Poultry meat and eggs represent a fundamental source of high-quality protein for the global population, due to the high production efficiency (i.e., low feed conversion ratio) and the broad consumer acceptance (Castro et al., 2023). Nowadays, in addition to productivity and cost, other aspects are gaining increasing attention from both consumers and public authorities, including environmental sustainability, animal welfare, meat safety, and the so-called “nutraceutical” properties (Verbeke et al., 2010; Di Pasquale et al., 2014). Feed composition clearly plays a pivotal role in all these domains. Notably, feed costs account for 60-80% of the total production costs in poultry farming, and the procurement of raw materials has a considerable environmental, and therefore social, impact (Elahi et al., 2022). Moreover, feed ingredient availability is subject to marked price fluctuations and geopolitical uncertainties. European policies are increasingly aimed at achieving raw material independence for feed production by reducing imports from foreign countries. In this context, the development and evaluation of alternative feed sources have become a major research focus. Oilseeds and oilseed meals, such as those derived from rapeseed, linseed, canola, and camelina, are important sources of energy, crude protein, and fats, including essential polyunsaturated fatty acids (PUFA) that can accumulate in animal tissues and improve the nutritional quality of the meat (Zając et al., 2020; Hajiazizi et al., 2024). These sources offer remarkable environmental and economic benefits, as they typically involve undemanding cultivars in terms of fertilization and irrigation. They are also resistant to diseases and parasites, requiring minimal treatments, and are used in various industrial applications. Their byproducts, such as oilseed cakes, can be further valorized as animal feed, thus contributing to the principles of circular economy by reintroducing agricultural and industrial residues into the production cycle (Singh et al., 2023). Among these crops, camelina (Camelina sativa L. Crantz) and linseed (Linum usitatissimum L.) have attracted particular interest. Camelina, a member of the Brassicaceae family, is highly adaptable to diverse environmental conditions, requires low agronomic inputs, and has a relatively short growing cycle. Its seeds contain 30-49% oil, rich in omega-3 (ω3) and omega-6 (ω6) fatty acids (FAs), 24-31% protein, and noteworthy levels of tocopherols, phytosterols, and phenolic compounds (Berti et al., 2016; Boyle et al., 2018; Mondor and Hernández-Álvarez, 2022). The cake obtained after oil extraction provides about 35% protein, a balanced amino acid profile, and 10-22% residual oil with a high proportion of ω3 FAs (Aziza et al., 2014). Linseed cake, a co-product of linseed oil production, is likewise rich in nutrients, with around 36% protein, 7-10% residual oil, and significant levels of lignans, antioxidants, and minerals (Duliński et al., 2017). Nevertheless, these crops are often rich in antinutritional factors (ANFs), and their excessive inclusion in poultry diets may compromise animal performance (Bischoff, 2021).

Various insect species are currently being explored as promising alternative feed sources. Among them, Tenebrio molitor (TM) larvae have been primarily investigated in the form of full-fat or defatted meals as substitutes for conventional protein sources (Moula and Detilleux, 2019; Volek et al., 2021; Andrade et al., 2023). They can also be offered as live larvae, providing environmental enrichment and contributing to improved animal welfare (Bellezza Oddon et al., 2021; Colombino et al., 2021; Dalle Zotte et al., 2024). Nutritionally, TM larvae are rich in protein (approximately 50% of dry matter, DM) and contain high levels of essential amino acids. They also have a substantial fat content (around 30% of DM), which includes beneficial FAs, notably ω-3 and ω-6 PUFA (Kröncke et al., 2019; Trukhanova et al., 2022). In addition, TM larvae represent a valuable source of minerals and vitamins (Nowak et al., 2016). Their chitin-rich exoskeleton contains a bioactive polysaccharide with well-documented immunostimulatory and prebiotic properties (Swiatkiewicz et al., 2015; Elahi et al., 2022). Insect larvae also naturally produce antimicrobial peptides (AMP) as a defense mechanism against microbial infections (Elahi et al., 2022). These compounds have been shown to improve resistance to various pathogens, including zoonotic agents, potentially enhancing food safety and reducing the need for antimicrobial treatments (Hwang et al., 2022). The bioactive compounds present in insects have been reported to modulate the gastrointestinal microbiota when insects are used as full or partial replacements for conventional protein sources in chicken diets (Biasato et al., 2019; Moula and Detilleux, 2019). Similarly, oilseed cakes have been investigated as potential probiotic feed ingredients for livestock, with camelina cake shown to enhance the acid-forming activity of lactic acid bacteria and promote the growth of other beneficial bacterial populations (Mazanko et al., 2023). In addition, linseed cake, when combined with probiotics such as Lactobacillus acidophilus, has been reported to improve gut health in broiler chickens by supporting a more favorable intestinal microbiota and reducing pathogenic bacteria (Gheorghe et al., 2020). However, current knowledge on the effects of incorporating TM larvae and oilseed cakes into poultry diets, particularly regarding nutrient digestibility, gut morphology, immune function, and the microbiome, remains limited. Furthermore, direct comparisons between these two dietary supplements remain scarce, making it difficult to determine which provides the greater benefits for chicken’s health and performance.

The present study addresses these topics by comparing feed digestibility, morphometric parameters, inflammatory cell infiltration, gene expression, and microbiota composition across different intestinal segments of chickens fed conventional, oilseed cakes-enriched, or insect-enriched diets.

The present study investigated the effects of diets incorporating alternative protein sources and FA supplements, specifically camelina and linseed cakes as well as TM meal, on diet digestibility, intestinal morphology, immune responses, and microbiome composition in broiler chickens.

The apparent digestibility coefficients indicated that the CD and TD diets exhibited overall higher feed digestibility compared to the OD diet. This effect is likely attributable to the presence of ANFs in camelina and linseed cakes which can impair productive performance, particularly when included at high concentrations (Aziza et al., 2014; Oryschak et al., 2020; Hajiazizi et al., 2024). Indeed, camelina and linseed cakes are known to contain glucosinolates, phytic acid, sinapine, and tannins (Tan et al., 2011; Pojić et al., 2015; Tangendjaja, 2022). Elevated levels of phytates can reduce amino acid digestibility and inhibit digestive enzyme activity. Likewise, tannins may decrease protein digestibility by forming indigestible protein-tannin complexes (Kahindi et al., 2014). This explains the reduced CP digestibility in the OD group compared to the CD. Consistent with these results, previous studies have reported decreased nutrient digestibility in chickens fed diets containing oilseed cakes or seeds (Rebolé et al., 2002; Amerah et al., 2015; Zając et al., 2020). In particular, the high levels of mucilaginous material in linseed have been shown to impair fat digestibility. These compounds belong to the non-starch polysaccharides (NSPs), a group of substances known to increase intestinal viscosity and thereby reduce nutrient absorption by limiting the diffusion of digestive enzymes and nutrients (Longstaff and McNAB, 1991; Choct et al., 1996).

Therefore, appropriate levels of camelina and linseeds products in poultry rations should be taken into consideration because of presence of these factors and their effects on feed consumption and assimilation (Thacker and Widyaratne, 2012; Pekel et al., 2015; Hajiazizi et al., 2024).

In contrast to the effects observed with oilseed cake inclusion, the incorporation of TM larvae into chicken diets did not negatively affect apparent nutrient digestibility, being the CP the only exception. Schiavone et al (Schiavone et al., 2014). reported a reduction in CPd when 25% TM larvae meal was included in chicken diets, and a similar effect was observed by Bovera et al (Bovera et al., 2016). following complete replacement of soybean meal with TM meal. Accordingly, in the present study, a reduction in CPd was observed with the 10% inclusion of TM in the diet of broilers, most likely due to the presence of chitin. Chitin, indeed, is an indigestible polysaccharide that can negatively affect nutrient digestibility in poultry. Although chickens possess some chitinase activity, it is insufficient to effectively degrade chitin (Tabata et al., 2017). The indigestible chitin may increase digesta viscosity or slow intestinal transit, thereby limiting enzyme–substrate interaction and reducing nutrient absorption (Abenaim and Conti, 2025). The inclusion level used in the present study was sufficient to avoid compromising the apparent ileal digestibility of most nutrients, although it already caused a reduction in CPd. In contrast, the higher inclusion levels adopted in other studies may have been excessive, leading to a more pronounced decrease in nutrient utilization (Belluco et al., 2013; Schiavone et al., 2014). Thus, selecting an appropriate inclusion level may help mitigate the negative effects of chitin present in the larvae exoskeleton.

The administration of TM larvae did not significantly affect gut morphometric parameters, as no statistically significant differences were observed compared to the control diet with the exception of crypt depth in the ileum. However, a general trend toward increased villus length (except in the ileum) and reduced crypt depth was noted. These morphological features—longer villi and shallower crypts—are associated with improved nutrient absorption capacity in the gastrointestinal tract (Aziza et al., 2014). Previous studies have reported both no effect of TM administration in broilers and improved intestinal morphology in Japanese quails (Zadeh et al., 2019; Malematja et al., 2023). Conversely, reduced villus height has been observed in laying hens fed high levels of black soldier fly larvae and in Ross broilers fed high levels of mealworm (Biasato et al., 2018; Moniello et al., 2019). Interestingly, such negative effects were not observed with lower dietary inclusion levels. This evidence suggests a dose- and/or host-dependent effect, or more broadly, the contribution of multiple interacting factors. The intermediate dose provided in the present study may have not led to detrimental effect described for high dose, although causing lower, not statistically significant, positive effect of villi structure. Furthermore, the limited processing steps may have preserved the native structure and concentration of nutrients, potentially affecting their bioavailability (Elahi et al., 2022).

Overall, the insect-based diet can be considered beneficial, leading to improved or at least comparable intestinal morphometric features, which suggest a similar absorptive potential. Likewise, the observed pattern in crypt depth indicates an equal or reduced need for tissue regeneration.

The results of the oilseed-based diet appear more surprising, as despite its known beneficial properties, the presence of ANFs was also expected to play a role. A statistically significant increase in villus length and reductions in crypt depth were herein observed in most intestinal segments, resulting in a significantly higher villus-to-crypt ratio (V:C). However, a shortening of villus length had previously been reported in association with camelina-based diets (Aziza et al., 2014). Various factors may account for these findings, as previous studies on camelina administration have often yielded contradictory results, which have been attributed to differences in diet composition, feed formulation, bird genotype, and other experimental conditions. An increase in villus length has been reported in diets enriched with components such as dietary fibre, which is known to decrease overall feed digestibility (Jha and Mishra, 2021), and may represent a compensatory response to increase absorptive capacity (Liebl et al., 2022). The doses also often have a main role in the observed results, since previous studies revealed a detrimental effect associated particularly with high doses. A 5% inclusion of camelina seed in poultry diets had no adverse effects on growth performance. However, 10% inclusion was associated with reduced body weight (BW) and BW gain (Juodka et al., 2022). The effects of camelina cake supplementation are more variable: some studies reported that 8–16% inclusion enhanced BW and BW gain while others attributed reduced growth performance (Ryhänen et al., 2007; Oryschak et al., 2020). Moreover, the content and bioavailability of ANFs can be influenced by the specific camelina cultivar and processing methods applied (Singh et al., 2023). Therefore, it can be hypothesized that these variables also played a role in the present study. The direct effect of PUFA and antioxidants, as γ-tocopherol present in camelina and linseed and α-tocoferol abundant in TM larvae, or the indirect modulation of the microbiome could have favored enterocyte differentiation and maturation, improved villi development and reduced regeneration need. Different studies have confirmed the many favorable effects of dietary ω-3 PUFA, including anti-inflammatory or inflammation-reducing properties (Alagawany et al., 2019; Malematja et al., 2023). The higher expression of BCL-2 in both treatment diets might support their beneficial effects in reducing stresses and damages to the intestinal cells.

No effect on mucin composition has herein been reported, comparably to Colombino et al (Colombino et al., 2021). However, evidence of a dose dependent effect on mucus layer, potentially mediated by altered microbiota, was also described (Biasato et al., 2018; Biasato et al., 2019; Biasato et al., 2020). An analogous dose of TM of 10% was associated with lower mucin staining intensity (Biasato et al., 2019). A 10% camelina meal also resulted in reduction of goblet cell number (Aziza et al., 2014). The lack of effects in the present study may thus be due to other concomitant factors.

Despite the absence of significant histological differences in immune cell infiltration, some inflammatory mediators exhibited differential expression profiles among groups. The higher expression of IL-10, an anti-inflammatory cytokine, might reflect the beneficial effects of PUFAs or other anti-inflammatory and antioxidant compounds provided by camelina, linseed and TM. A reduced stimulation of the immune system by microbial components is also plausible. The presence of antimicrobial molecules, which are well documented in TM larvae, could also have contributed to this response. However, the higher expression of IL-1β, along with several other inflammatory mediators—although not statistically significant—in the camelina-fed group still suggests the presence of an inflammatory stimulus, which was not observed in the CD or TD groups. Certain ANFs, or their metabolites, may compromise the integrity of the mucosal barrier, thereby triggering an inflammatory response. In particular, some toxic glucosinolate derivatives, in addition to affecting feed palatability and nutrient assimilation and exerting systemic and hormonal effects, may directly irritate the gastrointestinal mucosa (Bischoff, 2021). However, extensive literature also provides evidence of the anti-inflammatory effects of several such molecules, complicating results interpretation (Bischoff, 2021).The specific—and sometimes conflicting—findings observed in the present and other studies may result from complex interactions between the specific composition of the feed and host-related factors, including the microbiota. Comparably, chitin, a component present in TM larvae is known for its positive effect on inflammation modulation, stimulating cytokine production (Swiatkiewicz et al., 2015; Elieh Ali Komi et al., 2018; Elahi et al., 2022; Malematja et al., 2023). However, in the present study, no marked pro-inflammatory pattern was observed. Comparably, a downregulation of IL-2, another pro-inflammatory mediator, has been reported by Colombino et al. (2021), in poultry fed with live TM, and was attributed to their lower chitin content (Colombino et al., 2021). A similar conclusion can be proposed in the current context, where a relatively low TM dose was administered. An alternative, non-contradictory hypothesis might be attributed to the reduced stimulation of the immune system resulting from the exclusion or limitation of pathogenic agents—possibly induced by antimicrobial peptides (AMPs), competitive exclusion by other bacteria, and short-chain fatty acid production (Zhang et al., 2023).

No remarkable differences were observed in the microbiome among the different diets in terms of alpha diversity (i.e., within-sample variability), and only minimal differences were detected in beta diversity (i.e., between-group variability). The proximal intestinal tract was dominated by Firmicutes, particularly Lactobacillaceae, Clostridiaceae, and Peptostreptococcaceae in the ileum, although some inter-individual variability and the presence of other bacterial groups were also observed; Proteobacteria were represented mainly by Enterobacteriaceae, in line with other studies (Oakley et al., 2014). As expected, the caecum displayed a much higher microbial heterogeneity (Rychlik, 2020). However, differently from what is often reported, Firmicutes remained largely dominant. Previous studies have shown that in the caecal microbiota of healthy adult hens, Gram-positive Firmicutes and Gram-negative Bacteroidetes are usually equally represented, each accounting for approximately 45% of the total microbial community (Rychlik, 2020). In the present study, regardless of the diet, Firmicutes still represent approximately 95% of the detected species. The analysis of rarefaction curves indicated adequate coverage of the taxa at the sequencing depth considered, supporting the reliability of the observed microbial composition rather than suggesting an artifact. Nevertheless, microbiome composition appears variable and other studies have also reported a much lower contribution of Bacteroidetes to the caecal microbiota (Wei et al., 2013; Oakley et al., 2014; Huang et al., 2021; Yin et al., 2023). Moreover, age, chicken genotype, geographical origin, and diet related factor, as well as colonization pathways, play a role (Singh et al., 2014; Siegerstetter et al., 2017; Huang et al., 2021; Yin et al., 2023). It has been reported that relative abundance shifts between Firmicutes and Bacteroidetes may be linked to feed efficiency. In humans, the Firmicutes/Bacteroidetes (F/B) ratio has been correlated with obesity (Bervoets et al., 2013) and a higher Firmicutes/Bacteroidetes ratio has been observed in poultry exhibiting greater feed conversion efficiency (Huang et al., 2021). Firmicutes are known to degrade host-indigestible polysaccharides, thereby promoting nutrient digestion and absorption. The balance among different taxa determines the overall metabolic output, including the production of short-chain fatty acids (SCFAs), which have a profound impact on host metabolism and physiology. Firmicutes are mainly butyrate producers, contributing to a butyrate-dominant energy supply in the gut. Lachnospiraceae family, significantly present in the caeca, is a well-known butyrate producer, along with Ruminococcaceae (Biasato et al., 2020). Accordingly, Loponte et al. (2019) found an increased amount of total VFAs (+45.6%), acetate (+40.3%), and butyrate (+64.6%) in broilers fed a TM larvae meal as a complete replacement of soybean meal (Loponte et al., 2019). Butyrate is considered the primary energy source for enterocytes (Bovera et al., 2011) and is also essential for the proper development of Gut-Associated Lymphoid Tissue (GALT), which may have contributed to the well-developed intestinal mucosa observed in this study. Moreover, van der Wielen et al. (2000) reported that increased butyrate concentrations were associated with reduced levels of Enterobacteriaceae (van der Wielen et al., 2000) and the high concentration of butyrate produced in the caeca of broilers fed the TD diet may play a key role in inhibiting E. coli and Salmonella colonization. Therefore, the detected microbiome composition can be considered beneficial for both animal and, indirectly, human health.

Among the individual taxonomic groups that were differentially represented compared to the diet control, Peptostreptococcaceae were significantly overrepresented in both the OD and TD diets. Peptostreptococcaceae contributes substantially to the butyrate pool in the chicken caecum. Several studies have reported that increased intestinal butyrate levels enhance gut barrier function, as evidenced by reduced translocation of bacterial endotoxins (LPS) from the lumen into the bloodstream and improved mucosal regenerative capacity. An anti-inflammatory effect has also been reported (Zhang et al., 2023). Desulfotomaculaceae, which were more abundant in the control diet group compared to the other experimental groups, produce hydrogen sulfide (H₂S) as the final product of their metabolism. At high concentrations, H₂S is cytotoxic to intestinal epithelial cells, as it inhibits mitochondrial enzymes and can induce apoptosis or cell death. An increase in Desulfotomaculaceae in the poultry microbiota is generally considered unfavorable from an immunological perspective, as it tends to promote a pro-inflammatory state (Onrust et al., 2015). Therefore, the decrease in this population in OD—and in TD group compared to the control, although not reaching statistical significance—might have contributed to the observed anti-apoptotic status, the reduced expression of some pro-inflammatory cytokines, and the higher levels of the anti-inflammatory IL-10. However, a higher presence of this taxon in TD compared to OD was also observed, suggesting that they only play a part in the overall regulation of caecum health and immunity regulation. Overall, the complexity of the potentially underlying determinants and the presence of multiple confounding factors require caution in the assessment of causal associationns. Finally, a reduction in Enterobacteriaceae was observed in the OD group compared to the control. This family, belonging to the phylum Proteobacteria, includes Gram-negative facultative anaerobes such as Escherichia coli, Salmonella, Klebsiella, and other genera relevant to the poultry microbiota. Unlike many obligate anaerobes, they do not produce appreciable amounts of propionate or butyrate. Consequently, a bloom of Enterobacteriaceae in the caecum does not enhance the production of short-chain fatty acids (SCFAs) beneficial to the host; on the contrary, it is often associated with a reduction in butyrate-producing fermenters and decreased energetic yield from the diet. Enterobacteriaceae are also known to exert predominantly pro-inflammatory effects in the intestinal tract. Their lipopolysaccharide (LPS) is among the most potent immune activators (Xi et al., 2023). Therefore, a reduction in their abundance might be considered beneficial and desirable.

Overall, the inclusion of oilseed cakes significantly reduced the digestibility of most nutrients, likely due to the higher fiber content and the presence of residual antinutritional factors which can interfere with nutrient absorption. In contrast, the inclusion of TM larvae affected mainly CPd. These findings suggest that the levels of inclusion of such raw materials may need to be carefully evaluated to avoid negative effects on nutrient digestibility. Despite these reductions, the camelina and linseed-based diet, led to improvements in intestinal morphometry and seems to positively affect immune parameters. Similarly, the TM-based diet appeared to promote a lower inflammatory status. Although no striking changes were observed in the microbiome composition, the main differences included an increase in taxonomic groups associated with butyric acid production in the experimental diets compared to control, along with a reduction in less beneficial or potentially pathogenic bacteria. Therefore, a likely combination of increased availability of antioxidants, polyunsaturated fatty acids (PUFAs), and other bioactive compounds—together with a modest modulation of the microbiome and its short-chain fatty acid (SCFA) metabolism—may have contributed to enhanced intestinal functionality and modulation of immune-related gene expression. These findings, combined with broader socio-economic considerations, suggest that such raw ingredients may offer advantages for poultry health and, indirectly, for human health, supporting their suitability for more consistent inclusion in poultry feed.