BackgroundAs a result of climate change, dairy cows even in confinement systems are exposed to high ambient temperatures and environmental factors inducing heat stress. However, there are indications that chronic heat stress with reduced feed intake initially stimulates a systemic inflammatory response and progressively reduces the immunocompetence. This finally increases the disease susceptibility. This study aimed to elucidate the effects of chronic heat stress or equivalent reduced feed intake via pair-feeding at thermoneutrality on the daily hematological profile, leukocyte NF-κB p65 signaling pathway, immune function, and metabolism in peripheral blood mononuclear cells (PBMC) of dairy cows. MethodsPrimiparous, mid-lactating Holstein cows (n = 30) were assigned to heat-stressed (HS; temperature-humidity index (THI) 76, 28°C, relative humidity (RH) 50%), control (CON; THI 60, 16°C, RH 69%), or pair-fed (PF; THI 60, 16°C, RH 69%) group for 7 days. ResultsHS cows showed a lower number of erythrocytes, platelets, lymphocytes, hemoglobin, hematocrit, and iron concentration and increased endotoxin concentration compared to PF cows. The presence of NF-κB p65 in the nucleus of leukocytes was lower in HS than in the two other groups on day 5, while it was higher in HS cows on day 6. Furthermore, on day 6, RNA sequencing of PBMC showed an enrichment of higher expressed genes in pathways of platelet activation, coagulation cascade, leukocyte transendothelial migration, and focal adhesion in HS cows compared to both non-heat-stressed groups. However, T cell receptor signaling pathway, intestinal immune network for IgA production, antigen processing and presentation, and metabolic processes were expressed lower in HS compared to CON cows. ConclusionThe results suggest that heat stress induces dynamic changes of the red blood cell and lymphocyte profiles but only transiently activating the leukocytic NF-κB p65 signaling pathway while suppressing T cell signaling, both likely in response to the increased circulating endotoxin concentration. The activation of platelets and the coagulation cascade were likely due to heat-stress-induced microvascular injuries, altered hematocrit, or vasodilatation. Altered blood coagulation and immune responses need to be considered in the management of heat-stressed dairy cows.

Glutamine (Gln) supplementation has been shown to improve bodyweight (BW) in suckling low birthweight (LBiW) pigs. However, it is not known if Gln has lasting effects on growth and stress resilience beyond the supplementation period in LBiW suckling pigs. Therefore, we explored if post-Gln supplementation LBiW pigs show reduced stress during weaning and a lipopolysaccharide challenge. Twenty pairs of male German Landrace littermate piglets, originating from 14 L with 12 to 22 (live and stillborn) piglets, were selected at birth (0 d of age). One littermate was LBiW (mean 1.10 ± 0.02 kg; n = 20; below the lowest BiW quartile of the FBN pig facility) and the other normal (NBiW; mean 1.48 ± 0.02 kg; n = 20; within the middle 50th BiW quartile) birthweight. At 24 h postfarrowing, litter sizes were standardized to 12 piglets, and experimental LBiW and NBiW piglets assigned to Gln (1 g/kg BW/d) or alanine (Ala: isonitrogenous control to Gln; 1.22 g/kg BW/d) supplementation groups (LBiW-Gln, LBiW-Ala, NBiW-Gln, and NBiW-Ala). Piglets were orally supplemented 3 times daily (0700, 1200, and 1700), from 1 to 12 d of age, and remained with their birth litter until weaning at 28 d of age. At 55 d of age, pigs were challenged with lipopolysaccharide (i.p. 100 µg/kg BW) and euthanized at 61 d of age. The piglets were weighed, and their abdominal circumference, crown-rump length, body mass index and ponderal index were determined. Plasma metabolites, amino acids, TNF-α, and white blood cell counts were also measured. At birth, LBiW pigs were lighter (P = 0.002), had a shorter crown-rump length (P = 0.02), a smaller abdominal circumference (P = 0.02), a lower body mass index (P < 0.001), and plasma glucose (P = 0.07) but higher inositol (P = 0.001) levels, than their NBiW littermates. From pre- (27 d) to postweaning (32 d), the lymphocyte percentage decreased, and the segmented neutrophil percentage and the neutrophil to lymphocyte ratio increased in LBiW-Ala (P < 0.001), NBiW-Ala and NBiW-Gln (P < 0.05) pigs. Postlipopolysaccharide-challenge, TNF-α was lower at 1 h in Gln than Ala-supplemented pigs (P < 0.05). In conclusion, LBiW piglets had zootechnical and metabolic markers associated with impaired development at birth, and supplementation with Gln moderately improved immune markers of stress during weaning, and reduced the TNF-α peak in LBiW and NBiW pigs during a lipopolysaccharide challenge. However, no effect on LBiW piglet bodyweight was observed.

The milk replacer feeding regime in dairy calves has a great impact on metabolic and immunological functioning and affects animal welfare and lifetime performance. The feeding regime influences the rumen microbial composition, and epithelium-associated microbes may interact with the immune system of the host. We examined the correlations between blood leukocyte counts and the rumen epithelium-associated microbiome in dairy calves fed 2 different milk replacer feeding intensities and if these factors related to metabolic traits. Fourteen newborn female dairy calves were allocated to a group receiving either 10% (n = 7) or 20% (n = 7) milk replacer of their body weight (on average 41 kg) and provided ad libitum access to grass hay and concentrate pellets. At 3 weeks of life, all calves were fitted with a rumen cannula. Calves were weaned at 12 weeks of life and received a total mixed ration for ad libitum intake. Pre- (8-10 weeks of life) and post-weaning (21-23 weeks of life), methane production was measured in respiration chambers, and rumen epithelium and blood were sampled for 16S rRNA sequencing and leukocyte analyses, respectively. Pre-weaning, the reduced milk replacer feeding intensity was accompanied with higher concentrate intake but lower growth performance (P < 0.001), a higher abundance of amylolytic and lower abundance of cellulolytic epimural microbes. The group fed a low milk replacer intensity had also greater portions of monocytes (P = 0.031), CD8+ (P < 0.001), and CD14(+) (P = 0.044) leukocytes, suggesting elevated inflammatory conditions. Correlations between CD8(+) T cells and rumen methanogens, Ruminococcaceae, and Lachnospiraceae were recorded, but these were not consistent throughout maturation. Post-weaning, differences in feed intake and rumen microbial composition converged among milk replacer groups, while differences in growth performance (P = 0.040) and CD8+ cells (P < 0.001) were still present. In conclusion, a reduced milk replacer feeding intensity in dairy calves compromised growth performance and immunity and this effect persisted in the long-term. Significant correlations between the proportion of leukocytes and distinct epimural microbe taxa indicated an interplay between rumen epimural colonization and immune functioning of the host. However, further research is required addressing this interplay between rumen epimural microbes and immune functioning in dairy calves. (c) 2024 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Background In current German guidelines, the use of video laryngoscopy is one of the main quality features in the preclinical airway management of patients with severe injuries. The aim of the study is to evaluate the nationwide used emergency dataset MIND for the prediction of video laryngoscopy use in prehospital trauma patients requiring emergency anaesthesia with the help of machine learning and considering various application frequencies. Methods From a southwest German register we selected real-life datasets pertaining to severely injured adults who had received airway management from emergency physicians in the years between 2018 and 2020. With machine learning, the use of video laryngoscopy (VL) in preclinical adult trauma patients was modeled with a random forest algorithm and also clustered with k-means algorithm. Based on the original dataset (ratio 1:2,6), datasets with a 1:1 and a 2:1 ratio VL / no VL were created at random. Attributes chosen were initial vital signs, experience in airway management as well as injury pattern, auscultation findings, capnography, non-invasive ventilation, supraglottic airway devices, muscle relaxant, immobilisation of the cervical spine, declaration of a difficult airway situation and thoracic drainage insertion. Results Out of a total of 1,451 emergency anaesthesias, VL was applied in 277 of the 992 cases evaluated (28 %). With simu-lated growing usage frequency, sensiti-vity increased from 0.66 (original) up to 0.77 (2:1) and a positive predictive value rose from 0.5 (original) to 0.82 (2:1). The specificity and the negative predictive value decreased from 0.82 (original) to 0.58 (2:1) and from 0.9 (original) to 0.51 (2:1), respectively. The area under the receiver operating characteristic varied from 0.78 (original) to 0.77 (2:1). The cluster "VL" contained the attributes bronchial spasm, dyspnoea or cyanosis, non-invasive ventilation, age, Glasgow Coma Scale (GCS) and vital parameters. Conclusion The attributes of MIND could be used to develop a preliminary prediction model for video laryngoscopy usage in the field of prehospital trauma care. Also, clustering revealed a characteristic type of usage. In order to increase stability, further attributes like device type, airway evaluation or airway manage-ment approach could be included in future models.

Fistulation is a helpful procedure in animal nutritional research and also common practise in human medicine. However, there are indications that alterations in the upper gastrointestinal tract contribute to intestinal immune modulations. The present study aimed to investigate effects of a rumen cannulation in week 3 of life on the intestinal and tissue specific immune system of 34-week old heifers. Nutrition influences the development of the neonatal intestinal immune system to a high extent. Therefore, rumen cannulation was investigated in combination with different pre-weaning milk feeding intensities (20% (20MR) vs. 10% milk replacer feeding (10MR). Heifers of 20MR without rumen cannula (NRC) showed higher cluster of differentiation (CD)8+ T cell subsets in mesenteric lymph nodes (MSL) compared to heifers with rumen cannula (RC) and 10MRNRC heifers. CD4+ T cell subsets in jejunal intraepithelial lymphocytes (IELs) were higher in 10MRNRC heifers compared to 10MRRC heifers. CD4+ T cell subsets in ileal IELs were lower and CD21+ B cell subsets were higher in NRC heifers compared to RC heifers. CD8+ T cell subsets in spleen tended to be lower in 20MRNRC heifers compared to all other groups. Splenic CD21+ B cell subsets were higher in 20MRNRC heifers compared to RC heifers. Splenic toll like receptor (TLR) 6 expression was increased and IL4 expression tended to be increased in RC heifers than NRC heifers. Splenic TLR2, 3 and 10 gene expression was higher in 20MR compared to 10MR heifers. Jejunal prostaglandin endoperoxide synthase 2 expression was higher in RC heifers than NRC heifers, and MUC2 expression tended to increase in 20MR heifers compared to 10MR heifers. In conclusion, rumen cannulation modulated T and B cell subsets in the down streaming gastrointestinal tract and spleen. Pre-weaning feeding intensity seemed to affect intestinal mucin secretion and T and B cell subsets in MSL, spleen and thymus until several month later. Interestingly, in MSL, spleen and thymus the 10MR feeding regime evoked similar modulations of T and B cell subsets like rumen cannulation.

Ovarian cycle is controlled by circulating levels of the steroid hormone 17-beta-estradiol, which is predominantly synthesized by the granulosa cells (GCs) of ovarian follicles. Our earlier studies showed that unsaturated fatty acids (USFs) downregulate and saturated fatty acids (SFAs) upregulate estradiol production in GCs. However, it was unclear whether pituitary gonadotropins induce accumulation of free fatty acids (FFAs) in the follicular fluid since follicle-stimulating hormone induces and luteinizing hormone inhibits estradiol production in the mammalian ovary. Interestingly, we show here the gas chromatography analysis of follicular fluid revealed no differential accumulation of FFAs between pre-and post-luteinizing hormone surge follicles. We therefore wondered how estradiol production is regulated in the physiological context, as USFs and SFAs are mutually present in the follicular fluid. We thus performed in vitro primary GC cultures with palmitate, palmitoleate, stearate, oleate, linoleate, and alpha-linolenate, representing >80% of the FFA fraction in the follicular fluid, and analyzed 62 different cell culture con-ditions to understand the regulation of estradiol biosynthesis under diverse FFA combinations. Our analyses showed co -supplementation of SFAs with USFs rescued estradiol produc-tion by restoring gonadotropin receptors and aromatase, antagonizing the inhibitory effects of USFs. Furthermore, tran-scriptome data of oleic acid-treated GCs indicated USFs induce the ERK and Akt signaling pathways. We show SFAs inhibit USF-induced ERK1/2 and Akt activation, wherein ERK1/2 acts as a negative regulator of estradiol synthesis. We propose SFAs are vital components of the follicular fluid, without which gonado-tropin signaling and the ovarian cycle would probably be shat-tered by USFs.

Ovulation is an inflammation-like process, and cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E2 (PGE2) is its key mediator. Balanced regulation of inflammatory processes in high-yielding dairy cows may be essential for physiological ovulation and fertility. This study aimed to elucidate the mechanisms underlying ovulation failure and cyst development after disturbing intrafollicular inflammatory cascades. Therefore, nonselective (indomethacin and flunixin-meglumine), COX-2 selective (meloxicam), and highly COX-2 selective (NS-398) inhibitors were injected into preovulatory follicles 16 h after administration of GnRH, and ovulation was monitored via ultrasound examination. Additionally, follicular fluid was collected after injection of indomethacin, meloxicam, and NS-398. Moreover, primary granulosa cell cultures from preovulatory follicles were prepared and treated with indomethacin, meloxicam, and NS-398. The concentrations of 17β-estradiol, progesterone, and prostaglandin E2 (PGE2) in the follicular fluid and cell supernatant were estimated. Indomethacin and flunixinmeglumine blocked ovulation, even at low doses, and led to ovarian cyst development. The selective and highly selective COX-2 inhibitors meloxicam and NS-398 were not effective in blocking ovulation. However, indomethacin, meloxicam, and NS-398 significantly and comparably reduced PGE2 concentration in vivo and in vitro (P < 0.05) but had no effect on estradiol or progesterone production. This may contradict the generally accepted hypothesis that PGE2 is a key mediator of ovulation and progesterone production. Our results suggest a connection between ovarian disorders and inflammatory actions in early postpartum cows.

Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.

Sialylated milk oligosaccharides and glycoconjugates have several positive effects on the mucosal barrier, the gut microbiome, and an effective immune system. For this reason, they are important biomolecules for mammary gland health and optimal development of offspring. In milk, the major sialic acid, N-acetylneuraminic acid (Neu5Ac), can be attached as monosialyl-residues or as polymers. To investigate the sialylation processes during lactation of German Holstein cows, we analyzed udder tissue in addition to milk at different time points of lactation. The analysis of the milk samples revealed that both the levels of Neu5Ac and its polymer, polysialic acid (polySia), rapidly decreased during the first three days of lactation, and a high interindividual variance was observed. In mature milk, however, the sialylation status remains relatively constant. The results indicate that mammary gland epithelial cells are one source for milk polySia, since immunohistochemistry of udder tissue exhibited strong polySia staining in these cells. Furthermore, both polysialyltransferases, ST8SiaII and ST8SiaIV, are expressed. Based on known functions of monosialyl residues and polySia, we discuss the potential impact of these biomolecules and the consequences of the heterogeneous sialylation status of milk in relation to udder health and offspring health.

Aquaculture management involves regular handling procedures, but these can evoke stress responses in farmed fish. We compiled an extensive list of published parameters that indicate the most likely handling-induced physiological deviations from the norm. However, since these parameters are based almost exclusively on studies of rainbow trout and Atlantic salmon, we conducted a handling-challenge experiment with maraena whitefish ( Coregonus maraena ). This salmonid fish was sampled at either 3 or 24 h after a single 1-min handling or after 10 days of daily repeated 1-min handling. The cortisol levels were strongly elevated in some individuals at 3 h after the single handling challenge, but these elevations were not significantly different between the challenged and control cohorts. The phagocytic capacity of myeloid head-kidney cells stimulated with fluorophore-labeled, inactivated Aeromonas salmonicida was significantly decreased in maraena whitefish at 3 h after the handling challenge compared to control fish. Microarray analysis of head-kidney samples from the challenged and control fish revealed 12 differentially expressed genes at 3 h and 70 at 24 h after the single handling episode, but only 5 differentially expressed genes after 10 days of repeated daily handling. The identified genes were assigned to numerous stress- and immune-relevant functional pathways, including “glucocorticoid receptor signaling” (3 h post-challenge), “HIF1A signaling” (24 h post-challenge), or “complement system” (10 days of repeated challenge). Our data reveal the tight interconnection of immune and stress pathways in the head kidney of maraena whitefish and corroborate several parameters previously found regulated in other tissues of handling-stressed rainbow trout. These findings indicate that handling may compromise the health and welfare of maraena whitefish in aquaculture.

Background Patients after cardiac arrest developing acute liver failure (ALF) show higher fatality rates and worse outcomes. As machine learning is able to support physicians in their decision-making with the help of big data in health records, the aim of this study is to evaluate decision-tree models for the prediction of ALF after resuscitation. Patients and methods The study is a secondary analysis of 347 Belgian patients after successful resuscitation from 2007 to 2015, whose anonymised dataset was published under the Creative Common Attribution Licence. For machine learning, a J48 decision-tree model as well as its AdaBoost variation and a random forest model were created. Evaluation was performed by sensitivity, specificity, positive / nega-tive predictive value (PPV / NPV) and by the area under the receiver operator curve (AUC-ROC). Results 184 patients (53 %) developed ALF after resuscitation. The main split points in J48 were the attributes international normalised ratio ad admission (INR, threshold 1.48) and total bilirubin ad ad-mission (threshold 0.99 mg/dl). Further splitting was performed in chronic renal and heart failure, administered amount of epinephrine during resuscitation, and the use of beta-lactam antibiotics. J48 yielded a sensitivity of 0.8 and a speci-ficity of 0.72 (0.73 PPV / 0.79 NPV, AUC-ROC 0.83). AdaBoost yielded a compa-rable sensitivity (0.79) and a higher PPV (0.75) and specificity (0.74, NPV 0.77, AUC-ROC 0.85). Random forest reached a sensitivity of 0.86 (PPV 0.74) and a specificity of 0.74 (NPV 0.84, AUC-ROC 0.87). The random forest achieved a significantly higher test quality in AUC-ROC than the other models. Conclusion Machine learning with decision-tree models seems to be suitable for the prediction of ALF after resuscitation. The clinical risk of developing ALF could be increased in conditions with elevated INR and total bilirubin, chronic renal and heart failure as well as prolonged resuscitation. Based on the applied at-tributes and developed models, further studies in large cohorts could lead to an advancement of the prediction models and therefore support physicians in diagnosis and decision-making.

Background: Pre-existing pre-operative anaemia is associated with an adverse post-operative outcome. The aim of this study was to analyse the influence of a pre-operative anaemia, especially one that is caused by iron-deficiency, on the post-operative outcome after major non-cardiothoracic surgery. Methods: This was a retrospective study with 1,192 patients who had undergone non-cardiothoracic surgery. The patients were divided into three groups according to their pre-operative haemoglobin levels (group I: Hb >12 g/dl (n=795), II: Hb 10-12 g/dl (n=254), III: Hb <10 g/dl (n=143)). MCH and MCV were used as a screening parameter to classify iron deficiency anaemia. The number of transfused RBCs (red blood cell concentrates), the length of stay as well as postoperative adverse events and in-hospital mortality were evaluated. Results: More RBCs were transfused in patients with pre-existing anaemia (p< 0.001). Post-operative adverse events were more likely in patients with anaemia (p <0.001). In-hospital mortality was higher in patients with severe pre-operative anaemia (p =0.002). Conclusion: Pre-existing anaemia in non-cardiothoracic surgery patients is associated with more transfusions, a higher level of adverse outcomes and a higher in-hospital mortality. Using MCH and MCV as screening parameters helps to detect patients with an iron deficiency and start a patient blood management programme prior to surgery. This may help to reduce adverse outcomes after major surgery.

In der Notfallmedizin ist die Durchführung der inhalativen Therapie auch durch Rettungsfachpersonal vorgesehen. Standard Operating Procedures (SOP) und Handlungsempfehlungen zum Einsatz sind nicht bundeseinheitlich und liegen im Falle der Vernebelung von Notfallmedikamenten nicht in allen Bundesländern und Rettungsdienstbereichen vor. Es existieren auch keine Empfehlungen, welcher Verneblertyp genutzt werden soll. Die Medikamentendepositionsraten von Jet- und Mesh-Verneblern unterscheiden sich aber erheblich; klinisch ist die Verwendung von Mesh-Systemen mit einem besseren Patienten-Outcome assoziiert. Die vorliegende Umfrage sollte die Praxis des Vernebelns von Notfallmedikamenten im süddeutschen Rettungsdienst evaluieren. Ein besonderes Augenmerk wurde auf den eingesetzten Verneblertyp sowie den Einfluss von SOP und Handlungsempfehlungen auf die Anwendung gelegt. Insgesamt 4800 in Baden-Württemberg, Bayern und Rheinland-Pfalz tätige Notfallsanitäter*innen und Rettungsassistent*innen erhielten insgesamt 17 Fragen zur Durchführung der Medikamentenvernebelung in ihrer alltäglichen Praxis. Der Jet-Vernebler ist mit Abstand der am häufigsten eingesetzte Verneblertyp. Die Depositionsraten sowohl des Jet- als auch des Mesh-Verneblers werden deutlich überschätzt. Nur 67,3 % aller Befragten vernebelten im Rahmen von SOP oder Handlungsempfehlungen. Das Vorhandensein dieser war allerdings maßgeblich für den Einsatz der Vernebelung ohne Beisein eines*er Notarzt*ärztin und die gefühlte Sicherheit beim Vernebeln. Der flächendeckende Einsatz von Mesh-Verneblern könnte die Therapie von Notfallpatient*innen homogenisieren und möglicherweise das Outcome verbessern. SOP und Handlungsempfehlungen zur Anwendung von Vernebelung in der Notfallsituation könnten die inhalative Therapie verbessern und homogenisieren.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

The functional spectrum of the teleostean head kidney covers haematopoietic, immune and endocrine signalling pathways with physiological effects that are likely to conflict if activated at the same time. An in vivo experiment on the salmonid fish maraena whitefish (Coregonus maraena) revealed that the head kidney shows a remarkably strong response after injection of Aeromonas salmonicida within 48 h. In order to investigate the potential influence of endocrine signalling on the initiation of immune responses, we established a primary culture of head-kidney cells of maraena whitefish. For the characterisation of this model system, we used flow cytometry complemented with an extensive panel of immunological/haematological and stress-physiological/neuroendocrinological qPCR assays. More than one third of the cells expressed the characteristic signature of myeloid cells, while more than half of the cells expressed those genes typical for lymphocytes and monocytes. In parallel, we quantified the expression of genes encoding endocrine receptors and identified ADRA2D as by far the most highly expressed adrenergic-receptor gene in head-kidney cells. The stimulation of the head-kidney cells with toll-like receptor ligands induced the expression of typical immune genes (IL1B, CXCL8, TNF, SAA) after only 1 h. The incubation with the stress hormones cortisol, adrenaline and noradrenaline also had an immune-activating effect, though less pronounced. However, cortisol had the strongest suppressive effect on the stimulation-induced immune response, while adrenaline exerted a comparably weaker effect and noradrenaline was almost ineffective. Moreover, we found that cortisol reduced the expression of genes coding for adrenergic and some glucocorticoid receptors, while noradrenaline increased it. In conclusion, the primary head-kidney cells of maraena whitefish reflect the immunological and neuroendocrinological diversity of the entire organ. This in vitro system allowed thus identifying the correlative changes between the activities of hormones and immune factors in salmonid fish in order to contribute to a better understanding of the regulation circuit between stress and immune defence.

Conjugated linoleic acid (CLA) isomers are known for their health-promoting effects in mammals and metabolic functions in dairy cows and are synthesized in the forestomach depending on essential fatty acid (EFA) intake. The current preliminary study investigated effects of a maternal fatty acid supplementation (MFAS) during late pregnancy and early lactation with coconut oil (CON, control), CLA (Lutalin®), or CLA + EFA (Lutalin® linseed oil; safflower oil) on plasma fatty acid composition and T and B cell subsets in mesenteric lymph nodes (MLN) and the small intestine of 5-day-old calves. MFAS of CLA + EFA increased α-linolenic, eicosapentaenoic, docosapentaenoic, and n -3 fatty acid proportions in calf plasma fat on days 1 and 5 after birth ( P < 0.05). On day 5, CLA and CLA + EFA calves showed higher plasma fat trans -10, cis -12 CLA proportions, and CLA calves had higher plasma cis -9, trans -11 CLA proportions compared with CON calves ( P < 0.1). MFAS of CLA tended to increase CD4 + T cell subsets in MLN and increased CD21 + B cell subsets in ileal lamina propria compared with CON but decreased CD2 + T cell subsets in jejunal lamina propria ( P < 0.05). CLA + EFA decreased CD4 + T cell subsets in MLN compared with CLA ( P < 0.05). MFAS of CLA seemed to affect the intestinal adaptive immune system of calves, but additional EFA supplementations reversed CLA effects. Possible direct CLA and EFA effects or whether changes in milk composition affected this immune modulation must be clarified in further studies.

The aim of this study was to establish a model to induce cystic ovarian follicles (COFs) in cattle using the cyclooxygenase inhibitor, indomethacin. Eighteen Holstein-Frisian cattle were synchronized with prostaglandin F2alpha (PGF2α) and gonadotropin-releasing hormone (GnRH). Ultrasound-guided transvaginal intrafollicular injections were performed in 23 preovulatory follicles with different concentrations of indomethacin 16 h after GnRH administration. An injection of 0.2 ml 35 µM indomethacin solution (resulting in a final concentration of 8 µg/ml in the follicular fluid) was the minimal dosage leading to COF formation. The induced COFs reached a maximum mean diameter of 36.9 ± 4.5 mm eleven days after injection. The estrous cycle was extended to 25–39 days. Luteinization was first observed 4 days after injection, accompanied by a slight increase in plasma progesterone concentration. The bioactivity of indomethacin was demonstrated by the decrease of prostaglandin E2 in the follicular fluid of three animals. The method presented here is minimally invasive and allows for the generation of defined COFs for further investigations.

Synovial mesenchymal stem cells (SMSCs) have become a great cell source for musculoskeletal stem cell research, especially related to cartilage and bone tissue regeneration, due to their superior cell proliferation properties and multidifferentiation potential into various cell lineages. This study revealed isolation methods, culture conditions, and morphological and molecular characterization of SMSCs derived fibrous synovium (FS) and adipose synovium (FP) of two pig breeds differing in growth performance [German Landrace (DL), and fat deposition (Angeln Saddleback (AS)]. Herein, FS possessed nucleated cell numbers nearly twice as high as those of FP at Passage 0. SMSCs derived from different types of synovial membrane and genetic background show similar cell morphologies and immunophenotypes, which were assessed by cell surface epitopes and multilineage differentiation potential, but differ significantly in their molecular characteristics. In addition, transcripts of SMSCs from AS were more enriched in IGF-1 signaling and VEGF ligand receptor, while SMSCs from DL were more enriched in growth hormone signaling and bone metabolism. The results indicate that genetics and tissues play significant roles for SMSC characteristics so that SMSCs can be traced back to the original cell donor and be used for fine turning in applications of medical research and therapies.

Here we assessed the effects of dietary essential fatty acids on the developmental competence of oocytes in cows and on the functionality of follicular granulosa cells (GC). Lactating German Holstein cows were supplemented from week 9 ante partum (ap) until week 8 post-partum (pp) in four dietary groups designed as (i) control (CTRL: coconut oil), (ii) essential fatty acid (EFA: linseed and safflower oil), (iii) conjugated linoleic acid (CLA: Lutalin®), and (iv) EFA+CLA (mixture of linseed oil, safflower oil and Lutalin®). EFA, CLA or EFA+CLA supplementation did not improve in vitro embryo production. However, higher proportions of α-linolenic acid (ALA) and cis-9, trans-11 CLA were observed in the follicular fluid suggesting the exposure of GC to relatively high levels of ALA and cis-9, trans-11 CLA. Consequently, we tested different concentrations of ALA and cis-9, trans-11 CLA in a bovine GC culture model for their effects on steroid production, marker gene expression and viability. Both fatty acids upregulated CD36 and downregulated the expression of FOXL2, while ALA significantly increased SOX 9 transcript levels. Both ALA and cis-9, trans-11 CLA reduced the CCND2 expression and cis-9, trans-11 CLA induced apoptosis. ALA and cis-9, trans-11 CLA significantly down-regulated the expression of STAR, CYP19A1, FSHR, LHCGR and decreased the 17β-Estradiol (E2) and progesterone (P4) production. In conclusion, dietary lipids did not improve in vitro embryo production, while ALA and cis-9, trans-11 CLA affected the morphology and functionality of GC. This could suggestively lead to compromised follicle development and ovarian cyclicity in dairy cows.

The cellular glycocalyx of vertebrates is frequently decorated with sialic acid residues. These sialylated structures are recognized by sialic acid-binding immunoglobulin-type lectins (Siglecs) of immune cells, which modulate their responsiveness. Fifteen Siglecs are known to be expressed in humans, but only four Siglecs are regularly present in fish: Siglec1, CD22, myelin-associated glycoprotein (MAG), and Siglec15. While several studies have dealt with the physiological roles of these four Siglecs in mammals, little is known about Siglecs in fish. In the present manuscript, the expression landscapes of these Siglecs were determined in the two salmonid species Oncorhynchus mykiss and Coregonus maraena and in the percid fish Sander lucioperca. This gene-expression profiling revealed that the expression of MAG is not restricted to neuronal cells but is detectable in all analyzed blood cells, including erythrocytes. The teleostean MAG contains the inhibitory motif ITIM; therefore, an additional immunomodulatory function of MAG is likely to be present in fish. Besides MAG, Siglec1, CD22, and Siglec15 were also expressed in all analyzed blood cell populations. Interestingly, the expression profiles of genes encoding Siglecs and particular associated enzymes changed in a gene- and tissue-specific manner when Coregonus maraena was exposed to handling stress. Thus, the obtained data indicate once more that stress directly affects immune-associated processes.