Search Results (61)

The Lidia cattle breed is featured in several traditional popular bullfighting festivals throughout Spain, including the “Toro de Cuerda” event, in which the animals are subjected to intense physical exercise. However, the physiological impact and welfare implications of these activities remain poorly characterized. This study aimed to evaluate the stress response and muscle damage in Lidia breed bulls during roping bull celebrations through comprehensive blood analysis. Blood samples were collected from 53 adult male Lidia bulls before and after a standardized 45 min continuous running exercise during traditional roping bull events in four Spanish autonomous regions. Hematological parameters, muscle enzymes (creatine kinase, lactate dehydrogenase, lactate), and stress hormones (cortisol and ACTH) were analyzed. Significant increases (p < 0.05) were observed in leukocytes, lymphocytes, monocytes, eosinophils, neutrophils, erythrocytes, hematocrit, hemoglobin, and post-exercise platelets. Muscle enzymes showed marked elevations, with creatine kinase increasing up to 10-fold above baseline values. Stress hormones, cortisol and ACTH, also demonstrated significant increases. Despite the magnitude of these changes, all parameters remained within established reference ranges for the bovine species. This study provides the first physiological assessment of Lidia cattle during popular bullfighting celebrations, establishing baseline data for evidence-based welfare evaluation and management protocols. Full article

Naturally occurring prostaglandin E₂ (PGE₂) influences cytokine production regulation in bovine neutrophils exposed to Staphylococcus aureus Rosenbach. Here, we employed bovine neutrophils as the primary experimental system, and administered specific inhibitors targeting various receptors, which were subsequently exposed to S. aureus. Cytokine expression levels in dairy cow neutrophils induced by S. aureus via the endogenous PGE₂-EP2/4 receptor pathway were investigated, and its effects on P38, extracellular signal-regulated kinase (ERK), P65 activation, and phagocytic function in Staphylococcus aureus Rosenbach-induced dairy cow neutrophils, were examined. Blocking cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) enzymes substantially decreased PGE₂ production and release in S. aureus-exposed bovine neutrophils. Cytokine output showed significant reduction compared to that in SA113-infected controls. Phosphorylation of P38, ERK, and P65 signaling molecules was depressed in the infected group. Pharmacological interference with EP2/EP4 receptors similarly diminished cytokine secretion and phosphorylation patterns of P38, ERK, and P65, with preserved cellular phagocytic function. During S. aureus infection of bovine neutrophils, COX-2 and mPGES-1 participated in controlling PGE₂ biosynthesis, and internally produced PGE₂ molecules triggered NF-κB and MAPK inflammatory pathways via EP2/EP4 receptor activation, later adjusting the equilibrium between cytokine types that promote or suppress inflammation. This signaling mechanism coordinated inflammatory phases through receptor-mediated processes. Full article

In swine reproduction, immune-mediated mechanisms such as neutrophil extracellular trap (NET) formation can affect sperm function and reduce fertility outcomes. This study evaluated the capacity of antioxidant and reproductive compounds—butylated hydroxytoluene (BHT), prostaglandin E₂ (PGE₂), bovine serum albumin (BSA), and seminal plasma (SP)—to modulate NETosis in co-cultures of swine neutrophils and cryopreserved spermatozoa. NET formation was quantified by nuclear area expansion and validated by digital cytometry and immunofluorescence. BHT (0.5 mM) and PGE₂ (10 µM) produced the most significant inhibitory effects, reducing NETotic cell percentages from 34.5 ± 2.7% (sperm-exposed controls) to 12.2 ± 1.3% and 14.5 ± 2.1%, respectively (p < 0.01). SP at 20% decreased NETosis to 16.8 ± 1.8%, while BSA (0.5%) achieved a moderate reduction to 21.3 ± 2.5%. Flow cytometry revealed reduced peroxynitrite levels in sperm treated with SP and BSA. Two NET phenotypes (aggNETs and sprNETs) were identified. BTS medium enhanced NET formation, whereas DNase I degraded NETs effectively. These findings identify porcine NETosis as a redox-sensitive pathway modulated in vitro, suggesting an immunological role in enhancing sperm preservation for swine artificial insemination. Full article

Bovine viral diarrhea (BVD) is caused by bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus and in the family Flaviviridae. According to some studies, the disease incurs USD 1.5–2.5 billion per year and USD 0.50 to USD 687.80 per cow loss in beef and dairy farms, respectively. Using vaccines is among the strategies to prevent the disease. However, complete protection requires vaccines that target both the humoral and cellular immune responses of the adaptive immune system. A comprehensive literature review was made to provide insights into the interaction of BVDV with host immunity, vaccine applications, and the limitation of the currently available vaccines, as well as explore strategies used to advance the vaccines. BVDV causes immunosuppression by interfering with the innate and adaptive immune systems in a manner that is species and biotype-dependent. Interferon production, apoptosis, neutrophil activity, and antigen-processing and presenting cells are significantly affected during the viral infection. Despite maternal antibodies (MatAbs) being crucial to protect calves from early-age infection, a higher level of MatAbs are counterproductive during the immunization of calves. There are numerous inactivated or modified BVDV vaccines, most of which are made of cytopathic BVDV 1 and 2 and the BVDV 1a subgenotypes. Furthermore, subunit, marker, DNA and mRNA vaccines are made predominantly from E2, E^rns, and NS₃ proteins of the virus in combination with modern adjuvants, although the vaccines have not yet been licensed for use and are in the experimental stage. The existing BVDV vaccines target the humoral immune system, which never gives the full picture of protection without the involvement of the cell-mediated immune system. Several limitations were associated with conventional and next-generation vaccines that reduce BVDV vaccine efficiency. In general, providing complete protection against BVDV is very complex, which requires a multi-pronged approach to study factors affecting vaccine efficacy and strategies needed to improve vaccine efficacy and safety. Full article

K. pneumoniae is a major cause of bovine mastitis worldwide, making it difficult to control due to its resistance to multiple drugs. L. paraplantarum has been explored as a promising new approach to fighting bovine mastitis. In this study, the probiotic potential and safety of L. paraplantarum SDN1.2, as well as its ex vivo and in vivo anti-inflammatory effects against K. pneumoniae-induced mastitis, were comprehensively investigated using bioinformatics analyses and experimental validation methods. The results revealed that L. paraplantarum SDN1.2 exhibits non-hemolytic activity, is not cytotoxic, lacks virulence genes (e.g., adhesion factors, toxins, and invasion factors) and antibiotic resistance genes (e.g., beta-lactamases and tetracycline resistance genes), as supported by whole-genome sequencing, and significantly inhibits the growth of K. pneumoniae, as evaluated by antimicrobial tests. Following further validation in vitro, L. paraplantarum SDN1.2 demonstrated the capability to inhibit the adhesion and invasion of K. pneumoniae to bMECs. In a mouse model of K. pneumoniae-induced mastitis, L. paraplantarum SDN1.2 reduced the extent of neutrophil infiltration and inflammatory lesions. Furthermore, L. paraplantarum SDN1.2 pretreatment significantly reduced myeloperoxidase (MPO) activity and the expression of inflammatory cytokines (IL-6, IL-1β, and TNF-a) in mouse mammary gland tissue. In K. pneumoniae-infected bMECs, L. paraplantarum SDN1.2 significantly lowered lactate dehydrogenase (LDH) levels and expression of inflammatory cytokines such as IL-6, IL-1β, and TNF-α. The results demonstrated that the newly isolated L. paraplantarum SDN1.2 from bovine sources exhibits promising characteristics as a safe probiotic for the alleviation of bovine mastitis due to its safety profile and anti-inflammatory and antibacterial properties. Full article

The skeletal muscle of cattle is the main component of their muscular system, responsible for supporting and movement functions. However, there are still many unknown areas regarding the ranking of the importance of different types of cell populations within it. This study conducted in-depth research and made a series of significant findings. First, we trained 15 bovine skeletal muscle models and selected the best-performing model as the initial model. Based on the SHAP (Shapley Additive exPlanations) analysis of this initial model, we obtained the SHAP values of 476 important genes. Using the contributions of these 476 genes, we reconstructed a 476-gene SHAP value matrix, and relying solely on the interactions among these 476 genes, successfully mapped the single-cell atlas of bovine skeletal muscle. After retraining the model and further interpretation, we found that Myofiber cells are the most representative cell type in bovine skeletal muscle, followed by neutrophils. By determining the key genes of each cell type through SHAP values, we conducted analyses on the correlations among key genes and between cells for Myofiber cells, revealing the critical role these genes play in muscle growth and development. Further, by using protein language models, we performed cross-species comparisons between cattle and pigs, deepening our understanding of Myofiber cells as key cells in skeletal muscle, and exploring the common regulatory mechanisms of muscle development across species. Full article

Klebsiella pneumoniae (K. pneumoniae), a kind of zoonotic bacteria, is among the most common antibiotic-resistant pathogens, and it causes nosocomial infections that pose a threat to public health. In this study, the roles of synthetic bovine neutrophil β-defensin-5 (B5) in regulating inflammatory response and metabolic response against multidrug-resistant K. pneumoniae infection in a mouse model were investigated. Mice were administrated intranasally with 20 μg of B5 twice and challenged with K. pneumoniae three days after B5 pretreatment. Results showed that B5 failed to directly kill K. pneumoniae in vitro, but it provided effective protection against multidrug-resistant K. pneumoniae via decreasing the bacterial load in the lungs and spleen, and by alleviating K. pneumoniae-induced histopathological damage in the lungs. Furthermore, B5 significantly enhanced the mRNA expression of TNF-α, IL-1β, Cxcl1, Cxcl5, Ccl17, and Ccl22 and obviously enhanced the rapid recruitment of macrophages and dendritic cells in the lungs in the early infection phase, but significantly down-regulated the levels of TNF-α, IL-1β, and IL-17 in the lungs in the later infection phase. Moreover, RNA-seq results showed that K. pneumoniae infection activated signaling pathways related to natural killer cell-mediated cytotoxicity, IL-17 signaling pathway, inflammatory response, apoptosis, and necroptosis in the lungs, while B5 inhibited these signaling pathways. Additionally, K. pneumoniae challenge led to the suppression of glycerophospholipid metabolism, the phosphotransferase system, the activation of microbial metabolism in diverse environments, and metabolic pathways in the lungs. However, B5 significantly reversed these metabolic responses. Collectively, B5 can effectively regulate the inflammatory response caused by K. pneumoniae and offer protection against K. pneumoniae. B5 may be applied as an adjuvant to the existing antimicrobial therapy to control multidrug-resistant K. pneumoniae infection. Our study highlights the potential of B5 in enhancing pulmonary bacterial clearance and alleviating K. pneumoniae-caused inflammatory damage. Full article

Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (<30 min) in both B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis. Full article

Supplementing trace minerals is common in managing bovine respiratory disease (BRD) in post-weaned cattle; however, its influence on host immunity and metabolism in high-risk cattle remains unclear. We aimed to assess the impact of three supplementation programs on liver and serum trace element concentrations and blood gene expression. Fifty-six high-risk beef steers were randomly assigned to one of three groups over 60 days: (1) sulfate-sourced Cu, Co, Mn, and Zn (INR), (2) amino acid-complexed Cu, Mn, Co, and Zn (AAC), or (3) AAC plus trace mineral and vitamin drench (COMBO). Serum and liver biopsies for Cu, Co, Mn, and Zn at d0, d28, and d60 were analyzed from cattle free of BRD (n = 9 INR; n = 6 AAC; n = 10 COMBO). Differences and correlations of mineral concentrations were analyzed via generalized linear mixed models and Spearman’s rank coefficients, respectively (p < 0.05). Whole blood RNA samples from healthy cattle (n = 4 INR; n = 4 AAC; n = 4 COMBO) at d0, d13, d28, d45, and d60 were sequenced and analyzed for differentially expressed genes (DEGs) via glmmSeq (FDR < 0.05), edgeR (FDR < 0.10), and Trendy (p < 0.10). Serum and liver Cu and Co concentrations increased over time in all groups, with higher liver Cu in COMBO (487.985 μg/g) versus AAC (392.043 μg/g) at d60 (p = 0.013). Serum and liver Cu concentrations (ρ = 0.579, p = 6.59 × 10⁻⁸) and serum and liver Co concentrations (ρ = 0.466, p = 2.80 × 10⁻⁵) were linearly correlated. Minimal gene expression differences were found between AAC versus COMBO (n = 2 DEGs) and INR versus COMBO (n = 0 DEGs) over time. AAC versus INR revealed 107 DEGs (d13–d60) with increased traits in AAC including metabolism of carbohydrates/fat-soluble vitamins, antigen presentation, ATPase activity, and B- and T-cell activation, while osteoclast differentiation and neutrophil degranulation decreased in AAC compared to INR. Our study identifies gene expression differences in high-risk cattle fed inorganic or amino acid-complexed mineral supplements, revealing adaptive immune and metabolic mechanisms that may be improved by organically sourced supplementation. Full article

Bovine spermatozoa are highly susceptible to oxidative stress (OS), and it is known to affect their cellular functions. The main leukocyte producers of reactive oxygen species (ROS) in mammalian semen are polymorphonuclear neutrophils (PMN). PMN activation can result in the formation of neutrophil extracellular traps (NETs), which have been shown to affect the motility and function of spermatozoa. However, OS effects on bull spermatozoa derived from individual NETs components have not been investigated. The hypothesis of this study was that specific NETs components might generate OS on bull spermatozoa. Bovine sperm cells were incubated with five NETs-associated molecules, including 30 μg/mL histone 2A (H2A), neutrophil elastase (NE), 1 μg/mL myeloperoxidase (MPO), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), for a time course ranging from 15 to 240 min. Fluorescence microscopy was used to evaluate the coincubation of bovine PMN and sperm cells. Within 15 min, H2A, NE, and LL-37 caused membrane disruption, while MPO and Cat-G caused OS on bull spermatozoa after 1 h of coincubation. NET formation was observed within 15 min of coincubation in co-cultures of bovine PMN/sperm cells. This study is the first to report on the role of cytotoxic OS effects caused by NETs-derived components in bovine sperm in vitro. Full article

Traditionally, non-aureus staphylococci and mammaliicocci (NASM) were not considered significant players in bovine mastitis. This study investigated the involvement of NASM (Staphylococcus hominis and Staphylococcus chromogenes) and lactic acid bacteria (LAB) strains (Weissella paramesenteroides) through bovine neutrophil responses. Bovine neutrophils displayed minimal apoptosis upon NASM and LAB challenge. Neutrophils expressed high TLR2 after challenge, but TLR6 expression varied and remained low in NASM pathogen recognition. Bovine neutrophils effectively engulfed and killed LAB, but their activity was significantly impaired against NASM. This was evident in S. chromogenes, where reduced TLR6 recognition and a weakened phagocytic response likely contributed to a lower bactericidal effect. Regardless of the bacteria encountered, intracellular ROS production remained high. S. chromogenes-challenged neutrophils displayed upregulation in genes for pathogen recognition (TLRs), ROS production, and both pro- and anti-apoptotic pathways. This response mirrored that of Weissella. except for CASP9 and BCL2, suggesting these bacteria have divergent roles in triggering cell death. Our findings suggest that S. chromogenes manipulates bovine neutrophil defenses through coordinated changes in functional responses and gene expression, while LAB strains have a weaker influence on apoptosis. Full article

High levels of non-esterified fatty acids (NEFAs) during the transition period lead to increased oxidative stress and immunosuppression in cows. Feeding them a vitamin-E-supplemented diet reduces reactive oxygen species (ROS) levels in the blood and diminishes immunosuppression in the transition period. However, whether the restoration of immune cell function occurs through the direct action of vitamin E in cells is still a topic that requires further discussion. Therefore, in this experiment, we aimed to investigate the effect of NEFAs on peripheral blood leukocytes (PBLs) and whether vitamin E mitigates the impact of NEFAs. We employed three groups: (1) blank, (2) NEFA only, and (3) pre-culturing with vitamin E before NEFA treatment (VENEFA). In peripheral blood mononuclear cells (PBMCs), there were no differences in vitamin E content among the three groups. However, in the vitamin E pre-treatment group, the vitamin E levels of polymorphonuclear neutrophils (PMNs) were significantly higher than those in the other two groups. NEFA levels increased malondialdehyde (MDA) levels in PBMCs, but pre-treatment with vitamin E reduced accumulation of MDA levels. Regarding the expression of proinflammatory genes, NEFAs increased the expression of interleukin-1β in PBMCs and colony-stimulating factor 2 in PMNs. Vitamin E pre-treatment restored the increase in interleukin-1β levels caused by NEFAs in PBMCs. None of the groups affected the phagocytosis of PMNs. Few studies have confirmed that NEFAs cause oxidative stress in bovine PBLs. In summary, this study found that NEFAs induce oxidative stress in PBLs and alter the expression of inflammation-related genes; meanwhile, vitamin E can reduce some of the effects caused by NEFAs. This result may suggest that vitamin E can assist bovine PBLs in resisting the immune suppression caused by an NEB during the transition period. Full article

The production of milk by dairy cows far exceeds the nutritional needs of the calf and is vital for the economical use of dairy cattle. High milk yield is a unique production trait that can be effectively enhanced through traditional selection methods. The process of lactation in cows serves as an excellent model for studying the biological aspects of lactation with the aim of exploring the mechanistic base of this complex trait at the cellular level. In this study, we analyzed the milk transcriptome at the single-cell level by conducting scRNA-seq analysis on milk samples from two Holstein Friesian cows at mid-lactation (75 and 93 days) using the 10× Chromium platform. Cells were pelleted and fat was removed from milk by centrifugation. The cell suspension from each cow was loaded on separate channels, resulting in the recovery of 9313 and 14,544 cells. Library samples were loaded onto two lanes of the NovaSeq 6000 (Illumina) instrument. After filtering at the cell and gene levels, a total of 7988 and 13,973 cells remained, respectively. We were able to reconstruct different cell types (milk-producing cells, progenitor cells, macrophages, monocytes, dendritic cells, T cells, B cells, mast cells, and neutrophils) in bovine milk. Our findings provide a valuable resource for identifying regulatory elements associated with various functions of the mammary gland such as lactation, tissue renewal, native immunity, protein and fat synthesis, and hormonal response. Full article

Neutrophils are innate immune cells that respond quickly to sites of bacterial infection and play an essential role in host defense. Interestingly, some bacterial pathogens benefit from exuberant neutrophil inflammation. Salmonella is one such pathogen that can utilize the toxic mediators released by neutrophils to colonize the intestine and cause enterocolitis. Because neutrophils can aid gut colonization during Salmonella infection, neutrophils represent a potential host-directed therapeutic target. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein that plays an essential role in many neutrophil effector responses. We hypothesized that inhibition of MARCKS protein would alter bovine neutrophil responses to Salmonella Typhimurium (STm) ex vivo. We used a MARCKS inhibitor peptide to investigate the role of MARCKS in neutrophil responses to STm. This study demonstrates that MARCKS inhibition attenuated STm-induced neutrophil adhesion and chemotaxis. Interestingly, MARCKS inhibition also enhanced neutrophil phagocytosis and respiratory burst in response to STm. This is the first report describing the role of MARCKS protein in neutrophil antibacterial responses. Full article

Our previous study showed that not only bovine lactoferrin (LF), the protein of milk and neutrophils, but also the human species forms complexes with oleic acid (OA) that inhibit tumor growth. Repeated injections of human LF in complex with OA (LF/8OA) to hepatoma-carrying mice decelerated tumor growth and increased animals’ longevity. However, whether the effect of the LF/8OA complex is directed exclusively against malignant cells was not studied. Hence, its effect on normal blood cells was assayed, along with its possible modulation of ceruloplasmin (CP), the preferred partner of LF among plasma proteins. The complex LF/8OA (6 μM) caused hemolysis, unlike LF alone or BSA/8OA (250 μM). The activation of neutrophils with exocytosis of myeloperoxidase (MPO), a potent oxidant, was induced by 1 μM LF/8OA, whereas BSA/8OA had a similar effect at a concentration increased by an order. The egress of heme-containing proteins, i.e., MPO and hemoglobin, from blood cells affected by LF/8OA was followed by a pronounced oxidative/halogenating stress. CP, which is the natural inhibitor of MPO, added at a concentration of 2 mol per 1 mol of LF/8OA abrogated its cytotoxic effect. It seems likely that CP can be used effectively in regulating the LF/8OA complex’s antitumor activity. Full article