Coordination of the primary defense mechanisms against pathogens relies on the appropriate expression of pathogen recognition receptors (PRRs) triggering the early release of effector molecules of the innate immune system. To analyze the impact of this system on the counteraction of infections of the mammary gland (mastitis), we characterized the bovine gene encoding the key PRR Toll-like receptor 9 (TLR9) and mapped its precise position on chromosome BTA22. The sequence information was used to establish real-time PCR quantification assays to measure the mRNA abundances of TLR9, TLR2, and TLR4 together with those of ␤-defensin 5 (BNBD5), an early bactericidal effector molecule of the innate system, in healthy and infected mammary glands. Mastitis strongly increased (4-to 13-fold) the mRNA abundances of all of these genes except TLR9. Slight subclinical infections already caused a substantial increase in the copy numbers, though they did so the least for TLR9. Induction was not systemic, since mRNA abundance was low in uninfected control quarters of the udder but high in the severely infected quarters of the same animal. The number of TLR2 copies correlated well with those of TLR4, indicating coordinated regulation of these two PRRs during infection of the udder. Their coordinated regulation explains our unexpected observation that pure Staphylococcus aureus infections caused a strong increase also in TLR4 mRNA abundance. In situ hybridizations revealed that BNBD5 is expressed predominantly in the mammary epithelial cells (MEC) of the infected gland. Our data therefore suggest a significant contribution of the innate immune system to counteract mastitis and attribute a prominent effector function to the MEC.

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Epithelial cells of the endometrium secrete prostaglandins to regulate the bovine oestrous cycle and form a functional barrier to microbes. However, bacterial infection of the endometrium commonly causes infertility in dairy cattle by disrupting endometrial physiology. Epithelial cell cultures are used to study the mechanisms of physiology and pathology, but 2D cultures may not reflect the 3D complexity of the epithelium. In this study, a polarised epithelial cell transwell culture was developed, using transepithelial resistance (TER), to monitor epithelial integrity. Polarised epithelial cells were treated with oxytocin and arachidonic acid to test physiological function and with lipopolysaccharide (LPS) to mimic bacterial infection. Supernatants were analysed for prostaglandin E 2 (PGE), prostaglandin F 2a , the chemokine interleukin-8 (IL8) and the ability of supernatants to induce neutrophil migration. Confluent epithelial cells established polarity when TER was O1800 Ucm 2 and predominantly released prostaglandins basolaterally. In contrast, IL8 from epithelial cells accumulated apically and the supernatants were highly chemotactic for neutrophils. The striking exception was when the epithelial cells were treated with LPS in the apical or basolateral compartment independently, which led to the release of IL8 towards the treated compartment. Although stromal cells also accumulated PGE and IL8 in response to treatment, co-culture of stromal cells in the well below polarised epithelial cells did not influence cellular responses. In conclusion, polarised endometrial epithelial cells vectorially released prostaglandins and chemokines to reflect their respective mechanistic roles in physiology and pathology.

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The task of spermatozoa is to transport its DNA-load as efficiently and safely as possible from the male organism to the female. Before it reaches its destination, it has to pass almost through the entire female reproductive tract, a potentially hostile environment. During passage, it is confronted by a sophisticated system that provides sperm storage sides but also possibly facilitates selection. The present review attempts to summarize the current knowledge of sperm interactions during that journey. A better understanding of the highly complex processes taking place between insemination and fertilization will be necessary to improve the efficiency of conventional reproductive techniques as well as for enabling the development and establishment of new ones.

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The somatotropic axis is a key metabolic pathway during transition from late pregnancy to early lactation in dairy cows. The first objective of this study was to determine the feasibility of selecting cows with persistent differences in total insulin-like growth factor 1 (IGF-1) concentration by taking only a single antepartum blood sample. The second objective was to elucidate the underlying causes of differences in peripheral IGF-1 concentrations throughout late pregnancy and whether hormonal axes also differed in dairy cows with low versus high IGF-1. Twenty clinically healthy Holstein Friesian cows were chosen based on their plasma IGF-1 concentration at 244 to 254 d after artificial insemination (AI) and other selection criteria (health status, body condition score, number of lactations). These cows were selected from a large-scale farm, transported to the clinic, and monitored daily from 261 to 275 d after AI. The concentrations of IGF-1, growth hormone, IGF binding proteins 2, 3, and 4, insulin, cortisol, thyroid hormones, progesterone, and estradiol were measured. Ultimately, 7 IGF-1-low and 7 IGF-1-high cows were statistically analyzed. Additionally, a liver biopsy was taken on d 270 ± 1 after AI for analysis of gene expression of somatotropic family members, liver deiodinase 1, and suppressor of cytokine signaling-2. It was possible to select cows with different IGF-1 concentrations based upon only 1 blood sample collected in late pregnancy. Concentrations of IGF-1 in IGF-1-low versus IGF-1-high animals (n=7 each) remained significantly different between groups from the day of selection of the animals until d 275 after AI. Second, the differences in total plasma IGF-1 concentration between experimental groups may be attributed to differences in hepatic production of acid labile subunit. The ability of IGFBP-3 to bind IGF-1 declined before calving in all cows. Furthermore, in addition to decreased mRNA expression of growth hormone receptor 1A and IGF-1 relative to calving, serum binding capacities for IGF-1 also decreased. Insulin-like growth factor binding protein 4 mRNA expression was higher in cows with low IGF-1 concentrations; this binding protein inhibits IGF-1 action at the tissue level and therefore may reduce IGF-1 bioavailability. Finally, other endocrine end points (e.g., insulin and thyroid hormones) differed between the 2 groups.

Impaired function of polymorphonuclear neutrophilic leukocyte (PMNL) during the peripartal period is a major reason for increased susceptibility of dairy cows to infections in this critical interval. Factors dysregulating PMNL function are widely unknown. Insulin-like growth factor (IGF-I) enhanced PMNL functions in vitro. The objective of this study was to investigate the influence of IGF-I and, additionally, β-hydroxybutyrate and nonesterified fatty acid concentrations on phagocytic activity (PA, percentage of viable PMNL) and phagocytic capacity (PC, mean fluorescence intensity of phagocytic PMNL) assessed by flow cytometry. Antepartum (i.e., wk -3, -2, -1; before calving), plasma concentrations of IGF-I were high (80-110 ng/mL) without significant differences between primiparous and pluriparous cows (n=18 and n=41, respectively). Concentrations of IGF-I declined toward the week of calving (wk 1). Postpartum (i.e., wk 2, 3, and 4; after calving), IGF-I remained lower than before parturition, with concentrations higher in primiparous compared with those of pluriparous cows. The PA was constant in primiparous cows throughout the study period. Conversely, PMNL of pluriparous cows had a significantly increased and higher PA in wk 2 and 3 postpartum compared with that of primiparous cows. The PC decreased significantly only in primiparous cows the week of calving, whereas the number of PMNL in primiparous cows exceeded that of pluriparous cows significantly. The phagocytic power (PP, a product of PA by PC), but not the phagocytic overall performance (POP, a product of PA, PC, and PMNL number), differed between primiparous and pluriparous cows in wk 3 postpartum. No significant differences in POP were found, except in wk 4 after calving between the primi- and pluriparous cows. In both groups, POP increased in the week of calving (wk 1). In contrast to β-hydroxybutyrate, which was weakly positive correlated with PA and PP in pluriparous cows in the transition period (wk -3 antepartum to wk 4 postpartum), pluriparous animals had weak negative correlations of PMNL number, PA, PP, POP, and IGF-I concentration in this period. In primiparous animals, only PP and PC were weakly negatively correlated with IGF-I in the transition period. Increased plasma IGF-I concentrations were not associated with enhanced phagocytosis function of bovine blood PMNL ex vivo and, thus, can not be regarded as a suitable predictor for this function.