NIC, together with scientists from China Agricultural University, has elucidated a novel in vivo antioxidant mechanism of Lewis acids.

Aging is a complex process, with cellular oxidative damage being one of its primary causes. Oxidative damage refers to the harm inflicted on cells by free radicals and other oxidizing agents. Free radicals are highly reactive molecules that can interact with other cellular components, leading to oxidative injury. Under normal physiological conditions, the production and scavenging of reactive oxygen species (ROS) maintain a dynamic equilibrium to sustain vital functions. However, in the balance between the body’s antioxidant defenses and ROS, ROS often prevail, compromising animal health. In livestock production, oxidative stress frequently triggers various diseases, such as ovarian atresia in laying hens, diarrhea, enteritis, and liver disorders in broilers, ketosis and mastitis in dairy cows, sudden death syndrome in weaned piglets, adverse pregnancy outcomes in sows, and vaginitis and oophoritis.

Flavonoids are a class of polyphenolic secondary metabolites widely found in plants and the diet. They possess a 15-carbon structural framework comprising two benzene rings and one heterocycle, as shown in Figure 1. Flavonoids exhibit remarkable antioxidant activity; researchers have demonstrated that they not only exert antioxidant effects but also lower blood glucose and lipids and possess antitumor properties, with their antioxidant capacity likely underpinning these physiological benefits. Studies have confirmed that flavonoids primarily enhance the body’s antioxidant defenses and immune function by activating the Keap1-Nrf2/ARE signaling pathway, leading to their widespread use in livestock and poultry production. This review summarizes the antioxidant mechanisms of flavonoids and their applications in animal production, aiming to provide a theoretical basis for their further utilization.

Figure 1. Basic structure of flavonoid compounds

Oxidative stress is a major focus in animal health and nutrition research. The Kelch-like ECH-associated protein 1–nuclear factor erythroid 2-related factor 2/antioxidant response element (Keap1–Nrf2/ARE) signaling pathway is the most well-established mechanism for regulating the antioxidant system in animals. It controls the transcription of numerous antioxidant genes, enabling the organism to defend against harmful stimuli and maintain cellular homeostasis and normal physiological function—such as stress resistance, anti-apoptosis, and modulation of inflammatory responses—thereby playing a crucial role in protecting animal health. Keap1 consists of five domains: the N-terminal region (NTR), the BTB domain (BTB), the intervening region (IVR), two glycine-rich repeat domains (DGR), and the C-terminal region (CTR), each of which is essential for inhibiting Nrf2 activity. Nrf2 contains seven functional domains, Neh1 through Neh7, which are involved in regulating Nrf2 stability or transcriptional activity. The ARE, also known as the electrophilic response element, is located upstream of gene sequences encoding proteins with cytoprotective functions and phase II metabolic enzymes. ^[28] Under oxidative stress conditions, the stabilized Nrf2 translocates into the nucleus, forms heterodimers with the transcription factor Maf, and activates the transcription of downstream genes. Common downstream gene products include quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), and glutamate-cysteine ligase catalytic subunit (GCLC), among others.
 

Under normal physiological conditions, Keap1 binds to Nrf2 in the cytoplasm, leading to continuous ubiquitination and proteasomal degradation of Nrf2. Structural changes in Keap1 result in loss of its inhibitory function, halting ubiquitination of Nrf2; consequently, newly synthesized Nrf2 accumulates and becomes activated. Activated Nrf2 translocates into the nucleus, inducing transcription of a battery of cytoprotective genes and ultimately activating the cellular defense system. Subsequently, Nrf2 dimerizes in the nucleus, binds to ARE sequences, and activates transcription of downstream antioxidant enzymes and target protein genes, notably γ‑glutamylcysteine synthetase (γ‑GCS), HO‑1, and NQO1, thereby modulating antioxidant enzyme activity and exerting free radical scavenging, antioxidant, and anti-inflammatory effects. When the organism returns to a normal physiological state, Nrf2 dissociates from ARE sites and retranslocates to the cytoplasm, where it is ubiquitinated and degraded, actively terminating its activity and maintaining it at low levels. Nrf2 partners with other proteins to jointly regulate target gene transcription, restoring intracellular homeostasis. This process is essential for maintaining normal cellular function and physiological balance.

Figure 2 Regulatory mechanisms of nuclear factor erythroid-2-related factor (Nrf2) activation

3.1 Antioxidant Mechanisms of Flavonoids

When biological organisms are subjected to adverse stimuli, the levels of free radicals within the body increase. Due to their high reactivity, free radicals can induce lipid peroxidation reactions in unsaturated fatty acid side chains associated with phospholipids, enzymes, and membrane receptors, as well as in nucleic acid macromolecules, generating harmful substances such as malondialdehyde (MDA). This alters membrane permeability and fluidity, leading to changes in cellular structure and function, and ultimately causing cellular degeneration or even death. When the body’s endogenous antioxidant capacity is insufficient to repair such damage, it becomes necessary to obtain nutrients from external dietary sources to mitigate oxidative stress–induced injury. The antioxidant mechanisms of flavonoid compounds include: ① direct scavenging of reactive oxygen species. By directly neutralizing reactive oxygen species, flavonoids protect cells from free radical–mediated damage and maintain intracellular redox homeostasis, thereby supporting normal cellular function and physiological status. ② inhibition of enzymes involved in free radical generation. Xanthine oxidase catalyzes the oxidation of xanthine and hypoxanthine, promoting the production of peroxide radicals. Studies have shown that flavonoids can significantly enhance the body’s ability to inhibit xanthine oxidase activity. ③ chelation of metal ions. Flavonoids also suppress free radical formation through metal ion chelation. Most transition metal ions possess unpaired electrons, which accelerate free radical formation. However, flavonoids can complex with these metal ions, inhibiting free radical generation, reducing intracellular free radical load, and maintaining redox balance. ④ promotion of antioxidant substance synthesis. Multiple studies indicate that flavonoids increase the body’s antioxidant capacity by enhancing the activity of antioxidant enzymes in livestock and poultry, thereby reducing MDA levels and alleviating stress responses.

3.2 Regulatory Effects of Flavonoids on the Keap1-Nrf2/ARE Signaling Pathway

The cellular antioxidant capacity of flavonoids is primarily mediated by modulating the Keap1–Nrf2 pathway. Under basal conditions, Nrf2 is sequestered in the cytoplasm through its association with Keap1 and targeted for degradation by the ubiquitin ligase Cul3. Upon oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it forms heterodimers with other bZIP proteins and binds to AREs. This binding activates the expression of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), NADPH, NQO1, and HO-1. Thus, flavonoids exert their antioxidant effects mainly by regulating the Nrf2–Keap1 signaling axis to upregulate the expression of key downstream factors and proteins.

Figure 3 Regulatory effects of flavonoids on antioxidant pathways

3.3 Applications of Flavonoids in Livestock and Poultry Production

Flavonoids possess beneficial biological activities and are therefore widely used in livestock and poultry production. Studies have shown that flavonoids such as naringin, daidzein, quercetin, and mulberry leaf flavonoids can effectively improve growth performance, reproductive performance, immune function, and antioxidant capacity in livestock and poultry.

Flavonoids can enhance the body’s immune function, improve reproductive and growth performance in livestock and poultry, and exhibit estrogen-like effects. They can influence the gonadal or growth axes in livestock and poultry, thereby affecting the levels of related hormones. Zhang Ling et al. found that adding 30 g/t of soybean flavones to the diet of laying hens during the late production phase significantly reduced the feed-to-egg ratio, increased the egg-laying rate and Haugh units, and elevated serum triiodothyronine (T ₃ ), follicle-stimulating hormone (FSH), luteinizing hormone (LH) 、 Progesterone (P ₄ ) and estradiol (E ₂ ) levels. Study E found that adding genistein to the diet of laying hens in the late production phase significantly increased egg production rate and reduced feed-to-egg ratio, enhanced eggshell strength, and simultaneously elevated plasma total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) activity while decreasing malondialdehyde (MDA) content in the ovary and yolk. Furthermore, it upregulated the expression of Nrf2, SOD1, and CAT genes, with 120 mg/kg yielding the most pronounced effects.

Flavonoids can enhance sow reproductive performance and lactation, antioxidant capacity, and immunity, as well as improve the growth performance of weaned piglets, meat quality, and the body’s overall antioxidant capacity. Wei Xihui et al. found that, compared with the control group, supplementing the diet of approximately 85-day‑pregnant Yorkshire sows with 8 mg/kg or 5 mg/kg soybean isoflavones significantly increased both individual and litter weights of nursing piglets at 20 days of age, and also significantly increased individual weight and average litter weight of weaned piglets at 28 days of age. Cheng Zhonggang et al. reported that feeding soybean flavones to growing–finishing pigs can increase their average daily gain and serum T ₃ , Tetraiodothyronine (T ₄ ) levels, as well as SOD and GSH-Px activities, while reducing feed-to-gain ratio, serum urea nitrogen content, and MDA content. Hu Youjun et al. found that supplementing the diet with 40 mg/kg of soy isoflavones increased average daily gain and reduced feed-to-gain ratio in weaned piglets (6–20 kg), promoted growth, and enhanced the animals’ antioxidant capacity. Adding 40 mg/kg of soy isoflavones to the diet also improved average daily gain during the growing-finishing phase (60–90 kg), improved meat quality, and increased muscle antioxidant enzyme activity.

Adding it to feed can enhance the lactation and reproductive performance of ruminants; however, some active constituents of flavonoids are readily degraded in the rumen, limiting their application and research in ruminants. Ai Xiajie et al. found that feeding soybean flavones to Holstein dairy cows 10 days before calving significantly increased protein and fat concentrations in colostrum, while also elevating these components in mature milk, thereby improving colostrum quality. Xiao Fan’s study demonstrated that supplementing calf diets with 600 mg/day of soybean flavones markedly improved weight gain in Holstein calves, reduced the incidence of diarrhea, and enhanced production performance. Additionally, this treatment promoted the synthesis and secretion of serum immunoglobulins, lowered serum tumor necrosis factor-α levels, and boosted immune function. It also increased the activity of serum antioxidant enzymes, decreased serum malondialdehyde (MDA) content, regulated growth-related hormone levels, and improved the internal metabolic milieu of nutrients.

Summary

 

The growth and development, reproductive performance, and meat quality of livestock and poultry are all influenced by the organism’s redox status. During the defense against oxidative stress–induced damage, the Keap1–Nrf2/ARE signaling pathway plays a crucial role in protecting cells from injury. When reactive nitrogen species (RNS) or reactive oxygen species (ROS) trigger an oxidative stress response, this pathway is activated: it negatively regulates Keap1, causing its dissociation from Nrf2. Nrf2 then translocates to the nucleus, where it recognizes and binds to the antioxidant response element (ARE) to exert antioxidant effects. Flavonoids can modulate the Keap1–Nrf2/ARE pathway to enhance the host’s antioxidant capacity, thereby improving production performance, boosting immunity, and enhancing the quality of meat, eggs, and milk. This offers a new avenue for addressing the widespread issues of oxidative stress and inflammation currently faced in the livestock and poultry industries.

Written by: E Xiaodi

Review: Hu Youjun