Full-fatted Hermetia illucens larva as a protein alternative: effects on weaning pig growth performance, gut health, and antioxidant status under poor sanitary conditions

ABSTRACT

We investigated the effectiveness of full-fatted black soldier fly larvae (BSF) supplementation on growth performance, gut health, and antioxidant capacity of weaned pigs under poor sanitation. Eighty 28-d-old piglets were divided into four groups (five replicates of four pigs each): control group comprising pigs under hygienic conditions without BSF addition (PC); control group comprising pigs under poor sanitary conditions without BSF addition (NC); and BSF6 and BSF12 groups comprising pigs supplemented 6% and 12% BSF, respectively. Pigs were fed the diets in two feeding programs: Phase I (1–14 d) and II (15–28 d) containing 3,300 and 3,265 kcal/kg of metabolisable energy and 22.5% and 21.5% of crude protein, respectively. Compared to the NC, the BSF6 and BSF12 increased body weight and average daily feed intake in Phase II (P < 0.05). The BSF exhibited greater nutrient digestibility, immunoglobulin A and glutathione peroxidase levels, cecum weights, duodenal villus height, duodenal villus-to-crypt depth ratio, and caecal Lactobacillus spp. (P < 0.05). Furthermore, the BSF6 and BSF12 showed significantly decreased diarrheal rate, tumour necrosis factor-alpha, small intestine weight, caecal pH, and duodenal crypt depth unlike the NC (P < 0.05). The BSF12 had lower malondialdehyde concentration and Escherichia coli than the NC (P < 0.05). In conclusion, the addition of 12% BSF improved growth performance and nutrient utilisation, thereby reducing gut inflammation and modulating antioxidant capacity in post-weaning piglets.

KEYWORDS:

• Insect protein
• black soldier fly larva
• husbandry practice
• weaning piglets
• gut health

Introduction

Post-weaning challenges include poor hygienic housing, which makes nursery piglets susceptible to disease occurrence, low feed intake, impairments in nutrient digestibility, and weight loss (Jayaraman and Nyachoti 2017). Poor sanitary conditions also induce a systematic inflammatory response by activating an acute phase protein called haptoglobin, causing oxidative stress (Sierżant et al. 2019). In addition, oxidative stress is caused by reactive oxygen species (ROS), which lead to inflammation in disease-prone piglets (Sierżant et al. 2019). Endogenous antioxidants and dietary modifications can both inhibit the ROS cascade (Chia et al. 2020). Therefore, it is critical to provide digestible diet that not only maintains mucosal development but also acts as an immunostimulant for piglets under uncertain conditions.

Black soldier fly (Hermetia illucens L.) (BSF) is an attractive ingredient for livestock feed because of its comparable amounts of crude protein (53%), amino acid profiles (2.3% – 2.6% lysine), lipids (58%), and calcium (7%) to those of conventional protein ingredients (Ewald et al. 2020). The BSF larva is normally enriched with monounsaturated fatty acids (MUFAs), particularly lauric acid (C:12), which constitutes about 52% of the total MUFAs (Ewald et al. 2020). It potentially improved nutrient availability and antimicrobial action (Jackman et al. 2020; Rebucci et al. 2021), thus improving the intestinal health and performance of weaned pigs. Yu et al. (2019) reported this with 4%−8% BSF larva supplementation. The non-biodegradable BSF chitin promotes hindgut fermentation of short-chain fatty acids (SCFAs) and has immunomodulatory effects on immune function. Taken together, BSF-derivative antimicrobial peptides completely inhibited pathogen growth at the genetic level (Xia et al. 2021). However, there is limited information on the use of full-fatted larva to minimize the weaning stress of piglets under poor hygienic conditions. Therefore, we hypothesised that the optimum level of full-fatted larvae as an attractive protein ingredient will provide adequate nutrients and other immunostimulants to promote the gut health of weaning pigs. We aimed to investigate the effect of various BSF levels on the growth performance, nutrient digestibility, intestinal morphology, proinflammatory cytokine, and antioxidant status of weaning pigs housed under poor sanitary conditions.

Material and methods

All procedures of animal handling were reviewed and approved by the Institutional Animal Care and Use Committee of Khon Kaen University (Khon Kaen, Thailand) under authorised no. 124/64 (approved on November 18, 2021).

Black soldier fly preparation

The BSFs were fed a mixture of agricultural by-products from corn meal, rice bran, and banana, including broiler feed of 20% crude protein mixed with tap water (approximately 60–70% moisture). The BSF larva was harvested at 15 ± 4 ds, then directly dried at 65 ^oC for 48 h to constant weight, and grind prior uses for nutrient analysis and feed formulation.

Animals, treatments, and management

At weaning (28 ± 2 d), a total of 80 ([Landrace × Large White] × Duroc) piglets, with an average weight of 7.37 ± 0.23 kg, from 20 litters were chosen for a 28-d feeding period. All piglets were assigned into 20 pens (2.03 m width × 2.13 m length each), with four piglets per pen (barrows: gilts of 1:1). Following a randomized complete design, the piglets were divided into four groups based on their initial weight and sex: positive control (hygienic condition, disinfectant used (Biostop^TM) with water at the ratio of 1:100 before entering piglets to the feeding trial, and daily cleaning with tap water, group PC); negative control (poor sanitation without cleaning and disinfection for the entire 28-d experiment, group NC); and experimental groups (group BSF6 and BSF12 with BSF larva at 6% and 12%, respectively, under poor sanitary conditions).

The PC piglets were grouped in a separate house, for a total of five, in a non-environmentally challenged housing, an open-housed nursery room of 33 °C during three weeks of feeding and 29 °C afterwards, to avoid pathogenic contamination from the NC-BSF piglets (15 pens in total). All nutrient contents in a mash diet were formulated following the NRC guidelines (2012), which was divided into two phases: phase I (ds 1–14 post-weaning) and phase II (ds 15–28 post-weaning) (Table 1). All piglets were supplied experimental diets and freshwater on an ad libitum basis as a welfare standard.

Table 1. Ingredients and composition values of the experimental diets (as-fed basis)

Ingredient	Phase I (d 1-14)					Phase II (d 15-28)
	PC	NC	BSF6	BSF12		PC	NC	BSF6	BSF12
Maize	39.46	39.46	39.22	39.18		50.35	50.35	50.33	50.55
Soybean meal (43.8%)	30.02	30.02	24.16	18.38		32.06	32.06	26.22	20.21
Broken rice	10.00	10.00	10.00	10.00		6.00	6.00	6.00	6.00
Full-fat soybean meal	7.00	7.00	7.00	7.00		3.50	3.50	3.50	3.50
Whey powder	6.00	6.00	6.00	6.00		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	6.00	12.00		0.00	0.00	6.00	12.00
L-lysine (78%)	0.15	0.15	0.19	0.21		0.21	0.21	0.21	0.29
Dicalcium phosphate	0.66	0.66	0.98	0.94		1.08	1.08	1.19	1.19
Limestone	0.86	0.86	0.60	0.44		0.95	0.95	0.70	0.41
NaCl	0.35	0.35	0.35	0.35		0.35	0.35	0.35	0.35
Premix^1	0.50	0.50	0.50	0.50		0.50	0.50	0.50	0.50
Total	100.00	100.00	100.00	100.00		100.00	100.00	100.00	100.00
Calculated value (%)
Crude protein	22.5	22.5	22.5	22.5		21.5	21.5	21.5	21.5
ME (kcal/kg)	3,300	3,300	3,300	3,300		3,265	3,265	3,265	3,265
Ca	0.80	0.80	0.80	0.80		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	1.43	1.43	1.43	1.43		1.38	1.38	1.38	1.38
Methionine + Cysteine	0.65	0.65	0.65	0.65		0.60	0.60	0.60	0.60
Tryptophan	0.25	0.25	0.23	0.23		0.23	0.23	0.23	0.23
Threonine	0.92	0.92	0.92	0.92		0.86	0.86	0.84	0.88
Ether extract	3.61	3.61	5.47	7.33		3.30	3.30	5.15	7.02
Crude fiber	3.41	3.41	3.02	2.63		3.68	3.68	3.29	2.89
Chitin	0.00	0.00	0.26	0.51		0.00	0.00	0.26	0.51
Analysed value (%)
Crude protein	22.5	22.5	22.5	22.5		21.5	21.5	21.5	21.5
Ether extract	3.58	3.58	5.13	7.52		3.33	3.33	5.82	7.18
Crude fiber	3.36	3.36	3.06	2.68		3.49	3.49	3.16	2.87
GE (kcal/kg)	4,122	4,122	4,132	4,140		4,060	4,060	4,076	4,083

ME = metabolizable energy, GE = gross energy

¹ Each kilogram contains: vitamin A, 7.2 MIU; vitamin D3, 1.36 MIU; vitamin E, 24,000 IU; vitamin K3, 0.8 g; vitamin B1, 1.2 g; vitamin B2, 2.4 g; vitamin B6, 1.6 g; vitamin B12, 14 mg; nicotinic acid, 12 g; choline, 120 g; pantothenic acid, 8 g; biotin, 0.06 g; folic acid, 0.4 g; cobalt as CoSO₄, 0.2 g; copper as CuSO₄, 56 g; ferrous as FeSO₄, 64 g; manganese as MnSO₄, 24 g; zinc as ZnSO₄, 40 g; iodine as KI, 0.4 g; and selenium as Na₂SeO₃, 0.1 g.

² Chitin content was calculated from 4.26%

Growth performance and diarrheal occurrence

The body weight (BW) and feed consumption of each piglet were recorded at 06:00 on 1, 15, and 29 d to evaluate average daily gain (ADG), average daily feed intake (ADFI), and the ratio of ADG to ADFI (G:F). Diarrheal assessment was recorded daily at 06:00 to determine diarrheal rate by calculating the number of diarrheal pigs/total number of pigs (per pen).

Metabolic trial

Twenty castrated barrows (10.43 ± 0.89 kg BW) were used to determine the apparent total tract digestibility (ATTP) in a completely randomized design. During a 7-d adaptation period and a 5-d collection period, piglets were fed mash diets twice daily (every 12-h interval) at a feeding quantity close to that of the ad libitum to avoid any errors in digestibility measurements. The Cr₂O₃ and Fe₂O₃ (0.2%) were the initial and terminal indigestible markers, respectively, which were included in the diet during the collection period. Pooled faecal samples and experimental diets were dried at 65 °C for 72 h, ground (0.88-mm sieve), and subsequently analysed for their nutrient contents. Dry matter (DM, #930.15) was evaluated after oven-drying, crude protein (CP, #984.13, N × 6.25) was analysed by the Kjeldahl procedure, ash (#942.15) was analysed by overnight incineration at 550 °C, and ether extract (EE, #920.39) was analysed using the Soxhlet apparatus (AOAC 2000). The average values of the nutrient composition in triplicate sampling each were used for the ATTP calculation, following the methods of Adeola (2001).

Immunity, inflammation, and oxidative stress

On 29 d, the fasting pigs were individually weighed, and pigs (n = 20, five pigs per group) having a BW closest to the average BW were chosen for euthanasia by intravenous barbiturate overdose. Blood samples were sampled by jugular punctures from the 20 euthanasia pigs and transferred to vacutainer serum tubes (Greiner Bio-one, Chonburi, Thailand, 5 mL/tube) until centrifugation (1,872 x g, 4 °C, 15 min). These samples were aliquoted and later quantified for the antibody (immunoglobulin A [IgA] and M [IgM]) and proinflammatory cytokine (interleukine-1β [IL-1β], IL-6, and tumour necrosis factor-alpha [TNFα]) contents using enzyme-linked immunosorbent tests (R&D System, Minneapolis, MN, USA). For the antioxidant capacity analyses; the total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) concentrations were determined using commercial kits (Sigma Aldrich, St. Louis, MO, USA) following the manufacturer protocols. Each sample was tested in triplicate to avoid variation.

Visceral organ sampling

The abdominal cavity was excised following blood collection, and the visceral organs, including the heart, liver, kidneys, stomach, spleen, and gastrointestinal tract, were immediately weighed for organ weight calculations. The caecum and colon were cleaned with deionized water, whereas the small intestine was flushed with phosphate-buffered saline (PBS) to eliminate fat and connective tissue, which were later used to determine the hindgut pH using a portable pH meter (AP 110, Fisher Scientific, Pittsburgh, PA, USA) and histological measurements, respectively.

Histological measurements

Intestinal tissue samples of the duodenum (20 cm distal from the pyloric junction), jejunum (3 cm proximal to ileocecal junction) and ileum (15 cm proximal to the ileocecal valve) from the 20 slaughtered pigs were immediately collected. Before PBS washing, tube pieces were placed in neutral-buffered (pH 7.0) paraformaldehyde (4% vol/vol) for a 48-h fixation. The sampled intestinal tubes were dehydrated with ethanol and xylene, cleared, and embedded in paraffin. These tissues were transversely cut into 5 µm-thick sections per intestinal site using a microtome (Leica RM2235, Wetzlar, Germany) and subsequently stained with haematoxylin and eosin (H&E staining, Sigma-Aldrich, St. Louis, MO, USA) for histological assays. For each trait, 15 intact well-oriented samples were obtained to determine the villus height (VH, from the tip to the villus-crypt junction) and crypt depth (CD, from the bottom of the villus to the mucosa) using a light microscope at a magnification of ×4, and then the VH/CD was calculated using the obtained values.

Microbiological assay

A total of 20 samples (approximately 3 g) from the rectum of the euthanasia pigs were collected to prevent cross-contamination of the microbial counts. The samplings were suspended in NaCl solution (0.9%, w/v), homogenized in a mixer for 2 min, and diluted in a 10-fold dilution series. Relevant dilutions were spread-plated in triplicate on MacConkey and Salmonella – Shigella agars and were subsequently incubated at 37 °C for 24 and 48 h for Escherichia coli and Salmonella spp. determinations, respectively. For the Rogosa and Sharpe agar, the plate was enumerated at 37 °C for 48 h to detect Lactobacillus spp. The growth of each microbe was log-transformed and represented as log 10 colony-forming unit (CFU)/g of faeces.

Statistical analysis

Data were analysed using the GLM procedure of SAS (version 9; SAS Inst. Inc., Cary, NC, USA). Each pen was the experimental unit for the growth criteria, and individual pigs were the experimental unit for other criteria investigated (nutrient digestibility, blood measurement, organ weight, hindgut pH, and histological and microbial assays). Treatment effects were assessed by Duncan’s new multiple range test at P < 0.05.

Results

The BSF composition

The nutrient composition of BSF is presented in Table 2. It is composed of 42.62% crude protein, 26.38% ether extrac,t and 5,823.24 kcal/kg gross energy. The major component of essential and non-essential aminal acids was lysine and arginine, respectively which accounted for 1.67% and 2.14%, respectively. Furthermore, the total amino acid recovery accounted for 0.41%.

Table 2. Nutrient composition of black soldier fly larva (dry matter basis)

Nutrients	Content (g/100 g)
Crude protein	42.62
Ether extract	26.38
Gross energy (kcal/kg)	5,823.24
Essential amino acids
Isoleucine	0.57
Leucine	1.16
Lysine	1.67
Methionine	0.65
Phenylalanine	1.07
Threonine	0.83
Tryptophan	0.14
Tyrosine	1.12
Histidine	0.68
Valine	1.24
Total	9.13
Non-essential amino acids
Alanine	1.57
Arginine	2.14
Aspartic acid	0.68
Cystine	0.07
Glutamic acid	0.81
Glycine	0.88
Serine	1.04
Proline	1.17
Total	8.36
Total amino acid recovery^1	0.41

¹ Amino acid recovery = total amino acid / % crude protein

Growth performance and diarrheal rate

The pigs under poor sanitation (NC) had detrimental effects on lighter BW and lower ADFI during 15–28 d in comparison to those in sanitation conditions (PC, P < 0.05; Table 3). However, the improvement in ADFI showed greater effects with BSF6 addition in Phase I and II (P = 0.045 and P = 0.001, respectively) than those fed NC, which ultimately increased BW by 6.03% in Phase II (P = 0.008). The BSF-supplemented diet also improved BW and ADFI compared to PC during Phase II and decreased diarrheal rate compare to the NC diet (P < 0.05). The ADG and G:F were unaffected among dietary treatments.

Table 3. Effect of full-fatted black soldier fly larva addition on the growth performance and diarrheal rate in weaning pigs under poor sanitary conditions

	Item	Black soldier fly larva (%)				SEM	P-value
		PC	NC	BSF6	BSF12
D1-14	BW d0 (kg)	7.37	7.39	7.37	7.36	0.228	0.998
	BW d14 (kg)	10.80	10.51	10.95	10.88	0.283	0.709
	ADG (g)	245.29	222.64	255.21	251.57	27.622	0.839
	ADFI (g)	332.01^ab	322.20^b	356.99^a	346.60^ab	8.098	0.045
	G:F	0.742	0.693	0.714	0.728	0.085	0.979
D15-28	BW d28 (kg)	17.29^a	16.24^b	16.99^a	17.44^a	0.210	0.008
	ADG (g)	463.50	409.36	431.79	468.51	27.598	0.419
	ADFI (g)	680.21^b	666.61^c	691.21^a	696.00^a	3.337	0.001
	G:F	0.682	0.615	0.624	0.673	0.041	0.584
d1-28	ADG (g)	354.39	316.00	343.51	360.04	12.821	0.126
	ADFI (g)	523.19	534.40	523.99	541.61	17.760	0.862
	G:F	0.679	0.594	0.656	0.668	0.028	0.186
	Diarrhoea rate (%)	7.92^ab	11.96^a	6.13^b	3.69^b	1.773	0.038

Abbreviations: PC = sanitation; NC = poor sanitation; BSF6 = NC + 6% black soldier fly larva; BSF12 = NC + 12% black soldier fly larva; BW = body weight; ADG = average daily gain; ADFI = average daily feed intake; G:F = gain-to-feed ratio.

^a,b,c Different superscripts within a row represent a significant difference at P < 0.05.

Apparent total tract digestibility

Decreased ATTP values for the DM (P = 0.005), CP (P = 0.012), and EE (P = 0.039) were observed in the NC group compared to those in the PC or BSF group, but there was a comparable difference between the PC and BSF diets (Table 4). No significant difference was detected in ash digestibility among the dietary treatments.

Table 4. Effect of full-fatted black soldier fly larva addition on the apparent total tract digestibility in weaning pigs under poor sanitary conditions

	Item	Black soldier fly larva (%)				SEM	P-value
		PC	NC	BSF6	BSF12
ATTP (%)	Dry matter	86.79^a	78.57^b	90.89^a	89.40^a	1.884	0.005
	Crude protein	79.17^a	53.19^b	86.76^a	85.76^a	6.134	0.012
	Ash	41.23	49.79	51.09	47.65	12.349	0.942
	Ether extract	71.48^ab	59.34^b	81.04^a	87.56^a	5.950	0.039

Abbreviations: PC = sanitation; NC = poor sanitation; BSF6 = NC + 6% black soldier fly larva; BSF12 = NC + 12% black soldier fly larva; ATTP = apparent total tract digestibility.

^a,b Different superscripts within a row represent a significant difference at P < 0.05.

Immunity, pro-inflammation, and oxidative stress

The pigs provided NC treatment had poorer IgA and higher TNFα secretions than those in the PC group (P < 0.05; Table 5). However, the BSF12 diet improved IgA (P = 0.002), SOD (P = 0.039), and GPx levels (P = 0.023), including lower TNFα (P = 0.023) and MDA (P = 0.027) levels, than the control diets. However, there were no differences in the serum IgM, IL1β, IL6, and TAC levels among all groups.

Table 5. Effect of full-fatted black soldier fly larva addition on the blood-related gut health in weaning pigs under poor sanitary conditions

	Item	Black soldier fly larva (%)				SEM	P-value
		PC	NC	BSF6	BSF12
Gut health	IgA (g/L)	0.89^b	0.64^c	0.97^ab	1.09^a	0.061	0.002
	IgM (g/L)	2.27	2.07	2.13	2.35	0.183	0.692
	IL1β (pg/mL)	316.20	335.97	308.96	287.13	25.521	0.613
	IL6 (pg/mL)	126.79	138.67	119.15	124.21	10.299	0.601
	TNFα (pg/mL)	47.89^b	68.03^a	50.54^b	47.13^b	4.594	0.023
	TAC (U/mL)	12.57	10.88	15.32	17.84	2.406	0.237
	SOD (U/mL)	70.46^b	61.86^b	82.12^ab	99.80^a	8.377	0.039
	GPx (U/mL)	630.36^bc	558.65^c	864.99^ab	906.67^a	80.236	0.023
	MDA (nmol/mL)	7.36^a	8.17^a	6.01^ab	4.48^b	0.775	0.027

Abbreviations: PC = sanitation; NC = poor sanitation; BSF6 = NC + 6% black soldier fly larva; BSF12 = NC + 12% black soldier fly larva; IgA = immunoglobulin A; IgM = immunoglobulin M; IL1β = interleukine-1β; IL6 = interleukine-6; TNFα = tumor necrosis factor alpha; TAC = total antioxidant capacity; SOD = superoxide dismutase; GPx = glutathione peroxidase; MDA = malondialdehyde.

^a,b,c Different superscripts within a row represent a significant difference at P < 0.05.

Organ weight and hindgut pH

The pigs that received PC and BSF-supplemented diet had significantly lighter small intestine (P = 0.028) and heavier cecum (P = 0.038) weights tha those that received the NC (Table 6). The BSF12 group had decreased hindgut pH in the cecum (P = 0.045), proximal (P = 0.004), and distal (P = 0.047) colons compared to that in the NC group. No significant differences were detected in weights of the heart, liver, kidney, stomach, spleen, and colon after dietary supplementation.

Table 6. Effect of full-fatted black soldier fly larva addition on the organ weight and digesta pH in weaning pigs under poor sanitary conditions

	Item	Black soldier fly larva (%)				SEM	P-value
		PC	NC	BSF6	BSF12
Organ weight (g/kg BW)	Heart	4.12	4.07	4.43	4.74	0.455	0.708
	Liver	31.72	30.07	32.04	34.98	2.686	0.639
	Kidney	4.28	4.87	4.48	3.99	0.372	0.432
	Stomach	9.09	8.90	8.25	9.83	0.891	0.667
	Spleen	2.23	2.21	2.63	2.43	0.185	0.368
	Small intestine	29.76^b	36.64^a	29.77^b	31.11^b	1.575	0.028
	Colon	14.38	12.86	14.62	15.03	1.333	0.686
	Cecum	2.74^a	2.12^b	2.84^a	2.69^a	0.164	0.038
Hindgut pH	Cecum	6.34^ab	6.67^a	5.13^b	4.97^b	0.448	0.045
	Proximal colon	7.03^a	6.47^ab	5.45^bc	4.43^c	0.419	0.004
	Middle colon	5.62^a	5.99^a	4.12^b	4.88^ab	0.355	0.013
	Distal colon	5.64^ab	6.14^a	4.75^ab	4.26^b	0.448	0.047

Abbreviations: PC = sanitation; NC = poor sanitation; BSF6 = NC + 6% black soldier fly larva; BSF12 = NC + 12% black soldier fly larva.

^a,b,c Different superscripts within a row represent a significant difference at P < 0.05.

Intestinal morphological structure

Intestinal morphology was unaffected between both the control diet groups (Table 7). However, inclusion of the BSF diet led to a longer VH and shorter CD in the duodenum than in those fed the NC diet (P = 0.014 and P = 0.031, respectively). The BSF6 addition also increased the duodenal VH/CD by 55.6% and 25.1% compared to that of the NC and PC treatment groups, respectively (P = 0.005). However, there was no change in the intestinal morphology of the jejunum and ileum after BSF supplementation.

Table 7. Effect of full-fatted black soldier fly larva addition on the intestinal morphology in weaning pigs under poor sanitary conditions

	Item	Black soldier fly larva (%)				SEM	P-value
		PC	NC	BSF6	BSF12
Duodenum	Villus height (µm)	615.93^ab	555.58^b	694.45^a	650.98^a	25.199	0.014
	Crypt depth (µm)	344.39^ab	391.73^a	313.78^b	338.72^b	15.967	0.031
	VH/CD	1.79^bc	1.44^c	2.24^a	1.93^ab	0.123	0.005
Jejunum	Villus height (µm)	525.86	554.11	613.87	611.31	52.403	0.576
	Crypt depth (µm)	248.53	269.16	251.31	233.41	26.039	0.813
	VH/CD	2.26	2.07	2.48	2.64	0.276	0.489
Ieum	Villus height (µm)	461.78	454.07	472.32	515.14	35.085	0.625
	Crypt depth (µm)	217.21	221.84	178.69	196.61	18.558	0.369
	VH/CD	2.41	2.07	2.74	2.67	0.306	0.438

Abbreviations: PC = sanitation; NC = poor sanitation; BSF6 = NC + 6% black soldier fly larva; BSF12 = NC + 12% black soldier fly larva; VH/CD = villus height to crypt depth ratio

^a,b,c Different superscripts within a row represent a significant difference at P < 0.05.

Microbiological assay

Escherichia coli was increased in pigs housed in the NC condition as compared to those in the PC and BSF12 groups (P = 0.043; Table 8). A significant decrease in Lactobacillus spp. was also found in the NC group compared to that observed in the BSF group (P = 0.028). However, all dietary treatments did not exhibit Salmonella spp. colonisation.

Table 8. Effect of full-fatted black soldier fly larva addition on the microbial count (log colony-forming unit [CFU]/g faeces) in weaning pigs under poor sanitary conditions

	Item	Black soldier fly larva (%)				SEM	P-value
		PC	NC	BSF6	BSF12
Microbe	Escherichia coli	5.84^b	8.47^a	6.98^ab	6.61^b	0.573	0.043
	Salmonella spp.	5.48	7.03	6.34	5.91	0.602	0.351
	Lactobacillus spp.	7.54^ab	6.67^b	8.96^a	9.39^a	0.604	0.028

Abbreviations: PC = sanitation; NC = poor sanitation; BSF6 = NC + 6% black soldier fly larva; BSF12 = NC + 12% black soldier fly larva.

^a,b Different superscripts within a row represent a significant difference at P < 0.05.

Discussion

Piglets exposed to a poor housing environment along with drastic changes in gut morphology and feed items showed decreased feed intake post-weaning (Jayaraman and Nyachoti 2017), consequently, reducing performance and gut integrity (Wan et al. 2017; Selenius et al. 2018). In addition, weaned pigs in any environmental condition can adapt to stress; however, pigs in sanitary conditions can recover better than those in a poor sanitary environment (Pastorelli et al. 2012), which can impair the growth performance of pigs, leading to lower ADFI and BW, as seen in the NC pigs in this study. This could be attributed to the limitations of pigs in the temporary changes in nutrient metabolism and partitioning to supply energy for metabolic-stressed response (Pastorelli et al. 2012). This negative effect can be overcome by 10% BSF supplementation (Biasato et al. 2019). According to Shumo et al. (2019), BSF contains considerable amounts of glutamine and its metabolites (about 6–8 mg/g), which could be attributed to the increased feed palatability of BSF under housing-related stressors, serving conditional essential amino acids to improve weanling pigs’ growth performance (Ji et al. 2019). The BSF also has a greater capacity to deliver essential and non-essential amino acids for protein synthesis, improving pig performance (Kar et al. 2021). Furthermore, the weaned pigs can better utilize a diet with lower fibre than those who consume a high-fibre diet (3.49% vs. 2.87%), which increases BW in Phase II. However, without the insanitation, there was no growth improvement observed in pigs fed the BSF diet (Spranghers et al. 2018). This suggests that the BSF addition can compromise feed intake reduction and poor growth performance in this critical period. In addition, the lowered diarrheal rate in piglets that consumed a BSF-supplemented diet may contribute to faster adaptation of intestinal functions in nutrient utilisation and maintaining gut health (Crosbie et al. 2021), resulting in better nutrient digestibility and decreased diarrheal occurrence observed in this study.

Diet improvements were traditionally assumed to be driven by changes in both intestinal development and the degree of nutrient absorption and utilisation. Piglets in unsanitary conditions generally require greater amounts of amino acids than those housed in hygienic conditions. The optimal standardised ileal digestibility of threonine and lysine increased from 65% to 66.5% in those housed under good sanitation (Jayaraman et al. 2015), but no difference in the digestibility of DM and CP after a two-week housing challenge was observed (Cho et al. 2021). However, the positive outcomes of BSF addition in the increased DM, CP, and EE digestibility are largely correlated with the increased growth performance. Full-fatted BSF has been reported as an excellent source of digestible energy (4,927 vs. 3,941 kcal/kg), metabolizable energy (4,569 vs. 3,396 kcal/kg), and predicted net energy (3,477 vs. 2,640 kcal/kg) as compared to defatted BSF meal, including ileal amino acid digestibility, particularly digestible Lys (88%), as compared to animal-based protein ingredients, indicating that using full-fatted BSF in nursery pig feed could be a more efficient approach to increase nutrient digestibility (NRC 2012; Crosbie et al. 2020). The improved CP digestibility is consistent with the findings of Neumann et al. (2018) when 21% full-fatted BSF was supplemented in the nursery diet. Previous studies demonstrated that the amount of crude fat (32.5%) derived from full-fatted BSF is enriched in lauric acids (up to 70% of total saturated fatty acid content), which has increased energy availability in the small intestine for better performance (Crosbie et al. 2020). Conversely, Spranghers et al. (2018) did not observe any changes in nutrient digestibility in pigs fed dietary full-fatted BSF up to 8%. One possible reason is that heating and drying processes of BSF above 60 °C cause negative impacts on the nutrient value and availability, owing to the changes in chemical and physical functions of lipids and protein structure (DiGiacomo and Leury 2019). This suggests that full-fatted BSF meal may have a positive effect on nutrient absorption by altering gut health.

Poor hygiene favours increased disease and subclinical disease occurrence, which induce the systemic inflammatory response and subsequently cause oxidative stress. Superoxide radicals are ROS that suppress immune cells initially during inflammation (Sierżant et al. 2019). Our findings revealed that NC conditions increased TNF and decreased antioxidant enzyme (SOD and GPx) levels, resulting in altered lipid peroxidation via increased MDA secretion. However, two metabolites (i.e. methionine sulfoxide and sarcomine) of the BSF-based diet caused regulatory effects on the expression of genes correlated with peroxisome proliferator-activated receptor (PPAR), toll-like receptor, and cytokine signalling pathway (Yu et al. 2020; Kar et al. 2021). This outcome is established as the homeostatic regulation of cellular differentiation and activation of the inflammatory response in enterocytes (Kar et al. 2021). The presence of conjugated linoleic acids in BSF larva may not only regulate proinflammatory cytokines but also activate IgA secretions, thus regulating pathogen invasion and allergen absorption (Liu 2015; Ewald et al. 2020; Murru et al. 2021). Consistently, Yu et al. (2020) demonstrated that the inclusion of 2% BSF larva significantly decreased TNFα levels in weaned pigs. Moreover, Ravi et al. (2021) observed that the presence of total phenolic compounds (about 32–35 mg/g DM) in BSF may play an important role in antioxidant capacity. The metal chelators (Fe, Cu, Mn, and Zn) found in relatively high levels in BSF-larva meal, has superior action as co-factor of antioxidant enzymes in scavenging ROS (Chia et al. 2020). These studies were in line with the improvements in intestinal health and decreased pro-inflammatory cytokine secretion in weaning pigs fed a diet containing BSF up to 12% under poor sanitary conditions.

Changes in intestinal weight can determine absorptive capacity. The lighter small intestine weights may be contributed to the faster intestinal development of digestive capacity during the immature gastrointestinal tract, which response in an adaptive manner (Khonyoung and Yamauchi 2015). From this aspect, the protein and fatty acid contents of medium chain  – and polyunsaturated fatty acids may assume to be efficiently absorbed and accelerate micelle formation, thus providing a higher surface area for nutrient absorption. This is in line with the greater improvement of CP and EE digestibility, as well as heavier BW by the BSF-supplemented diet. Furthermore, the BSF pigs had positively increased caecum weights, which could be associated with a higher capacity of hindgut fermentation. It is well-established that BSF larva is rich in chitin, which is undegradable in the small intestine and can enter the large intestine for microbial fermentation (Yu et al. 2019), yielding short-chain fatty acids (SCFAs). The SCFAs, particularly butyric acid, are used as the main energy sources for the host colonic epithelial cells to modulate the proliferation of beneficial bacteria and induce anti-inflammatory action (Yu et al. 2019). The increase of caecum weight in this study is expected with increasing hindgut fermentation, which was consistent with the higher Lactobacillus spp. counts in pigs fed the dietary BSF larva. This is in agreement with the study of Metzler-Zebeli et al. (2019), wherein increased hindgut fermentation not only promoted microbial activity but also SCFA production, which consequently provided acidic conditions in the digesta. The possible action is that under a luminal pH of around 6–7, both lactate and SCFAs are commonly ionised and therefore require monocarboxylate transporters, which are transporter proteins in the intestine and colon of pigs for transcellular absorption, whereas protons remain in the intestinal lumen (Sepponen et al. 2007). As a result, the digesta acidity increase, which is beneficial for the adaptation of intestinal microbes that may increase SCFA production. Therefore, acidification in the hindgut pH was observed in fed dietary fibre for a longer period, resulting in a stronger pH decrease (Metzler-Zebeli et al. 2019). Interestingly, we did not observe any significant difference in the liver weight of the weaning pigs in both conditions. This might be due to the ability of the pigs to maintain metabolic activity in the liver under uncertain conditions. Therefore, this assumption may be further quantified by the metabolic profiles of weaning pigs at different times.

Poor husbandry practice altered immunity and provoked inflammation (Jayaraman and Nyachoti 2017; Chatelet et al. 2018). It may impair piglet growth due to nutrient competition between structural tissues and pig immune response (Jayaraman et al. 2015). In addition to reduced feed intake during weaning, poor sanitary conditions result in changes in intestinal structure, such as a shorter VH and, to a lesser extent, diminished CD (Jayaraman and Nyachoti 2017), as well as impaired brush-border enzyme function and absorptive capacity (Lallès et al. 2004). However, the potential effect of dietary BSF on intestinal morphology was previously demonstrated (Biasato et al. 2019). The underlying mechanism is unclear, but it could be contributed to the lauric acid content (52%, Ewald et al. 2020), which is rapidly absorbed and hydrolysed more efficiently in the small intestine due to the chemical polarity and a shorter carbon chain structure, thereby increasing nutrient uptake and absorption (Jackman et al. 2020). Furthermore, lauric acid effectively activates the renewal rate of enterocytes (Liu 2015). In this study, the nutrients derived from a BSF-supplemented diet may be used as an energy source to support enterocyte proliferation and intestinal mucosa, which can promote gut health and nutrient availability to pigs under poor sanitary conditions.

The BSF contains several active components, including chitin, lauric acid, and antimicrobial peptides (AMPs, defensin-like peptides, decropin, sacrocin, and attacin; Ewald et al. 2020; Xia et al. 2021), which benefit the intestinal integrity by inhibiting the adhesion of pathogens, whereas activating the shedding of beneficial bacteria, particularly Bifidobacterium spp. and Lactobacillus spp. (Kar et al. 2021). One reason for C:12 function is that it has a low critical micelle concentration (600 µmol/L) and higher pKa (about 14), thereby surviving longer in the stomach for the greater membrane-disruptive activity of pathogenic bacteria, having both bacteriostatic and bactericidal properties (Yoon et al. 2018; Jackman et al. 2020). For BSF-derived AMPs, the unabsorbable compounds in the gut and the action at the genetic level of the bacterial targets have a preferential affinity for Gram-negative bacteria rather than Gram-positive bacteria (Xia et al. 2021). We assumed that the higher BSF-derived chitin (0.26% to 0.51%) may act as the chitin-glucan complex in inhibiting the growth of Escherichia coli and promoting Lactobacillus spp., which was confirmed by previous studies in pigs fed 4% to 8% of BSF larva (Wan et al. 2017; Selenius et al. 2018). These positive effects suggest that the diarrheal rate can be used to predict the gut outcome of weaning pigs (Jayaraman and Nyachoti 2017), which is consistent with our findings.

Conclusion

Pigs with poor sanitation showed lighter weight, diarrheal susceptibility, decreased nutrient utilization, higher crypt depth, and TNFα secretion. However, BSF addition at 12% to the diet could minimize these effects via increased feed intake, nutrient utilisation, and immune and antioxidative stress, which could promote gut health and growth performance of weaning pigs under poor sanitary conditions.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data and model availability statement

The datasets underlying our findings are reported in the manuscript.

Additional information

Funding

This work was supported by the Program Management Unit for Human Resources and Institutional Development under Research and Innovation.