Immune responses against spermatozoa: Role of Antisperm antibodies in infertility in bovines

Introduction

Infertility in bovines refers to a condition of reduced reproductive efficiency characterized by failure or delay in the production of a viable calf (Gautam et al., 2025). Over the past several decades, a progressive decline in the fertility of lactating dairy cattle has been reported globally, despite substantial advancements in reproductive management practices (Lonergan et al., 2016; Funeshima et al., 2021). Infertility in dairy cattle may result from a wide range of physiological, anatomical, nutritional and management-related factors, many of which can be readily identified through routine clinical and reproductive evaluations. However, in a considerable proportion of cases, infertility persists despite the absence of overt abnormalities, indicating the involvement of less apparent mechanisms. Among these, immunologically mediated infertility remains poorly understood, frequently overlooked and often misdiagnosed, thereby contributing to unexplained or persistent reproductive failure (Parkinson, 2018). Immunoinfertility primarily involves immune sensitization against antigens associated with reproductive function. These antigens may originate from reproductive hormones, such as luteinizing hormone-releasing hormone (LHRH), gonadotropin-releasing hormone (GnRH), gonadal steroids, prostaglandin F₂-α and oxytocin, as well as from gameteand semen-derived components, including spermatozoa, seminal plasma proteins and ova. (Cooper and Larsen, 2006). Immunoinfertility in cattle, predominantly mediated by antisperm antibodies (ASAs), represents a significant yet frequently underrecognized cause of reproductive failure, contributing to approximately 10-30% of infertility cases in bovines depending on breed, management practices and diagnostic sensitivity (Gupta et al., 2023) [21,31,20,37,11,23] . ASAs impair bovine fertility by targeting sperm surface antigens and disrupting essential processes including sperm motility, capacitation, acrosome reaction and spermoocyte interaction, ultimately compromising fertilization and early embryonic development. Clinically, ASAs associated immune responses are linked to repeat breeding, extended interestrus intervals, early embryonic loss, delayed uterine involution, prolonged calving intervals, Research in cattle are limited and fragmented (Zodinsanga et al., 2015; Hernández et al., 2022) and because of that a comprehensive understanding of ASAs-mediated infertility in bovines is not revealed, despite its direct relevance to reproductive efficiency and herd productivity. In this context, the present review critically synthesizes existing evidence on antisperm antibodies in cattle, with emphasis on their etiology, mechanisms of action, diagnostic advancements and current strategies for treatment, control and prevention [53,24] .

Basic immunology of sperm in cattle Sperm as an antigen in bulls

In bulls, spermatozoa are considered foreign to the immune system because they are produced only after puberty, once immune self-tolerance has already been established. As a result, sperm antigens are not recognized as self during early immune development. Under physiological conditions, these antigens remain sequestered; however, when immune privilege is compromised, sperm antigens may elicit immune responses, leading to the production of antisperm antibodies (Romrell and Michael, 1978) [41] .

Mechanism of immunological tolerance in bulls

The blood-testis barrier (BTB) is established at puberty through the development of highly specialized intercellular junction between adjacent Sertoli cells, presence of gap junctions, basement membrane of seminiferous tubules and surrounding peritubular myoid cells, collectively creating a highly selective and protective microenvironment for germ cell development. Functionally, the BTB divides the seminiferous epithelium into a basal compartment containing spermatogonia that remains accessible to systemic factors and immune cells and an ad-luminal compartment that encloses meiotic and post-meiotic germ cells and is completely secluded from immune surveillance. By restricting the movement of immune cells and circulating antibodies into the ad-luminal compartment, the BTB safeguards immunogenic germ cells from immune-mediated injury. Additionally, it prevents the leakage of germ cell and sperm antigens into the systemic circulation, thereby minimizing antigen presentation and subsequent activation of antisperm immune responses. Beyond its physical barrier function, the BTB contributes to immune privilege by maintaining a locally immunosuppressive environment, as sertoli cells secrete immunomodulatory factors that suppress local immune activation, ensuring immune tolerance (Rode et al., 2015) [40] . Further, multiple cellular and molecular mechanisms act in concert in bull to maintain immune tolerance toward developing germ cells. Sertoli cells play a central role by actively phagocytosing apoptotic and degenerating germ cells, thereby preventing the release of intracellular sperm antigens into the interstitial space and systemic circulation, which minimizes antigen leakage and immune sensitization (Fijak et al., 2018).

In parallel, the submucosa of the male reproductive tract is enriched with suppressor T cells, which actively downregulate immune activation, inhibit effector T-cell responses and prevent inappropriate immune reactions against sperm antigens, thereby reinforcing local immune suppression (Milovanovic et al., 2005). Seminal plasma further contributes to immune protection by acting as an immunosuppressive milieu that masks sperm antigens and inhibits immune recognition, with high concentrations of polyamines such as spermine and spermidine suppressing lymphocyte proliferation through oxidative metabolites; their degradation products, including acrolein and putrescine, amplify local immunosuppression, while soluble FcγRIII (CD16) modulates antisperm immune responses, with reduced levels reported in ASAs-positive individuals (Cheema et al., 2016) [33,6,14,6] . Testicular macrophages predominantly exhibit an alternatively activated M2 phenotype, which is characterized by potent anti-inflammatory and immunoregulatory functions essential for maintaining testicular immune privilege. These macrophages constitutively secrete high levels of immunosuppressive cytokines, particularly interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which act synergistically to dampen local immune activation. Whereas, testicular dendritic cells are predominantly maintained in an immature and tolerogenic state, which is essential for preserving immune tolerance toward germ cell antigens. These cells efficiently capture sperm and germ cell antigens within the testicular microenvironment but exhibit low expression of MHC class II molecules and co-stimulatory signals such as CD80 and CD86, thereby limiting their capacity to activate effector T-cell responses (Ferrer, 2019). Additional protection is provided by complement-regulatory proteins expressed on spermatozoa and reproductive tissues, including decay-accelerating factor, membrane cofactor protein and CD59, which collectively inhibit complement activation and prevent membrane attack complex-mediated lysis of sperm cells (Cheema et al., 2016). Moreover, paracrine factors such as testosterone, TGF-β1, activin A and IL-10 synergistically sustain an immunosuppressive testicular niche, ensuring immune privilege and uninterrupted spermatogenesis (Ferrer, 2019) [6,14] .

Sperm as an antigen in cows

Spermatozoa from bulls are perceived by the female reproductive tract as allogeneic antigens because they originate from a genetically distinct individual and are therefore immunologically foreign to the female immune system. Upon exposure, these antigens can stimulate local innate and adaptive immune responses within the female reproductive tract, resulting in the recruitment and activation of immune cells such as neutrophils, macrophages and lymphocytes. In certain cases, this immune activation progresses to the generation of anti-sperm antibodies (Nagaria et al., 2011) [35] .

Mechanism of immunological tolerance in cows

The mucosal lining of the female reproductive tract constitutes a crucial first line of immunological regulation by establishing a physical and functional barrier that limits direct contact between spermatozoa and immune cells. This barrier is formed by epithelial cells interconnected through tight junctions, giving rise to an organized mucosal epithelium that operates as a transiently immunoprivileged site during sperm transport. It restricts antigen penetration, modulates immune cell access and ensures controlled exposure to paternal antigens, thereby facilitating immune tolerance (Vlasova and Saif, 2021). During natural mating, the female reproductive tract is routinely exposed to spermatozoa; however, this exposure does not normally elicit a deleterious immune response against sperm cells due to the presence of multiple immunoinhibitory factors. Immunosuppressive substances such as 19-hydroxy prostaglandin E, polyamines, transglutaminase and high-molecular-weight Fc receptorbinding proteins are present in seminal plasma as well as in follicular and uterine fluids and cervical mucus, actively modulate local immune activity. These factors suppress immune cell activation and inhibit antibody-mediated reactions (Lazarevic et al., 2013) [49,29] . Macrophages and dendritic cells residing in the bovine female reproductive tract predominantly exhibit a tolerogenic antigen-presenting phenotype that is essential for preventing immune reactions against spermatozoa. Instead of eliciting robust inflammatory or classical antigen-presenting responses, these cells limit antigen presentation, downregulate the production of pro-inflammatory cytokines and preferentially activate regulatory immune pathways. Such tolerogenic programming ensures that sperm antigens are not perceived as pathogenic, thereby preventing immune-mediated sperm elimination (Vlasova and Saif, 2021). Additionally, upon exposure to sperm antigens, regulatory T cells undergo controlled activation and limited expansion. They actively suppress the activation and proliferation of potentially harmful effector T lymphocytes through tolerogenic signalling pathways and the release of regulatory cytokines. This finely coordinated immune response prevents immune-mediated damage to spermatozoa. The interaction of spermatozoa with oviductal epithelial cells induces a significant upregulation of transforming growth factor-β₁ (TGF-β₁) expression. Together with the prominent localization of TGF-β₁ and interleukin-10 (IL-10) within the oviductal epithelium and the surrounding vascular endothelium, this response establishes a strong antiinflammatory and immunoregulatory microenvironment. These cytokines directly modulate local antigen-presenting cells, particularly macrophages and dendritic cells, promoting their differentiation toward a tolerogenic phenotype. As a result, sperm antigen presentation occurs in a noninflammatory context, preferentially driving the induction and expansion of regulatory T cells rather than effector immune responses, thereby maintaining immune tolerance to spermatozoa. Besides, estrogen plays a pivotal role in modulating immune activity within the female reproductive tract by regulating cytokine expression and immune cell function, thereby establishing a dynamic and tightly controlled immune zone. In contrast, progesterone exerts potent immunosuppressive effects by attenuating inflammatory responses and limiting excessive activation of immune effector cells. Acting synergistically, these ovarian hormones orchestrate an immunotolerant microenvironment that restrains immune recognition of sperm antigens, protects spermatozoa from immune-mediated damage (Yousef et al., 2016) [49,29,51] .

Formation of antisperm antibodies in bulls

Blood-testis barrier may disrupt due to mechanical injury, infectious, inflammatory or degenerative insults, leads to the abnormal exposure of immunogenic sperm antigens to the systemic immune system. Such antigenic leakage initiates an immune response that may progress from an initial regulatory or anti-inflammatory phase to the activation of humoral immunity, culminating in the production of antisperm antibodies (Ferrer et al., 2015). Structural similarities between microbial antigens and sperm surface proteins can result in molecular mimicry, whereby the immune system fails to discriminate between pathogenic and self-antigens. Consequently, immune responses directed against infectious agents may inadvertently target spermatozoa, leading to the generation of cross-reactive antibodies (Vickram et al., 2019) [16,3] . Frequent semen collection or repeated inflammatory episodes lead to prolonged and cumulative exposure of sperm antigens to the immune system. This sustained antigenic stimulation enhances immune surveillance and increases the likelihood of antigen recognition and presentation leads to elevated antisperm antibody levels. In some bulls, antisperm antibodies develop in the absence of any clearly identifiable pathological or environmental trigger. This phenomenon is thought to be associated with inherent genetic variations affecting immune regulation, which may predispose certain individuals to heightened immune reactivity. Hormonal influences can further modulate immune responses, altering the balance between tolerance and activation. Additionally, defects in normal immune tolerance mechanisms may allow sperm antigens to be recognized as non-self, ultimately leading to spontaneous ASAs production (Gupta et al., 2023) [23] . [38,42,5,22] . Dead or membrane-compromised sperm lose membrane integrity, leading to the release and surface exposure of intracellular antigens that are normally concealed from immune recognition. Once exposed, these antigens are readily detected by immune cells within the female reproductive tract. This triggers antigen presentation and local inflammatory or humoral immune responses. Consequently, such sperm become highly immunogenic and may contribute to antisperm antibody formation (Hunter, 1989) [25] . Estrus cycle dependent hormonal fluctuations markedly regulate mucosal immune responsiveness in the bovine reproductive tract. During estrus, estrogen dominance enhances immune cell recruitment, antigen presentation and local antibody production, increasing immune vigilance. In contrast, during diestrus, progesterone predominance exerts immunosuppressive effects by dampening inflammatory responses and reducing immune cell activation, thereby favouring sperm survival and potential embryo implantation. Despite extensive research, a significant proportion of antisperm antibody (ASAs) cases in cows lack a clearly identifiable cause. Approximately 15-20% of cows develop ASAs in the absence of recognized predisposing factors, suggesting the involvement of complex interactions among genetic predisposition, individual immune regulatory differences and subtle environmental or management-related influences (Gupta et al., 2023) [23] .

Formation of antisperm antibodies in cows

Sperm antigens, antisperm antibody classes and their localization on spermatozoa

During the early phase of the primary immune response following exposure to an antigenic stimulus, IgM is the first immunoglobulin class to be synthesized. Upon subsequent or repeated exposure to the same antigen, class switching occurs, leading to a secondary immune response characterized predominantly by the production of IgG and IgA antibodies (Batard et al., 1993). IgM, IgG and IgA have been implicated in immunologically mediated infertility. Among these, IgG is the principal antibody detected in blood serum, whereas IgA predominates in genital tract secretions. Both antibody classes are present in the reproductive fluids of male and female bovines, where they may interact with sperm antigens and impair sperm function, transport or fertilizing capacity (Kim et al., 1999). Antisperm antibodies exert their deleterious effects on spermatozoa in a site-specific manner, depending on their binding location along the sperm surface. Antibody binding to the sperm head region interferes with critical fertilization events by blocking sperm-zona pellucida interaction, preventing oocyte binding, inhibiting the acrosome reaction and impairing sperm-oolemma fusion. When ASAs localize to the midpiece, they disrupt mitochondrial function, leading to reduced ATP production and compromised energy metabolism, ultimately shortening sperm longevity. In contrast, antibody attachment to the tail region predominantly affects sperm motility by restricting forward progressive movement, reducing migration efficiency within the female reproductive tract, and impairing penetration of cervical mucus. Collectively, these region-specific actions of ASAs significantly compromise sperm functional competence and contribute to immunologically mediated infertility (Kuntareddi et al., 2024) [4,27,28] .

Table 1: Sperm antigens and their location

Antigens on bull spermatozoa Location on spermatozoa References Hyaluronidase Acrosomal matrix and inner acrosomal membrane of the sperm head (Robertson and Sharkey, 2001) [39] Acrosin Acrosomal vesicle, predominantly bound to the inner acrosomal membrane (Robertson and Sharkey, 2001) [39] Neuraminidase Outer acrosomal membrane and acrosomal contents (Robertson and Sharkey, 2001) [39] Corona-dispersing enzyme Acrosomal cap of the sperm head (acrosomal contents) (Robertson and Sharkey, 2001) [39] ADAM1, ADAM2 and ADAM3 Sperm head (Evans, 2002) [13] PH-20, PH- [8,44]

ACTL7a

Acrosome and tail of mature spermatozoa (Fu et al., 2016) [19] Impact of antisperm antibodies on cattle reproduction ASAs negatively influence key sperm functions, including motility, viability, capacitation and the acrosome reaction, as discussed in the following sections.

Impact of ASAs on sperm motility:

Antisperm antibodies directed against the sperm head and principal piece markedly impair the ability of spermatozoa to penetrate cervical mucus (Coddington et al., 1997). IgG and IgA isotypes of ASAs present in cervical mucus have been shown to induce a characteristic "shaking" phenomenon, wherein progressive sperm movement is converted into a non-progressive, immobilized state upon contact with mucus. Evidence suggests that ASAs binding to the sperm head or main tail segment effectively inhibit cervical mucus penetration, whereas antibodies targeting only the tail tip do not exert a similar effect (Wang et al., 1985). Sperm immobilization further promotes agglutination, leading to clumping of spermatozoa and restricted motility within cervical mucus (Lazarević et al., 2013). Elevated concentrations of IgG and IgA ASAs in the cervical mucus of crossbred cows have also been associated with antigen-antibody interactions on the sperm surface, resulting in head-to-head, head-to-tail or tailto-tail agglutination (Vikrant et al., 2016). The lower ASAs levels have more improved mucus penetration capacity, higher mitochondrial membrane potential, and superior sperm kinematic parameters, including straightness, linearity, curvilinear velocity, straight-line velocity and average path velocity (Archana et al., 2021) [9,50,29,48,2] .

Impact of ASAs on sperm viability: Antisperm antibodies are known to alter the permeability and fluidity of the sperm plasma membrane, thereby compromising sperm viability. The circulating ASAs have negative effect on sperm viability. The number of membrane intact spermatozoa are more with lower level of ASAs (Ahuja et al., 2016) [1] .

Impact of ASAs on sperm capacitation

Antisperm antibodies bound to the sperm surface markedly impair the capacity of bovine spermatozoa to undergo normal membrane fluidity changes during capacitation. This disruption adversely affects sperm-oocyte interactions and limits the ability of spermatozoa to penetrate the zona pellucida, ultimately resulting in fertilization failure (Ferrer et al., 2017) [1] .

Impact of ASAs on sperm acrosome reaction

Antisperm antibodies in the uterine fluid of cows can cause the induction of a premature acrosome reaction (Ahuja et al., 2016). The zona pellucida-triggered acrosome reaction can be suppressed by the majority of ASA-positive sera (Francavilla et al., 1997). The findings indicate that antisperm antibodies exert a variable influence on sperm capacitation and the acrosome reaction; certain ASAs impair the ability of spermatozoa to undergo these processes, whereas others appear to have no significant effect [1,18] .

Broader reproductive impact beyond sperm functions

Beyond their direct effects on sperm function, antisperm antibodies exert broader adverse consequences on reproductive performance at the herd level. The presence of ASAs has been associated with fertilization failure and early embryonic death, ultimately contributing to reduced conception rates and the development of repeat breeding syndrome. These reproductive inefficiencies lead to extended calving intervals and a marked decline in the success of artificial insemination programs. Collectively, such outcomes translate into substantial economic losses for livestock production systems. Thus, ASAs represent not only a biological impediment to fertilization but also a significant factor compromising overall reproductive efficiency and productivity in cattle (Gupta et al., 2023) [23] . [23] .

Treatment of antisperm antibodies

Research on the treatment of antisperm antibodies remains at an early and evolving stage. Existing clinical and experimental efforts are largely centered on three complementary strategies.

By reducing ASAs production

One of the principal strategies for managing antisperm antibodies involves reducing their production by addressing underlying pathological and immunological triggers. Prompt diagnosis and treatment of genital infections or inflammatory conditions using appropriate antibiotics or anti-inflammatory agents can help control local inflammation and thereby limit the induction of ASAs. In addition, immunosuppressive interventions, corticosteroids can substantially reduce ASAs levels. However, such approaches must be applied with caution, as corticosteroid therapy is associated with potential adverse effects, including endocrine disturbances and negative impacts on semen quality (Turek and Lipshultz, 1994) [46] . Consequently, while suppression of immunemediated ASAs production remains a viable strategy, careful consideration of risk-benefit balance is essential in clinical and herd fertility management.

By laboratory techniques for ASAs removal

Several laboratory-based approaches have been explored to minimize the impact of antisperm antibodies on sperm function. Sperm washing combined with density-gradient centrifugation is a routinely employed method that helps eliminate ASAs-associated components while selectively enriching highly motile spermatozoa for use in artificial insemination. Immunomagnetic separation represents a more targeted approach, wherein magnetic microbeads coated with anti-immunoglobulins bind ASA-positive sperm, allowing their selective removal through magnetic cell sorting and thereby improving the quality of the processed semen sample (Vigano et al., 1991) [47] . In addition, experimental enzymatic or protease-based surface treatments, using agents such as chymotrypsin or IgA-protease, have been investigated to detach antibodies bound to the sperm membrane (Mazumdar and Levine, 1998) [32] . Collectively, these techniques aim to reduce ASA burden and enhance the functional competence of spermatozoa, although their practical applicability and efficacy require further validation.

Assisted Reproductive Technology

The application of preparative procedures in assisted reproductive technologies such as intrauterine and intracervical insemination, in vitro fertilization, gamete intrafallopian tube transfer, subzonal sperm injection and intracytoplasmic sperm injection minimizes the exposure of gametes to antisperm antibodies, thereby enhancing gamete functionality and improving fertilization potential (Naz, 2004) [36] .

Conclusions

Antisperm antibodies represent a significant yet frequently underestimated cause of reproductive failure in bovine species. Despite spermatozoa being immunologically nonself, both bulls and cows normally maintain immune tolerance through specialized regulatory mechanisms that prevent pathological immune recognition. Disruption of this balance most commonly due to compromise of the bloodtestis barrier, genital tract infections, mucosal injury, underlying reproductive pathologies or repeated exposure to sperm antigens which can lead to the production of ASAs in both sexes. Functionally, different immunoglobulin classes exert region-specific effects on spermatozoa; IgG and IgA preferentially bind to distinct sperm domains, thereby impairing motility, capacitation, acrosome reaction and sperm-oocyte interaction. The presence of ASAs can be identified using direct assays that detect antibodies bound to sperm surfaces or indirect methods that measure circulating antibodies in seminal plasma, serum or reproductive tract secretions. However, despite advances in diagnostic approaches, therapeutic strategies for managing ASAassociated infertility remain limited and are still in an early stage of development, underscoring the need for further targeted research in bovine reproductive immunology.

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