This study was reported in accordance with ARRIVE guidelines and was conducted in strict accordance with the guidelines of the National Research Council (NRC) of Thailand, and the protocols were approved by the Institutional Animal Care and Use committee of Khon Kaen University (Khon Kaen, Thailand; approval number 125/64 on 18 November, 2021).
Black soldier fly larvae and probiotics
The BSFL (Hermetia illucens) were obtained locally (Ban Dangnoi, Khon Kaen, Thailand), reared on broiler feed substrate, and harvested on day 13 of larval development. BSFL samples were dried at 65 °C for 72 h, and their nutrient composition was analysed before they were added to the diet formulation. The main components of BSFL were CP (35.88%), EE (EE, 30.58), lysine (2.31%), total essential amino acids (15.81%), non-essential amino acids (18.59%), lauric acid (9.17%), total saturated fatty acids (21.09%), unsaturated fatty acids (11.62%), and chitin (4.26%, Table 1). The multi-probiotics included Bacillus subtilis (1 × 1011 cfu/kg), Bacillus licheniformis (1 × 109 cfu/kg), and Saccharomyces cerevisiae (1 × 109 cfu/kg).
Table 1 Analysed values of BSF larvae (Hermetia illucens, dry matter basis)1
Item
|
Amount (g/100 g)
|
Item
|
Amount (g/100 g)
|
Dry matter
|
92.36
|
Saturated fatty acids (SFA)
|
|
Total ash
|
11.74
|
Butyric acid (C4:0)
|
ND
|
Crude protein
|
35.88
|
Caproic acid (C6:0)
|
ND
|
Ether extract
|
30.58
|
Caprylic acid (C8:0)
|
ND
|
Crude fibre
|
6.58
|
Capric acid (C10:0)
|
0.33
|
Neutral detergent fibre
|
30.62
|
Undecanoic acid (C11:0)
|
ND
|
Acid detergent fibre
|
16.22
|
Lauric acid (C12:0)
|
9.17
|
Acid detergent lignin
|
1.85
|
Tridecanoic acid (C13:0)
|
0.02
|
Nitrogen free extract
|
7.58
|
Myristic acid (C14:0)
|
2.29
|
Calcium
|
3.59
|
Pentadecanoic acid (C15:0)
|
0.07
|
Phosphorus
|
0.70
|
Palmitic acid (C16:0)
|
7.81
|
Chitin
|
4.32
|
Heptadecanoic acid (C17:0)
|
0.07
|
Essential amino acids
|
|
Steric acid (C18:0)
|
1.33
|
Isoleucine
|
1.61
|
Unsaturated fatty acids (USFA)
|
|
Leucine
|
2.61
|
Palmitoleic acid (C16:1n7)
|
1.08
|
Lysine
|
2.31
|
cis-10-heptadecanoic acid (C17:1n10)
|
0.05
|
Methionine
|
0.58
|
cis-9-oleic acid (C18:1n9)
|
6.33
|
Phenylalanine
|
1.51
|
cis-9,12-linoleic acid (C18:2n6)
|
3.65
|
Threonine
|
1.47
|
α-linolenic acid (C18:3n3)
|
0.28
|
Tryptophan
|
0.42
|
γ-linolenic acid (C18:3n6)
|
ND
|
Tyrosine
|
1.77
|
cis-11,14-eicosadienoic acid (C20:2)
|
ND
|
Histidine
|
1.09
|
cis-11,14,17-eicosatrienoic acid (C20:3n3)
|
0.06
|
Valine
|
2.44
|
Eicosapentaenoic acid (C20:5n3)
|
0.14
|
Total
|
15.81
|
Erucic acid (C22:1n9)
|
ND
|
Non-essential amino acids
|
|
Docosadienoic acid (C22:2)
|
ND
|
Alanine
|
3.24
|
Docosahexaenoic acid (C22:6n3)
|
0.01
|
Arginine
|
1.45
|
Nervonic acid (C24:1n9)
|
0.02
|
Aspartic acid
|
3.25
|
Total SFA
|
21.09
|
Cystine
|
ND
|
Total USFA
|
11.62
|
Glutamic acid
|
4.39
|
|
|
Glycine
|
2.08
|
|
|
Serine
|
1.71
|
|
|
Proline
|
2.47
|
|
|
Total
|
18.59
|
|
|
GE = gross energy; SFA = saturated fatty acids; USFA = unsaturated fatty acids; ND = not detected
Animals, treatments, and management
A total of 80 piglets [(Landrace × Large White) × Duroc] weaned at 28 ± 3 day of age (7.34 ± 0.21 kg body weight, BW) were divided into four experimental groups and fed the following diets: PC, basal diet supplemented with 0.02% amoxicillin; NC, basal diet without supplementation; BSFL12, basal diet supplemented with 12% full-fat BSFL; and BSFL + Pro, basal diet supplemented with BSFL + 0.1% multi-probiotics. Each treatment had five replicates. Each animal pen had an equal number of gilts and barrows (1:1 ratio) in a randomized complete block design with initial body weight as the blocking factor. Pigs were housed in pens with slatted concrete flooring (20 pens, 1.6 × 2.1 m; stocking density, 0.8 m2/pig) furnished with a low-pressure nipple drinker, stainless trough, and heating lamp. Rice straw and gunny bags were supplied as bedding materials during the 14-day post weaning period and were changed twice daily (0600 and 1900) following the guidelines of EU Directive 2010/63/EC for animal experiments. All pigs were vaccinated against Aujeszky’s disease, salmonellosis, and transmissible gastroenteritis. Mash diets were formulated to meet or exceed the nutrient requirements for pigs weighing 11–25 kg. The diets were formulated in two phases (Phase I, 1–2 weeks post-weaning; Phase II, 3–4 weeks post-weaning) as suggested by the NRC [49] (Table 2). All pigs had access to feed and water throughout the experimental period.
Table 2 Ingredients and nutrient values of the experimental diet (% as fed basis)1,2
Ingredient
|
Phase I (day 1 to 14)
|
|
Phase II (day 15 to 28)
|
PC
|
NC
|
BSFL12
|
BSFL + Pro
|
|
PC
|
NC
|
BSFL12
|
BSFL + Pro
|
Corn
|
40.39
|
40.41
|
40.07
|
39.88
|
|
50.57
|
50.59
|
50.84
|
50.67
|
Soybean meal (43.8%)
|
28.88
|
28.88
|
17.22
|
17.31
|
|
31.39
|
31.39
|
19.80
|
19.87
|
Broken rice
|
10.00
|
10.00
|
10.00
|
10.00
|
|
6.00
|
6.00
|
6.00
|
6.00
|
Full-fat soybean meal
|
8.00
|
8.00
|
8.00
|
8.00
|
|
3.50
|
3.50
|
3.50
|
3.50
|
Whey powder
|
5.00
|
5.00
|
5.00
|
5.00
|
|
2.00
|
2.00
|
2.00
|
2.00
|
Skimmed milk
|
5.00
|
5.00
|
5.00
|
5.00
|
|
3.00
|
3.00
|
3.00
|
3.00
|
Black soldier fly larva
|
0.00
|
0.00
|
12.00
|
12.00
|
|
0.00
|
0.00
|
12.00
|
12.00
|
Bacillus probiotic
|
0.00
|
0.00
|
0.00
|
0.10
|
|
0.00
|
0.00
|
0.00
|
0.10
|
Amoxicillin
|
0.02
|
0.00
|
0.00
|
0.00
|
|
0.02
|
0.00
|
0.00
|
0.00
|
L-Lysine HCl (78%)
|
0.29
|
0.29
|
0.29
|
0.29
|
|
0.34
|
0.34
|
0.34
|
0.34
|
DL-Methionine (99%)
|
0.09
|
0.09
|
0.09
|
0.09
|
|
0.11
|
0.11
|
0.11
|
0.11
|
Dicalcium phosphate
|
1.39
|
1.39
|
1.39
|
1.39
|
|
1.56
|
1.56
|
1.56
|
1.56
|
Limestone
|
0.09
|
0.09
|
0.09
|
0.09
|
|
0.66
|
0.66
|
0.00
|
0.00
|
Sodium chloride
|
0.35
|
0.35
|
0.35
|
0.35
|
|
0.35
|
0.35
|
0.35
|
0.35
|
Vitamin-mineral premix
|
0.50
|
0.50
|
0.50
|
0.50
|
|
0.50
|
0.50
|
0.50
|
0.50
|
Total
|
100
|
100
|
100
|
100
|
|
100
|
100
|
100
|
100
|
Calculated values (%)
|
|
|
|
|
|
|
|
|
Metabolizable energy (kcal/kg)
|
3,300
|
3,300
|
3,300
|
3,300
|
|
3,300
|
3,300
|
3,300
|
3,300
|
Crude protein
|
22.50
|
22.50
|
22.50
|
22.50
|
|
21.50
|
21.50
|
21.50
|
21.50
|
Ca
|
0.68
|
0.68
|
0.68
|
0.68
|
|
0.80
|
0.80
|
0.80
|
0.80
|
Total phosphorus
|
0.66
|
0.66
|
0.66
|
0.66
|
|
0.68
|
0.68
|
0.68
|
0.68
|
Lysine
|
1.45
|
1.45
|
1.45
|
1.45
|
|
1.38
|
1.38
|
1.38
|
1.38
|
Methionine + Cysteine
|
0.65
|
0.65
|
0.65
|
0.65
|
|
0.65
|
0.65
|
0.65
|
0.65
|
Tryptophan
|
0.22
|
0.22
|
0.22
|
0.22
|
|
0.23
|
0.23
|
0.23
|
0.23
|
Threonine
|
0.87
|
0.87
|
0.87
|
0.87
|
|
0.86
|
0.84
|
0.84
|
0.88
|
Crude fat
|
3.83
|
3.83
|
7.53
|
7.53
|
|
3.30
|
3.72
|
3.72
|
7.02
|
Crude fibre
|
3.41
|
3.41
|
3.40
|
3.40
|
|
3.64
|
3.64
|
3.66
|
3.66
|
Chitin
|
0.00
|
0.00
|
0.52
|
0.52
|
|
0.00
|
0.00
|
0.52
|
0.52
|
1Provided (per kg of complete diet): vitamin A 8,000 IU; vitamin D3 1,600 IU; vitamin E 34 IU; biotin 64 g; riboflavin 3.4 mg; calcium pantothenic acid 8 mg; niacin 16 mg; vitamin B12 12 g; vitamin K 2.4 mg; Se as Na2SeO3 0.1 mg; I as KI 0.32 mg; Mn as MnSO4 25.2 mg; Cu as CuSO4 53.9 mg; Fe as FeSO4 127.3 mg; Zn as ZnSO4 83.46 mg, and Co as CoSO4 0.28 mg.
2Calculated values were obtained the nutrient composition data from actual analysis and NRC [49]
3Probiotic mixture consisted of Bacillus subtilis, B. licheniformis, and Saccharomyces cerevisiae
Measurement of growth performance and diarrheal rate
On days 1, 15, and 29 post-weaning, each pig was weighed individually at 0600. Following this, pen-based feed disappearance was recorded to determine the ADG, ADFI, and G:F (calculated as ADG/ADFI). The severity of diarrhoea was measured visually using a faecal consistency score: 0, firm and shape faeces; 1, soft and shaped faeces; 2, loose faeces; and 3, watery faeces. Scores of 0 and 1 represented normal faeces, whereas scores of 2 and 3 represented diarrhoea. Diarrheal rate (%) was calculated using the following formula: number of pigs with diarrhoea / (total number of pigs by treatment × days with diarrhoea) × 100.
Apparent total-tract digestibility
A total of 20 barrows (initial average BW, 10.26±2.36 kg) were chosen separately from the feeding trial and assigned to each dietary treatment in five replicates in a completely randomized design. The pigs were housed individually in cages (0.58 m × 0.83 m) equipped with a grid and slurry pit for a 7-d adaption and 5-d faecal collection period. The experimental diets were offered at every 12-h interval in an amount equalling 3× the maintenance energy requirement (106 kcal of metabolizable energy per kg of BW0.75 [49] NRC, 2012). During the faecal collection period, chromic oxide and ferric oxide were homogenously mixed in all experimental diets (5 g/kg) as the indigestible indicator at the first and last meal, respectively. The collection of faecal output started when the initial marker appeared and ended when the final marker appeared in the faeces. Pooled faeces were weighed and dried in a forced air-drying oven (60 °C for 72 h) and subsequently ground in a hammer mill using a 0.88 mm screen. Representative samples of pooled faeces and diets were used to measure the levels of DM (method #930.15), CP (method #984.13), total ash (method #942.15), and EE (method #920.39) using standard AOAC protocols [50]. These values were used to calculate ATTP of these components [51].
Blood collection and analyses
On day 29, one healthy pig per pen (n = 20) weighing the average weight per pen was selected for blood sampling (10 mL) through the anterior vena cava. Blood samples were collected in serum-coated tubes with silica (Grener Bio-one, Chonburi, Thailand), allowed to clot at room temperature for 60 min, centrifuged for 10 min at 4 °C at 13,000 ×g, and then frozen at -20 °C. Serum concentrations of IgA, IgG, IgM, IL1β, IL6, and TNFα were quantified using porcine enzyme-linked immunosorbent kits (Abcam, Cambridge, UK). TAC and the levels of SOD, GSH-Px, and MDA were determined using commercial kits (Sigma-Aldrich, St. Louis, MO). All assays were performed as outlined by the manufacturer and conducted in triplicates to control for variations.
Organ weight and digesta pH
After blood collection, all selected pigs were slaughtered after 12 h of fasting. The digestive tract was eviscerated to harvest the heart, liver, kidney, stomach, and spleen. Each organ was separately flushed with 0.9% phosphate buffer saline solution, blot-dried, and weighed using a digital scale. Segments of the colon, cecum, and small intestine were also collected for later examination. The cecum and colon (proximal, middle, and distal portions) tissues collected were immediately used to measure the hindgut pH with a pH meter (AP 110, Fisher Scientific, Pittsburgh, PA, USA).
Intestinal morphology
Segments of the small intestine were collected immediately prior to euthanasia. Longitudinal dissections of the duodenum (50 cm caudal to pyloric sphincter), jejunum (5 cm from the pyloric sphincter), and ileum (20 cm from the ileocecal orifice) were performed carefully. Each sample was rinsed with saline solution and fixed with a neutral buffer (pH 7.0) and formaldehyde solution (10% vol/vol) for 72 hours. The tissue samples were embedded with ethanol and xylene and then transversely cut into a 5-µm-thick sections using a rotary microtome (Leica RM2235, Wetzlar, Germany). The tissue sections were placed on glass slides and stained with haematoxylin-eosin (H&E staining, Sigma-Aldrich, St Louis, MO, USA). In total, 20 well-oriented villi (per stained section) and crypt columns were used to determine VH (from the tip of the villus to the basolateral membrane) and CD (from the villus–crypt junction and the submucosa) using an optical light microscope at 10× magnification. Average VH and CD values were recorded, and the VH:CD was calculated.
Microbial count
Faecal samples were collected by rectal massage of the pigs (5 samples per treatment) and suspended in 0.9% (w/v) sodium chloride solution at 1:10 dilution. A 0.1 mL aliquot of each dilution was spread-plated in triplicate onto Rogosa and Sharpe, MacConkey, and Salmonella–Shigella agar for the determination of Lactobacillus spp., Escherichia coli, and Salmonella spp., respectively following the manufacturer’s guidelines. The average growth of each microbe was log-transformed and represented as log10 colony-forming unit (CFU)/g of faeces.
Statistical analysis
Data were analysed with general linear models in SAS (version 9.4, SAS Inst. Inc., Cary, NC, USA) using a randomized complete block design with “pen” as the experimental unit for growth performance and diarrhoea rate, and “each pig” as the experimental unit for digestibility, blood analyses, organ weight, hindgut pH, intestinal morphology, and microbial counts. The statistical model was as follows:
where Yij is the jth observation of the ith treatment; µ is the overall mean; αi is the effect of the ith block (i = 1–5); βj is the effect of the jth treatment (j = 1–4); and εij is the error term. Duncan’s new multiple range test was used to test for significant differences among dietary treatments. All data were represented as the mean ± standard error of the mean (SEM), and P < 0.05 was considered statistically significant.