Full-Fat Insect Meal as a Protein and Energy Source for Weaned Piglets: Effects on Growth Performance, Nutrient Digestibility, Gastrointestinal Function and Microbiota

Abstract

Background: Weaning is associated with growth decline and morphological changes in the gastrointestinal tract of the pig. Insects, such as the Black soldier fly larvae (BSFL), are suggested as a sustainable novel protein source in diets for pigs. They contain chitin, medium chained fatty acids, and anti-microbial peptides, which could improve the gastrointestinal function and health in the post-weaning pig. The objective of this study was therefore to investigate the effect of increasing inclusion of full-fat BSFL in diets for post-weaning pigs on growth performance parameters, digestibility, gut morphology, and the microbial community in the colon. Eighty crossbred weanling pigs were weaned at approximately 32 days of age, with an average weaning weight of 10.6 ± 0.8 kg. For four weeks, the pigs were fed one of four dietary treatments: a control diet or one of three diets containing an increasing amount of full-fat BSFL meal: 4.76% (BSFL5), 9.52% (BSFL10), and 19.06% (BSFL20).

Results: Increased level of full-fat BSFL in the diet did not affect growth performance or fecal consistency. A reduction in apparent total tract digestibility (ATTD) of crude protein (P = 0.035) was found for increased inclusion of BSFL, whereas the ATTD of crude fat increased (P<0.001). Jejunal, ileal, or colonic morphometry was not affected by the BSFL inclusion.

Some changes in the colon microbiota were observed, but no differences in the short-chain fatty acid concentrations were detected between the dietary treatments. At the phylum level, dietary treatment affected the relative abundance of Bacteroidota, Firmicutes, Campilobacteria, and Thermoplasmatota, but there was no clear pattern relationship with the BSFL inclusion level. At the genus level, the inclusion of BSFL in the diet reduced the relative abundance of Lactobacillus (P = 0.015) compared to the control.

Conclusions: Collectively, the results indicate that up to 19.06% of full-fat BSFL meal could be included in a balanced diet for PW pigs without affecting growth performance, gut function, or health. 

Background

The world is facing a growing food demand by an ever-increasing number of people. To meet this challenge, new resources must be considered by using innovative solutions. Insects have been proposed as a high quality, efficient, and sustainable alternative protein source [1]. Black soldier fly larvae (BSFL), Hermetia illucens, is an easily reared species, capable of efficient conversion of a wide range of organic materials [2], and do not accumulate pesticides or mycotoxins [3, 4]. The BSFL has an overall favorable amino acid profile but is limiting in sulfur-containing amino acids and contains a high amount of minerals, especially calcium [5].

A well-functioning gastrointestinal tract (GIT) is of importance to the overall growth performance and health of pigs in all stages, and especially for the newly weaned pigs. If not defatted, BSFL is high in fat, especially in lauric acid (12:0) [5] which is categorized as a medium-chain fatty acid (MCFA) with antimicrobial effects, especially against gram-positive bacteria [6]. Insects and insect larvae also contain chitin, a dietary polysaccharide that can function as a prebiotic and an immunostimulant [7]. Finally, anti-microbial peptides, which are part of the insect immune system, are effective antimicrobial agents with low risk of development of bacteria resistance. Therefore, the anti-microbial peptides have good potential as health promoters in livestock, even though there is limited information about the in vivo effects [8].

Based on the content of these constituents, insects are interesting to investigate both as a feed ingredient and as a functional ingredient with potential health beneficial effects. Recently there has been a high focus devoted to the potential of insect meals in diets for monogastric farm animals [9–12], however, there is a scarcity of literature on the use of full-fat BSFL in diets for pigs. The objective of this study was to investigate the effect of increased inclusion of full-fat BSFL in diets for post-weaning (PW) pigs, focusing on growth performance parameters, digestibility, gut morphology, and microbial community in the colon.

Materials And Methods

A 27 days experiment was performed in February 2019 at the Center for livestock production (SHF, NMBU, Ås, Norway), which is an animal experimental unit approved by the National Animal Research Authority (permit no. 174). All pigs were handled under the applicable laws and regulations controlling experiments with live animals in Norway (regulated by the “Animal Welfare Act” and “The Norwegian Regulation on Animal Experimentation” derived from the “Directive 2010/63/EU on the protection of animals used for scientific purposes.

Results

Discussion

The interest of using insects in animal feed is increasing [35, 36], and inclusion of BSFL have been investigated in diets for pigs [9], chicken [12] and fish [11, 37], but to this date, there is limited information about the effect of high dietary inclusion levels. The present study demonstrated that up to 19.06% full-fat BSFL meal can be included as an alternative protein and energy source in pelleted diets for weaned piglets without affecting growth performance.

Dietary inclusion of BSFL did not alter the ADG, ADFI, or FCR for a four-week period PW. There was, however, a tendency for decreased FCR with increased inclusion of BSFL the first 14 days PW. These results are partly consistent with Yu et al. [38], which reported a linear improvement in both ADG and FCR when feeding increasing BSFL inclusion from 0 to 4% in the two first weeks PW, whereas no differences were found for a four-week feeding period. Contrary, another study showed no differences in performance when feeding up to 8% full-fat BSFL for 15 days, after weaning at 21 days of age [9].

Insects contain varying levels of chitin, depending on both types of insects and life stage [5]. This polysaccharide is the major component of the insect’s cuticle and creates a strong skeleton together with minerals and proteins. The exoskeleton of insects is different from crustaceans by having more amino acids strongly bound to the chitin [39, 40] and the degree of chitin deacetylation is dependent on the insect life stage [41]. Inclusion of chito-oligosaccharide (derivates from chitosan) in a corn-soybean meal diet has been shown to reduce the incidences and score of diarrhea in piglets weaned at 16 days of age [42], and in piglets weaned at 21 days of age and challenged with Escherichia coli [43]. However, no difference in fecal score or fecal DM were found between the dietary treatments in this study and may be due to the complexity of the chitin in the insect cuticle and the low degree of acetalization and solubility compared to the chito-oligosaccharides [44]. The increased fecal score in the first week PW is in accordance with the critical period for PW diarrhea [45]. The increase in fecal score in the last experimental week could be related to increased stress from sampling, as individual fecal samples for digestibility were collected daily in this period.

There is scarce information regarding the nutrient digestibility of insects and only a few studies including BSFL. Newton et al. [46] found similar ATTD of CP in five weeks old barrows fed 33% BSFL meal compared to a soybean meal control. In the present study, there was reduced ATTD of CP with increased inclusion of BSFL, but no difference in trypsin activity was found. A decline in ATTD of CP was also reported by Yu et al. [38] when including low amounts of BSFL (< 4%). Reduced CP digestibility with increased dietary inclusion of BSFL has also been reported for broiler chickens [47], Atlantic salmon [37, 48], and rainbow trout [49]. The CP digestibility is found to negatively correlate with chitin content in BSFL, in vitro [50]. Because of the nitrogen-rich chitin, the Kjeldahl-N method overestimates the digestible protein content of insects [51]. This is supported by the fact that the AID of total amino acids were not affected by dietary treatment. Janssen et al. [52], suggested using a conversion factor of 5.6 to avoid the overestimation of protein content in BSFL.

Limited information is available about the amino acid digestibility of BSFL in pig diets. However, the amino acid digestibility of full-fat BSF prepupae was recently investigated by Tan et al. [53]. The AID coefficients for amino acids were found to be between 0.641 and 0.821, and the SID coefficients to be between 0.767 and 1.177. During the diet optimization in the present study, the SID coefficients of all amino acids were set to 0.83. AID of lysine tended to decrease with increased inclusion of BSFL. Tan et al. [53] reported a SID coefficient of 0.776 for lysine in BSFL. Overestimation of the SID coefficient in the diet formulation of lysine might be the cause of the tendency of reduced AID of lysine with increased BSFL. Moreover, AID of alanine and proline tended to increase with increased inclusion of BSFL. The SID values for BSFL published by Tan et al. [53] were close to the one used in the diet formulation, but Tan et al. [53] also reported a higher AID coefficient for alanine in BSFL than soybean meal. Proline is high in endogenous losses, attributed to low reabsorption of mucin [54], but no information about BSFL affecting endogenous losses was found in the literature. In general, the AID of amino acids in the control diets was slightly lower compared with the control diet in a previous experiment with this age pigs [55]. Especially the AID of tyrosine was in general low and differed between the dietary treatments. The reported SID coefficient for tyrosine in BSFL (82.4%; Tan et al. 2020) is only slightly lower than the coefficient used in the diet formulation (83%). In the method used for amino acid analysis, glucosamine, resulting from hydrolyzed insect chitin, give a ninhydrin-positive peak close to the tyrosine peak in the chromatogram, which may cause a higher uncertainty for accurate tyrosine analyses. It is important to remember that even with standardized feeding time before sampling, the AID calculations with the slaughtering method are snapshots compared to methods with continuous collections or pooled collections over several days. This might contribute to why the trend in AID and ATTD are the same, but only the ATTD results are significant.

There was a linear increase in AID and ATTD of CF with increased inclusion of BSFL, whereas lipase activity measured in the oral part of jejunum showed no difference. The fat from BSFL amounted to be 19–69% of the total dietary fat and was significantly affecting the fatty-acid profile of the diets. Saturated fatty acids increased with increased BSFL inclusion, whereas the total amount of polyunsaturated acids decreased. The increase in saturated fatty acids was dominated by lauric acid (C12:0). The MCFA lauric acid is a major constituent of the BSFLs fat. It is synthesized by the larvae, and therefore present at a high level independent of diet [56, 57]. Finke [5] reported that lauric acid constituted 42% of the total fatty acids in the BSFL. The MCFAs are rapidly absorbed in the mucosa and, unlike long-chain fatty acids, directly into the portal vein for transportation to the liver [6, 58]. The differences in fatty acid composition between the diets are therefore believed to be the cause of differences in ATTD of CF. Higher digestibility of fat was also reported with 8% [9] and 33% inclusion of BSFL [46]. Contrary, Yu et al. [38] found a lower ATTD of CF when including 4% full-fat BSFL. Their reported ATTD of CF was overall low (< 70%). Different feed ingredients causing a different fatty acid profile of the diets, and low inclusion level of BSFL limiting the differences in fatty-acid profiles between diets could explain the contradictory result by Yu et al. [38].

Gilts showed higher AID and ATTD of CP, CF, and ash, as well as for AID of several amino acids, compared to barrows. Higher ATTD of CP in gilts is previously reported by [59], including a nonsignificant increase in ATTD of ether extract. The use of wood shavings as bedding material also increased the numerical ATTD except for NDF. This is probably a result of the pigs eating the wood shavings. Dietary fiber is known to decrease ATTD [60], but when not included in the dietary composition it can disturb digestibility calculations by reducing the relative amount of the nutrient in the digesta.

The MCFA can directly supply the enterocytes with energy and thereby improve morphological changes in periods with nutrient deficiency such as weaning [6]. It is well known that weaning causes villus atrophy. However, both in this study and the study by Spranghers et al. [9], no effect of BSFL inclusion was found on intestinal morphometry. The effect of MCFA on morphology could be more evident the first days PW as the VH in the small intestine seems to recover within two weeks PW [61]. Biasato et al. [10] also reported no difference in intestinal morphometric indices after feeding pigs diets with up to 10% defatted BSFL. In accordance with our results, they also observed greater morphometric indices in the jejunum than the ileum.

The gut health was mildly suboptimal due to the observation of inflammation and edema in the jejunum and colon, but the morphological disorders did not appear to have a strong association with any of the dietary treatments. The morphological changes in edema and inflammation could be related to the edema disease outbreak. The results are consistent with Biasato et al. [10] which replaced defatted BSFL with soybean meal, concluding that up to 10% BSFL inclusion does not negatively affect gut morphology or histological features.

The microbiota of the gut is involved in the nutritional, physiological, and immunological functions of the pig [62]. Variation in the diet composition is one of the most important factors affecting the GIT microbial ecosystem of the pig [63]. The microbial community in the colon was dominated by Bacteroidota and Firmicutes, also reported by Yu et al. [64]. Yu and coworkers found no differences in phyla abundances when including 4 or 8% full-fat BSFL in diets for finishing-pigs. By contrast, the relative abundance of Bacteroidota, Firmicutes, Campilobacteria, and Thermoplasmatota differed between the dietary treatments in the present study, but there was no clear dose-effect of the dietary BSFL inclusion. However, the highest relative abundance of Firmicutes and lowest relative abundance of Bacteroidota was found in the pigs fed the control diet. In accordance with Yu et al. [64], no significant differences in Shannon indices were observed.

Prevotella was the most abundant genus in the colon of the pigs regardless of dietary treatment. This is in correspondence with several authors reporting Prevotella to be the most abundant genus in the fecal microbiome in the PW period [65–67]. Lactobacillus was more abundant in the colon of the control piglets. This result is contrary to what recently was reported by Yu et al. [64], where inclusion of 4% BSFL increased the abundance of Lactobacillus. In the study by Yu et al. [64], finishing-pigs were fed corn-based diets contrary to our PW pigs, which were fed wheat and barley-based diets. Also, when they increased the inclusion to 8% BSFL, Yu et al. [64] did not find the same beneficial effects on the colonic microbiota as when feeding the 4% inclusion of BSFL. Lactobacillus is a genus of gram-positive lactic acid-producing bacteria in general considered favorable in the gut microbiome. It is known that lauric acid, abundantly found in BSFL fat, has antimicrobial effects, especially against gram-positive bacteria [6]. Spranghers et al. [9], observed inhibition of the growth of lactobacilli in an in vitro study with BSFL fat but did not observe differences in vivo in the small intestine. They also observed that a high amount of the lauric acid was absorbed already in the stomach and therefore had little opportunity to modulate the colon microbiome. In rainbow trout, it is shown that the BSFL life stage and lipid content of the BSFL are important factors influencing the gut microbiome [68].

It is important to mention the treatment with antibiotics of all pigs in the second week of the experiment. The effect of the antibiotic on the gut microbiota is antibiotic specific [62], and it is therefore difficult to know what impact the antibiotic treatment of the pig in this experiment had on their gut microbiota. Indeed, the antibiotic treatment could have affected microbiota results and be the reason for the small differences in the colon microbiome between the dietary treatments. Because of the antibiotic treatment in the second experimental week, it might be that the feeding period was too short to cause significant changes in the gut microbiome. Also, therapeutic doses of antibiotics might suppress the innate immune defenses and contribute to increased disease susceptibility [62]. Almost half of the pigs, independent of dietary treatment, had reddening of the colonic mucosa, indicating some inflammation in the colonic intestinal wall. However, the overall Shannon diversity indices reported were similar to what was reported by Yu et al. [64] in finishing pigs.

Conclusion

To conclude, up to 19.06% of full-fat BSFL meal could be included in a balanced diet for PW pigs without affecting performance results. Increased inclusion of BSFL decreased ATTD of CP and increased ATTD of CF but did not affect the general gut function, as assessed by enzyme activity, morphometry, and histological evaluation. Some changes in the colon microbiota composition were observed, such as decreased relative abundance of Lactobacillus with dietary inclusion of BSFL. However, there are limited information and contradictive results on the effect of BSFL on the gut microbiome in pigs, thus further investigations are needed.

Abbreviations

ADF: Acid detergent fiber

ASV: Amplicon sequence variant

AID: Apparent ileal digestibility

ATTD: Apparent total tract digestibility

ADF: Average daily gain

ADFI: Average daily feed intake

BSFL: Black soldier fly larvae

CF: Crude fat

CP: Crude protein

CD: Crypt depth

DM: Dry matter

FCR: Feed conversion ratio

GIT: Gastrointestinal tract

MCFA: Medium-chain fatty acid

NDF: Neutral detergent fiber

PW: Post-weaning

PCoA: Principal coordinate analyses

SCFA: Short-chain fatty acid

SEM: Standard error of the mean

SID: Standardized ileal digestibility

VH: Villus height

Declarations

Ethics approval and consent to participate

The experiment was conducted at an animal experimental unit approved by the National Animal Research Authority (permit no. 174). The authors state that all piglets were handled under the applicable laws and regulations controlling experiments with live animals in Norway, and in accordance with the EU Directive 2010/63/EU for animal experiments.

Consent for publication

Not applicable

Availability of data and materials

The datasets analyzed in the present study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Foods of Norway, a Center for Research-based Innovation (The Research Council of Norway; grant number 237841/030) and FeedMileage, Efficient use of Feed Resources for a Sustainable Norwegian Food Production (The Research Council of Norway; grant no. 310 233685/E50).

Authors' contributions

IMH, JØH, MØ, and LTM contributed to the conceptualization and study design. IMH, JØH, LTM, GHG, and RMÅ contributed to sample collections. RMÅ conducted enzyme activity analyses, 16s rRNA extraction, and sequencing. IMH and GHG performed statistical analysis on growth performance, liver index, fecal consistency, and digestibility data. IMH performed statistical analysis on pH, enzyme activities, short-chain fatty acids, and raw sequence data. IMH wrote the original draft. All authors critically reviewed the original draft and approved the final manuscript.

Acknowledgement

The authors would like to thank the staff at the Center for Livestock Production (SHF), NMBU, Ås, for taking care of the daily animal management during the experiment, and members of the Foods of Norway team for their help during sampling. The authors also want to thank Elvis, Amir, and Thijs for contributing to sample collection and histological evaluation of intestinal tissue.

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