (FCR) using (Aprianto et al., 2023). Index performance (IP) was calculated at the end of the experiment.IP=(100−D)×BBFCR×(AU)×100

D=(mortality+culled)initialpopulation×100%

AU=Σ(U×P)totalharvestedpopulation

Notes: IP: Performance index; D: Depletion (%); BB: average body weight at harvest (g); FCR: feed conversion ratio; A/U: average age of harvested chicken (days); U: age of chicken; P: total population of harvested chickens.

On d 35, 2 chickens per replicate, for a total of 50 chickens with body weights close to the median, were collected and slaughtered. The animals were manually slaughtered by severing the airway, alimentary canal, and blood vessels, followed by dipping in hot water, feather removal, viscera removal, and cutting into carcasses (Aprianto et al., 2023). The carcasses were cut after removing the fur, head, neck, shank, and viscera. The abdominal fat was collected and weighed. The pH of the digestive tract was measured using a digital pH meter (Hanna HI-8314-1; Hanna Instruments Inc., Smithfield, RI).

Blood Profiles

On the 35th d, blood was collected from 25 chickens, one sample per chick (5 chickens per treatment, with one chicken per replicate), generating a total of 25 blood samples after a fasting period of 6 h. Blood samples (1 mL) were collected from the jugular vein in the morning and placed in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes. The EDTA tubes were placed in a cool box and hematology tests were performed immediately after blood collection. Reference values for poultry blood were based on (Weiss and Wardrop, 2010; Campbell, 2015).

Histomorphology of Jejunum

Measurements were taken after slaughtering five broilers from each treatment unit (n = 25). For histomorphological analysis, each slaughtered chicken was not immersed in hot water, and the jejunum was collected immediately to minimize damage. The jejunum was separated from the end of the duodenum up to 1 cm proximal to Meckel's diverticulum. A 6 cm long jejunum was obtained from the middle of the jejunum and cut into 3 equal parts. Jejunum was fixed with buffered formalin (Merck, Germany), dehydrated using a series of alcohol (70, 80, 90, and 100%), cleared, infiltrated (xylene solution and paraffin solution), embedded and trimmed (2 µm using a Leitz-1512 Microtome, Leitz, Wetzlar, Germany) and placed on slides for staining with hematoxylin (Merck KGaA 64271 Darmstadt, Germany) and eosin (Merck KGaA 64271, Darmstadt, Germany), and observed with a light microscope (Dono, 2013) at 10 times magnification. The number, length, and depth of the villous crypts were determined based on (García et al., 2007) and (Tekeli et al., 2010). Histomorphological tests for mucus production in goblet cells were performed with Alcian blue (Sigma-Aldrich, Germany) staining (Bennett et al., 2024), and observed with an Olympus microscope (Olympus Corporation, Japan).

Microflora Analysis of Jejunum and Cecum

Measurements were performed by slaughtering 3 broilers per treatment unit (n = 15). Microflora analysis was performed by calculating the total plate count (TPC). The pour plate method followed the protocol described by Bridson (2006) for Lactobacillus acidophilus, and the spread plate method for Escherichia coli and Salmonella thypimurium. All the three bacterial strains were obtained from the Food and Nutrition Culture Collection (FNCC) PAU UGM, Yogyakarta. The jejunal and cecal digesta were collected in sterile bottles containing a cryoprotectant and vortexed. The TPC calculation media used were peptone glucose yeast (PGY) for LAB with the same material composition as de-Man's rogosa sharpe agar (MRSA) (Sigma-Aldrich, Germany), Briliance E. coli or coliform selective agar media for total E. coli and Salmonella shigella agar (Merck, Germany) media for Salmonella. Bacterial counts were determined manually.

Statistical Analysis

The research data were analyzed using one-way ANOVA for the five treatments. The mathematical formula is as follows:Yij=μ+Ti+εij

Notes: Yij = observation values in treatment-i and replication-j; µ = general median value; Ti = the effect of treatment-i; εij = error in treatment-i and replication-j.

If significant differences were found, further analysis was conducted using Duncan's test with the RStudio software version 4.0.5 package (RStudio Team, 2021).

RESULTS

Growth Performance and Carcass Characteristics

The drinking water consumption of all treatments showed comparable results, whereas the consumption of lauric acid bioactives showed significant differences. The administration of BSFO SNEDDS up to a dose of 30 mL/L improved broiler feed conversion (P < 0.05). The highest performance index was achieved with BSFO SNEDDS, with IP values exceeding 300. A performance index of >300 was considered good. Adding BSFO SNEEDS to drinking water increased the percentage of carcasses as well as the weight of the breasts, thighs, and wings in broiler chickens. BSFO SNEDDS treatment increased the carcass percentage between 70% and 72%. The performance of BSFO SNEDDS in drinking water is presented in Table 3.Table 3 Performance of broiler chickens with a self-nanoemulsifying drug delivery system of black soldier fly oil (BSFO SNEDDS) in drinking water.

Table 3Treatments1	P1	P2	P3	P4	P5	SEM	P-value	
Water intake (L/bird)	6.57±0.59	6.63±0.52	6.75±0.27	7.06±0.37	7.20±0.18	0.09	0.105	
Bioactive (µL/bird)2	0.00 ±0.00e	165.70±13.07d	1755.52±69.70c	3670.16±193.92b	5619.12±136.73a	438.44	<0.001	
Feed intake (g/bird/d)	112.85±5.29	116.17±3.89	112.54±4.44	112.78±7.08	110.70±5.65	1.05	0.619	
Body weight (g/bird)	1932.93±54.58	1911.75±47.30	1940.67±35.63	1967.00±101.74	1938.00±54.48	12.04	0.741	
Body weight gain (g/bird)	1787.67±55.24	1766.88±47.28	1793.98±35.37	1820.91±103.39	1792.90±55.48	12.16	0.765	
Feed conversion ratio	1.82±0.12ab	1.83±0.10a	1.71±0.09abc	1.70±0.07bc	1.69±0.07c	0.02	<0.05	
Performance index	290.94±31.27bc	286.75±33.42c	324.15±21.63ab	331.97±23.65a	328.35±15.85a	6.19	<0.05	
Carcass (%)	62.78±2.80b	65.18±1.37b	71.85±2.92a	70.83±1.80a	72.12±3.90a	0.93	<0.001	
Breast yield (g)	515.60±46.19b	520.02±43.24b	597.72±56.89a	619.38±48.69a	601.10±30.35a	12.27	0.003	
Thigh yield (g)	423.62±32.40b	463.92±27.14b	549.90±28.79a	527.66±8.33a	539.72±45.33a	11.46	<0.001	
Wing yield (g)	138.30±7.91b	146.38±13.08b	167.96±7.16a	159.80±4.19a	160.58±8.80a	2.71	<0.001	
Abdominal fat (%)	1.13±0.056c	1.10±0.11bc	1.43±0.31ab	1.55±0.31a	1.66±0.23a	0.06	<0.01	
1 Distinct superscripts within the same row indicate significant differences: ordinary drinking water without feed additives as the negative control (P1), drinking water with 25 mg/L bacitracin as the positive control (P2), and drinking water with 10 mL/L (P3), 20 mL/L (P4), and 30 mL/L (P5) BSFO SNEDDS.

2 The mentioned bioactive are particularly bacitracin and lauric acid.

pH, Histomorphology, Goblet Cell, Immunoglobulin, and Hematology

The use of BSFO SNEDDS decreased (P < 0.001) the pH of the jejunum of chickens, resulting in increased acidity, whereas doses up to 30 mL/L did not reduce the pH of the cecum. Adding BSFO SNEDDS to drinking water increased villus height (P < 0.01). The addition of BSFO SNEDDS at doses of 20 and 30 mL/L resulted in the largest goblet cell diameter (P < 0.01). IgY showed significant differences (P < 0.01), whereas IgA showed comparable results. Leukocytes showed significant results (P < 0.01), with a BSFO SNEDDS dose of 20 mL/L reducing the number of leukocytes comparable to bacitracin. The results of pH, histomorphology, goblet cells, Ig, and hematology of 35-days-old broiler chickens treated with the self-nano-emulsifying drug delivery system of BSFO SNEDDS in drinking water are presented in Table 4. The cellular architecture of the jejunal villi is shown in Figure 1. Histomorphological evaluation demonstrated that the jejunal villi of birds differed based on their height following treatment. After treatment with >20 mL/L BSFO SNEDDS, height increased significantly (P < 0.01). These findings were supported by the histological evaluation results, which focused on goblet cells with Alcian blue staining (Figure 3) and revealed that P4 and P5 had much larger heights. Similarly, visualization using SEM showed the corresponding results for the villi after various BSFO SNEDDS treatments (Figure 2).Table 4 pH value, histomorphology, goblet cell, immunoglobulin, and hematology of 35-day-old broiler chickens with a self-nanoemulsifying drug delivery system of black soldier fly oil (BSFO SNEDDS) in drinking water.

Table 4Treatments1	P1	P2	P3	P4	P5	SEM	P-value	
Jejunum (pH)	6.79±0.51a	6.70±0.23a	5.83±0.28b	6.08±0.02b	6.21±0.02b	0.09	<0.001	
Cecum (pH)	6.89±0.28	6.77±0.25	6.62±0.20	6.85±0.09	6.74±0.15	0.04	0.32	
Villi height (µm)	1206.40 ±88.99bc	1198.20 ±53.76bc	1166.40 ±37.49c	1255.40 ±12.76ab	1315.40 ±77.05a	15.33	<0.01	
Villi width (µm)	82.62±40.58b	72.67±29.85b	87.06±16.35b	87.02±20.11b	134.55±46.64a	7.43	<0.1	
Crypt depth (µm)	54.85±23.80	52.97±7.27	63.13±17.21	62.38±15.77	47.22±13.38	3.23	0.52	
Villi height: crypt depth	25.06±9.52	22.90±2.70	20.10±7.73	21.07±4.77	29.81±8.87	1.49	0.26	
Jejunal goblet cell (µm)	2240.5±143.87b	3101.1±647.74b	2853.6±138.77b	27235.7±30864.41a	40446.4±23618.22a	4525.59	<0.01	
Immunoglobulin Y (IgY)	4.38±0.78c	6.44±0.50a	5.05±1.10bc	5.94±1.22ab	6.04±0.54ab	0.22	<0.01	
Immunoglobulin A (IgA)	0.44±0.14	0.54±0.09	0.49±0.14	0.50±0.10	0.39±0.16	0.03	0.60	
HEMATOLOGY								
 Hemoglobulin	8.90±0.63	9.13±0.57	9.01±0.41	8.48±0.25	8.04±1.56	0.17	0.24	
 Hematocrit (%)	29.60±2.41	30.60±2.70	29.40±2.07	27.40±1.14	26.40±4.83	0.62	0.18	
 Erythrocytes	2.96±0.32	2.78±0.12	2.95±0.19	2.79±0.27	2.52±0.62	0.07	0.31	
 Leukocytes	21.70±3.29a	13.90±5.78c	16.10±2.10bc	13.30±3.63c	18.70±1.44ab	0.91	<0.01	
 TPP (g/dL)	3.98±0.35	3.74±0.17	3.73±0.55	3.81±0.15	4.70±1.44	0.15	0.21	
 MCV (fL)	100.52±7.27	110.56±12.69	99.72±7.65	98.64±7.94	106.80±12.13	2.03	0.27	
 MCH (pg)	30.24±1.96	32.96±2.55	30.62±2.51	30.58±3.29	32.44±3.37	0.56	0.44	
 MCHC (%)	30.12±1.33	29.92±1.39	30.76±1.74	30.98±1.40	30.38±0.65	0.26	0.72	
 Heterophiles	10.09±2.38b	6.10±4.09b	7.69±1.98b	8.65±1.91b	18.86±10.85a	1.35	<0.05	
 Basophils	0	0	0	0	0	-	-	
 Eosinophils	1.88±1.52	1.68±1.23	1.0±0.57	1.97±2.05	3.67±2.78	0.37	0.24	
 Limphocytes	9.43±1.17a	5.9±1.15c	7.51±1.62bc	7.62±0.57b	8.20±1.36ab	0.323	<0.01	
 Monocytes	0.30±0.24	0.23±0.24	0.62±0.73	0.52±0.37	0.87±1.16	0.13	0.56	
 H/L ratio	1.08±0.27b	1.03±0.63b	1.07±0.39b	1.14±0.28b	2.37±1.51a	0.18	<0.1	
1 Distinct superscripts within the same row indicate significant differences: ordinary drinking water without feed additives as the negative control (P1), drinking water with 25 mg/L bacitracin as the positive control (P2), and drinking water with 10 mL/L (P3), 20 mL/L (P4), and 30 mL/L (P5) BSFO SNEDDS. The parameters measured include Haemoglobin (g/dL), Erythrocytes (106/µL), Leukocytes, Heterophils, Basophils, Eosinophils, Lymphocytes, and Monocytes (103/µL).

Figure 1 Histomorphology of jejunal villi from broiler chickens with haematoxylin eosin staining (ordinary drinking water without feed additives, negative control (P1; A), drinking water + 25 mg/L bacitracin, positive control (P2; B), drinking water + 10 mL/L BSFO SNEDDS (P3; C), drinking water + 20 mL/L BSFO SNEDDS (P4; D), drinking water + 30 mL/L BSFO SNEDDS (P5; E)).

Figure 1

Figure 2 SEM results of jejunal villi of broiler chickens treated with BSFO SNEDDS in drinking water ((ordinary drinking water without feed additives, negative control (P1; A), drinking water + 25 mg/L bacitracin, positive control (P2; B), drinking water + 10 mL/L BSFO SNEDDS (P3; C), drinking water + 20 mL/L BSFO SNEDDS (P4; D), drinking water + 30 mL/L BSFO SNEDDS (P5; E)).

Figure 2

Figure 3 Histology of goblet cells with alcian blue staining. Goblet cells are shown in blue circles ((ordinary drinking water without feed additives, negative control (P1; A), drinking water + 25 mg/L bacitracin, positive control (P2; B), drinking water + 10 mL/L BSFO SNEDDS (P3; C), drinking water + 20 mL/L BSFO SNEDDS (P4; D), drinking water + 30 mL/L BSFO SNEDDS (P5; E)).

Figure 3

Microflora Analysis of Jejunum and Cecum

Adding BSFO SNEDDS to drinking water reduced the population of E. coli bacteria (P < 0.01) in the jejunum of chickens. The visualization results of the present study against diverse bacterial species were illustrated using total plate count experiments (Figure 4, Figure 5, Figure 6). As shown in Figure 4, the total bacterial population was lowest when treated with the highest concentration of BSFO SNEDDS (P5). Conversely, LAB proliferation was the highest in this treatment group (Figure 6). No single S. typhimurium was detected in this assay (Figure 5). A dose of 10 mL/L of BSFO SNEDDS increased the abundance of L. acidophilus (P < 0.01). Lactobacillus acidophilus and E. coli in the cecum showed comparable results. S. typhimurium was not detected in any of the treated samples. The results of the jejunal and cecal microflora analyses are shown in Table 5.Figure 4 Total plate count (TPC) result for chicken jejunum digesta microflora against E. coli bacteria (ordinary drinking water without feed additives, negative control (P1; A), drinking water + 25 mg/L bacitracin, positive control (P2; B), drinking water + 10 mL/L BSFO SNEDDS (P3; C), drinking water + 20 mL/L BSFO SNEDDS (P4; D), drinking water + 30 mL/L BSFO SNEDDS (P5; E)).

Figure 4

Figure 5 Total plate count (TPC) result for chicken jejunum digesta microflora against S. typhimurium bacteria (ordinary drinking water without feed additives, negative control (P1; A), drinking water + 25 mg/L bacitracin, positive control (P2; B), drinking water + 10 mL/L BSFO SNEDDS (P3; C), drinking water + 20 mL/L BSFO SNEDDS (P4; D), drinking water + 30 mL/L BSFO SNEDDS (P5; E)).

Figure 5

Figure 6 Test results for chicken jejunum digesta microflora against lactic acid bacteria (LAB) (ordinary drinking water without feed additives, negative control (P1; A), drinking water + 25 mg/L bacitracin, positive control (P2; B), drinking water + 10 mL/L BSFO SNEDDS (P3; C), drinking water + 20 mL/L BSFO SNEDDS (P4; D), drinking water + 30 mL/L BSFO SNEDDS (P5; E)).

Figure 6

Table 5 The microflora of jejunal and cecum with a self-nanoemulsifying drug delivery system of black soldier fly oil (BSFO SNEDDS).

Table 5Treatments (log10 cfu/g)	
Parameters	P1	P2	P3	P4	P5	SEM	Pvalue	
Jejunum	
E. coli	4.14 ±0.38a	3.51±0.37ab	3.84±0.50ab	3.31±0.33b	2.30±0.15c	0.186	<0.01	
S. typhimurium	ND1	ND	ND	ND	ND	-	-	
L. acidophilus	3.97±0.20c	4.92±0.60b	6.05±0.83a	5.09±0.39b	5.77±0.20ab	0.224	<0.01	
Cecum	
E. coli	7.11±0.89	7.04±0.74	7.92±0.76	7.15±1.11	6.67±0.16	0.206	0.455	
S. typhimurium	ND	ND	ND	ND	ND	-	-	
L. acidophilus	0.85±0.05	0.85±0.04	0.90±0.04	0.85±0.07	0.82±0.01	0.012	0.467	
Distinct superscripts within the same row indicate significant differences: ordinary drinking water without feed additives as the negative control (P1), drinking water with 25 mg/L bacitracin as the positive control (P2), and drinking water with 10 mL/L (P3), 20 mL/L (P4), and 30 mL/L (P5) BSFO SNEDDS.

DISCUSSION

BSFO SNEDDS administered via drinking water is a potential technology for facilitating the application of BSFO as an alternative antibiotic. SNEDDS can improve the solubility, absorption mechanism, bioavailability, and efficacy of essential oil-based formulations (Masoud et al., 2013; Savale, 2015), as well as control the release rate of less water-soluble drugs in the intestine (Wang et al., 2009a). Poultry requires water to maintain body temperature and metabolic processes (Kleyn and Chrystal, 2020). Drinking water consumption is influenced by feed consumption (Maroof et al., 2017). The feed and drinking water consumption results were comparable. Increased consumption of bioactive lauric acid can positively affect the balance of microflora in the small intestine and support the growth of intestinal villi. Adding BSFO SNEDDS up to 30 mL/L significantly improved broiler feed conversion (P < 0.05). Maximum absorption improves the growth performance of chickens. Lauric acid, a fatty acid in BSFO SNEDDS, provides better digestive and health conditions that affect feed conversion and indirectly improves the performance index. The mechanism of action of the antibacterial activity of fatty acids includes increasing membrane permeability, disrupting membrane enzyme activity and the electron transport chain, and releasing oxidative phosphorylation (Yoon et al., 2018). The addition of BSFO SNEDDS increased the percentage of carcasses to 70 to 72%. Aprianto et al. (2023) showed that the supplementation of BSFO calcium salt in broiler feed resulted in a lower carcass percentage (69%). This proves that the use of nanotechnology in the form of SNEDDS with nanosized particles facilitates absorption in the digestive tract. The available micronutrients can be optimally utilized, resulting in better growth performance compared to the use of BSFO in its biomass form. In the present study, a notable increase in abdominal fat was recorded at 1.55 and 1.66% for P4 and P5, respectively, possibly due to the absorption of lauric acid in the small intestine in the form of triglycerides. Classified as a medium-chain fatty acid (MCFA), lauric acid is directly transported to the liver, which is not the case with long-chain fatty acids. In the liver, lauric acid is rapidly metabolized to energy or transformed into ketone bodies. Therefore, once the maintenance energy requirements are fulfilled, lauric acid is stored directly in the form of fat. Lauric acid in black soldier fly larvae and pupae is stored in the muscles and liver (Fischer et al., 2022). It has a mode of action similar to that of bacitracin, which inhibits or disrupts cell-wall synthesis in the wall (cell membrane). Lauric acid functions as an antibacterial agent that increases villus growth and reduces the population of pathogenic bacteria in the cell walls of the small intestine. Lauric acid contains a hydroxyl (–OH) group that functions as an antibacterial agent. The cationic H+ group binds strongly to the surface of anionic bacteria through electrostatic attraction, causing friction that generates energy and heat that causes a shift and damage to the bacterial cell membrane, resulting in lysis and death (Li et al., 2001). Lauric acid is the most potent MCFA (Ullah et al., 2022). MCFA are known to promote metabolism. Bacteria require more energy to survive, thereby depleting their energy and leading to death. Optimal growth of villi increases the number of pores (adsorptive cells) and enhances the absorption of micronutrients from the digesta of the small intestine into the intestinal wall. This is supported by Pelicano et al. (2005), who stated that an increased micronutrient absorption capacity maximizes the amount of nutrients used to meet the body's daily metabolism.

BSFO SNEDDS contain lauric acid, which can lower the pH of the digestive tract. These organic acids support the growth of L. acidophilus in the digestive tract. An increase in L. acidophilus population leads to higher lactic acid production and causes a decrease in pH in the digestive tract, thus supporting the growth of non-pathogenic bacteria and inhibiting pathogenic bacteria (Fascina et al., 2012). The presence of H+ creates an acidic environment that pathogenic bacteria cannot tolerate. Acidic pH in the gastrointestinal tract protects the gastrointestinal tract from colonization and translocation of pathogenic microbes that are harmful to the host body (Tiihonen et al., 2010). Toxin production and secretion from the epithelial mucus by goblet cells are also reduced (Sklan, 2004). The height of jejunal villi increased with BSFO SNEDDS treatment. This proves that the use of SNEDDS reduces pathogenic bacteria, facilitates the absorption of bioactive compounds, and results in better villus growth than studies using biomass forms. Intestinal morphology is related to the nutrient absorption capacity of the small intestine (Kim et al., 2021). Reduced pathogenic bacteria reduce harmful toxic substances that can reduce nutrient absorption by epithelial cells (Hosseini et al., 2017). Lauric acid modulates the microbiome to enhance intestinal health (Duarte-Silva et al., 2020). Decreased mucosal secretion causes villi cells in the small intestinal wall to grow optimally, increases the number of pores in the villi cells, and maximizes absorption, which improves the growth performance of livestock (Pelicano et al., 2005). A higher ratio of villus height to crypt depth indicates better nutrient absorption owing to an increased surface area. Goblet cells are mucin producers that coat the intestinal surface of the digestive tract, protect against pathogenic infections, and prevent intestinal inflammation (Yang and Yu, 2021). Mucin is the first barrier that covers the mucosal surface and protects the epithelial cells from chemical, enzymatic, microbial, and mechanical damage (Linden et al., 2008; Ariyadi et al., 2013). An increase in the number and diameter of goblet cells is associated with higher immunity levels. The increase in the number of goblet cell nodules was assumed to be due to an increase in the immune system (Yang and Yu, 2021). The addition of lauric acid to the diet can significantly increase villus height and number of goblet cells (Ullah et al., 2022). BSFO SNEDDS doses of 20 and 30 mL/L yielded the best results, with the highest goblet cell diameters. Ig levels are another measure of immunity. The IgY results were significant, with the range of numbers still within the normal standard for poultry Ig, except for the negative control, which was below the standard. The standard for poultry IgY is 5 to 7 (Karspers et al., 2021). IgA results of this study were within the range of normal values where the standard of poultry IgA is 0.35 to 0.65 (Karspers et al., 2021). BSFO SNEDDS treatment increased broiler immunity, as evidenced by the hematological results. Hemoglobulin results were still within normal values, and the hemoglobin reference value for chickens is 7 to 13 g/dL (Weiss and Wardrop, 2006). Leukocyte counts decreased with the administration of up to 20 mL/L BSFO SNEDDS. Leukocytes were at normal values where the reference value of chicken leukocytes is 12 to 30 103 /µL (Weiss and Wardrop, 2006). The reference value of heterophils for chickens is 3 to 6 103 /µL (Weiss and Wardrop, 2006). The heterophil/lymphocyte (H/L) ratio is an indicator of stress and resistance to stressors. The higher the H/L ratio, the higher the stress level in poultry. In the present study, the application of nanotechnology through an SNEDDS requires cautious dosage management. This is critical because an excessive dosage can affect the in vivo outcomes. SNEDDS possesses a characteristic zeta potential with an electrical charge, indicating that an overdose can lead to toxicity. A high concentration of surfactants in SNEDDS can disrupt the cellular balance and damage the cellular field, potentially increasing stress in chickens and affecting the H/L ratio. Therefore, BSFO SNEDDS should not exceed 20 mL/L (P4).

The results of the jejunum and cecum digesta microflora tests showed that the administration of BSFO SNEDDS reduced the pathogenic bacteria E. coli and the administration of BSFO SNEDDS at a dose of 10 mL/L increased the non-pathogenic bacteria L. acidophilus. The use of black soldier fly larvae partially reduces harmful microbes and may be regarded as beneficial in terms of gut health (Hartinger et al., 2022). In the cecum digesta test results, pathogenic microflora showed comparable results, whereas S. thypimurium bacteria were not detected in either the jejunum or cecum because the study was conducted with strict biosecurity and sanitation.

In conclusion, the traditional antibiotic BSFO SNEDDS improved the development of jejunal villi structure in broilers, increased goblet cells, decreased pathogen microbes, and may have improved broiler growth performance. Further studies should be conducted to evaluate the effects of BSFO on immunity-related gene expression in broiler chickens.

ACKNOWLEDGMENTS

The authors would like to acknowledge the Institute for Research and Community Service of Universitas Sebelas Maret for research funding through a research scheme PENELITIAN DISERTASI DOKTOR (PDD-UNS) RKAT PTNBH Universitas Sebelas Maret for the 2024 fiscal year with assignment agreement letter No. 194.2/UN27.22/PT.01.03/2024. We also would like to address gratitude to Animal Science Learning Center (ASLC) and Closed House Department of Animal Production, Faculty of Animal Science of Universitas Gadjah Mada for providing facilities in this study.

Author Contributions: All authors discussed the results and contributed to the final manuscript. RD: Conceptualization, Supervision, Funding acquisition, Data curation, Investigation, Writing-original draft, review and editing. BA, RM: Data curation, Methodology, Investigation. ZZ: Supervision, Data curation Writing-review and editing.

DISCLOSURES

All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version.

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process: During the preparation of this work the author(s) used Editage Service (Bahasa to English (AI) + Human Bilingual Review) in order to translate the manuscript. After using this service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.
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