Necrotic enteritis (NE), caused by the anaerobic bacterium Clostridium perfringens (or C. perfringens), is responsible for major economic losses in the poultry industry due to impaired bird performance and increased morbidity and mortality rates [1]. Due to the ban on the preventive use of antibiotics and antibiotic growth promoters in the EU (Regulation (EC) No. 1831/2003), health problems have arisen in broiler chicken flocks, including an outbreak of Clostridium perfringens infections. The acute form of the disease leads to increased mortality in broiler flocks [2]. In the subclinical form, there is damage to the intestinal mucosa caused by C. perfringens, leading to reduced digestion and absorption and consequently to lower body weight gain and increased feed conversion ratio. Importantly, C. perfringens in poultry poses a risk of transmission to humans through the food chain [3].

In recent years, the poultry industry has made significant progress in breeding broiler chickens, resulting in shorter fattening times and faster weight gain. However, the rapid growth of birds results in histological and biochemical modifications of the muscle tissue, the occurrence of myopathies, and ischemic and degenerative changes in the muscles [4]. Histopathological lesions in myopathies include necrosis and atrophy of fibers, split fibers, giant fibers and connective tissue hyperplasia [5].

With the aim of minimizing the consumption of pharmaceuticals, possibilities have been sought to replace drugs with health-promoting preparations that are not medicines but exert similar effects. One bioactive substance that has been looked at in great detail is cannabidiol (CBD). Research on CBD has discovered a new, so-called endocannabinoid system, which regulates not only brain function but also the immune system. Studies examining the effects of cannabinoid-based drugs on immunity have shown that many cellular and cytokine mechanisms are suppressed by these agents [6]. This leads to the hypothesis that the aforementioned drugs may be of value in the treatment of chronic inflammatory diseases [7]. The mechanism underlying CBD action has not yet been fully elucidated, but it may represent biological activity in the regulation of inflammation through close affinity to the processes mediating necrosis and intestinal inflammation in chickens. Since NE is a serious problem in the poultry industry, CBD could be an interesting solution in prevention strategies. Therefore, additives that can help to improve birds’ health while maintaining performance indicators are currently of great interest [8]. While it is overall consensus that the abuse of the most abundant bioactive substance found in Cannabis plants—tetrahydrocannabinol—is usually associated with toxicity to the host, current findings indicate that CBD, the second active compound in cannabis plants in terms of abundance (but without psychedelic effects on the host), is one of the most promising cannabinoids [9,10]. On the other hand, reports indicating CBD toxicity based on preclinical and clinical studies, as well as experiments involving animals and in vitro models, also exist [11]. More recently, an increasing body of evidence obtained using different models has suggested that CBD exposure (depending on dose) may affect the mitochondrial respiration efficiency of immune cells as well as mitochondrial fission and fusion, which are of key importance during response to high metabolic stress [12].

Selenium (Se) is another bioactive substance that has caught the attention of poultry producers. Selenium has been recognized as an essential trace element to animals and humans that is actively involved in oxidative stress resistance, reproductive performance and immune function [13]. Selenium is an important antioxidant mineral in animals and is known to affect feather production and maintain cellular integrity in bird tissues. Various Se forms are available for supplementation in animal and poultry feeds, e.g., inorganic sodium selenate (Na₂SeO₄) and sodium selenite (Na₂SeO₃) [14,15]. The organic form, such as that of selenized yeast, is similar to Se compounds found in cereals and feeds. However, selenium concentrations in feed ingredients vary widely depending on the plant species and, in particular, the Se status of the soil. Therefore, Se supplementation is necessary in poultry nutrition to provide a safety margin against its deficiency and to maintain production efficiency [16]. With the development of nanotechnology, the effect of particles in “nano” form has begun to be studied. Nanoscale selenium is of great interest as a food additive, especially for individuals with selenium deficiency, but also as a therapeutic agent without significant side effects in medicine. The nano form of selenium has attracted even more attention due to its high bioavailability and low toxicity compared with inorganic and organic forms, where inorganic compounds are more toxic than organic ones [17]. Studies have demonstrated the antimicrobial and antifungal activity of nano-Se [18]. In the context of potential bioactivity/toxicity of Se, it was evidenced, using Lilly Laboratories culture-Porcine Kidney 1 cell lines, that Se produced significant protection against Cd-induced apoptosis by mediating oxidative stress, which in turn had ameliorative effects on mitochondrial dysfunction [19]. In another study, it was shown that Se was effective in the prevention of mitochondrial damage caused by Adriamycin in Sprague–Dawley rats [20]. Eventually, Se nanoparticles were used to investigate their potential in malignant ascites treatment using a mouse model. It was revealed that Se could influence mitochondrial function and induce cell apoptosis [21]. Taken together, the results suggests that both molecules (CBD and nano-Se) have a close affinity to mitochondrial function; thus, based on this interaction, their beneficial/toxic effects on the host can be assessed. Nano-Se can be obtained via chemical synthesis, physical methods or even in a biological way (green synthesis), using microorganisms or plant extracts, which indeed determine their biological action to a high extent [22]. Among others, nano-Se obtained via chemical synthesis seems to be the most suitable component for nutritional applications, because in this form, it is positively charged, biocompatible, non-immunogenic, nontoxic, pH sensitive and biodegradable [22].

To date, research has focused on the effects of cannabidiol and nano-selenium on the improvement of intestinal barrier function and bacterial enzyme activity in chickens. This study investigated the bioactive properties of CBD and nano-Se in broilers with potential beneficial effects on gut health and function. Konieczka et al. [8] found that CBD and nano-Se could modulate the response of chickens to C. perfringens infection, which in turn may provide time for effective intervention. The beneficial effects of both agents on host physiology were manifested in supporting intestinal barrier function through increased expression of genes that control intestinal integrity (tight junction proteins; TJPs). CBD and nano-Se promoted changes in extracellular bacterial enzyme activity toward increased energy uptake in challenged chickens but showed no counter-effects on mediating the host response to infection. Recently, an increasing number of studies have indicated a close relationship between intestine condition and processes determining the physicochemical properties of meat in poultry [23,24,25]. The metabolism of key elements, including peptides, fatty acids and amino acids, in the host depends to a high extend on gut barrier function [26]. As reported, both CBD and nano-Se manifest direct and indirect actions on the same mechanisms of the gut physiological status, particularly the modulation of gut bacterial composition and activities; thus, we speculated that both CBD and nano-Se could be more effective in supporting gut integrity and should thus affect meat properties, especially in challenged birds, as this condition leads to fermentation disturbance, as proven in various models [27,28,29]. For instance, chickens challenged with C. perfringens were shown to have a higher abundance of several foodborne pathogens in the gut, including C. jejuni, E. coli and L. monocylogenes; this treatment also affected the transcript levels of many genes regulating host metabolism [30]. In our previous study [31], in a turkey model, we found that challenging birds with C. perfringens had a significant effect on the sarcoplasmic protein profile of the breast muscle, indicating a strong association between C. perfringens infection and the function of glycolytic enzymes in turkeys, which could lead to significant consequences in cell metabolism [32]. In line with this, [33] showed a possible linkage between reduced vessel density and ultrastructural alterations in chicken breast muscle in early-stage wooden breast myopathy development, and the enlargement of the sarcoplasmic reticulum and greater severity of mitochondrial morphological alterations due to osmotic imbalance. The authors of [34] reported that skeletal muscles, including breast muscle, in chickens and turkeys can be affected by C. perfringens due to immune system failure caused by different stressors. In another report [35], it was shown that C. perfringens impairs the muscle regeneration process due to induced necrosis via the disturbance of collagen deposition in injured tissue, alternations in capillary vessels and nerves in infected muscle, as well as changes in the transcript levels of gene mediators of the inflammatory response and fibrosis in infected muscle. Both CBD and nano-Se may have the potential to interact with all of the mentioned pathogenesis factors.

However, the most significant correlation between host intestinal response and meat quality in chickens was found in our recent study, in which dietary CBD in C. perfringens-infected chickens reduced meat volatile compound levels correlated with bacterial activity [36]. In the latter study, we showed that the only group of birds with a different VOC profile (including spoilage markers) was the C. perfringens-challenged group, indicating a strong impact of C. perfringens infection on meat properties. This could be attributed to the fact that C. perfringens affects lipid metabolism by downregulating the expression of fatty acid catabolism-related genes, including peroxisome proliferator-activated receptor-alpha, carnitine palmitoyl transferase 1 and acyl CoA oxidase 1 [37], which play important roles in lipid metabolism and thus strongly contribute to meat sensory properties. Eventually, Bień et al. [38] demonstrated that using different forms of selenium, including the nanosized form, in chicken diet was effective in the modulation of the fatty acid profile, and lipid and enzymatic indices of fatty acid metabolism in breast muscle as well as in the liver in birds.

Corresponding to the above reported findings, the aim of this study was to verify whether the use of CBD and Se in the form of nanoparticles in the diet of broiler chickens subjected to necrotic enteritis caused by Clostridium perfringens affected the degree of blood supply to the superficial breast muscle and the extent of pathological changes. At the same time, the study verified whether the use of the tested substances in the diet of chickens exerted toxic effects on selected markers of intestinal barrier function and mitochondria.