Global poultry meat production is projected to increase by 32% by 2030 and 59% by 2050 from production in 2012, with low- and middle-income countries making up most of this demand (1). The cost of feed, particularly proteins, and availability of feedstuffs are by far the most important factors in poultry production sustainability and competitiveness. Indeed, soybean meal (SBM) is the preferred source of protein in broiler diets, as it is the only economically feasible protein source currently used in many countries. However, the continuous increase in global demand for high-quality animal protein, which consequently needs an improved animal production and abundant animal feeds, may inevitably surpass the SBM supply. This may, in turn, affect the inflation and the market price of SBM, which is linearly rising. Furthermore, there has also been a precedent set to limit animal-derived protein sources for animal feed in the European Union via Regulation (EC) No. 999/2001 (2). Thus, numerous innovative and effective approaches will need to be implemented to breach this looming protein gap to keep up with the worldwide demand for poultry meat.

Reducing crude protein (CP) in broiler diets has been extensively studied, yet the underlying issues of increased fat deposition and decreased performance are still prevalent (3–5). Implementing the ideal protein concept, computer least-cost formulation, and commercially produced crystalline amino acids allow for the lowest dietary CP diet thus far (6, 7). The reduction of CP with supplemental crystalline amino acids alleviates the demand for SBM, yet alternative protein sources will also be important for meeting the anticipated increase in demand for protein in animal and poultry feed. Alternatives for SBM in broiler diets have been investigated, including traditional and novel options such as canola meal, blood meal, meat and bone meal, fish meal, peameal, sunflower meal, insect meal, and numerous algae species (8–12). Many of the trials involving algae in broiler feed have focused on growth performance or meat quality with limited mechanistic explanations for their beneficial effects. Algae inclusion rates of up to 100 g/kg have maintained or increased performance parameters in chickens reared under conventional conditions (13–15).

Spirulina (Athrospira sp.) platensis is a filamentous spiral-shaped blue-green cyanobacterium that grows naturally in warm and alkaline aquatic media and is particularly interesting as a possible animal feed protein source due to its high level of CP (43–70%) and balanced amino acid profile. In addition to essential fatty acids, vitamins, minerals, and pigments (16), Spirulina contains several compounds shown to have antioxidant, anti-inflammatory, immune-modulating, and probiotic properties (17–20). Spirulina is also considered a nutritionally safe feed ingredient with no risk of mineral toxicity and free of the major algal toxin, microcystins (21, 22). Spirulina use as a feed ingredient for multiple species has been outlined (23), and one of the confounding issues put forth is the underlying mechanisms by which Spirulina impacts them. The purpose of this study was, therefore, to determine hematological parameters, inflammatory markers, and oxidative stress in broilers fed a low CP diet with and without Spirulina.

Inflammation is a response to stress and can occur in multiple levels of severity, from immediate acute responses to long-term chronic conditions. There is an intertwining relationship between inflammatory response and oxidative stress, in which inflammation elevates free radicals to levels exceeding cell threshold, thus inducing oxidative stress (26). Stress-induced inflammation can be associated with a plethora of factors, including dietary nutrient composition, bacterial or viral infection, environmental factors, obesity, aging, and numerous diseases (27–33).

There is limited information on how low CP diets and Spirulina impact inflammatory biomarkers in broiler chickens. It is important to remember that “low” or “high” CP level is relative regarding species, age, environment, and other factors. Excessive dietary protein intake can cause undigested protein or amino acids to enter the hindgut, increasing the amounts of opportunistic bacteria such as Clostridium perfringens in the ileum and ceca of broilers (34). Excess dietary nutrients/metabolites could cause metabolic (meta)-inflammation, which is different from pathological and physiological inflammation, and typically occurs in modern broilers production systems due to several factors such as ingredients used in the diet, nutrient excess in the diet, and high feed intake (35). All forms of inflammation lead to an upregulation of certain inflammatory biomarkers (36). However, the mechanisms mentioned earlier could not be the case in our study, as, surprisingly, the LCP diet led to an increase in basophil concentration and inflammation-associated biomarkers.

Basophil concentrations vary widely between avian species but are more highly concentrated in birds than their mammalian counterparts (37). Basophils are highly granular leukocytes that are chemo-attracted to inflammation sites and undergo degranulation to release histamine and Th2 cytokines and increase expression of toll-like receptors (38). The significant increase in basophil counts in our study indicates a systemic inflammation that might be a result of increased bacterial translocation that was observed in the LCP-fed birds. This is supported by an increase in the expression of inflammatory cytokines and chemokines, including IL-3, IL-4, IL-6, IL-10, CCL-20, and TNF-α in response to the LCP diet. The increase in liver bacterial translocation might be the underlying cause of the observed inflammatory stress. Reducing dietary CP can increase the broiler's intestinal permeability and leakage, which in turn induces bacterial translocation (39–41). Bacterial translocation increases pro-inflammatory cytokine production (42), which adversely impacts growth performance, as nutrients are directed toward immune responses rather than growth (43). This is evidenced in our experimental conditions by lower bodyweight, bodyweight gain, and a 15-point increase in feed conversion ratio in LCP-fed birds compared with their SCP counterparts in unpublished data.

It is worth noting that Kamely et al. (44) reported no differences in inflammatory cytokine TNF-α and IL-1β expression in the abdominal cavity exudate of Ross 708 broilers. The discrepancy between the present study and Kamely et al. (44) suggests that a low protein diet might affect the inflammatory system in a tissue-specific manner [systemic in our experimental conditions vs. local or abdominal cavity in Kamely et al. (44)]. The difference in the experimental approaches (diet formulation, amino acid levels, etc.) might also contribute to the discrepancy mentioned earlier.

Supplementation of Spirulina to LCP diets alleviated these negative effects and reduced inflammation as evidenced by amounts of IL-3, IL-4, IL6, IL-10, CCL-20, C3, and NLRP3 mRNA that were not different from those in the SCP control diet. This was in conjunction with a seven-point increase in feed conversion ratio in algae-LCP fed birds compared with LCP in unpublished data from this trial. Qureshi et al. (19) noted that Spirulina enhances the immune response in chickens fed a standard CP diet, and numerous trials have shown that algae ameliorate the expression of inflammatory molecular signatures in other species (45–47).

Inflammasome-forming NLRP3 and non-inflammasome-forming NLRs (NLRC3, NLRC5, and NLRX1) help mediate inflammatory responses (48), such as the one witnessed in the LCP-fed birds. The NLRs are activated in response to pathogen-associated molecular patterns and damage-associated molecular patterns; however, the exact underlying mechanisms for many NLRs in chicken have yet to be elucidated. In mammals, for instance, it is known that following microbial stimuli, NLR detects molecular patterns in the cytosol and activates the NLRP3 inflammasome, which in turn activates caspase 1, resulting in the production of IL-18 and IL-1β (49). Chen et al. (50) suggested that NLRP3 inflammasome activation occurred via the TLR2-MyD88-nuclear factor-kappa B (NF-κB) signaling pathway when chickens were challenged with Mycoplasma gallisepticum. The upregulation of MyD88 expression in this study supports the involvement of the previously described pathway. MyD88 acts as a downstream signaling adaptor for all TLRs except TLR-3 (51). MyD88 binds to TLRs and IL-1 receptor families to activate mitogen-activated protein kinases, activator protein 1, and NF-κB pathways (52). NF-κB plays an important role in producing pro-inflammatory cytokines after stress induced by gram-negative bacteria infection, reactive oxygen species, oxidized low-density lipoprotein, and multiple other factors (53).

Previous studies in mammals described several additional secondary signals, including plasma membrane disruption for bacterial toxins, lysosome destabilization, and liposome-triggering mitochondrial reactive oxygen species (ROS), leading to inflammasome oligomerization and activation (54–56). Although the mechanisms by which Spirulina reduces bacterial translocation and reverses the pro-inflammatory state induced by LCP are not known at this time point, it is conceivable that Spirulina might enhance gut integrity and merits further in-depth investigations. In addition to enhanced intestinal barrier integrity, Spirulina contains high levels (180 mg/g) of the biliprotein phycocyanin, which has been shown to have antioxidant, antibacterial, and anti-inflammation properties (57–60). Hao et al. (61) recently described a regulatory mechanism by which phycocyanin inhibits NF-κB expression and inflammation in human lung cancer cells through downregulating toll/IL-1 receptor domain-containing adaptor protein. Park et al. (17), on the other hand, showed that Spirulina modulates the antioxidant system in broilers. In our experimental conditions, Spirulina was also shown to modulate the systemic antioxidant defense system by lowering GPx-1 and upregulating GPx-3 and SOD-2, which is quite intriguing. GPx catalyzes the reduction of various hydroperoxides to H₂O via oxidation of reduced GSH into its disulfide form (62). SOD2, also known as mitochondrial manganese-dependent SOD, transforms toxic superoxide (a byproduct of the mitochondrial ETC) into hydrogen peroxide and diatomic oxygen (62). The differential expression between GPx-1 and GPx-3 suggests a potential compensatory mechanism; in other words, GPx-3 expression was upregulated by Spirulina to compensate for the lack of GPx-1, which might be depleted due to elevations in ROS production. The upregulation of SOD2 could result from increased xanthophyll content or perhaps suggests enhanced cytoprotection and clearance of mitochondrial ROS by C-phycocyanin derived from Spirulina (63), which might explain at least partly the reduction of inflammation and cytokine expression. Spirulina also has the xanthophyll lutein, which can increase SOD2 irrespective of ROS levels (64). In support of our hypothesis, phycocyanin is considered a principal component responsible for antioxidant activity in algae by scavenging hydroxyl radicals (65). Furthermore, carotenoids such as β-carotene also protect cells from oxidative stress through quenching singlet oxygen damage (66).

In conclusion, this is the first report, to our knowledge, defining potential molecular targets by which Spirulina inclusion reduces systemic inflammatory activity, including cytokines, chemokines, bacterial translocation, and proportions of basophils in broilers fed a low protein diet.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by the University of Arkansas Animal Care and Use Committee (#21002).

GM, MK, WB, and SD: conceptualization. GM, GT-I, MK, and SD: methodology. GM, NE, and GT-I: software. GM, EG, NE, and SD: validation. GM, EG, GT-I, and GE: formal analysis. GM, EG, GT-I, MK, and SD: investigation. WB, MK, GT-I, GE, and SD: resources. GM, NE, and SD: data curation. GM: writing—original draft preparation. NE and SD: writing—review and editing. SD: visualization and supervision. WB, MK, and SD: project administration. WB, SD, and MK: funding acquisition. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.