1. IntroductionDomestic Muscovy ducks (Cairina moschata) are favored in many countries for their high meat yield, distinct flavor, and low-calorie content. Commercial duck production is associated with various stresses that reduce growing ducklings’ productive and reproductive performance; most of these stresses are associated with oxidative stress [1]. Newly hatched ducklings require a strong antioxidant defense mechanism, with an abundance of polyunsaturated fatty acids in the lipid part of their tissues. Their bodies are constantly attacked by free radicals, produced as a natural byproduct of regular metabolic activity and as part of the immune system’s defense mechanism against invading microorganisms [2]. Infectious disorders and/or diseases are the most common forms of stress in poultry production, with phagocytic immune cells producing free radicals in the process of combating pathogens. The sources of potential stress vary for every farm; nevertheless, overproduction of free radicals and the critical necessity for antioxidant protection sources are common concerns on all farms [3]. The viability of chicks has long been recognized as a crucial determinant in the poultry industry’s profitability. Oxidative stress is considered to be a critical molecular process underlying cell damage [4]. Oxidative stress produces free radicals, highly reactive unstable species capable of causing cell death and damaging a wide range of biologically significant components, including proteins, DNA, carbohydrates, lipids, and structural tissues [4,5].Supplementing the chick feed with a natural supply of antioxidants is a useful nutritional tool for dealing with various challenges encountered in poultry production [1]. Diets are widely established for their importance in maintaining animal health, reproductive and productive performance in poultry farms. Natural antioxidants are more important than other dietary components in the commercial chicken industry for maintaining immunological competence, high growth levels, and reproduction. Understanding the role of antioxidants in reducing the harmful effects of free radicals and toxic metabolites in animals underpins this principle [3]. The antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) make up the newly hatched chick’s antioxidant system. As it catalyzes the conversion of H2O2 into water and oxygen, CAT is a crucial antioxidant enzyme.Similarly, glutathione peroxidase acts as a co-factor in the catalysis of the conversion of H2O2 into non-toxic molecules and aids in preventing lipid peroxidation [6]. Two types of SOD were detected in the chicken liver; Mn-SOD was localized in the mitochondria, while Cu and Zn-SOD were found in the cytosol, but nuclear genes code both. In addition, superoxide dismutase and catalase are essential components of the cell’s enzymatic defense. Vitamin E, selenium, carotenoids, phytochemicals, and silymarin are all examples of dietary antioxidants that can boost SOD and CAT expression [6,7]. Upregulation of defensive molecules (CAT, reduced glutathione (GSH), Cu/Zn-SOD, and Mn-SOD) expression in response to stress is a helpful tool for stress management as an adaptive mechanism to reduce reactive oxygen species (ROS) production [1].Silybum marianum L. (SM), also known as milk thistle, is a well-known herbal medicinal plant used for chronic liver diseases. Silymarin is the active component present in fruits (seeds) of this plant containing various flavonolignans (Taxifolin, Silybin A, Silybin B, Isosilybin A + B, Silychristin, and Silydianin). The antioxidant properties of SM are thought to be responsible for its beneficial activities [8,9]. With the established effect of SM on antioxidant parameters, many previous studies focused on the influence of components in SM fruits on the growth performance, oxidative condition, and health status of broiler rabbits, ducks, and chickens [9,10]. In all vertebrates, the pituitary gland produces GH; it aids in various differentiating processes across multiple target tissues, including functional growth, development, and maturation [11]. Differential expression of the GH gene in Muscovy ducks and fish has already been proposed as a molecular predictor of growth performance, nutritional status, maturity, and hormonal activity [12,13,14]. In this regard, the objective of the present study was to assess the protective and enhancing effect of dietary SMS supplementation at different levels (0, 4, 8, and 12 g kg−1 diet) on growth performance, mortality rate, and various physiological responses of Muscovy ducklings during the brooding period. GH, CAT, SOD1, and SOD2 gene expression in the pituitary and liver tissues of Muscovy ducklings fed diets containing different SMS levels was evaluated using qRT-PCR at the end of the experiment. Furthermore, a comet assay was used to investigate the favorable effect of varying SMS levels on decreasing nuclear DNA fragmentation. 4. DiscussionBecause of the bioactive ingredients, such as phenolics, flavonoids, and pigments, dietary supplementation with natural herbs or their extracts is becoming the most useful and practical way to improve animal feeding for their various benefits, including growth promotion, antioxidant quality, cell energy, and appetite stimulation, as well as showing immunostimulants [9,10]. Dietary supplementation with 8 or 12 g kg−1 SMS diet positively affected Muscovy ducklings’ growth during the brooding period in the present study. When compared to the group of birds fed diets supplemented with the low dose (4 g kg−1 diet) and the control group, the birds fed diets supplemented with those two SMS doses showed substantial (p35]. Similarly, [14] found that fish fed a diet supplemented with 7.5 and 10 g kg−1 SMS diet recorded the highest FBW, WG, SGR, PER, APU, and the best FCR values compared to the control group. The study of [36] in Cumene Hydroperoxide (CH)-challenged ducks revealed that dietary supplements with 200 mg kg−1 SMS increased protein concentration, health status and improved the absorption capacity of the intestines. Alhidary et al. [37] found that supplementing broiler diets with silymarin reduced the harmful effects of aflatoxicosis, which harmed feed intake, weight increase, feed efficiency, serum biochemistry, and immunological status.Liver enzymes: Aspartate transaminase (AST), alanine transaminase (ALT), and gamma-glutamyltransferase (γ-GT), are the most commonly employed diagnostic markers. Elevations of serum ALT, AST, and γ-GT can be found in liver, biliary, and pancreas diseases that are used as the primary indicator of liver function. Generally, the addition of SMS at different levels accompanied an improvement in liver function compared to the control group, especially the 12 g kg−1 diet SMS group. Compared to the control, ALT, AST, and γ-GT reduced considerably in response to the high dose. The obtained results are in agreement with [38], which reported that silymarin is a potent antioxidant due to its solubilizing nature, antioxidant hydrophilic (phenolic compounds and vitamin C), and lipophilic (carotenoids and vitamin E) nature. The hepatoprotective properties of silymarin, due to its ability to scavenge free radicals and raise the cellular content of glutathione, led to lipid peroxidation inhibition [39]. The high antioxidant action of silymarin could be attributed to the hydrophobicity of silymarin flavonoid components, which act as an electron donor or reducing agent with the OH− radical or superoxide anion quickly [40].Under the influence of 8 and 12 g kg−1 diet SMS dosages, the total protein level and their fractions (Albumin and Globulin) increased, reflecting improved hepatic function and maximum dietary protein utilization. The previous investigations of [18,19,30] stated that SMS could improve growth performance, liver function, and health status in different poultry species. Other mechanistic explanations for the antioxidant properties of silymarin have been proposed, including (a) limiting the generation of free radicals by inhibiting certain ROS-producing enzymes or increasing mitochondrial integrity under stressful situations (b) blocking the nuclear factor B (NF-B)-dependent pathways, lowering inflammatory responses by 10 and (c) activating a variety of non-enzymatic antioxidants and antioxidant enzymes to maintain an optimum redox equilibrium in the cell [21,22]. Many studies have found that silymarin affects the induction of cellular antioxidant defense via modulating numerous transcription factors such as Nrf2, NF-B, and the downstream production of antioxidant genes and proteins [41].The current study found that a high dose of SMS (12 g kg−1 diet) increased the endogenous antioxidant defense system (SOD and CAT) while decreasing oxidative stress indicators (GSSG, and NO). The results of a previous study [36] indicated that Cumene Hydroperoxide (CH)-induced ducks fed a diet containing 200 mg SMS kg−1 had higher SOD, CAT, and GST levels. The decreased NO concentration in SMS-treated groups may be due to its anti-inflammatory properties through different mechanisms. The most prevalent anti-inflammatory pathway of silymarin is attributed to its antioxidant action, which decreases NO radicals or the membrane-stabilizing effect. It leads to cell membrane protection and inhibits inflammatory mediators such as arachidonic acid [42]. According to recent studies, silymarin in SMS influences the activation of cellular antioxidant defense via the regulation of numerous transcription factors, including Nrf2, NF-κB, and the downstream expression of antioxidant genes and proteins [41]. The current findings reveal a reduction in NO levels in SMS-treated groups, which might be attributable to anti-inflammatory characteristics via several pathways.Silymarin has different mechanisms for increasing cell energy and decreasing its metabolites. Silibinin protects mitochondria against pathological events by improving the electron transport chain, reducing ROS, and stimulating mitochondrial bio-energetics, a pro-survival cell signaling mechanism [43]. Current results agree with [44] that silibinin boosted ATP levels considerably, linked to better cell membrane potential. Other studies showed that silibinin reduced the signs of oxidative stress in the liver tissue and increased mitochondrial ATP production compared to the control livers [45]. Indeed, decreased intracellular ATP is a significant marker for increased oxidative stress. Accordingly, using silymarin promoted increased ATP and decreased ADP and AMP. The increase in ATP utilization may be attributed to the ability of Silibin complexes with phospholipids, which preserve hepatic mitochondrial bioenergetics and prevent mitochondrial proton and ATP leakage [46]. It is worth mentioning that silymarin can increase ATP and ameliorate cell 11 and mitochondrial faction via decreasing ROS production and inhibiting NF-κB activation. These data agree with the in vitro study showing the keeping of ATP from the depletion of glial cells against peroxide-induced ROS formation [47]. Serotonin (5HT) is the primary neurotransmitter responsible for appetite and is structured with L-tryptophan as a precursor monoamine [48]. Silymarin is known to elevate some neurotransmitter concentrations in the brain [49]. Moreover, ref. [50] reported a dose-dependently neuroprotection effect of silymarin against oxidative stress in the brain. Indeed, the mechanism of action of silymarin treatment to improve biogenic amines, especially 5HT, may be summarized in two pathways; the first may be attributed to the ability to suppress monoamines oxidase activity which may be responsible for enhanced brain monoamine levels; the second may be due to its most potent antioxidant capacity which increases neuronal cell membrane protection, and subsequently decreases MDA, free radicals and ROS. DNA is the most significant target of oxidative stress, widely contributing to damaging DNA and accelerating the mutation. Therefore, our results suggest a beneficial effect of SMS at high doses, which may decrease ROS and subsequently reduce cell damage and DNA metabolism, which yielded a decrease in 8OHdG. The reduced level of 8OHdG may be due to the link between silymarin, which activates ATP production, and DNA stabilization [51]. In addition, it enhances GSH and other antioxidant defense system markers, enhancing neuronal cell function for neurotransmitter secretion at the presynaptic cleft. Extensive data support the notion that silymarin has a serotonergic effect, which may increase the appetite and the utilization of feed intake. Generally, the impact of SMS to improve 5HT was observed only in response to the medium and highest dose. The amelioration of serotonin activity during brooding period stress may be hypothesized by the possible entry into the central nervous system coupled with antioxidant properties [52]. Obtained data are consistent with [53], which found that silymarin increased biogenic amines in mice intoxicated with reserpine, decreasing biogenic amines and enhancing dopamine depletion. Increasing the number of biogenic amines at the synaptic cleft agreed with [52], who reported that Silymarin increased norepinephrine, dopamine, and serotonin levels in specific brain areas.The GH, a primary pituitary gland hormone encoded by the GH gene, is a multiple-functional gene that plays an essential role in the hypothalamus-12 pituitary target-organ growth axis. It influences protein synthesis and increases somatic cell number and size, thus stimulating growth and development [54]. Results confirm that GH regulates growth in a tissue-specific manner, particularly in the early postnatal age, distinguishing it from any other pituitary hormones [36,37]. The results show that the GH expression levels in the grower duck pituitary were significantly higher than in the liver [55,56]. The GH mRNA level increased in the pituitary and liver of ducks fed a diet supplemented with 8 g kg−1 SMS followed by diets supplemented with 12 g kg−1 SMS. Furthermore, our results are consistent with those presented in previous studies, which provide evidence of the possible expression and physiological role of GH in many tissues besides the pituitary, which may also contribute to the tissue-specific effects of GH in Muscovy duck and early chick embryos [12].It is known that antioxidant enzymes in poultry are usually affected by using various nutritional supplements [57]. SOD and CAT genes codifying for the anti-oxidative enzymes are playing a vital role in the enzymatic defense of the cells. Accordingly, Silymarin has been reported to enhance gene expression of antioxidant enzymes (SOD and CAT) as protection mechanisms against free radicals [58]. In the present study, increasing the transcripts levels of antioxidant markers (CAT, SOD1, SOD2) in the pituitary and liver of ducks fed different levels of SMS compared with those fed a control diet indicates the possible use of SM ground seeds as a promising antioxidant agent in diets. Following the same pattern, ref. [14] recorded a bifacial effect of dietary SM seeds addition to fish diet on the expression profile of SOD and CAT genes, causing an increase in the activities of oxidative enzymes due to the high active silymarin flavonolignans contents in SM seeds. These findings result from vitamin E and flavonoids in the active silymarin contents in SM seeds, which have extremely efficient scavenging free radicals within tissues [12,40]. Additionally, ref. [59] found that SOD and CAT genes expression in the liver of two broiler strains was influenced by heat stress showing up-regulation of CAT mRNAs, whereas SOD transcripts levels remained unaffected. Ref. [60] demonstrated a significant upregulation of the SOD1 gene in the liver of C. moschata groups that received two levels of dietary cadmium (Cd) contamination and catalase (CAT) gene under the effect of the highest level of Cd (C10).Poultry is exposed to oxidative stress in intensive farming systems that can damage cell lipids, proteins, and DNA, which can cause a reduction in performance and health [5]. Oxidative DNA damage caused by free radicals has been identified as an oxidative stress index [43,44]. The current study results prove that active silymarin content in S. marianum dry seeds significantly protects against DNA damage induced by oxidative stress. These results are in agreement with [61], who determined that SMS supplementation was effective in suppressing DNA damage in rats induced by NDEA by showing a significant decrease in the comet assay parameters, % DNA in tail, commet tail length (TL) and Tail moment (TM), and with Saravanan and [62], who reported that the SM with alcohol administration significantly decreased the DNA damage comparing with rats which were treated with alcohol alone. Additionally, [63] proved that treatments with SM only, or in combination with either melatonin or CGA, were influential in deteriorating the oxidative damage of DNA and apoptosis in rat cardiac tissue caused by the toxic effects of carbon tetrachloride (CCl4). This protective effect of SM can be elucidated by its ability to scavenge free radicals before they cause damage to nuclear DNA.