INTRODUCTION
The global population increase and the development of meat consumption cultures may significantly impact animal feed use and land demand [1]. A recent report shows that pork consumption accounted for 24% of global meat consumption in 2023, and poultry and beef took 40% and 22%, respectively [2]. For the world’s pig industry, in 2023, almost 1.3 billion pigs will be produced, and the production of pig feed is predicted to be 323 million metric tons [3]. To meet the continuously increasing demand for pig feed, the Food and Agriculture Organization suggested finding alternative raw materials to conventional feed ingredients for a stable feed supply [4]. Animal proteins (fish meal, plasma protein [PPR], etc.) are the most costly and have been used extensively in pig diet formulas. However, these conventional animal protein sources will intensify competition between human and animal feed chains [5]. Therefore, developing new, sustainable, high-quality protein sources is a crucial challenge for the pig industry.
With an estimated 1,900 species of edible insects, including the black soldier fly (BSF) larvae, the future of sustainable protein sources for animal feed looks promising [6]. The nutritional value of BSF larvae, rich in protein, lipids, vitamins, and minerals, has been well-documented [7]. The defatted processing of BSF larvae products used in swine research is classified into three types: full-fat BSF larvae, partially defatted BSF larvae, and defatted BSF larvae, with the primary difference being in fat content [8]. Defatted and full-fat BSF larvae boast a rich amino acid profile, particularly leucine, lysine, and valine [9]. Unlike other insects, BSF larvae can be reared easily on several organic wastes, with a higher feed conversion rate, survival rate, and rearing costs [10]. Given these nutritional benefits, BSF larvae present a promising alternative protein source that could help address the global food challenges.
While several studies have evaluated the inclusion of BSF larvae in weaning diets, particularly in terms of growth performance and nutrient digestibility [11] and modifications to blood profiles [12], as well as potential gut health implications [13,14], the majority of these studies have focused on replacing soybean meal. However, the effects of BSF larvae replacing animal protein sources in diet formulas remain unexplored, highlighting the need for more research.
Therefore, the present study aimed to investigate the effects of BSF larvae replacing PPR on growth performance, blood profiles, immune response, and diarrhea incidence in weaning pigs.
MATERIAL AND METHODS
Experimental animals and management
A total of 192 weaning ([Yorkshire×Landrace])×Duroc) pigs (8.12±0.01 kg body weight [BW]) were allotted to one of four treatments based on sex and initial BW, with six replicates of eight pigs per pen in a randomized complete block design. All pigs were housed in slotted plastic floor pens equipped with a feeder and a nipple waterer and allowed ad-libitum access to feed and water throughout the experimental period (Phase I: 0–2 weeks and Phase II: 3–4 weeks). The temperature in the experimental house was maintained at 30°C in the first week, decreased by 1°C every week, and was 27°C in the last week.
Experimental design and diet
The BSF larvae replacing PPR used in this study were provided by Foodyworm. The nutritional composition of the BSF larvae meal and PPR is presented in Table 1. The treatments for early and late weaning phases were as follows: 1) Control: corn-soybean-based diet, 2) BSF25: corn-soybean-based diet with 25% of PPR replaced by BSF larvae, 3) BSF50: corn-soybean-based diet with 50% of PPR replaced by BSF larvae, 4) BSF100: corn-soybean-based diet with 25% of PPR replaced by BSF larvae. All nutrients in the experimental diets were formulated to meet or exceed the NRC [15] requirements. The formulation of the experimental diets was presented in Tables 2, 3.
Growth performance
BW and feed intake data were collected at the end of each phase to calculate average daily gain (ADG), average daily feed intake (ADFI), and the gain to feed (G:F) ratio. In addition, the amount of feed eaten by all piglets was recorded each day, and waste feed in the feeder was recorded at the end of each phase.
Blood sampling and analyses
Blood samples were taken from the jugular vein of twelve pigs with nearly average BWs in each treatment after 3 h of fasting to measure albumin (ALB), creatinine (CRE), glucose, total protein, globulin, urea, types of cholesterol, and immune response (IgA, IgG) when BW was recorded. The blood samples were centrifuged for 15 min at 1,207×g and 4°C (centrifuge 5810R; Eppendorf). The sera were transferred to 1.5 mL plastic tubes (BD vacutainer SSTTMII advance; Becton-Dickinson) and stored at −20°C until analysis. Serum IgG and IgA concentrations were analyzed by ELISA assay by the manufacturer’s protocols (ELISA Starter 87 Accessory Package, Pig IgG ELISA Quantitation Kit, Pig IgA ELISA Quantitation Kit; Bethyl). Total protein concentration (modular analytics, PE; Roche) and glucose (enzymatic kinetic assay; Roche) concentrations were analyzed using a blood analyzer. Total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), ALB, and CRE were measured using spectrophotometric kits following the manufacturer’s instructions (TBA-120FR; Toshiba Medical Systems Corporation). Globulin was analyzed with the automatic analyst Olimpus AU 600 (Diagnostica).
Incidence of diarrhea
The diarrhea incidence was determined at 8:00 am for 28 days. Data were recorded by pen during phases 1 and 2. The incidence of diarrhea was scored on a 4-point scale according to the condition of the feces and diarrhea (0 = normal feces; 1 = moist feces; 2 = mild diarrhea; 3 = severe and watery diarrhea). Slightly wet feces on the rump area was designated as contaminated. After recording these data, the watery diarrhea was cleaned away.
Statistical and chemical analyses
All the collected data were carried out using least squares mean comparisons and evaluated using SAS’s general linear model procedure [16]. Each pen was used as a single unit in the feeding trial, and individual pigs were used as experimental units for blood profiles, immune response, and incidence of diarrhea. Orthogonal polynomial contrasts were performed to determine linear and quadratic effects of inclusion levels of BSF larvae. A one-way ANOVA analysis was performed to compare the control to other treatments. Statistical differences were considered highly significant differences at p<0.01, significant differences at p<0.05, and tendencies between p≥0.05 and p≤0.10.
RESULTS
Growth performance
The effects of BSF larvae replacing PPR on growth performance are presented in Table 4. In phase I, the ADG and G:F ratio decreased when the BSF larvae level increased (p<0.05; p≤0.05). In comparison with other treatments, the highest BW was observed in the BSF25 group (p<0.05). During all phases, a decreased trend of ADG was shown as the BSF larvae level increased (p≤0.1). However, the final BW (4 weeks) from all treatments did not show any differences.
Blood profiles
The effects of BSF larvae levels on blood profiles are presented in Table 5. In phase I, the pigs from the control and BSF100 groups showed a higher trend of HDL cholesterol levels than those from the BSF25 and BSF50 groups (p≤0.1). In phase II, ALB level decreased as BSF larvae level increased (p<0.05), and pigs from the control and BSF25 treatments had higher values in ALB levels than BSF50 and BSF100 groups (p<0.05). During all phases, there were no significant differences in CRE, glucose, total protein, globulin, urea, or TC levels.
Immune response
The effects of BSF larvae replacing PPR on immune response are presented in Table 6. The study found no significant difference in IgA when pigs were fed BSF larvae meal during the experimental period. However, in phase II, the IgG concentration in the BSF25 group showed a higher trend than other groups (p≤0.1), and then a quadratic trend was also observed as the amount of BSF larvae replacing PPR increased (p≤0.1).
Diarrhea incidence
The effects of BSF larvae replacing PPR on the incidence of diarrhea are presented in Table 7. The change of diarrhea rate was not observed during the whole experimental period.
DISCUSSION
According to previous studies, the BSF larvae used in swine diets can be divided into three types, which depend on the fat content [8]. Full-fat BSF larvae contain 35.9%–48.1% of crude protein (CP) and 36.8%–48.1% of crude fat (CF) [17]. An average of 59% CP and 9% CF was found in the partially defatted BSF larvae meal [18], and defatted BSF larvae meal showed the CP content ranged from 40.8%–60.7% and the CF content ranged from 7.97%–12.28% [19]. This study provided defatted BSF larvae meal, which possessed 59.34% of CP, 4.83% of CF, and 11.19% of crude fiber content. These nutritional differences may be related to the growth phase of BSF larvae, harvest point, and defatted-processing technology of BSF larvae meal [20,21]. Compared to PPR, BSF larvae had a poor amino acid profile, especially most of the essential amino acids related to the growth of weaning pigs (arginine, lysine, and threonine). Therefore, amino acid balance and or indispensable amino acid: CP ratios must be considered in diet formulation when adding BSF larvae meal to replace the PPR source fully. On the other hand, BSF larvae contained 8.42% chitin as potential functional constituents, which may positively enhance the health of monogastric animals [22,23]. If BSF larvae products achieve a degree of reliability in large-scale production, they will have great potential to partially replace animal protein sources in animal feeds.
In recent years, the supplementation of edible insects replacing traditional protein sources in animal diets has drawn more and more attention [9,24], which mainly focused on the inclusion levels that affect the growth performance in weaning pigs [18,25]. In the present study, during phase I, replacing 25% of PPR with BSF larvae improved the BW, but the ADG and G:F ratio linearly decreased as the ratio of BSFL replacing PPR increased. A similar result was obtained by Liu et al [14], who reported that a small quantity of BSF larvae supplemented (replacing 25% of soybean meal) could improve BW, and ADG and G: F ratio was decreased as the replaced amount exceeded 25% from weaned day 1 to 14. Spranghers et al [11] found that the addition of full-fat (4% and 8%) and defatted BSF (5.4) prepupae to diets had no significant differences in daily gain, feed intake, and feed-to-gain ratio compared to the control diet. Crosbie et al [26] also reported that 25% and 50% of the animal protein sources replaced by full-fat BSF larvae in weaning diets had no significant changes in BW, ADG, and ADFI overall in the weaning period. Ipema et al [27] found that supplementing with BSF larvae did not affect piglets’ ADG or final BW, nor did it impact their feed efficiency or energy efficiency. In this study, we found that adding BSF larvae replacing PPR had no detrimental effect on BW, ADG, ADFI, and G:F ratio at the final phase. The reasons for the growth differences may be related to the life stage of BSF larvae, rearing conditions, the replacement ratio, or fat content in BSF larvae [19]. Secondly, the total chitin content in the experimental diet could also affect growth performance. Chitin, a nitrogen-containing fiber that is relatively difficult to digest, may decrease the growth rate of piglets fed by BSF larvae due to an immature digestive system after weaning [14].
Limited information is available about the change in blood profiles when pigs are fed different BSF larvae levels. The current study observed only statistically significant differences in ALB and HDL concentrations at phase I or II. ALB regulates blood and body fluid volume through osmoregulation, accounting for over half of serum protein [28]. The normal serum ALB level in piglets is between 2–4 g/dL [29], and ALB levels are used to assess liver function, inflammation, and malnutrition [30]. Our study showed no significant differences in haematochemical parameters, except for the ALB concentrations, which showed that the control and BSF25 groups had the same statistical value and were higher than those in the BSF50 and BSF100 groups. This finding was in agreement with a study by Biasato et al [18], who reported that the addition of BSF larvae at 0%, 5%, and 10% to the diet did not result in significant differences in levels of globulin, CRE, total protein, or TC. Moreover, HDL cholesterol concentrations increased as pigs were fed with increased BSF larvae, replacing PPR. HDL cholesterol is considered the “positive” form of cholesterol, and higher levels within the normal range are associated with beneficial effects on cardiovascular health [31]. Chia et al [32] reported that serum lipid parameters, such as HDL and LDL concentrations, did not change among pigs fed with different levels of BSF larvae. However, in the present study, as the inclusion level of BSF larvae increased, HDL cholesterol levels also increased, suggesting an improvement in the blood lipid profile of piglets. This beneficial effect may be correlated with the change in intestinal microbiota due to the abundance of subdoligranulum [14]. Van Hul et al [33] reported that subdoligranulum was positively correlated with HDL levels in the blood.
IgG and IgA mainly represent the immune status of the body [34]. IgG is the most critical immunoglobulin in humoral immunity to prevent the invasion of infections or harmful bacteria. IgA is the body’s most common and secreted immunoglobulin, prohibiting the adhesion and colonization of pathogens in the intestines. In the current study, BSF25 groups had higher serum IgG concentrations than other groups in phase I, which contributed to improved pig performance, as shown in Table 4. The improvement of the immune response might be related to the chitin contained in BSF larvae. As an active compound, chitin has a positive effect on the immune response [35]. However, weaning pigs do not fully digested high levels of chitin to produce chitosan [36] and possess anti-nutritional properties [37]. In our study, the total chitin intake for each treatment can be calculated as follows: Control group, 0 mg; BSF25 group, 0.75 mg; BSF50 group, 1.46 mg; BSF100 group, 2.88 mg. Yousef et al [38] reported that the level of chitin exceeding the critical value harmed the immune response. Meanwhile, a study by Liu et al [14] also reported that BSF larvae as a replacement for SBM at 25% had higher IgM and IgG concentrations in the ileum of weaning pigs compared with the control or BSF larvae replacing 100% SBM.
Numerous studies have reported that incorporating insect protein into weaning pig diets can improve intestinal health, thereby reducing diarrhea [39,40]. Boontiam et al [39] reported that 12% of BSF larvae decreased diarrhea rate compared to the 6% of BSF larvae in weaning diets because the higher BSF-derived chitin (0.26% to 0.51%) may inhibit the growth of Escherichia coli and promote Lactobacillus spp. in the gut. Jin et al [40] also confirmed that BSF larvae supplementation of up to 8% can change gut microbiota distribution, increase lactobacillus’ relative abundance, and improve intestinal health. However, in the current study, there were no significant differences in diarrhea rates among the treatments. There was an agreement with the study by Liu et al [14], who reported that the rate of diarrhea in piglets from all treatments did not show any differences as the ratio of BSF larvae replacing SBM increased. Phaengphairee et al [41] also found that the incidence of diarrhea was unaffected by BSF larvae supplemented up to 12% in weaning pig diets compared to the control. This result predicted that weaning pigs could not secrete enough chitinase to produce chitooligosaccharides as prebiotics to improve gut health.
CONCLUSION
In the current study, replacing PPR up to 25% with BSF larvae in weaning diets had a positive effects on growth performance, blood profiles, and immune response; BW or IgG concentration was especially improved in the early (0–2 weeks) or late weaning phase (4 week). However, the ratio of BSF larvae replacing protein plasma exceeded 50%, negatively affecting ADG and the G:F ratio. Therefore, it is possible to substitute up to 25% PPR by BSF larvae in the diet of weaning pigs.
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