**3. Selenium Supplementation**

Selenium can affect the immune and antioxidant systems of animals through GPx and selenoproteins with various biological functions. Dietary Se can be supplemented in animals from two important sources: organic and inorganic. Organic Se is more bioavailable than inorganic Se [Table 2]. In recent years, the application of Se in nanoforms (Table 2) has attracted more attention, mainly due to the possibility of using Se in a zero-oxidation state (Se0), which presents low toxicity and better bioavailability compared to other oxidation states [49]. Moreover, supplementation of NPs can improve the delivery and absorption of the trace elements in animals and humans while causing no environmental damage.

#### *3.1. Selenium Absorption and Bioavailability*

The efficiency of Se absorption is affected by the form of dietary selenium and differs between ruminants and non-ruminants [50]. It has been found that organic forms of Se are actively absorbed in the intestinal tract via an amino acid transport mechanism, unlike inorganic Se, which is absorbed by a simple diffusion process [51]. Selenomethionine (SeMet) is essential for humans and animals and cannot be synthesized in the body [52]. Selenocysteine (SeCys) is a structural component (cofactor) of selenoenzymes; therefore, it is important for their catalytic activity. These selenoenzymes play a key role in redox homeostasis in mammals; their active component is SeCys which is synthesized in the body de novo [53]. GPx and other selenoenzymes are the major Se-containing antioxidants in the body that help to neutralize ROS [54]. The expression of selenoproteins is specific to various tissues and depends on Se availability in feed [55]. Selenoproteins help animals resist oxidative stress, which can be caused by, heat, decreased productive performance, and various diseases (e.g., mastitis and intramammary infections in dairy cattle). The concentration of GPx is typically analyzed in blood plasma to assess selenium deficiency and to evaluate antioxidant status or oxidative stress [56]. In the case of Se deficiency, GPx concentration tends to be low. When oxidative stress is high and Se contention in feed is limited, L-SeMet is released from the proteins due to the protein turnover and provides Se for the production of selenoproteins. SeCys, the same as inorganic Se, does not play a significant role as a nutritious Se source [57]. SeMet represents the storage form of Se in animal and human tissues [58]. Dietary selenomethionine can be incorporated into muscle protein in place of methionine and become a rich store of selenium; it represents a highly available substrate for many proteins and can substitute methionine in the protein structure [58,59]. All dietary Se sources, except for organic L-SeMet present in SYs or naturally derived in plants, are primarily metabolically transformed to selenide in the liver and then used for SeCys synthesis. Non-ruminant animals are not able to

synthesize selenomethionine from inorganic forms of selenium [60] but can convert it to another essential amino acid—selenocysteine [16]. The mineral (inorganic) forms of Se are reported to have some disadvantages such as relatively high toxicity, low transfer to animal products (milk, eggs, and meat), and the inability to facilitate Se storage in the body [16,61]. In ruminants, microbial digestion by ruminal microorganisms (RMOs) proceeds before digestion in the abomasum and small intestine [62]. The ruminal microbes reduce most of supplemented inorganic Se to unabsorbable selenium sources (selenide and elemental selenium), thereby decreasing Se bioavailability to 20–25% [51]. The absorption of inorganic Se in the form of sodium selenite in the small intestines of monogastric animals and poultry is approximately 80%, while in ruminants, this range is only 29%, and for organic Se in monogastric species and poultry, it is greater than 90% [19,63]. This difference appears to be the result of as reduction in dietary Se forms by microbes in the rumen. The organic forms of selenium naturally occur in plant-based feedstuffs, and selenized yeas<sup>t</sup> (SY) are selenoaminoacids (selenomethionine and selenocysteine), which contain a selenol group in place of the sulfur-containing thiol group [18]. SeMet is considered a metabolically effective organic Se and is traditionally supplemented in the form of selenized yeas<sup>t</sup> (SY), which has been grown in a high selenium medium. In animals' intestinal tracts, proteins from SYs are broken down into small peptides and then amino acids. Thus, SeMet can be absorbed in the intestine the same way as amino acid methionine and build selenium deposition in the body. However, the binding of selenium by microbial cells highly depends on the concentration of Se in the cultivation medium and cultivation conditions [64]. Selenium can be found in all cells and tissues, but the level of Se and its distribution in the body tissues is influenced by the dietary Se form and intake. After oral selenium supplementation (organic SY and inorganic sodium selenite), the Se uptake in sheep was only 34%, whereas, in pigs, it was 85% [51]. Some studies demonstrated increased Se concentrations in meat after SY dietary inclusion compared to inorganic Se [65,66]. In their study, Paiva et al. [66] demonstrated the increase in muscle Se in lambs supplemented with organic Se (0.2, 0.4, and 0.8 mg/kg DM) compared to the inorganic Se form. Selenium content in the muscle was higher with more Se inclusion in the diet linearly. Organic Se sources demonstrated a higher capacity to accumulate Se in muscles than inorganic Se. Hepatic GPx activity was found to be higher in animals supplemented with sodium selenite (SS), which refers to the metabolic pathway of inorganic Se. Sodium selenite biotransformed to selenide for further GPx synthesis more quickly. Similar results were obtained by Steen et al. [67]. Se concentrations in the muscles and blood of lambs receiving organic Se were significantly higher compared to the inorganic group. Improved beef meat quality parameters (e.g., color stability) and increased Se concentration in the muscles of Charolais bulls supplemented with organic Se (0.2 mg/kg DM) for 60 days were observed by Grossi et al. [57]. The selenium concentration in the meat of Nellore cattle was higher in animals supplemented with organic Se (0.3, 0.9, and 2.7 mg/kg DM) compared to organic Se [68]. Results obtained by Hall et al. [69] and Galbraith et al. [70] also showed better bioavailability of the organic forms of Se compared to the inorganic form. An increase in Se in the blood serum and GPx activity in lambs was demonstrated after organic and inorganic Se (0.15 mg/kg DM) supplementation; no significant difference between the organic and inorganic Se forms was observed [71]. Various species of ruminant animals (i.e., dairy cows, beef cattle, calves, and lambs) were supplemented with the ten times maximum permitted Se (in the organic form of SY) dosage of 0.568 mg/kg DM, and there was no adverse health effect observed. Moreover, it was observed to increase Se in the blood and milk samples [72]. The digestive system of animals is more adapted to the organic form of Se, which naturally occurs in feeds, and it has better assimilation [73]. Organic Se in high doses can also be toxic, but SeMet does not produce free radicals when reacting with glutathione. However, the molecular mechanism of selenium toxicosis is not well understood and indeed needs further investigation. Selenium supplementation (SY and SS at the dose 0.4 mg/kg DM) increased GPx activity, regardless of the selenium source [74]. Some other studies also did not confirm the greater biological effects of organic Se [75–77].

Postpartum and pregnan<sup>t</sup> animals are generally more susceptible to Se deficiency and often require Se supplementation due to oxidative stress, which can be caused by pregnancy itself. Dietary organic Se supplementation in postpartum mice enhanced Se deposition in the liver, blood, and mammary gland, in addition to an increase in GPx activity [78]. In dairy cattle, oxidative stress during pregnancy and postpartum can increase intramammary diseases and mastitis cases. Long-term Se deficiency may also lead to thyroid-related diseases such as autoimmune thyroiditis [25]. Se supplementation in pregnan<sup>t</sup> and lactating ewes showed a significant increase in the Se concentration in the blood and an increase in thyroid hormones compared to the control group [79]. These results sugges<sup>t</sup> that organic Se supplementation can improve the antioxidant status of pregnan<sup>t</sup> animals. More efficient transfer of Se from organic Se dietary sources to dairy cattle milk in comparison with inorganic Se was demonstrated [80–82]. Due to the fact of these controversial results, additional investigations in this research area are strongly recommended.

Many studies have described the bioavailability of different chemical forms of selenium [51,56,63,64], e.g., diets enriched with the organic Se increased Se concentrations in animal tissues compared to animals supplemented with inorganic Se.

The selenium-enriched microalgae, *Chlorella vulgaris*, was used as an organic source of dietary Se [83,84]. The effects of the supplementation of sodium selenite and selenized microalgae biomass on fish mortality, growth, and the accumulation of Se in the muscles and liver were observed in common barbel [83]. The results showed more accumulation and bioavailability of Se in muscles and liver in barbel supplemented with Se-enriched microalgae than animals supplemented with inorganic sodium selenite. Marounek et al. [85] also observed better accumulation of Se in tissues of rabbits supplemented with organic Se yeasts and Se microalgae. Similar results were obtained by Hassan et al. [86,87]. Reports on the concentration of SeMet in *Chlorella vulgaris* are controversial. De Alcantara et al. [88] found that 70% of intracellular Se in *Chlorella vulgaris* is in the form SeMet.

In contrast, Neuman et al. [89] found 24–30% of SeMet from the total amount Se accumulated in algae cells, but the concentration of SeCys in algae cells was higher (48.76%). Supplementation in animals with Se-enriched microalgae can benefit from the presence of antioxidants, vitamins, and other biologically active compounds. On the other hand, microalgae cultivation is costly, and the accumulation of intracellular SeMet can be various depending on the cultivation method and conditions.

#### *3.2. Selenium and Vitamin E*

The most studied beneficial health effect of Se has been studied in connection with vitamin E. Vitamin E and Se have interrelated functions in animals and human organisms. Inadequate amounts in the diet leads to similar adverse effects. The synergetic interaction between Se and vitamin E can enhance GPx synthesis, an important part of the antioxidant pathway in the body. Selenium deficiency is often characterized by low concentrations of both Se and vitamin E [90]. Thus, optimum Se and vitamin E levels are necessary to minimize the oxidative damage of cells and tissues in the body [12]. Supplementing dairy cows with low doses of both vitamin E and Se (Se injection 1 mg/kg bw; vitamin E 32 g/d) reduced the duration of clinical mastitis symptoms by 62%, in cows supplemented only with vitamin E mastitis, the duration of symptoms was reduced by 44%, and in cows supplemented with Se by 46% [91]. These results demonstrate the beneficial interaction of Se–vitamin E (SeE) dietary inclusion in reducing the duration of clinical mastitis, which can improve dairy cows' production. Zahrazadeh et al. [92] evaluated the influence of SeE injection (0.5 mg of Se in the form of sodium selenite; 56 IU of vitamin E) on body condition, lactation performance, and oxidative status in dairy cows. SeE injections showed a beneficial effect on body score, antioxidative parameters, and lactating performance in Holstein cows. The mechanism of the beneficial effect of vitamin E and SeE supplementation on mastitis is not fully understood, and this needs further research. Hogan et al. [93] observed that vitamin E deficiency was associated with a reduction in neutrophils that had

bactericidal activity and increased mastitis. Ali et al. [94] reported that SeE injections (75 mg of vitamin E per day per animal; 2800 mg of Se) improved the reproductive performance of lambs compared to vitamin E supplementation alone. In another study, Se supplemented alone had a better effect on the semen quality of boars than the group supplemented with SeE [95]. Kappel et al. [96] did not observe the improvement in reproductive efficacy in cows injected with SeE (680 IU of vitamin E and 50 mg of Se in the form of sodium selenite). The daily nutritional requirement of vitamin E for adult cattle recommended by the National Research Council (NRC) is 15–60 international units (IUs). The daily nutritional need for nursing calves ranges between 40 and 60 IUs [34]. Supplementation of 0.3 mg/kg bw of organic Se and 100 mg/kg of vitamin E improved the immune status of broiler chickens, but there was no difference found compared to the animal group supplemented with Se alone [97]. In recent years, only a few studies have been related to the toxic effects of vitamin E supplementation. In general, vitamin E is considered to be one of the least toxic of the vitamins [37]. A study on higher doses (500, 1000, and 2000 mg/kg bw) of vitamin E oral supplementation over 30 days in albino rats showed a negative effect on the liver and kidneys. Still, no deaths were recorded [98]. Hale et al. [99] reported on the toxicity of intravenous vitamin E (33.5 mg/kg bw) supplementation in neonatal piglets. Rapid intravenous injections of vitamin E resulted in sepsis and abnormal pulmonary function. Further studies are needed to investigate the possible undesirable effect of vitamin E supplementation and to determine appropriate dosage levels.

#### **4. Selenium Nanoparticles in Animal Nutrition**

An appropriate animal diet and living environment play a key role in animal health and performance. Thus, optimizing these factors is important for increasing rearing efficiency, which can positively determine the quality of production of animal origin. Over the last decade, nanotechnology has received the attention of many researchers due to its promising agricultural and food applications. Nanotechnology provides new "intelligent" solutions in animal nutrient delivery and health protection, and, indeed, it has the large potential to improve animal production systems [100]. This interest is mainly caused by the unique physicochemical properties of nanoparticles (NPs), which refers to their small size (1–100 nm), high stability, hydrophobicity, and large surface area. NPs' hydrophobicity is important for good dispersion in water or serum and is also required to enhance their interaction with cell membranes [101]. The NPs' size affects the cellular intake and allows them to easily pass through the stomach wall and diffuse into body cells quicker than common elements with larger particle sizes. The in vitro absorption of NPs with a diameter of 0.1 μm was found to be higher than 1 and 10 μm NPs [102]. The thickness of gastric mucus layers (total mucus), which continuously cover the gastrointestinal tract's (GIT) surface, varies from 200 μm in the small intestine to 480–800 μm in the large intestine [14] and could allow the transport of NPs through the layer. According to Corbo et al. [103], NPs, especially nanominerals (e.g., Se and Zn), have a higher surface-area-to-volume ratio, providing more surface area for contact with the mucosal tissues and cells. Better absorption of NPs into the mucosal surface increases the particle residence time in the GIT. When a nanomineral is introduced into a biological medium, such as blood or mucus, proteins adsorb on its surface, giving it a unique "biological identity", a so-called protein corona, which can have an impact on the NPs' distribution as well as their potential toxicity [104]. Nanoparticles have been used in animal nutrition for their antibacterial, antifungal, and antioxidant properties as well as probiotics and to maintain general animal performance and health. The antimicrobial activities of metallic NPs (e.g., ZnO, CuO, and AgNPs) and SeNPs have been demonstrated by different researchers [105–108].

Selenium nanoparticles (SeNPs) are nano-sized (generally <60 nm in diameter) elemental selenium particles with excellent nano-properties [109]. For the NPs' synthesis, there are two main strategies used: bottom-up (including chemical vapor deposition, hydrothermal and solvothermal methods, chemical reduction, and green synthesis) and top-down (including mechanical milling, laser ablation, etching, sputtering, and electro-

explosion). The top-down strategy involves the mechanical breaking down of the bulk material into nanostructured materials. In contrast, the bottom-up method uses chemical reactions to break bulk into several parts to form NPs [110]. Methods of NPs synthesis can also be divided into physical, chemical, and biological (the so-called "green way" or "green synthesis"). The chemical methods of nanoparticle synthesis are the most common approaches commercially employed in various areas of NP applications. Concurrently, plenty of research indicates a potential environmental threat of nanotechnology related to NP toxicity [111–115]. The chemical approach to NP synthesis is related to the use of toxic chemicals, which are hazardous to humans and the environment [116]. Designing NPs via a green route using biological and eco-friendly materials reduces the negative environmental impact [117].

Various studies have investigated the possibilities of using selenium nanoparticles as a new source of selenium (Table 3). For instance, sodium selenite NPs coated with methacrylate polymers were orally supplemented with ruminants, improving selenium absorption [62]. Shi et al. [118] stated that dietary nano-sized Se improved Se content in the blood and tissues and enhanced ruminal fermentation and feed utilization in sheep, which were fed a basal diet supplemented with 0.3, 3, and 6 g/kg DM of nano-Se. Kojouri et al. [119] reported the positive effect of dietary SeNPs inclusion (0.1 mg/kg DM for 60 days) on the antioxidant activity and weight gain of young lambs. In another experiment, the inclusion of 1 mg/kg DM of nano-sized Se into sheep's diet exhibited a better antioxidative effect after 20–30 days of supplementation [120]. Xun et al. [121] also reported enhanced rumen fermentation and feed conversion efficiency in sheep supplemented with 4 mg/kg DM of nano-sized Se compared with selenium yeas<sup>t</sup> (SY). In another experiment, supplementation with 0.5 mg/kg DM of nano-sized Se improved hair follicle development and promoted growth in Cashmere goats [122]. Experiments with nano-Se inclusion in broiler chicken diets conducted by Gangadoo et al. [48,123] demonstrated improved gu<sup>t</sup> health and general animal performance; the best results in both experiments were obtained with an SeNP supplementation of 0.9 mg/kg DM with no toxic effect occurring. Previous studies have demonstrated the benefits of using SeNPs in broiler feed, with increased absorption and diffusion of material into organs and tissues, increased antioxidant capacity, and meat quality.

In contrast, Wang et al. [124] did not observe any beneficial effect of SeNP supplementation in terms of enhancing the oxidative status in broilers, but Se improved the survival rates. Gulyas et al. [125] reported changes in the proteome profile in chickens after SeNPs supplementation. These results could be related to the specific patented method of NP preparation used in this study. Several studies reported improvements in the growth performance [126–128], intestinal health [129,130], and antioxidant status [131] of aquatic animals supplemented with SeNPs. Se supplementation alleviated the antioxidant balance and enhanced kidneys cells' resistance to oxidative damage in grass carp [132]. In another study, SeNP supplementation improved intestinal health, feed utilization, and growth performance in Nile tilapia [130]. The enhancement of the growth performance and feed efficiency after SeNP supplementation (0.4–0.8 mg) in Nile tilapia was also observed by Ibrahim et al. [133]. Markedly, the nanoform of Se can enhance growth performance in fish. The recommended dosage of SeNP dietary inclusion ranges from 0.15 to 4 mg/kg depending on the fish species [134].

**Table 3.** Effect of SeNP supplementation on animal health and performance.



**Table 3.** *Cont.*

