The inflammatory environment mediated by a high-fat diet inhibited the development of mammary glands and destroyed the tight junction in pregnant mice.

Long-term intake of a high-fat diet seriously affects the health of pregnant women and leads to increased levels of inflammation in the mammary gland. Therefore, to further explore the effect of a high-fat diet on mammary gland development and the tight junction (TJ) during pregnancy, we placed mice into two groups: a high-fat diet group and a control group. We detected the expression of proteins related to fat synthesis in the mammary gland by western blotting. The results showed that a high-fat diet could lead to an increase in fat synthesis in the mammary gland. Then, the inflammatory levels and acinar cell morphology in the mammary gland were detected by ELISA and H&E staining. We also measured the levels of MAPK and NF-κB signal pathway-related proteins by western blotting. The results showed that a high-fat diet activated the MAPK and NF-κB signaling pathways and promoted the expression of inflammatory factors. Finally, the development of the mammary gland and the integrity of the TJ were determined by immunohistochemistry, immunofluorescence and western blotting. The results showed that a high-fat diet inhibited the development of the mammary gland and the expression of tight junction proteins (TJs). Our study showed that a high-fat diet could promote the expression of inflammatory factors by activating the MAPK and NF-κB signaling pathways and could reshape the microenvironment through extramammary inflammation. Finally, a high-fat diet inhibited the development of the mammary gland during pregnancy and destroyed the integrity of the TJ.

Weighing the Risk: effects of Obesity on the Mammary Gland and Breast Cancer Risk.

Obesity is a preventable risk factor for breast cancer following menopause. Regardless of menopausal status, obese women who develop breast cancer have a worsened prognosis. Breast tissue is comprised of mammary epithelial cells organized into ducts and lobules and surrounded by adipose-rich connective tissue. Studies utilizing multiple in vivo models of obesity as well as human breast tissue have contributed to our understanding of how obesity alters mammary tissue. Localized changes in mammary epithelial cell populations, elevated secretion of adipokines and angiogenic mediators, inflammation within mammary adipose tissue, and remodeling of the extracellular matrix may result in an environment conducive to breast cancer growth. Despite these significant alterations caused by obesity within breast tissue, studies have suggested that some, but not all, obesity-induced changes may be mitigated with weight loss. Here, we review our current understanding regarding the impact of obesity on the breast microenvironment, how obesity-induced changes may contribute to breast tumor progression, and the impact of weight loss on the breast microenvironment.

Lesions of anogenital mammary-like glands: Four cases including novel pathologic and immunohistochemical observations.

Anogenital mammary-like glands, formerly described as ectopic breast tissue, are currently considered to be normal histologic components of the anogenital region. Anogenital mammary-like glands can give rise to many lesions identical to counterparts in the native female breast. We describe four cases of such lesions, including fibroadenoma, gynecomastia-like hyperplasia, and ectopic mammary-type tissue with a spectrum of usual ductal hyperplasia, apocrine metaplasia, adenosis, and pseudolactational change. All four cases occurred in young women (ages 29-38) who presented with vulvar or perianal masses. Similar to previously reported cases, these lesions shared histologic and immunohistochemical characteristics identical to native female breast lesions. Novel findings in our cases included (1) the first case of gynecomastia-like change to be reported in the perianal area of a female, (2) Immunohistochemical staining identifying a 3-layered epithelium characterized by a population of CK14 and CK5/6 positive and hormone receptor negative superficial luminal cells, and (3) diffuse, strong positivity for GATA3 in all cases. Our study adds to the literature on these rare lesions and highlights findings which may be useful in understanding the pathogenesis and improving the diagnosis of anogenital mammary-like gland lesions.

Coordinated signals from PARP-1 and PARP-2 are required to establish a proper T cell immune response to breast tumors in mice.

Poly(ADP-ribose)-polymerase (PARP)-1 and PARP-2 play an essential role in the DNA damage response. Based on this effect of PARP in the tumor cell itself, PARP inhibitors have emerged as new therapeutic tools both approved and in clinical trials. However, the interactome of multiple other cell types, particularly T cells, within the tumor microenvironment are known to either favor or limit tumorigenesis. Here, we bypassed the embryonic lethality of dually PARP-1/PARP-2-deficient mice by using a PARP-1-deficient mouse with a Cd4-promoter-driven deletion of PARP-2 in T cells to investigate the understudied role of these PARPs in the modulation of T cell responses against AT-3-induced breast tumors. We found that dual PARP-1/PARP-2-deficiency in T cells promotes tumor growth while single deficiency of each protein limited tumor progression. Analysis of tumor-infiltrating cells in dual PARP-1/PARP-2-deficiency host-mice revealed a global change in immunological profile and impaired recruitment and activation of T cells. Conversely, single PARP-1 and PARP-2-deficiency tends to produce an environment with an active and partially upregulated immune response. Our findings pinpoint opposite effects of single and dual PARP-1 and PARP-2-deficiency in modulating the antitumor response with an impact on tumor progression, and will have implications for the development of more selective PARP-centered therapies.

Potential of breastmilk in stem cell research.

Breastmilk is a dynamic, multi-faceted, and complex fluid containing a plethora of biochemical and cellular components that execute developmental effects or differentiation program, providing nourishment and immunity to newborns. Recently, it was reported that breastmilk contains a heterogeneous population of naïve cells, including pluripotent stem cells, multipotent stem cells, immune cells, and non-immune cells. The stem cells derived from breastmilk possess immune privilege and non-tumorigenic properties. Thus, breastmilk may represent an ideal source of stem cells collected by non-perceive procedure than other available sources. Thus, this "maternally originating natural regenerative medicine" may have innumerable applications in clinical biology, cosmetics, and pharmacokinetics. This review describes the efficient integrated cellular system of mammary glands, the impressive stem cell hierarchy of breastmilk, and their possible implications in translational research and therapeutics.

Lycopene alleviates H2O2-induced oxidative stress, inflammation and apoptosis in bovine mammary epithelial cells via the NFE2L2 signaling pathway.

During the transition into lactation, bovine mammary epithelial cells (bMECs) are likely subjected to altered redox balance due to the high metabolic rate associated with the onset of lactation. In non-ruminants, lycopene (LYC), a naturally occurring hydrocarbon carotenoid, has attracted considerable attention as a potential natural agent against oxidative stress. The aim of this study was to investigate whether LYC alleviates oxidative injury in bMECs induced by H2O2 and the underlying molecular mechanisms. The primary bMEC and bovine MEC line MAC-T cells were treated with H2O2 (500 µM) and/or LYC (0.5, 1 or 2 µM) for 24 h. The results showed that treatment with LYC decreased H2O2-induced accumulation of intracellular reactive oxygen species (ROS), inflammatory cytokine (TNF-α, IL-6, and IL-1ß) expression and the apoptosis rate. These effects were associated with the activation of the NFE2L2-antioxidant response element (ARE) pathway coupled with inactivation of the nuclear factor-κB (NF-κB) inflammatory and caspase/Bcl2 apoptotic pathways. The bMECs were transfected with NFE2L2 siRNA for 48 h and/or treated with H2O2 (500 µM) and/or LYC (2 µM) for another 24 h. The fact that transfection with NFE2L2 siRNA abrogated the protection of LYC against H2O2-induced accumulation of intracellular ROS, inflammatory cytokine expression and apoptosis suggested that this antioxidant transcription factor is essential for the protective mechanism induced by LYC. These results suggest that LYC might be a potent antioxidant in vivo that could be administered to ruminant animals during stressful periods such as the transition into lactation.

Association between bovine leukemia virus proviral load and severity of clinical mastitis.

The purpose of this study was to clarify the effect of Bovine leukemia virus (BLV) infection on natural immunity in the bovine mammary gland and on the severity of clinical mastitis. We classified milk samples from clinical mastitic cows into BLV-positive (n=76) and BLV-negative (n=12). BLV-positive cows were further divided into cows with High BLV proviral load (H-PVL) (n=23) and Low BLV proviral load (L-PVL) (n=53). Severity of clinical mastitis was classified as MILD, MODERATE, or SEVERE. Multiple logistic regression analysis was performed on the host factors and environmental factors with severity of clinical mastitis as the objective variable. BLV proviral load (PVL) and season at onset of mastitis showed significant correlation with the severity of clinical mastitis. Binary logistic regression analysis was performed on natural immunity factors lactoferrin and lingual antimicrobial peptide (LAP) concentration in milk, with PVL as the objective variable. Of these natural immunity factors, LAP concentration in milk showed significant correlation with PVL. The results of the present study suggested that PVL and season are associated with severity of clinical mastitis, and that the immune function in the mammary gland is decreased in cows with H-PVL compared to that in cows with L-PVL.

Role of Liver X Receptor in Mastitis Therapy and Regulation of Milk Fat Synthesis.

Mastitis is important disease that causes huge economic losses in the dairy industry. In recent years, antibiotic therapy has become the primary treatment for mastitis, however, due to drug residue in milk and food safety factors, we lack safe and effective drugs for treating mastitis. Therefore, new targets and drugs are urgently needed to control mastitis. LXRα, one of the main members of the nuclear receptor superfamily, is reported to play important roles in metabolism, infection and immunity. Activation of LXRα could inhibit LPS-induced mastitis. Furthermore, LXRα is reported to enhance milk fat production, thus, LXRα may serve as a new target for mastitis therapy and regulation of milk fat synthesis. This review summarizes the effects of LXRα in regulating milk fat synthesis and treatment of mastitis and highlights the potential agonists involved in both issues.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis.

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows' mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down-regulation and location of bta-miR-26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real-time PCR, in situ hybridization and dual-luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta-miR-26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage.

Development of immune and microbial environments is independently regulated in the mammary gland.

Breastfeeding is important for mammals, providing immunological and microbiological advantages to neonates, together with the nutritional supply from the mother. However, the mechanisms of this functional diversity in the mammary gland remain poorly characterized. Here, we show that, similar to the gastrointestinal tract, the mammary gland develops immune and microbial environments consisting of immunoglobulin A (IgA) and the microflora, respectively, both of which are important for protecting neonates and the mother from infectious diseases. The IgA production and microflora development are coordinated in the gastrointestinal tract but seem to be independently regulated in the mammary gland. In particular, the chemokine (C-C motif) ligand 28 and poly-Ig receptor, crucial molecules for the IgA production in milk, were expressed normally in germ-free lactating mice but were almost undetectable in postweaning mothers, regardless of the microflora presence. Our findings offer insights into potentially improving the quality of breastfeeding, using both immunological and microbiological approaches.

Control mechanisms for producing antimicrobial factors in ruminant mammary gland.

Mastitis, a symptom of inflammation in mammary tissue by infection with various kinds of bacteria, causes huge economic losses in the milk industry. One of the popular methods for treatment of mastitis is antibiotics, although this prohibits milk shipping and sometimes causes resistant microbes. Therefore, a new strategy to treat mastitis without antibiotics is eagerly required around the world. Antimicrobial factors belong to innate immunity and can start their function extremely early after bacterial stimulation. These factors have antimicrobial activity for a broad spectrum of bacteria. Elucidation of causal mechanisms and functions of antimicrobial factors in the mammary gland is thought to result in suitable methods for prevention and treatment of mastitis. Therefore, this review introduces traits of some antimicrobial factors and the mechanisms for expressing, producing and secreting them in the mammary gland. For antimicrobial factors, lingual antimicrobial peptide (LAP), S100A7, cathelicidin and lactoferrin are controlled in different sites and different time courses, suggesting that antimicrobial factors play different roles for local defense against bacterial infection in the mammary gland. These findings will contribute to the development of prevention and treatment methods for mastitis.

Image analysis of immune cell patterns in the human mammary gland during the menstrual cycle refines lymphocytic lobulitis.

PURPOSE: To improve microscopic evaluation of immune cells relevant in breast cancer oncoimmunology, we aim at distinguishing normal infiltration patterns from lymphocytic lobulitis by advanced image analysis. We consider potential immune cell variations due to the menstrual cycle and oral contraceptives in non-neoplastic mammary gland tissue. METHODS: Lymphocyte and macrophage distributions were analyzed in the anatomical context of the resting mammary gland in immunohistochemically stained digital whole slide images obtained from 53 reduction mammoplasty specimens. Our image analysis workflow included automated regions of interest detection, immune cell recognition, and co-registration of regions of interest. RESULTS: In normal lobular epithelium, seven CD8[Formula: see text] lymphocytes per 100 epithelial cells were present on average and about 70% of this T-lymphocyte population was lined up along the basal cell layer in close proximity to the epithelium. The density of CD8[Formula: see text] T-cell was 1.6 fold higher in the luteal than in the follicular phase in spontaneous menstrual cycles and 1.4 fold increased under the influence of oral contraceptives, and not co-localized with epithelial proliferation. CD4[Formula: see text] T-cells were infrequent. Abundant CD163[Formula: see text] macrophages were widely spread, including the interstitial compartment, with minor variation during the menstrual cycle. CONCLUSIONS: Spatial patterns of different immune cell subtypes determine the range of normal, as opposed to inflammatory conditions of the breast tissue microenvironment. Advanced image analysis enables quantification of hormonal effects, refines lymphocytic lobulitis, and shows potential for comprehensive biopsy evaluation in oncoimmunology.

Activation of peroxisome proliferator-activated receptor gamma in mammary epithelial cells upregulates the expression of tumor suppressor Cyld to mediate growth inhibition and anti-inflammatory effects.

Several studies have implicated the downregulation of the tumor suppressor Cyld expression in breast cancer development. However, the mechanisms that regulate Cyld expression in mammary epithelial cells are largely unknown. In order to investigate them, a bioinformatic analysis of the promoter region of Cyld was performed and identified putative nuclear hormone receptor response elements that included peroxisome proliferator-activated receptor gamma (PPAR-γ)-responsive elements. In the present study, we showed that upon activation of the nuclear hormone receptor PPAR-γ by the agonist troglitazone (TZD), there was a significant increase in Cyld mRNA in human mammary epithelial cell lines. The effect of TZD could be attributed to the transactivation of the Cyld promoter as indicated by the upregulation of a luciferase reporter that was driven by the -1995 to +95 region of the human Cyld gene. Furthermore, the upregulation of Cyld expression by TZD was dependent on PPAR-γ since downregulation of PPAR-γ expression by RNAi compromised the induction of Cyld expression by TZD. CYLD induction mediated, at least in part, the TZD-mediated downregulation of tumor necrosis factor α (TNFα)-induced interleukin 8 (IL-8). In addition, downregulation of CYLD compromised the cytotoxic effects of TZD in immortalized mammary epithelial cells. Our results demonstrated that PPAR-γ is a novel regulator of Cyld transcription and identified CYLD as a mediator of the PPAR-γ-dependent anti-inflammatory and anti-proliferative activity in mammary epithelial cells, which underscores its potential to be used as a target for the development of breast cancer therapeutic approaches.

RETRACTED: Sophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstream pathways on LPS-induced mastitis in the mammary gland of mice.

Mastitis is defined as the inflammation of the mammary gland. LPS, which is widely used to induce mastitis models for the study of this disease, triggers similar inflammation as Escherichia coli. Sophocarpine, isolated from Sophora alopecuroides L., exhibits multiple biological properties. The aim of the present study was to determine the anti-inflammatory effect and mechanism of action of sophocarpine on mastitis within an LPS-induced mouse model. ELISA and western blotting were performed to detect protein levels. The qPCR was performed to detect mRNA levels. The ELISA and qRT-PCR results showed that sophocarpine inhibited the expression of TNF-α, IL-1ß and IL-6 in a dose-dependent manner. However, sophocarpine suppressed TLR4 expression. Further study showed that sophocarpine could suppress the phosphorylation of IκBα, p65 and p38. These results confirm that sophocarpine played an anti-inflammatory role in LPS-induced mastitis by regulating TLR4 and the NF-κB and MAPK signaling pathways in mammary gland tissues. Therefore, sophocarpine may be a potential therapeutic drug for the treatment of mastitis.

Macrophages: Regulators of the Inflammatory Microenvironment during Mammary Gland Development and Breast Cancer.

Macrophages are critical mediators of inflammation and important regulators of developmental processes. As a key phagocytic cell type, macrophages evolved as part of the innate immune system to engulf and process cell debris and pathogens. Macrophages produce factors that act directly on their microenvironment and also bridge innate immune responses to the adaptive immune system. Resident macrophages are important for acting as sensors for tissue damage and maintaining tissue homeostasis. It is now well-established that macrophages are an integral component of the breast tumor microenvironment, where they contribute to tumor growth and progression, likely through many of the mechanisms that are utilized during normal wound healing responses. Because macrophages contribute to normal mammary gland development and breast cancer growth and progression, this review will discuss both resident mammary gland macrophages and tumor-associated macrophages with an emphasis on describing how macrophages interact with their surrounding environment during normal development and in the context of cancer.

Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate.

Developing strategies to enhance cancer prevention is a paramount goal, particularly given recent concerns about surgical treatment of preinvasive states such as ductal carcinoma in situ. Promoting effective immunosurveillance by leukocytes that scan for nascent neoplastic transformations represents a potential means to achieve this goal. Because most breast cancers arise within the ductal epithelium, enhancing protective immunosurveillance will likely necessitate targeting one or more of the distinctive lymphocyte types found in these sites under normal conditions. Here, we have characterized the intraepithelial lymphocyte compartment of non-cancerous human breast tissue and identified a subset of T lymphocytes that can be pharmacologically targeted to enhance their responses to breast cancer cells. Specifically, Vδ2(+) γδ T cells were consistently present in preparations of mammary ductal epithelial organoids and they proliferated in response to zoledronic acid, an aminobisphosphonate drug. Vδ2(+) T cells from breast ductal organoids produced the antitumor cytokine IFNγ and efficiently killed bisphosphonate-pulsed breast carcinoma cells. These findings demonstrate the potential for exploiting the ability of Vδ2(+) γδ T cells to respond to FDA-approved bisphosphonate drugs as a novel immunotherapeutic approach to inhibit the outgrowth of breast cancers.

Genome-Wide Transcriptional and Post-transcriptional Regulation of Innate Immune and Defense Responses of Bovine Mammary Gland to Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is problematic for lactating mammals and public health. Understanding of mechanisms by which the hosts respond to severe invasion of S. aureus remains elusive. In this study, the genome-wide expression of mRNAs and miRNAs in bovine mammary gland cells were interrogated at 24 h after intra-mammary infection (IMI) with high or low concentrations of S. aureus. Compared to the negative control quarters, 194 highly-confident responsive genes were identified in the quarters with high concentration (109 cfu/mL) of S. aureus, which were predominantly implicated in pathways and biological processes pertaining to innate immune system, such as cytokine-cytokine receptor interaction and inflammatory response. In contrast, only 21 highly-confident genes were significantly differentially expressed in face of low concentration (106 cfu/mL) of S. aureus, which slightly perturbed the cell signaling and invoked corresponding responses like vasoconstriction, indicating limited perturbations and immunological evading. Additionally, the significant up-regulations of bta-mir-223 and bta-mir-21-3p were observed in the quarters infected by high concentration of S. aureus. Network analysis suggested that the two miRNAs' pivotal roles in defending hosts against bacterial infection probably through inhibiting CXCL14 and KIT. The significant down-regulation of CXCL14 was also observed in bovine mammary epithelial cells at 24 h post-infection of S. aureus (108 cfu/mL) in vitro. Integrated analysis with QTL database further suggested 28 genes (e.g., CXCL14, KIT, and SLC4A11) as candidates of bovine mastitis. This study first systematically revealed transcriptional and post-transcriptional responses of bovine mammary gland cells to invading S. aureus in a dosage-dependent pattern, and highlighted a complicated responsive mechanism in a network of miRNA-gene-pathway interplay.

Breast Tissue Composition and Immunophenotype and Its Relationship with Mammographic Density in Women at High Risk of Breast Cancer.

AIM: To investigate the cellular and immunophenotypic basis of mammographic density in women at high risk of breast cancer. METHODS: Mammograms and targeted breast biopsies were accrued from 24 women at high risk of breast cancer. Mammographic density was classified into Wolfe categories and ranked by increasing density. The histological composition and immunophenotypic profile were quantified from digitized haematoxylin and eosin-stained and immunohistochemically-stained (ERα, ERß, PgR, HER2, Ki-67, and CD31) slides and correlated to mammographic density. RESULTS: Increasing mammographic density was significantly correlated with increased fibrous stroma proportion (rs (22) = 0.5226, p = 0.0088) and significantly inversely associated with adipose tissue proportion (rs (22) = -0.5409, p = 0.0064). Contrary to previous reports, stromal expression of ERα was common (19/20 cases, 95%). There was significantly higher stromal PgR expression in mammographically-dense breasts (p=0.026). CONCLUSIONS: The proportion of stroma and fat underlies mammographic density in women at high risk of breast cancer. Increased expression of PgR in the stroma of mammographically dense breasts and frequent and unexpected presence of stromal ERα expression raises the possibility that hormone receptor expression in breast stroma may have a role in mediating the effects of exogenous hormonal therapy on mammographic density.

Genetic mutations potentially cause two novel NCF1 splice variants up-regulated in the mammary gland, blood and neutrophil of cows infected by Escherichia coli.

Neutrophil cytosolic factor 1 (NCF1) plays a crucial role in host defense against microbial pathogens. In this study, we investigated the potential alternative splicing patterns, expression and splice-relevant single nucleotide polymorphisms (SNPs) of the bovine NCF1 gene to increase insights into its potential role against bovine mastitis caused by Escherichia coli infection. Using RT-PCR and clone sequencing methods, we found two novel splice variants designed as NCF1-TV1 (retained intron 6) and NCF1-TV2 (retained part of intron 8), respectively, encoding two putative truncated proteins (239AA and 283AA). Two splice variants were drastically up-regulated in the mastitis-infected cows' mammary tissues, blood and neutrophils compared with these of healthy cows using real-time RT-PCR. Genomic sequencing analysis identified four novel SNPs g.10112 G>A, g.10766 T>C, SNPs g.12085 G>A and g.12430 T>C at the ends of intron 6 and intron 8 of NCF1. ESE motif predicted that the SNP (g.10766 T>C) might affect the binding with splicing-related factors and subsequently caused the production of aberrant splice variant NCF1-TV1, which is one of the potential reasons that the functional SNP was associated with increased milk somatic cell score in cow. Our results would help in better understanding the NCF1 gene function in the process against pathogen infection, and the effect of splicing-related SNP on the production of aberrant splice variant and careful functional characterization in dairy cattle.