Dietary supplementation with full-fat Hermetia illucens larvae and multi-probiotics as a substitute for antibiotics improves the growth performance, gut health, and antioxidative capacity of weaned pigs

Abstract

Background: The widespread use of antibiotics (such as amoxicillin) in the food animal production industry has led to the proliferation of multi-antibiotic resistant pathogens. In this study, we examined whether dietary supplementation with full-fat black soldier fly larvae (BSFL; alone or in combination with multi-probiotics) is an effective alternative to dietary antibiotics in weaning piglets. We also tested the effects of these diets on growth performance, nutrient digestion, intestinal morphology, and oxidative stress in weaned pigs. A total of 80 crossbred piglets [(Landrace × Large White) × Duroc] were randomly allotted to four diet groups: positive control (PC) diet supplemented with 0.02% amoxicillin; negative control (NC) diet without addition; BSFL12 diet (NC + 12% full-fat BSFL); and BSFL + Pro diet (BSFL + 0.1% multi-probiotics, including Bacillus subtilis, B. licheniformis, and Saccharomyces cerevisiae). All groups had five replicates, with four piglets per replicate.

Results: Dietary BSFL + Pro improved the final body weight, overall average daily gain, gain-to-feed ratio, and decreased the diarrhoea rate of piglets. Compared to the NC diet, the BSFL12 and BSFL + Pro diets improved nutrient digestibility (dry matter, crude protein, and ether extract) and increased the serum levels of immunoglobulin A and glutathione peroxidase, while reducing the levels of proinflammatory cytokines (interleukin-1β and tumour necrosis factor alpha) and serum malondialdehyde. The relative weight of the spleen was significantly higher and cecum pH was significantly lower in pigs fed the BSFL + Pro diet than in those fed the NC diet. Pigs fed the BSFL12 and BSFL + Pro diets had longer duodenal villi, a higher ratio of villus height to crypt depth, and shorter crypt depth. The BSFL + Pro diet also increased faecal Lactobacillus spp. counts and reduced Escherichia coli counts compared with PC and NC, respectively.

Conclusions: Dietary supplementation with BSFL or BSFL + multi-probiotics can improve the growth performance and intestinal health of pigs, and may be an effective strategy to replace antibiotic supplementation in weaned pigs.

Background

For several years, amoxicillin has been widely used to control post-weaning diarrhoea in piglets. However, the excessive use of antimicrobials in food animal production has led to concerns regarding the spread of multidrug-resistant pathogens. Since 28 January 2022, the use of amoxicillin in the food animal production industry has been regulated within the European Union [1] To curb this escalating problem, the bioactive properties of full-fat back soldier fly larva (BSFL) and multi-strain probiotics (i.e., Bacillus subtilis, B. licheniformis, and Saccharomyces cerevisiae) have been suggested as effective alternatives to in-feed antibiotics. The BSFL is an attractive protein source for monogastric animals, especially due to its high crude protein content (40.11%), ether extract (32.54%), essential amino acid profile (0.7% methionine, 2.3% lysine, and 1.8% arginine), and lauric acid content (64%) [2, 3]. BSFL-derived antimicrobial peptides (AMPs) and chitin also inhibit the proliferation of harmful bacteria and activate immunity [4, 5]. Previous studies have demonstrated that the BSFL can be used in diet formulations at a concentration of 4–8% without any detrimental effects on the growth performance of weanling pigs [6]. In addition, multi-strain probiotics are well-established to have better antibacterial, anti-inflammatory, and antioxidant properties than single-strain probiotics [7, 8]. Recently, a multi-strain Bacillus subtilis-based probiotic has been shown to improve the growth performance of pigs and the nutrient digestibility of essential amino acids [9]. Moreover, the inclusion of up to 0.3% probiotic mixture improves the growth performance of weaning pigs, inhibits the growth of harmful microbes, and reduces faecal NH₃ emissions [10].

To our knowledge, few studies have examined how BSFL and multi-strain probiotics (alone or in combination), when used as substitutes for amoxicillin, affect growth performance, nutrient digestibility, intestinal morphology, and oxidative stress in weaned pigs. We hypothesized that when use in combination, BSFL and multi-strain probiotics would interact to improve the growth performance and gut health of weaned pigs compared to in-feed antibiotics. This would help mitigate the problem of antimicrobial resistance due to the escalating use of amoxicillin.

Results

Growth performance and diarrheal rate

Compared to the negative control diet (NC; basal diet with no supplementation), the diet supplemented with BSFL and 0.1% multi-probiotics (BSFL + Pro) significantly improved the body weight (BW), average daily gain (ADG), and gain-to-feed ratio (G:F) of pigs by 11.27, 18.16, and 18.21%, respectively, during the experimental period (P < 0.05; Table 3). However, the outcomes of the BSFL + Pro diet were comparable to those of the positive control diet (PC, basal diet supplemented with 0.02% amoxicillin). The BSFL + Pro diet also reduced the diarrheal rate compared to NC (P = 0.049). However, average daily feed intake (ADFI) was unaffected by dietary treatments during Phases I and II (P > 0.05).

Table 3  Effect of dietary BSF and probiotic mixture to replace antibiotic on growth performance and diarrheal rate in weaning pigs^1,2

¹Abbreviations: PC = basal diet with amoxicillin 0.02%; NC =   basal diet without addition; BSFL12 = basal diet plus 12% black solder fly larva; BSFL + Pro = basal diet plus 12% black solder fly larva and 0.1% multiprobiotics

²Values shows means of five replicates (pen) per treatment.                                                                                        ^a,bValues within a row not sharing common lowercase superscripts differ significantly (P < 0.05).    

Apparent total tract digestibility

The apparent total-tract digestibility (ATTP) of dry matter (DM; P = 0.047), crude protein (CP; P = 0.022), and ethanol extract (EE; P = 0.019) in the BSFL12 (basal diet supplemented with 12% full-fat BSFL) and BSFL + Pro groups was significantly higher than that in the NC-fed group (Table 4), but comparable to that in the PC-fed group. However, there were no significant differences in the ATTP of total ash among dietary treatments (P > 0.05).

Table 4  Effect of dietary BSF and probiotic mixture to replace antibiotic on the apparent total tract digestibility in weaning pigs^1,2 

¹Abbreviations: PC = basal diet with amoxicillin 0.02%; NC =  basal diet without addition; BSFL12 = basal diet plus 12% black solder fly larva; BSFL + Pro = basal diet plus 12% black solder fly larva and 0.1% multiprobiotics

²Values shows means of five replicates (pen) per treatment.    

^a,bValues within a row not sharing common lowercase superscripts differ significantly (P < 0.05).    

Blood-related gut health and antioxidative stress

The levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and glutathione peroxidase (GSH-Px) were significantly higher in the BSFL12 and BSFL + Pro groups than in the NC group, whereas the levels of interleukin-1β (IL1β) and malondialdehyde (MDA) were significantly lower (P < 0.05; Table 5). The levels of tumour necrosis factor alpha (TNFα) were lower in the BSFL + Pro group than in the NC group (P = 0.044). However, the dietary treatments did not affect total antioxidant capacity (TAC) or the serum concentrations of immunoglobulin M (IgM), interleukin-6 (IL6), and superoxide dismutase (SOD) (P > 0.05).

Table 5  Effect of dietary BSF and probiotic mixture to replace antibiotic on the blood-related gut health and antioxidative stress in weaning pigs^1,2

¹Abbreviations: PC = basal diet with amoxicillin 0.02%; NC =  basal diet without addition; BSFL12 = basal diet plus 12% black solder fly larva; BSFL + Pro = basal diet plus 12% black solder fly larva and 0.1% multiprobiotics; IgA = immunoglobulin A; IgM = immunoglobulin M; IL1β = interleukine-1β; IL6 = interleukine-6; TNFα = tumor necrosis factor alpha; TAC = total antioxidant capacity; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; MDA = malondialdehyde. 

²Data are shown as group mean ± SEM (n = 5 per treatment)

^a-cValues within a row not sharing common lowercase superscripts differ significantly (P < 0.05).    

Organ weight and digesta pH

Pigs fed the BSFL + Pro diet had the highest spleen weight (P = 0.011; Table 6). Furthermore, the digesta pH in the cecum was lower in pigs fed the PC and BSFL + Pro diets than in those fed the NC diet (P = 0.021). However, the organ weights (including the heart, liver, kidney, and stomach) and colonic pH were unaffected by dietary treatments (P > 0.05).

Table 6  Effect of dietary BSF and probiotic mixture to replace antibiotic on the organ weight and digesta pH in weaning pigs^1,2 

¹Abbreviations: PC = basal diet with amoxicillin 0.02%; NC =   basal diet without addition; BSFL12 = basal diet plus 12% black solder fly larva; BSFL + Pro = basal diet plus 12% black solder fly larva and 0.1% multiprobiotics

²Data are shown as group mean ± SEM (n = 5 per treatment)

^a,bValues within a row not sharing common lowercase superscripts differ significantly (P < 0.05).    

Intestinal morphology

The VH, CD, and VH:CD values were higher in the BSFL12 and BSFL + Pro groups (P = 0.033, 0.017, and 0.004, respectively; Table 7). However, the intestinal morphology (including the jejunum and ileum) was similar among all treatment groups (P > 0.05).

Table 7  Effect of dietary BSF and probiotic mixture to replace antibiotic on the intestinal morphology in weaning pigs 

¹Abbreviations: PC = basal diet with amoxicillin 0.02%; NC =   basal diet without addition; BSFL12 = basal diet plus 12% black solder fly larva; BSFL + Pro = basal diet plus 12% black solder fly larva and 0.1% multiprobiotics

²Data are shown as group mean ± SEM (n = 5 per treatment)

^a,bValues within a row not sharing common lowerecase superscripts differ significantly (P < 0.05).    

Microbial count

The BSFL + Pro treatment significantly increased faecal Lactobacillus spp. by 29.26% and 64.90% compared with the PC and NC treatments, respectively (P = 0.008; Table 8). Simultaneously, faecal Escherichia coli decreased in the BSFL + Pro treatment in comparison to the NC group (P = 0.021). The faecal Salmonella spp. count was unaffected by dietary treatments.

Table 8  Effect of dietary BSFL and probiotic mixture to replace antibiotic on the microbial count                   (log colony-forming unit [CFU]/g faeces) in weaning pigs^1,2

¹Abbreviations: PC = basal diet with amoxicillin 0.02%; NC =  basal diet without addition; BSFL12 = basal diet plus 12% black solder fly larva; BSFL + Pro = basal diet plus 12% black solder fly larva and 0.1% multiprobiotics

²Data are shown as group mean ± SEM (n = 5 per treatment)

^a-cValues within a row not sharing common lowerecase superscripts differ significantly (P < 0.05).

Discussion

Growth performance and diarrheal rate

The extensive in-feed administration of antibiotics in swine production and lack of awareness regarding its effects are driving the accelerated and widespread emergence of antibiotic-resistant bacteria [11]. Dietary supplements, BSFL, and multi-probiotics have been proposed as substitutes for antibiotics for future generations of weanling pigs. However, the combined effects of these supplements on overall growth performance and diarrheal incidence in piglets have not well-established. Crossbie et al. [12] demonstrated that BSFL are excellent sources of essential amino acids and energy for weaning-to-growing pigs [12]. This was agreement with Jin et al. [13], who showed that dietary supplementation with 8% BSFL improved overall growth performance in a dose-dependent manner. However, BSFL-supplemented diets did not affect the growth performance of piglets when fed at a concentration of below 4% [14]. BSFL is relatively high in lauric acid (12.83% DM) [3], which accounted for ~1.54% of our BSFL-supplemented diet. Additionally, 53 genes associated with AMPs have been identified, and these AMPs show potential antibiotic activity when supplied at a minimal inhibitory concentration of 25 mg/mL. This is agreement with the observation of Zhang et al. [15], who showed that the injection of 2 mg/kg AMPs increased ADG and decreased diarrheal incidence in piglets. Taken together, these findings suggest that probiotic mixtures may have strong antimicrobial and growth-promoting effects when at an inclusion level of > 0.1% [10]. 

In the present study, we found that compared with the NC diet, supplementation with BSFL alone did not have a significant effect on the growth performance of pigs in any period. This may be due to the presence of fibrous chitin components in BSFL that are not digestible by monogastric animals [16] and therefore impair their performance. Therefore, it may be necessary to incorporate other substances in the diet that allow faster gut recovery and better nutrient uptake, while inhibiting pathogenic invasion in piglets fed an antibiotic-free diet. This study suggests that the combination of BSFL and probiotic mixtures controlled the diarrhoea rate and promoted growth performance in weaning pigs fed the antibiotic-free diet. 

Apparent total tract digestibility

The inclusion of insect protein (BSFL) and multi-probiotics has been shown to increase the nutrient digestibility of DM, CP, and EE in weaned pigs [17]. Previous studies have also reported that B. licheniformis and B. subtilis produce several extracellular enzymes that are beneficial to hosts, including α-amylase, proteinase, lipase, xylanase, cellulase, and pectinase [18]. Indeed, B. subtilis exceed B. licheniformis in producing glycosyl hydrolase, which assists the degradation of glycosidic linkages in complex sugars [19]. BSFL has high protein content and is a good source of amino acids that can further improve CP digestibility [20]. The addition of 12% BSFL in the diets did not impair CP digestibility due to the lower level of chitin (~0.52%). Furthermore, chitin and its derivatives have been reported to increase the activity of brush border digestive enzymes—especially maltase, lactase, sucrase, and proteinase—in the small intestine [21]. This increases the digestion and absorption of nutrients (Wang et al., 2020) in the dietary treatment supplemented with BSFL. The high fat content and degree of saturation of fatty acids may be a major reason for the improved digestibility of EE in BSFL-supplemented diets than in NC diets (7.02–7.53% vs. 3.72–3.83%). In this study, C¹²—which is rapidly utilized and passively absorbed by the animals—was the main component of saturated fatty acids in the BSFL12 diet. Combined with microbial lipase secretion from Bacillus-based probiotics, C¹² may have broad effects in the hydrolysed 1- and 3- positions of dietary triglycerides. Although the experimental dietary combinations showed improvement nutrient digestibility compared to the NC diet, their effects were comparable to those of the PC diet, suggesting their greater availability of nutrients beyond the antibiotic treatment.

Blood-related gut health and antioxidative stress

Serum immunoglobulins can be used to determine cellular responses and the ability of an animal’s body to recognize pathogenic invasion [22]. Our study showed that serum IgA concentrations increased in pigs fed the BSFL12 and BSFL+ Pro diets. Serum IgA can subsequently interact with lactoferrin and/or transferrin for bacteriostatic action by increasing the adhesion to epithelial cells (to improve their adhesion to the mucus) and neutralising bacterial toxins. Therefore, increased levels of serum IgA prompt the initial defence against infection by pathogens [23].  Serum IgG inhibits various stressors, including diseases and intestinal disorders, during the first week of post-weaning [24]. Therefore, higher levels of serum IgG can suppress invading pathogens and activate long-lasting immunity. The feedback regulation of IgG also induces the secretion of novel IgG antibodies through the activation of B lymphocytes [23]. This is consistent with previous reports showing that dietary inclusion of B. subtilis increased serum IgA and IgG levels in rabbits and neonatal piglets [25,26]. The higher C¹² content of BSFL-supplemented diets may also activate interleukin production, which in turn promotes the production of immunoglobulins [27]. However, the mechanism by which BSFL (alone or in combination with Bacillus-based probiotics) promotes immunoglobulin secretion is still unclear. It is possible that the binding site of Bacillus-based probiotics can bind with IgG Fc-receptors as additional ligands that subsequently influence immunoglobulin secretion [28].

Pro-inflammatory cytokines (such as IL1β, IL6, and TNFα) are known to be secreted as part of the innate immune response [29]. These factors have detrimental effects on intestinal mucosal injury and dysfunction, impair nutrient digestion and absorption, and subsequently lead to a poor growth rate [29]. However, dietary BSFL can activate intestinal development, immunity, and anti-inflammation [29,30] through the direct utilization of C¹² by enterocytes for energy production, thereby maintaining the integrity of the intestinal mucosa in young piglets [31]. Peptidoglycans in the insect skeleton can also attach to the binding sites of pathogens, thus triggering IgA release into the intestinal lumen and inhibiting the secretion of pro-inflammatory factors. Chitin and its derivatives have also been reported to polarize the faecal calprotectin, which is a sensitive and non-invasive marker of active inflammation in the gastrointestinal tract. They also regulate a main receptor for commensal recognition in gut innate immunity [32], thus reducing inflammation in the lower intestine. It is unclear how BSFL supplementation lowers the serum TNFα concentration. Possible factors regulating this mechanism include the molecular weight, degree of saturation, particle size, source, and purification level of the BSFL [33]. However, these factors need to be investigated further. The combined use of BSFL and Bacillus-based microbes considerably downregulated the secretion of serum TNFα. Bacillus subtilis in involved in innate immunity via β-defensin, which has broad antimicrobial effects and is the first defence mechanism of the adaptive immune response [26].

An increase in the antioxidant enzyme activity of GSH-Px is also associated with antioxidant defence mechanisms. GSH-Px effectively inhibits the harmful accumulation of intracellular hydrogen peroxide, thus preventing damage to DNA, proteins, and membrane lipids in the animal body. The BSFL has appropriate amount of total phenolic compounds (32–35 mg/gallic acid equivalents g DM), which contributes to the scavenging of reactive oxygen species [34]. This positive effect can further contribute to an increase in antioxidant enzyme activities and reduce oxidative stress, as shown in this study.

Organ weight and digesta pH

The spleen is an important immune organ that can be used to evaluate immune responses in weaned pigs. Changes in the immune organ index affect immune functions and resistance to pathogenic invasion in animals. In the present study, the relative weight of the spleen increased after 4 weeks of feeding with BSFL + Pro supplementation. Previous studies have reported an increase in the spleen weight following the dietary inclusion of B. subtilis (1 × 10⁶ cfu/g) or chitin and its derivatives [26,35]. This increase in spleen weight may enhance the immune functions of weaned pigs, help them overcome weaning stress, and therefore improve their overall growth performance. Furthermore, the digesta pH was significantly lower in the BSFL + Pro treatment group, indicating that these pigs were capable of generating acidic condition to allow microbial growth and colonization. The underlying mechanism of this may be related to the larger amount of chitin and its derivatives (rather than the fibre content) passing through the large intestine [36]. This causes a shift in hindgut fermentation and leads to the generation of short-chain fatty acids that supply energy for the growth of lactic acid-producing bacteria, thus lowering the number of pathogens [37]. This is consistent with our observation of faecal microbial counts in this study. However, the levels of short-chain fatty acids should be assessed to explore this positive effect in more detail.

Intestinal morphology

Changes in intestinal morphology—including VH, CD, and VH:CD—are indicative of gut health in pigs. A longer VH increases the mucosal surface area, thus allowing for improved digestion and absorption of available nutrients. Moreover, a shorter CD suppresses the rapid turnover of the intestinal epithelium, thus permitting the renewal of villi in response to normal sloughing or inflammation from pathogenic invasion. The VH:CD ratio is typically associated with increased epithelial turnover [38]. Our results indicated that dietary BSFL and BSFL + Pro increased duodenal VH in weaned pigs. This may be because BSFL-supplemented diets promoted the activity of digestive enzymes (including membrane-bound peptidases) and allowed more efficient utilisation of CP, amino acids, and EE [21]. This likely increased the availability of nutrients for the maturation of undifferentiated cells. Han et al. [39] reported that chitin derivatives enhanced enterocyte proliferation and diminished villous atrophy. Although the chitin content was high (~0.52%) in this study, it had no detrimental effects on nutrient availability. Kawasaki et al. [40] showed that piglets can partially utilise chitin through the activity of a chitin-degrading enzyme in the stomach. Tian et al. [41] also reported an increase in VH and VH:CD in weaned pigs fed Bacillus-based probiotics. Taken together, these findings indicate that the BSFL12 and BSFL + Pro diets have potential utility as an alternative to antibiotics, and can enhance the intestinal morphology of weaned pigs.

Microbial count

The presence of C¹² in the BSFL-supplemented diet (~9.17%) is beneficial for the intestinal microflora of weaning pigs, facilitates the semi-permeable membrane of pathogenic microbes, and improves the intestinal structure of piglets [42]. Therefore, in combination with chitin and it derivatives, C¹² may have desirable effects on the gut microflora of pigs. Chitin is a major fibrous compound in arthropod exoskeletons. It has highly attached with β-glucan in the chitin–glucan complex, which is selectively used as a fibrous substrate by host microorganisms for their own growth [43]. This promotes the adherence of beneficial microflora (such as Lactobacillus spp.) while reducing the number of faecal Escherichia coli in the hindgut [21,44]. A previous study demonstrated that the inclusion of lower amounts of chitin had a greater influence on colonic microbiota than chitin supplementation in high amounts [45]. In addition, Yu et al. [46] demonstrated that pigs fed 4% BSFL showed a significantly higher abundance of Lactobacillus spp. than those fed 8% BSFL (0.19% vs. 0.37% chitin, respectively). This may indicate that feeding low levels of BSFL has desirable effects on the faecal microflora of weaning pigs, comparable to those of the PC diet. However, our findings indicated that the BSFL + Pro diet had a greater influence on faecal Lactobacillus spp. than the BSFL12 diet. This result was consistent those of Wang et al. [47], who showed that the inclusion of 0.1% multi-probiotics (consisting of B. subtilis, B. licheniformis, and S. cerevisiae) had a beneficial effect on maintaining faecal microflora counts in growing pigs. One possible explanation for this outcome is that Bacillus-based probiotics are considered facultative anaerobes with high resistance to acidic conditions (such as the gastrointestinal tract of animals) [48]. This unique characteristic may allow these microbes to attach with the gut surface and produce various bacteriolytic proteins [18]. This notion was supported by the finding that the digesta pH was appropriate for the increased activity and proliferation of lactic acid-producing bacteria in this study.

Conclusion

Dietary supplementation with BSFL + Pro improved the growth performance, nutrient digestion, and intestinal health of weaned pigs to a higher degree compared with the non-supplemented diet. This combination also had potent effects on spleen weight and reduced lipid peroxidation in pigs compared to those fed antibiotics. These findings suggest that dietary supplementation with insect proteins and multi-probiotics is a viable alternative to antibiotic supplementation in nursery diets in the animal food production industry.

Materials And Methods

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  

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

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 BW^0.75 [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; α_i is the effect of the ith block (i = 1–5); β_j is the effect of the jth treatment (j = 1–4); and ε_ij 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. 

Abbreviations

BSFL, black soldier fly larva; AMP, antimicrobial peptide; NRC, National Research Council (of Thailand); CP, crude protein; EE, ether extract; ADG, average daily gain; ADFI, average daily feed intake; G:F, gain-to-feed ratio; PC, positive control diet (basal diet supplemented with 0.02% amoxicillin); NC, negative control diet (basal diet without supplementation); BSFL, basal diet supplemented with 12% full-fat BSFL; BSFL + Pro, basal diet supplemented with BSFL + 0.1% multi-probiotics; BW, body weight; ATTP, apparent total-tract digestibility; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; IL1β, interleukin-1β; IL6, interleukin-6; TNFα, tumour necrosis factor alpha; TAC, total antioxidant capacity; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; VH, villus height; CD, crypt depth; VH:CD, villus height-to-crypt depth ratio; SEM, standard error of the mean

Declarations

• Ethics approval and consent to participate: Animal trials were conducted in strict accordance with the guidelines of the National Research Council 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).
• Consent for publication                Not applicable
• Availability of data and materials            All dataset are available from the corresponding author on reasonable request
• Competing interests: All authors have no conflicts of interest to declare.
• Funding: The authors sincerely thank the Fundamental Fund of Khon Kaen University that received funding from the National Science, Research and Innovation Fund.
• Authors' contributions

Conceptualization, WB and PP; methodology, WB and JH; data curation, PP and WB; formal analysis, PP, WB, YYK and JH; investigation, PP and WB; project administration, PP and WB; funding acquisition, PP and WB; writing—original draft preparation, WB, JH, AW; writing - review and editing, WB, YH, AW, and YYK. All authors have read and agreed to the published version of the manuscript.

• Acknowledgements: Not applicable.

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