Broadly, mastitis can be categorized into two types: lactational and non-lactational mastitis (as shown in Figure 2). The most common form is lactational mastitis, which typically occurs during breastfeeding. This condition is infectious and presents with localized pain and swelling, accompanied by systemic symptoms. Although it can develop at any time during the lactation period, it most frequently occurs during the second or third week of postpartum. Non-lactational mastitis includes two primary forms: idiopathic granulomatous and periductal mastitis. Periductal mastitis, though rare, can affect non-lactating individuals, particularly those of reproductive age. It’s often linked to bacterial infection (52).

Lactational mastitis can be further classified as either clinical or subclinical intramammary inflammation, based on the presence or absence of visible symptoms. The primary causative agents in clinical cases are Gram-negative bacteria, with E. coli being the most common (53). Clinical condition is primarily categorized into two main presentations: acute and chronic. Acute mastitis is characterized by overt inflammatory signs, including erythema, localized hyperthermia, and tissue tumefaction at the affected site. In severe manifestations, systemic complications may emerge, including pyrexia, septicemia, and abscess formation. Conversely, chronic mastitis exhibits a more insidious progression, typically characterized by recurrent infections and progressive tissue deterioration (2).

In contrast, subclinical mastitis (SCM) represents a pathogenic infection that proceeds without overt clinical manifestations or systemic symptomatology. However, it is distinguished by diminished milk production, compromised quality parameters, and a marked increase in somatic cell count (SCC) (54, 55). This form is predominantly associated with Gram-positive bacterial infections, most notably S. aureus (53). Clinical mastitis is generally easy to diagnose due to visible symptoms, but SCM lacks obvious signs of inflammation, making it more challenging to detect. Diagnostic tools such as the California mastitis test (CMT), elevated SCC in milk, and microbial isolation and culture from milk samples aid in identifying SCM. Early detection is crucial in the dairy industry to minimize financial losses. Numerous microorganisms, primarily bacteria, have been identified as the causative agents of mastitis (56). SCC is a key marker for evaluating udder health and serves as a reliable method for detecting mastitis by quantifying immune cells, such as neutrophils, lymphocytes, and macrophages, in milk (57). When SCC levels exceed 200,000 cells/ml, it often indicates a bacterial infection. Both clinical and SCM can lead to significant changes in SCC level, highlighting the ongoing inflammatory response in the udder (58).

Environmental pathogens constitute a diverse group of microorganisms that originate from multiple reservoirs within the agricultural environment, including bedding substrates, arthropod vectors, housing infrastructure, and the bovine enteric microbiome, with E. coli representing a predominant example (59). The proliferation and transmission of these pathogens are significantly influenced by suboptimal husbandry conditions, including excessive stocking density, inadequate floor sanitation, insufficient ventilation systems, and elevated ambient temperatures coupled with high relative humidity (60). Contagious pathogens, primarily represented by S. aureus and S. agalactiae, exhibit host-adapted characteristics and are transmitted through direct inter-animal contact or via contaminated milking apparatus (2). Opportunistic pathogens demonstrate dual behavioral characteristics, functioning as either contagious or environmental agents depending on circumstances, and typically exploit periods of immunocompromised host status to establish intramammary infections (51).

Recent epidemiological studies have documented increased morbidity associated with mycotic mastitis in bovines. Notable fungal pathogens include zoonotic yeasts such as C. albicans and Kodamaea ohmeri, along with other Candida species: C. guilliermondii, C. famata, C. tropicalis, C. colliculosa, C. krusei, C. rugosa, C. glabrata, C. parapsilosis, and C. inconspicua. Additional fungal agents encompass Trichosporon species, Rhodotorula glutinis, Saccharomyces fragilis, Pichia kudriavzevii, and Cyberlindnera rhodanensis. Mold species, including Aspergillus amstelodami, A. fumigatus, and Geotrichum candidum, have also been implicated (61). Furthermore, yeast-like algae, specifically Prototheca zopfii and Prototheca blaschkeae, have been identified as causative agents (21). Yeast-like algae, including Prototheca zopfii and Prototheca blaschkeae, have also been implicated (62).

Viral infections contribute significantly to mastitis pathogenesis through direct and indirect mechanisms (63). Direct viral mastitis occurs with bovine herpesvirus 1 and 4 (clinical and subclinical presentations, respectively) (64–66), parainfluenza virus, and foot-and-mouth disease virus (67–69). Indirect viral contributions result from teat epithelial lesions caused by bovine herpesvirus 2, cowpox virus, pseudo-cowpox virus, vesicular stomatitis virus, papillomavirus, and bovine leukemia virus, which compromise barrier defenses and predispose to secondary bacterial invasion (21, 63, 70–72).

Mechanical trauma associated with automated milking systems represents a critical predisposing factor, as it disrupts the anatomical integrity of the udder quarter. Specifically, compromised keratin plug formation and mucosal damage to the teat sinus create portals of entry for pathogenic microorganisms (21, 73). Suboptimal milking hygiene protocols demonstrate a significant positive correlation with mastitis incidence, emphasizing the importance of standardized sanitation procedures (21). In addition to mechanical factors, genetic and phenotypic characteristics substantially influence mastitis susceptibility through multiple interconnected pathways. Breed-specific variations reveal differential susceptibility patterns, with high-producing Holstein-Friesian cattle exhibiting increased vulnerability relative to medium-yielding Jersey cattle (74), while low-yielding Rendena cattle demonstrate superior disease resistance (60). Moreover, parity effects indicate a heightened susceptibility in multiparous compared to primiparous animals, reflecting cumulative exposure and potential immunological changes (75).

Beyond genetic predisposition, immunological determinants play a fundamental role in disease susceptibility through variations in cytokine expression profiles and humoral immune responses (76). Critical immune effector mechanisms include antimicrobial peptides (such as lysozyme and lactoferrin), cellular immune components (macrophages and neutrophils), and hormonal receptor expression patterns, which collectively modulate the host defense capacity (77). Concurrently, anatomical predispositions include specific udder conformations, particularly pendulous udder structure and funnel-shaped teat morphology, which facilitate pathogen entry and retention (12). Additionally, age-related physiological changes in geriatric animals contribute to increased susceptibility through progressive teat canal dilation and enhanced mammary epithelial permeability (60).

Temporal physiological changes further complicate these intrinsic factors, as the periparturient transition period represents a critical vulnerability window characterized by profound metabolic and immunological alterations. During this phase, nutritional status has a significant influence on mastitis susceptibility, particularly given the substantial metabolic demands associated with colostrum synthesis and lactogenesis in dairy cattle (12). Consequently, a negative energy balance can precipitate deficiencies in essential proteins, trace minerals, and vitamins that are fundamental to optimal immune function (78). Therefore, maintaining an adequate nutritional status, including sufficient selenium, iron, copper, zinc, cobalt, chromium, essential amino acids, and vitamins A, E, and C, is paramount for both mastitis prevention and sustained lactational performance (79).

TCM has garnered significant attention as an effective alternative to conventional antibiotic treatments for mastitis, demonstrating therapeutic efficacy while minimizing risks associated with antimicrobial resistance and secondary complications. The comprehensive antibacterial, anti-inflammatory, immunomodulatory, and antioxidant properties of TCM, developed over centuries of use, position it as a viable alternative therapy for mastitis treatment (32, 41, 92, 93). Building upon this foundation, numerous TCM formulations have been systematically introduced for the treatment of various types of mastitis (94). These include a comprehensive range of therapeutic options, such as Chai Hu Qing Gan Tang (95), Yanghe decoction (96), and Chaihu Qinggan (38). Furthermore, other notable formulations include Tuoli Tounong Decoction (97), Yiqi Heying (98) and Gong Ying San (99).

Among the most extensively studied traditional formulations, Jingfang Granules (JF’s) demonstrate remarkable efficacy in treating LPS-induced mastitis through multiple therapeutic pathways. Specifically, these granules operate through nuclear factor κB (NF-κB), phosphatidylinositol 3-kinase (PI3K), Akt, mitogen-activated protein kinase/Extracellular signal-regulated kinase (MAPK/ERK), p38, and nucleotide-binding oligomerization domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) signaling cascades. Moreover, they maintain milk barrier integrity through the regulation of tight junction proteins and prevent cell apoptosis by modulating Bcl-2 and Bax expression (100). In parallel, Qicao Rukang powder has demonstrated comparable effectiveness in treating SCM, showing notable improvements in SCC, milk composition, and bacteriological cure rates. The powder’s therapeutic efficacy is attributed to its diverse active constituents, including polysaccharides, saponins, flavonoids, and terpenoids (101). Complementing these oral formulations, Pulsatilla saponin B4 injection protocols have shown significant effectiveness in treating clinical mastitis. These protocols achieve therapeutic benefits by reducing SCC, eliminating pathogenic bacteria, and lowering inflammatory markers, including CRP, SAA, HP, and various pro-inflammatory cytokines (102). Notably, the integration of traditional approaches has shown auspicious results when combined with modern therapeutic techniques. For instance, combined therapy using intramammary antibiotics and complementary acupuncture has demonstrated substantial efficacy in reducing bovine mammary inflammation in cases of SCM. This innovative approach, which targets specific points on affected mammary quarters, resulted in a significant reduction of N-acetyl-beta-D-glucosaminidase (NAGase) activity, thereby indicating improved healing of mammary epithelial cells (103).

Expanding beyond traditional Chinese formulations, a comprehensive evaluation of Tibetan herbal medicines has revealed additional therapeutic options. These include Swertia bimaculata, Gentiana urnula, Uncaria rhynchophylla, Aconitum flavum, Dracocephalum tanguticum, and Lagotis brachystachy, all of which demonstrated significant antibacterial activity against mastitis-causing Staphylococcus strains. Particularly noteworthy is Lagotis brachystachy, which demonstrated exceptional efficacy against MDR strains (104). Ultimately, clinical studies have provided robust validation of the benefits of TCM in the treatment of acute mastitis. These investigations have demonstrated significant improvements across multiple parameters, including clinical effectiveness, lactation rates, symptom relief, quality of life, and emotional well-being. Collectively, these findings provide strong evidence supporting the efficacy of TCM external therapy in both symptom alleviation and promoting recovery (105).

Contemporary research has increasingly focused on identifying and characterizing plant-derived bioactive compounds with substantial therapeutic potential for managing mastitis. Among the most promising candidates are Dimethyl itaconate, Polydatin, Sinomenine hydrochloride, and Jiawei Tounong powder, which have demonstrated significant efficacy through various molecular mechanisms (106–109). Of particular significance is Shikonin (SHI), a bioactive natural naphthoquinone constituent extracted from Lithospermum erythrorhizon, which shows remarkable anti-inflammatory and antimicrobial properties. Initially utilized in TCM for treating wounds and various skin conditions, SHI has subsequently emerged as a viable therapeutic alternative to conventional antibiotics in managing inflammatory conditions, most notably lipopolysaccharide-induced mastitis. The underlying mechanism of action involves the systematic inhibition of the NF-κB signaling pathway through the targeted suppression of p-IκBα and p-p65 proteins, thereby achieving a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 (110). Furthermore, SHI effectively alleviates oxidative stress through the activation of the Nrf₂/HO1 signaling pathway (111).

Complementing these findings, comprehensive essential oil studies have revealed significant bacteriostatic activity of traditional extracts, particularly those from lemon balm and peppermint oil, against prevalent mastitis pathogens, including S. aureus and E. coli (112). In parallel, Sodium houttuynia (SH), derived from Houttuynia cordata, has demonstrated considerable efficacy in inhibiting LPS-induced inflammatory responses in bovine mammary epithelial cells (bMECs). The therapeutic mechanism involves the sophisticated modulation of the NF-κB signaling pathway, resulting in markedly reduced pro-inflammatory cytokine expression (IL-1β, IL-6, TNF-α) and decreased levels of Toll-like receptor 4 (TLR4), inhibitor of nuclear factor kappa B (IκBα), and NF-κB p65 (113).

Building upon these observations, Zhang et al. (114) conducted comprehensive investigations into the protective effects of Salvia miltiorrhiza polysaccharides (SMPs) in S. aureus-induced mastitis models. Their findings convincingly demonstrated that SMP treatment significantly reduced bacterial load, inflammatory cell infiltration, and cytokine levels while simultaneously inhibiting activation of the NF-κB and MAPK pathways. These therapeutic effects were substantiated by notable histopathological improvements and significant reductions in MPO and NAGase activity (114). Correspondingly, quercetin, extracted from Ligustrum lucidum, has exhibited considerable promise in both the prevention and treatment of mastitis. Through sophisticated network pharmacological analysis, researchers identified seven active ingredients and 42 key molecular targets, with tumor necrosis factor (TNF), alpha serine/threonine kinase 1 (AKT1), and interleukin-6 (IL-6) serving as core therapeutic targets. Subsequent in vivo studies validated quercetin’s capacity to alleviate pathological changes and downregulate inflammatory markers through the modulation of the PI3K-AKT and NF-κB signaling pathways (115).

Furthermore, geraniol has emerged as an up-and-coming therapeutic alternative, demonstrating effective pathogen inhibition, probiotic enhancement, and maintenance of gut microbial diversity. Notably, geraniol treatment exhibited no detectable milk residues after four days of administration and, significantly, did not induce drug resistance during prolonged exposure periods (116). Concomitantly, Taraxacum mongolicum has been shown to exhibit substantial protective effects against S. aureus-induced mastitis through well-characterized anti-inflammatory mechanisms, including the targeted downregulation of TLR2 and the systemic inhibition of NF-κB and MAPK signaling pathways (117).

Of exceptional interest, Forsythiaside A (FTA) has established a pivotal role in mastitis treatment through multiple comprehensive studies (118–121). Recent research has successfully elucidated FTA’s sophisticated protective mechanisms, particularly its capacity to modulate mitophagy through the PINK1/Parkin signaling pathway. This pathway represents a crucial component for maintaining mitochondrial integrity, cellular energy production, and cell viability under conditions of mastitis-induced stress. FTA’s selective activation of mitophagy facilitates the targeted removal of dysfunctional mitochondria, thereby preserving mitochondrial integrity and reducing inflammatory responses. Additionally, FTA demonstrates remarkable effectiveness in lowering both cellular and mitochondrial reactive oxygen species (ROS), thereby mitigating oxidative damage, associated inflammation, and tissue injury. These synergistic mechanisms collectively contribute to reduced mastitis severity and improved dairy cow health and productivity (122).

Beyond these extensively characterized compounds, various other traditional therapeutic agents have demonstrated promising potential. Specifically, Tanshinone I and Tanshinone IIA/B exhibit significant inhibition of NF-κB activation in nMECs, proving particularly effective when combined with conventional antibiotics such as cephalosporins (123). Similarly, Artemisia argyi Leaves (ALE) have shown substantial therapeutic potential in LPS-induced mouse mastitis models, demonstrating the capacity to alleviate tissue damage, reduce oxidative stress, and regulate inflammation-associated gene expression (124). Moreover, Broadleaf Mahonia has been shown to exhibit significant anti-inflammatory properties by reducing pro-inflammatory cytokines, including IL-1β, CCL-5, and IL-6, in RAW264.7 cell cultures. This therapeutic effect is primarily mediated through systematic inhibition of NF-κB and MAPK signaling pathways, which represent crucial regulators of inflammatory responses. In cases of granulomatous lobular mastitis, preventive treatment with Broadleaf mahonia effectively reduces inflammation and promotes tissue homeostasis (125).

To comprehensively understand the underlying pathophysiological mechanisms, investigations into mastitis caused by S. aureus have revealed significant inflammatory responses characterized by elevated levels of IL-1β, TNF-α, and MPO activity, alongside increased ferroptosis markers such as Fe²⁺ and MDA levels. Decreased protective factors, including GSH, GPX4, and ferritin in mammary tissues, accompany these pathological changes (126). Remarkably, the strategic integration of traditional therapeutic approaches has yielded promising clinical results. In this context, Schisandrin B (SB) treatment has demonstrated considerable effectiveness in mitigating pathological changes by reducing both inflammation and ferroptosis. The underlying therapeutic mechanism involves the upregulation of SIRT1 and SLC7A11 expression, the inhibition of p53 and NF-κB activation, and the restoration of antioxidant defense systems. These therapeutic effects were confirmed through comprehensive histological analysis, demonstrating reduced tissue damage in SB-treated groups, thereby suggesting that SB’s therapeutic action occurs through the SIRT1/p53/SLC7A11 and NF-κB pathways (127). The molecular mechanisms underlying the protective effects of traditional Chinese medicine and plant-derived bioactive compounds against mastitis are schematically represented in Figure 3. Additionally, a comprehensive overview of research developments on traditional Chinese medicine and plant-derived bioactive compounds in mastitis therapy is summarized in Table 1 and Figure 4.

While plant-derived bioactive compounds show promising antimicrobial and anti-inflammatory effects against bovine mastitis pathogens in vitro and in mouse models (128), their use in dairy cattle remains limited due to several inherent barriers in large animal research. Research on Traditional Chinese Medicine and plant-derived compounds for mastitis treatment is still in its early stages, with most trials still in development using mouse models, resulting in insufficient foundational data to support progress to large animal studies. Conducting controlled clinical trials in dairy cattle involves significant economic challenges, requiring much larger sample sizes, longer observation periods, and higher operational costs compared to mouse models, often surpassing available research budgets.

The regulatory framework governing veterinary pharmaceuticals in food-producing animals requires comprehensive safety evaluations, including pharmacokinetic studies, tissue residue analyses, and the establishment of withdrawal periods for milk and meat products, leading to lengthy approval processes that deter initial research investments. The physiological complexity of ruminant digestive systems adds further challenges, as plant-derived compounds undergo extensive ruminal metabolism that can alter bioavailability and therapeutic effectiveness compared to monogastric models. Additionally, dairy industry stakeholders usually prioritize rapid-acting, standardized antimicrobial treatments that work with existing automated milking protocols and quality systems, which creates market resistance to traditional plant-based therapies that need more complex preparation, administration, and monitoring. These economic, regulatory, physiological, developmental, and practical factors collectively explain why the translation of Traditional Chinese Medicine and plant-derived compounds from promising laboratory results to field use in dairy cattle is limited, despite their demonstrated anti-inflammatory, antimicrobial, and immunomodulatory properties in experimental studies (129).