To the best of the authors’ knowledge, this is the first study to assess epigenetic and oxidative DNA changes occurring in the gut and blood and to examine in detail the structures and ultrastructures of the gastrointestinal tract in chickens challenged with C. perfringens bacteria and supplemented with cannabidiol and nano-selenium.

This study performed a histomorphometric analysis of the duodenum, jejunum, and ileum to assess the impact of infection and nutritional additives. Intestinal morphology is crucial for digestion and absorption, and indices such as villi height (VH), crypt depth (CD), villi width (VW), wall thickness (WT), villi area (VA), and villi height to crypt depth ratio (VC) are commonly used to assess intestinal health (39). However, limited evidence is available on how CBD affects gut function and anatomy in broiler chickens. This study shows that VH in the jejunum was significantly higher in the non-infected group with CBD and nano-Se supplementation compared to the control, C. perfringens, and infected group with nano-Se supplementation, and VH was lower in the nano-Se + C. perfringens group compared to the infected and non-infected group fed a diet with the two supplements. In contrast, neither treatment nor infection affected VH in the duodenum and ileum. As reported, longer villi are usually associated with improved absorption in the gut (40). In the case of VH, this result is consistent with the studies of Bami et al. (41) and Safdari et al. (42), who also showed that nano-selenium significantly increased VH. It can be concluded from this that cannabidiol may also have a gut-promoting effect. In contrast, the reduction in VH in the nano-Se + C. perfringens group compared to the infected group fed a diet with the two additives shows that, in this case, NE infection compromised investigated indices of the gut (43), and nano-selenium as the only additive did not improve the VH parameter. However, these results show that both compounds, combined under physiological or pathophysiological conditions, can act synergistically to improve intestinal barrier function. The lack of effect on VH in the duodenum and ileum is partly consistent with the study of Sopian et al. (44), who also observed no detectable effect of Cannabis sativa residues (CR) on this parameter. CD in the duodenum was significantly higher in the C. perfringens and infected groups fed a diet with CBD or nano-Se than in the control and infected and uninfected groups fed the two additives. This value was significantly lower in the jejunum in control, CBD + nano-Se, and CBD + nano-Se + C. perfringens groups compared to the C. perfringens and infected groups fed a diet with CBD or nano-Se additives. The crypt is a tubular gland formed by the small intestinal epithelium descending into the lamina propria at the root of the villi (45). The increase in CD in the infected group is consistent with a study by Guo et al. (46) showing that C. perfringens can damage the intestinal barrier. Additionally, the increase in CD indicates a larger necessity for cell proliferation to maintain normal intestinal health (1). This indicates that the increased depth of crypts in the infected groups may, on the one hand, be an indicator of active epithelial regeneration in response to damage. Still, on the other hand, it may also suggest chronic tissue damage and remodeling. The control group, which was neither infected nor received supplementation, showed a lower CD, which is expected in healthy intestines. Furthermore, this also indicates that the use of CBD or nano-Se alone in the diet did not show a deteriorated effect on the duodenum. The VC in the duodenum and jejunum was significantly higher in the uninfected group with CBD + nano-Se supplementation compared to the infected control group and the infected groups with CBD or nano-Se supplementation, and significantly lower in the ileum in the control group compared to the infected group with CBD and CBD + nano-Se supplementation. Higher VC indicates a higher rate of digestion and absorption function (47) but also suggests a greater need for cell proliferation to maintain intestinal integrity (48). This result is consistent with the studies of Sopian et al. (44) and Chen et al. (49), which showed that both nano-selenium and CR increased VC. In the current authors’ opinion, the presence of higher VC values indicates that the combination of CBD and nano-Se promotes improved intestinal integrity even in the absence of infection, mainly due to an increase in villi length with a similar rate of crypt depth in these groups. The lower values in the control group in the ileum indicate undisturbed mucosal indices. CBD and nano-Se in broiler diets affected the increase in VC in the ileum, which may indicate that there is a greater need for regeneration as a result of infection-induced damage in this segment and that these supplements improve the ability to regenerate the epithelium and maintain intestinal homeostasis in the presence of infection. Corresponding to the above, a reduction in VH and an increase in CD was found in the nano-Se + C. perfringens group in the present study. Shorter villi and deeper crypt may lead to decreased resistance to disease, lower growth performance, poor nutrient absorption, and increased secretion in the gastrointestinal tract (40, 50, 51). On the other hand, it should be noted that, as in the case of CD in jejunum, VC also did not differ between the control group and the infected group with two additives in duodenum and jejunum. This is important, as it reinforces the idea that combining CBD with nano-Se may show a protective beneficial effect on the gut barrier, protecting the chickens from the harmful effects of C. perfringens infection. An assessment of villi width showed that VW was lower in the CBD + nano-Se + C. perfringens group compared to the CBD + nano-Se group in both the duodenum and ileum. This is consistent with Zhao et al. (52), who showed a reduction in VW in C. perfringens-infected chickens. This shows that adding CBD and nano-Se can improve the functional status of the intestines under non-challenge conditions. On the other hand, Chen et al. (1) showed that birds with intestinal barrier failure had wider villi. According to this study, a widening of the villi indicates a smaller surface area for nutrient absorption and probably also a greater amount of gut-associated immune tissue proliferation and accumulation in the villi, which may be an indicator of impaired intestinal health. On the other hand, it is important to note that there was no significant difference between the control and the infected groups with the addition of CBD and nano-Se, supporting the above conclusion that the two components act synergistically and positively with each other under challenging conditions. Another indicator that showed significant differences was WT, which was significantly lower in the ileum in the C. perfringens group compared to the control group and the CBD-infected group. Reduced WT may directly result from the bacteria and their toxins, which can damage intestinal epithelial cells and lead to villous atrophy (53). The thickness of the intestinal wall under healthy conditions is the result of a balance between epithelial cell proliferation, migration, and exfoliation, which ensures the structural stability of the intestines. In contrast, the significantly greater wall thickness in the infected and supplemented group with CBD, which has anti-inflammatory and antioxidant effects, indicates a potential protective effect of this compound and may promote regeneration of damaged tissues and limit degradation of intestinal structures, leading to increased WT. In summary, gut region-specific effects have critical implications for the nutritional and immune status of broiler chickens. Changes in duodenal parameters in chickens infected and supplemented with CBD and nano-Se indicate the absence of adverse remodeling or chronic tissue damage, highlighting the protective effects of these supplements in the proximal intestine. Additionally, supplementation with the two ingredients in uninfected chickens indicates increased digestion and absorption capacity, potentially enhancing the role of the duodenum in nutrient uptake. In contrast, results in the jejunum, which is a critical site for nutrient absorption, suggest that this section benefits most from combined supplementation, particularly in maintaining intestinal integrity and absorption efficiency, in both infected and uninfected chickens. These improvements can lead to better energy and nutrient utilization, supporting growth and immune function. In contrast, the effect of the combination of CBD and nano-Se on the ileum, which is often involved in immune responses, suggests anti-inflammatory and regenerative benefits and appears to enhance immune defenses and structure in this section, which is essential for maintaining intestinal homeostasis and immunity in the presence of pathogens. This regional variability highlights the potential of CBD and nano-Se as synergistic agents to optimize nutritional and immune outcomes in broilers under physiological and pathophysiological conditions.

Light and electron microscopy analyses were performed to assess the histological structure and ultrastructure of the intestine. These studies showed that CBD + nano-Se supplementation is safe for the gut and does not cause negative qualitative changes in the histological structure of the small intestine and the ultrastructure of the jejunum. This result is partly consistent with a study by Gangadoo et al. (54), who showed in a histopathological analysis that nano-selenium did not cause harmful effects, revealing intact epithelial cells in the gastrointestinal tract in broiler chickens. This result also confirms the current authors’ previous study (32), which found that the combination of these two nutritional additives showed no opposite effect on mediating the host response to infection. This confirms that understanding the mechanisms of action of cannabidiol and nano-selenium is of great interest in developing new possibilities for promoting intestinal barrier function. In the present experiment, C. perfringens infection did not cause necrotic enteritis. The changes were moderate in character and mainly involved damage to the intestinal epithelium. This correlates with the clinical condition of the chickens, as none of the challenge tests induced overt clinical signs of NE. According to Timbermont et al. (16), NE is characterized by a strong inflammatory response in its early stages, where the most significant early changes are seen at the interface between the enterocyte domain and the lamina propria. An ultrastructural study by Olkowski et al. (55) shows that primary modifications occur at the level of basal and lateral domains of the enterocytes. In contrast, the apical domain of enterocytes remains intact even in the face of advanced necrotic changes. This indicates that mucosal necrosis does not result from direct damage to the mucosal epithelium. Rather, the necrotic death of enterocytes is a consequential effect of destroying lamina propria, the extra-cellular matrix, and intercellular junctions. This result is consistent with the study of Olkowski et al. (56), who also found no overt mucosal necrosis typical of NE field cases in a histological examination in C. perfringens-infected chickens. This is very important because the current study aimed to induce a moderate infection, mimicking a subclinical infection rather than the acute form. It should be mentioned here that increased collagenolytic enzyme activity in the mucosal and intestinal tissue environment is an integral component of the pathological process leading to NE (32, 55). However, none of the supplements administered had a remarkable effect on the morphological changes in the gut developed during infection. Perhaps it was too short a time to see changes at the microscopic level because, at the molecular level, treatment with nano-Se and CBD significantly increased the mRNA expression of genes such as junctional adhesion molecule 2 and zonula occludens-1, which are responsible for intestinal integrity (32, 33). In addition, in a previous study, the authors also showed that cannabidiol and nano-selenium in C. perfringens-infected chickens promoted changes in the extracellular activity of bacterial enzymes in the gut, which may indicate increased energy consumption under induced stress conditions (32).

In the current experiment, epigenetic, immunological and oxidative changes as a response to treatment in the blood and jejunum of chickens were investigated. TNF-α is an important pro-inflammatory cytokine that plays a key role in immune responses during infection. It helps initiate and regulate the immune system response by promoting inflammation, cell survival, and elimination of pathogens. However, dysregulation or overproduction of TNF-α may contribute to chronic inflammation (57, 58). In blood, significantly higher TNF-α in the CBD + C. perfringens group was found compared to the nano-Se + C. perfringens and CBD + nano-Se + C. perfringens groups. This may show a specific effect of this compound on the immune system. The increase in TNF-α levels may be related to an increase in the inflammatory response induced by the infection and suggests that the CBD supplement, rather than having an immunosuppressive effect, may have increased the inflammatory response in the context of this infection. Still, it should be noted that no significant difference was found between the control group and the infected group with one or two supplements. CBD is well known for its immunomodulatory effects (59, 60), but there are studies indicating that in the bacterial response, it may also induce an increase in the production of pro-inflammatory cytokines (61, 62). The increased TNF-α levels in the CBD-infected chicken group may be the organism's attempt to counteract the stress stimuli through an enhanced inflammatory response. In this study, TNF-α expression was not altered by the C. perfringens challenge (C vs. C. perfringens group), which agrees with previous studies (63–65). The difference in TNF-α levels between the group of chickens supplemented with CBD alone and the group supplemented with both nano-selenium and CBD may be due to the synergistic effects of the two compounds. Nano-selenium, through its antioxidant and immune response modulating effects, may have reduced the pro-inflammatory effects of CBD alone, explaining the lower TNF-α levels in the group with the combination of these supplements, which is consistent with the results of Sendani et al. (66). On the other hand, the lack of a significant difference between the control group and the infected group with the addition of both nano-selenium and CBD may suggest that the combination of these two compounds has a balanced effect on the immune system, mitigating the excessive inflammatory response caused by the infection, allowing for a more controlled immune response. On the other hand, in the gut, significant differences were shown in the expression of Casp 3, IgY, and % of methylated DNA. Caspase 3 is a key enzyme in the process of programmed cell death (apoptosis). Casp 3 activation is often triggered in response to various stressors, leading to controlled cell death that helps maintain homeostasis and eliminate damaged cells (67). In this study, Casp 3 levels were significantly lower in the control group compared to the C. perfringens, nano-Se + C. perfringens, and CBD + nano-Se groups. This result contrasts the study of Guo et al. (46), who found a lack of significance in Casp 3 levels in C. perfringens-infected chickens. In contrast, this result aligns with the study by Huang et al. (68), which showed that infections can significantly increase caspase 3. However, the increase in this factor in the CBD + nano-Se and nano-Se + C. perfringens groups is not easy to explain. The possible action was that nano-selenium at the dose used in the experiment exhibits a pro-apoptotic effect, perhaps to accelerate the elimination of damaged cells. It is important to note that no significant differences were shown between the control group and the CBD + C. perfringens and CBD + nano-Se + C. perfringens groups, which is consistent with a study by de Fillips et al. (69), which showed that CBD reduces inflammation in patients with LPS-induced colitis and lowers Casp 3 levels. It can also be concluded that the beneficial effect of nano-selenium, in this case, may depend on synergism with cannabidiol, or cannabidiol could have reduced the potential pro-apoptotic effect of nano-selenium. Evaluation of the immunoglobulin Y concentration showed that it was significantly lower in all test groups compared to the control and non-infected group with cannabidiol and nano-selenium. In poultry, IgY is a type of special immunoglobulin that is produced by stimulation of specific antigens in vivo and has a similar function to immunoglobulin G (IgG) in mammals (70). However, very limited information is available on this immunoglobulin and C. perfringens infection in poultry and its concentration dependence on nano-selenium and cannabidiol. Decreased IgY concentrations in chickens with bacterial infections may indicate an impaired immune response, favoring further infection and intestinal barrier damage. This is consistent with a study by Cui et al. (70), who showed reduced levels of IgY mRNA expression in the jejunum after LPS treatment of E. coli in chickens. Contemporary research confirms that IgY supplementation in the context of pathogen control may have a supportive effect. Still, it may sometimes be ineffective, which may explain the lower concentration of this immunoglobulin in the study groups (71, 72). Interestingly, IgY concentration in the control group and the non-infected group with the addition of CBD + nano-Se was at the same level. This indicates that the addition of cannabidiol and nano-Se does not, by themselves, reduce the concentration of IgY in chickens. The % of methylated DNA was significantly higher in the infected group compared to all other study groups, which is consistent with the results of Ognik et al. (11), which showed that C. perfringens infection increased the percentage of methylated DNA in the wall of the ileum in turkeys. DNA methylation is a key process regulating gene expression, which affects many cellular functions, including immune response, cell growth, and stress response. Evidence from in vivo and in silico studies shows that cannabidiol can regulate the activity of enzymes responsible for DNA methylation due to its direct binding to the enzymes and/or regulating their activity through neurotransmitter-mediated signaling (19, 73). In a rodent model of iron-induced neurodegeneration, CBD normalized mitochondrial DNA methylation levels in the hippocampus (73). Toubhans et al. (74) showed that selenium can modulate histone methylation, highlighting its important role in redox biology. Both assumptions are consistent with the current results, as the results in the infected groups with additives and the control group were at similar levels. Given the above, it may be concluded that the lack of significance in the assays of the other parameters indicative of epigenetic and oxidative changes may be related to two main facts: (i) the C. perfringens infection was not sufficient to induce the full changes in the parameters studied, and (ii) the exposure to challenge factor was too short to bring about the aforementioned changes fully.

In the current study, in order to evaluate the relationship between intestinal barrier functions and epigenetic and oxidative changes, a correlation analysis was performed of levels of diamine oxidase and lactic acid in the intestine and levels of the above-mentioned indicators in blood and jejunum. DAO is an intracellular enzyme in intestinal cells that catalyzes the breakdown of histamine in GIT. Its concentration under physiological conditions is relatively high in the intestine and very low in plasma (75). In contrast, lactic acid is the end product of glucose oxidation in aerobic glycolysis and can be synthesized in situ on the intestinal mucosa (76). Increased lactic acid production in the small intestine is a negative phenomenon, as it can lead to acidosis, which results in severe damage to the intestinal epithelium, resulting in excessive intestinal permeability, referred to as “leaky gut” (76). There were no differences between DAO concentrations and blood indices, which is consistent with the ELISA analysis results, as apart from TNF-α, no significant differences were found in the other parameters. In addition, DAO is a marker of intestinal barrier integrity, while epigenetic and oxidative indices may reflect the body's systemic response to various stressors (77). Interestingly, a statistically significant positive correlation was found between LA levels and superoxide dismutase levels. This is a beneficial effect because SOD, as an antioxidant enzyme, plays a key role in protecting cells from damage from free radicals (ROS), and there are reports that LA can affect a number of metabolic and immune processes in the body, including by affecting the redox status through the reaction of lactate dehydrogenase inducing reactive oxygen species and acting as an inhibitor of glucose breakdown (78). In the gut, the current study showed a significant positive correlation between DAO and IgY, and between LA and 8-OHdG. Schade et al. (79) highlight the roles of IgY in neutralizing toxins and pathogens, which helps maintain intestinal health. Increased DAO levels may indicate an intensification of enzymatic activity to protect against inflammation caused by pathogens or damage to the intestinal barrier, leading to activation of the local immune response, manifested by an increase in IgY. On the other hand, the association between LA and 8-OHdG may indicate that increased lactic acid production in the intestines of chickens leads to increased oxidative stress and cellular damage, as manifested by higher levels of 8-OHdG. Valavanidis et al. (80) report that 8-OHdG is one of the predominant forms of free radical-induced oxidative damage and is, therefore, widely used as a biomarker of oxidative stress and carcinogenesis. A negative correlation was also detected between DAO and IgA in the gut. Immunoglobulin A plays an important role in protection against gastrointestinal infections, supports intestinal homeostasis, and maintains the health of the intestinal microbiota (81). This may lead to the conclusion that high DAO activity may be an indicator of a healthy intestinal barrier, where protective mechanisms work effectively and reduce the need for a strong immune response in the form of sIgA. On the other hand, decreased DAO levels may suggest a weakened intestinal barrier, leading to increased production of sIgA to protect against potential pathogens.

Moreover, the results of the correlation analysis of histomorphometry vs. DAO and LA showed a significant negative correlation between villi width in the duodenum and DAO in the blood. DAO is released into the blood from mucosal cells due to damage to the intestinal barrier, leading to increased gastrointestinal permeability (46). This correlation may suggest that a reduction in the width of the intestinal villi may reflect the functional state of the barrier and is associated with damage to the intestinal wall, leading to an increase in intestinal permeability and escape of DAO into the bloodstream. A negative correlation was found between the concentration of LA in the blood and the crypt depth of the duodenum and villi width of the jejunum and ileum. Studies indicate that an increase in the synthesis of lactic acid in the intestines and the accompanying ejection of this compound into the blood can be observed in intestinal hypoxia, causing an increase in the anaerobic breakdown of glucose (82). On the other hand, correlation analysis between intestinal factor concentration and morphometry shows a positive correlation between DAO and villi height to crypt depth ratio and between LA and wall thickness and villi area of the duodenum. A positive correlation was also found in the ileum between villi width and crypt depth and DAO, as well as between villi width and LA. The correlation with DAO may be related to improved intestinal morphology and reduced intestinal barrier dysfunction and inflammation. The positive correlation between LA and parameters in the duodenum and ileum suggests that the presence of lactic acid in moderate amounts may support the healthy development and maintenance of intestinal villi, increasing intestinal absorptive capacity, and according to a study by Okada et al. (82), lactate may stimulate the proliferation of enterocytes and intestinal epithelial cells. The current study also indicated a negative correlation between DAO in the ileum and the villi height to crypt ratio in the ileum. This result is surprising and contrasts with the positive effect of DAO observed on parameters such as villi width and crypt depth. A high DAO concentration in the ileum may signal that inflammatory processes and intestinal damage are advanced enough to negatively affect the regenerative capacity and functionality of this intestinal segment, leading to a lower VH/CD ratio.

The current study evaluated the concentration of hemoglobin (Hb) in the blood of chickens. Hemoglobin is a key protein responsible for oxygen transport in chickens, and its function is fundamental to metabolic processes and homeostasis. In the context of poultry health research, measuring the concentration of hemoglobin in the blood can be an important indicator of the health status of animals, their ability to transport oxygen efficiently, as well as the body's response to various interventions, including infections or dietary supplementation. Ognik et al. (83) demonstrated that turkey hens with poorer health status were characterized by reduced hemoglobin levels, which may partly explain the significant differences between the infected group without and with nano-selenium or both supplements. The current results showed that hemoglobin concentration was significantly higher in the infected and supplemented groups with nano-selenium or the two nutritional supplements and in the uninfected group with CBD and nano-selenium, compared to the positive and negative control groups, which may suggest the beneficial effects of these nutritional supplements in increasing the blood's oxygen transport capacity and supporting erythrocyte production. Interestingly, all groups with nano-selenium supplementation had higher hemoglobin concentrations. This result is consistent with a study by Arain et al. (84), who showed increased Hb concentration in selenium-supplemented goats. However, Boostani et al. (85), who studied the effects of organic, inorganic, and nano-selenium on blood in broiler chickens exposed to oxidative stress, showed no significant differences in hemoglobin concentration between the study groups. The increased levels of the erythrocytic system markers may also be explained by the condensation of blood due to body dehydration resulting from diarrhea. However, in the current study, a subclinical infection was induced, which was not associated with clinical symptoms, such as diarrhea, which may also partly explain the lack of differences between the negative and positive control groups. On the other hand, there were no significant differences between the control group and the infected group with the addition of CBD alone. Data in the literature are quite contradictory about the effect of cannabidiol on parameters such as hemoglobin concentration. This result is consistent with several previous studies that also showed no significant differences in hemoglobin concentration and CBD supplementation in rats and goats (86, 87). However, previous studies in guinea pigs and rats demonstrated that hemoglobin concentration showed a consistent but non-significant decrease (88) or was significantly reduced (89) in the CBD groups compared to the control group. Therefore, further study is needed to determine the effects of CBD and nano-selenium on erythrocytes and hemoglobin production in chickens.

Finally, with reference to the results above, it is also important to present the performance response to treatment that we examined in our previous work (32). Body weight gain (BWG) in the experiments on days 9–23 and 24–35 was not significantly different between groups, while BWG calculated for the entire experimental period was significantly lower in the C. perfringens-infected group and in the CBD-infected group compared to the control group. Feed conversion ratio (FCR) was worsened in all experimental groups compared to the control group during the feeding period of days 9–23 but was not affected during other feeding periods. In contrast, feed intake was similar in all treatment groups during all periods of the study. This suggests that the response of chickens to infection is more complex than a simple increase or decrease in feed consumption and that mechanisms controlling feed utilization are implicated.

The main limitation of the present study is legislation. Currently, cannabidiol is not authorized for use as an animal feed additive in EU Member States. According to EU regulations, Cannabis sativa plants can be legally registered for cultivation when their tetrahydrocannabinol content is less than 0.2%. According to Commission Regulation (EU) 2022/1104 of July 1, 2022, amending Regulation (EU) No. 68/2013 on the catalog of feed materials, and with the Open Community Catalog of Feed Materials, it is permitted to use in animal feed such ingredients as hemp seed, hemp seed cake, hemp seed oil, hemp flour and hemp fiber from Cannabis sativa L. with a tetrahydrocannabinol content of less than 0.2% based on the method of quantification established by Regulation (EU) No. 639/2014. The European Food Safety Authority (EFSA) must approve all feed supplements. Cannabidiol is also regulated under novel food legislation. Therefore, due to a great discussion regarding application of Cannabis plant, future research, such as that showing the high potential of CBD, is needed to bring cannabidiol into legal use.

In conclusion, the current study provides evidence that cannabidiol and nano-selenium mediate epigenetic and oxidative DNA changes and affect intestinal development and functionality in broiler chickens at an early stage of infection with C. perfringens. Histomorphometry results indicate that cannabidiol and nano-selenium, in combination under physiological or pathophysiological conditions, can manifest a synergetic effect on robust intestinal barrier functions. Moreover, it was also found that the use of CBD or nano-Se alone or combined in the diet did not show an adverse effect or negative qualitative changes on the gut structures, as demonstrated by a study of ultrastructures and a histopathological examination. Moreover, it was also shown that C. perfringens infection did not cause necrotic enteritis, and the changes were moderate and primarily involved damage to the intestinal epithelium. This indicates that mucosal necrosis is not due to direct damage to the mucosal epithelium. Rather, necrotic death of enterocytes is a consequence of the destruction of the lamina propria, extracellular matrix, and intercellular junctions. On the other hand, a study of markers of epigenetic and oxidative DNA changes, such as TNF-α, Casp 3, IgY, or % of methylated DNA, found that the combination of these two compounds has a balanced effect on the immune system, mitigating the excessive inflammatory response caused by the infection, allowing for a more controlled immune response. This also indicates that the addition of cannabidiol and nano-Se does not reduce the concentration of IgY in chickens. In addition, the current study showed a significant correlation between DAO or LA and SOD, IgY, IgA, 8-OHdG, or histomorphometric parameters. Evaluation of hemoglobin levels demonstrates that all groups with nano-selenium supplementation had higher concentrations, and that the CBD-infected group did not differ from the control group. This study also found new mechanisms of action for cannabidiol and nano-selenium.