Highlights of the mini-symposium on extracellular vesicles in inter-organismal communication, held in Munich, Germany, August 2018.

All living organisms secrete molecules for intercellular communication. Recent research has revealed that extracellular vesicles (EVs) play an important role in inter-organismal cell-to-cell communication by transporting diverse messenger molecules, including RNA, DNA, lipids and proteins. These discoveries have raised fundamental questions regarding EV biology. How are EVs biosynthesized and loaded with messenger/cargo molecules? How are EVs secreted into the extracellular matrix? What are the EV uptake mechanisms of recipient cells? As EVs are produced by all kind of organisms, from unicellular bacteria and protists, filamentous fungi and oomycetes, to complex multicellular life forms such as plants and animals, basic research in diverse model systems is urgently needed to shed light on the multifaceted biology of EVs and their role in inter-organismal communications. To help catalyse progress in this emerging field, a mini-symposium was held in Munich, Germany in August 2018. This report highlights recent progress and major questions being pursued across a very diverse group of model systems, all united by the question of how EVs contribute to inter-organismal communication.

Gene expression profiling in pbMEC - in search of molecular biomarkers to predict immunoglobulin production in bovine milk.

BACKGROUND: Optimization of the immunoglobulin (Ig) yield in bovine milk used as therapeutic immune milk or whey for the prevention of Clostridium difficile-associated diarrhea in humans is of great importance to improve the economic efficiency of production. Individual dairy cows have diverse immune responses upon vaccination, resulting in a variable Ig yield in blood and milk. Therefore, it is advisable to pre-select cows with the best ability to produce and secrete high yields of specific Igs. RESULTS: The gene expression profile of pbMEC (primary bovine mammary epithelial cells), challenged with the gram-positive, non-mastitis, pathogen Clostridium difficile showed distinct and significant differences in the gene expression of effector molecules of the innate immune system. A number of genes were identified that could possibly serve as molecular biomarkers to differentiate high responder cows from low responder cows. These identified genes play key roles in the promotion of innate immunity. CONCLUSION: Using a gene expression profiling approach, we showed that upon others, especially the gene expression of the pro-inflammatory cytokines was altered between the high and low responder cows. Those genes are indicated as potential molecular biomarkers in the pre-selection of cows that are able to secrete high immunoglobulin yields in milk.

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary.

The aim of this study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol-17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1-2, 3-4, 6-7, >8). Experiment 3: Cows on days 8-12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.

Comparison of the immune competence of Turopolje, German Landrace × Turopolje, and German Landrace × Pietrain pigs after PRRSV vaccination.

The competences of the immune systems of the ancient pig breed Turopolje (T×T), German Landrace × Turopolje (L×T) and 'modern' pig breed German Landrace × Pietrain (L×P) were compared in this study. All pigs were immunized with a modified live vaccine against 'Porcine Reproductive and Respiratory Syndrome' (PRRS) virus (Ingelvac PRRS MLV(®)) to simulate an infection. Antibody production against PRRS MLV was evaluated in serum. Elimination of the viral infectious fragments during the experimental period was monitored in serum, leukocytes and tonsils by RT-qPCR. Furthermore relevant immune marker genes were quantified either on gene expression level using RT-qPCR [toll like receptor (TLR) 7, TLR8, TRAF6, CD163, SIGLEC1, CD4, CD8, CD14, CD19, tumor necrosis factor alpha (TNFα), interleukin (IL) 1, IL2, IL6, IL12], and on protein level using ELISA [interleukin (IL)-1, IL-2, IL-6, and IL-12]. The three breeds showed individual inactivation efficiencies as a reaction to the PRRS MLV vaccination. T×T eliminated the virus in serum within 16 days, followed by L×T (28 days) and L×P (36 days). The antibody titers against PRRS MLV of L×T and L×P were significantly higher compared to T×T (p<0.05). The gene expression data and protein analysis of interleukins revealed that T×T reacted with a type 1 immune response. In contrast, the two other breeds (L×T and L×P) showed a type 2 immune response, which resulted in the higher synthesis of B-cells and an increased concentration of specific anti-PRRS MLV antibodies.

Angiogenesis in The Ovary - The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow - An Overview.

In the ovary, the development of new capillaries from pre-existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), angiopoietin (ANPT) and hypoxia-inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5-5, 5-20, 20-180 and >180 ng/ml FF). The corresponding sizes of follicles were 5-7, 8-10, 10-13, 12-14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12 13-16 and >18 of the oestrous cycle and months 1-2, 3-4, 6-7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT-qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF-A, FGF-2, IGF-1 and IGF-2, ANPT-2/ANPT-1 and HIF-1-alpha was found during final follicle maturation and in CL during the early luteal phase (days 1-4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.

Effect of non-starch-polysaccharide-degrading enzymes as feed additive on the rumen bacterial population in non-lactating cows quantified by real-time PCR.

The effects of non-starch-polysaccharide-degrading enzymes, added to a maize silage- and grass silage-based total mixed ration (TMR) at least 14 h before feeding, on the rumen bacterial population were investigated. Six non-lactating Holstein Friesian cows were allocated to three treatment groups using a duplicate 3 × 3 Latin square design with three 31-day periods (29 days of adaptation and 2 days of sampling). Treatments were control TMR [69% forage and 31% concentrates on a dry matter (DM) basis] or TMR with 13.8 or 27.7 ml/kg of feed DM of Roxazyme G2 liquid with activities (U/ml enzyme preparation) of xylanase 260 000, ß-glucanase 180 000 and cellulase 8000 (DSM Nutritional Products, Basel, Switzerland). The concentrations of 16S rDNA of Anaerovibrio lipolytica, Fibrobacter succinogenes, Prevotella ruminicola, Ruminococcus flavefaciens, Selenomonas ruminantium and Treponema bryantii, and their relative percentage of total bacteria in rumen samples obtained before feeding and 3 and 7 h after feeding and from two rumen fractions were determined using real-time PCR. Sampling time had only little influence, but bacterial numbers and the composition of the population differed between the transition layer between rumen fluid and the fibre mat (fraction A) and the rumen fluid (fraction B) highlighting the importance to standardize sampling. The 16S rDNA copies of total bacteria and the six bacterial species as well as the population composition were mainly unaffected by the high levels of exogenous enzymes supplemented at all sampling times and in both rumen fractions. Occasionally, the percentages of the non-fibrolytic species P. ruminicola and A. lipolytica changed in response to enzyme supplementation. Some increases in the potential degradability of the diet and decreases in lag time which occurred collaterally indicate that other factors than changes in numbers of non-particle-associated bacteria are mainly responsible for the effects of exogenous enzymes.

Effect of magnetic stimulation on the gene expression profile of in vitro cultured neural cells.

Transcranial magnetic stimulation is a non-invasive tool in clinical diagnostics and therapy for physiological and psychological diseases and has an increased application in experimental neurophysiology. Despite this, the mechanisms of magnetic stimulation of the central nervous system remain still unclear. We applied sinus-shaped high frequency magnetic fields in different stimulation patterns and repeated treatments to cell cultures derived from frontal cortex of murine embryos (BALB/cOlaHsd mice) to elucidate the effects of repetitive magnetic stimulation on the gene expression of in vitro cultured neural cells. Gene expression profiling was performed by using qRT-PCR array and single qRT-PCR analyses. Our methodological approach using microelectrode arrays data recording and analysis minimizes variations in transcriptome analysis arising from cell differentiation status and tissue complexity. With 10 significant changes in gene expression out of 171 genes using Alzheimer disease and neurodegeneration related qRT-PCR arrays we demonstrate significant impact of repetitive magnetic stimulation on the mRNA transcript of neural cell cultures. Sixteen candidate genes were analyzed using single qRT-PCR in a replicated statistical design, which provided more precise estimates of differences in expression profiles. We discussed the utility of the experimental methods used for cell culture selection and the changes in gene expression considering physiological aspects.

Quantification noise in single cell experiments.

In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1ß, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis.

Influence of anabolic combinations of an androgen plus an estrogen on biochemical pathways in bovine uterine endometrium and ovary.

The application of anabolic steroids in food producing animals is forbidden in the EU since 1988, but the abuse of such drugs is a potential problem. The existing test systems are based on known compounds and can be eluded by newly emerging substances. The examination of physiological effects of anabolic hormones on different tissues to indirectly detect misuse might overcome this problem. Two studies were conducted with post-pubertal 24-months old Nguni heifers and pre-pubertal female 2-4 weeks old Holstein Friesian calves, respectively. The animals of the accordant treatment groups were administered combinations of estrogenic and androgenic compounds. The measurement of the gene expression pattern was undertaken with RT-qPCR. Target genes of different functional groups (receptors, angiogenesis, steroid synthesis, proliferation, apoptosis, nutrient metabolism and others) have been quantified. Several biochemical pathways were shown to be influenced by anabolic treatment. Both studies identified significant regulations in steroid and growth factor receptors (AR, ERß, LHR, FSHR, Flt-1, PR, IGF-1R, Alk-6), angiogenic and tissue remodeling factors (VEGFs, FGFs, BMPs, ANGPT-2, MMPs, TIMP-2, CTSB), steroid synthesis (S5A1, HSD17, CYP19A1), proliferation (TNFα, IGF-1, IGFBPs, p53, c-fos; CEBPD, c-kit), apoptosis (CASP3, FasL, p53) and others (C7, INHA, STAR). Several genes were regulated to opposite directions in post-pubertal compared to pre-pubertal animals. PCA for Nguni heifers demonstrated a distinct separation between the control and the treatment group. In conclusion, anabolics modify hormone sensitivity and steroid synthesis, and they induce proliferative effects in the whole reproductive tract (uterus and ovary) as well as anti-angiogenic effects in the ovary. However, the extent will depend on the developmental stage of the animals.

The potential of bovine vaginal smear for biomarker development to trace the misuse of anabolic agents.

In the European Union the use of anabolic hormones in meat production is forbidden since 1988 and this ban of anabolic agents in animal production is strictly controlled. New hormone cocktails passing the detection systems are attractive for the practice and so new approaches to discover their illegal use have to be developed steadily. Verifying physiological effects caused by anabolic steroids will be a new way to develop potential monitoring systems. One promising matrix in female animals will be vaginal smear containing vaginal epithelial cells, because the vaginal epithelium is a primary steroid hormone responsive organ. In this study we quantified the gene expression in vaginal smear of sexually mature cattle in order to observe physiological effects. Further we aimed to establish a new screening method by testing the effect of a combination of certain anabolic steroid hormones on physiological regulations of mRNA expression of selected genes. In an animal trial Nguni heifers were treated with the anabolic combination trenbolone acetate plus estradiol. Vaginal smear samples were taken at 4 different time points. Gene expression of 27 candidate genes, selected by screening the actual literature for steroidal effects on vaginal epithelial cells, were estimated using quantitative real-time RT-PCR. There were different expression changes observed at different time points. It could be shown that the applied anabolic combination significantly influenced the expression of the steroid receptor ERα, the keratinization factor CK8, the proinflammatory interleukins IL-1α and IL-1ß, the growth factors FGF7, EGF, EGFR, IGF-1R, TGFα and LTF, the oncogen c-jun and other factors like actinß and ubiquitin 3. Using biostatistical tools like principal components analysis or hierarchical cluster analysis, the potential to develop a gene expression pattern for targeting the illegal use of growth promoters could be demonstrated.

Effects of the prebiotics inulin and lactulose on intestinal immunology and hematology of preruminant calves.

Prebiotics are suggested as an alternative to antibiotics in animal rearing. Fermentable substances such as inulin or lactulose have been proposed to stimulate the immune system and health by modulation of the intestinal flora and its fermentation products. In this study, effects of inulin and lactulose on the intestinal health and hematology of calves have been investigated. Both prebiotics significantly decreased thrombocyte counts in peripheral blood. Only inulin was able to increase hemoglobin concentration and hematocrit. Total leukocyte count was decreased by lactulose while both prebiotics tended to lower monocyte proportions. mRNA expression of inflammation-related markers in the intestine was also affected by both prebiotics hinting at a decreased inflammatory status. This may be due to a possible decrease in intestinal pathogen load that remains to be verified. Only mRNA amounts of interleukin 8 were increased by lactulose in mesenteric lymph nodes. In the ileum, expression of a proliferation marker was increased by inulin while an apoptosis-related gene was increased by both prebiotics. The results of this study show a clear effect of prebiotics on certain parameters associated with animal health and performance that remain to be studied in detail in future investigations.

mRNA and microRNA quality control for RT-qPCR analysis.

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency. RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity.

Effects of inulin and lactulose on the intestinal morphology of calves.

For some time now prebiotics have been proposed to improve health by stimulation of beneficial bacteria in the intestine of humans and animals. The current study is aiming to show effects of feeding of either 2% inulin or 2% lactulose in milk replacer on performance and intestinal morphology of male Holstein-Friesian calves. After 20 weeks of feeding inulin led to significantly higher daily weight gains than lactulose while control animals ranged between the experimental feedings. Ingestion of milk replacer was reduced in lactulose treated animals. Additionally differences of villus height in jejunum (P = 0.07) and ileum (P = 0.03) could be found with an increase for lactulose treated animals and a decrease for inulin treated animals. In ileum the density of proliferative epithelial cells tended to be lower in inulin treated and higher in lactulose treated animals (P = 0.08). Both inulin and lactulose tended to decrease the quantity of goblet cells in the tips of ileal villi (P = 0.07). Both prebiotics can affect performance and intestinal morphology of calves and may as such affect animal health. But effects differ between substances.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle.

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

The immunomodulatory effect of lactulose on Enterococcus faecium fed preruminant calves.

Prebiotics and probiotics could represent an effective alternative to the use of synthetic antibiotics in nutrition. The mechanisms by which prebiotics affect the immune system have not yet been investigated in detail. Most effects have been attributed to increases in the innate and acquired immune responses. This study was conducted to elucidate the long-term effects of orally administered lactulose on the immune response in the intestinal tract of probiotic-fed calves. Preruminant calves were randomized to 3 feeding groups: milk replacer containing 1) no lactulose, 2) 1% lactulose, or 3) 3% lactulose. All 3 milk replacers contained 10(9) cfu Enterococcus faecium/kg. Messenger RNA expression of different cell activation markers, pro- and antiinflammatory cytokines, and IgA Fc receptor was investigated in the ileum, mesenterial lymph node, spleen, and white blood cells. A significantly greater number of blood lymphocytes were detected in the 3% lactulose group (P = 0.02) than in the control group. The expression results in male calves indicated that the transcription of IgA Fc receptor in the ileal mucosa of the 1% lactulose treatment group increased significantly (P = 0.04) and also tended to increase in the 3% lactulose group (P = 0.07). Furthermore, decreases in IL-10 and interferon-gamma mRNA expression were observed in the ileum (P = 0.04). The CD4-presenting lymphocytes were decreased significantly in the ileum (P = 0.04) and mesenteric lymph node (P = 0.01), whereas CD8-presenting lymphocytes were increased in the blood (P = 0.03) of females. Other proinflammatory cytokines (IL-1beta, IL-8, and tumor necrosis factor-alpha) and antiinflammatory cytokines (transforming growth factor-beta1) did not show significant differences in mRNA expression among treatment groups. The results indicate that additional lactulose feeding had an immunomodulatory effect on the composition of T-cell subsets in different immune compartments and had minor effects on pro- and antiinflammatory cytokine mRNA expression.

Gene expression in hair follicle dermal papilla cells after treatment with stanozolol.

Doping with anabolic agents is a topic in sports where strength is crucial, e.g. sprinting, weight lifting and many more. Testosterone and its functional analogs are the drugs of choice taken as pills, creams, tape or injections to increase muscle mass and body performance, and to reduce body fat. Stanozolol (17beta-hydroxy-17alpha-methyl-5alpha-androst-2-eno[3,2c]pyrazol) is a testosterone analogue with the same anabolic effect like testosterone but its ring structure makes it possible to take it orally. Therefore, stanozolol is one of the most frequently used anabolic steroids.Common verification methods for anabolic drugs exist, identifying the chemicals in tissues, like hair or blood samples. The idea of this feasibility study was to search for specific gene expression regulations induced by stanozolol to identify the possible influence of the synthetically hormone on different metabolic pathways. Finding biomarkers for anabolic drugs could be supportive of the existing methods and an additional proof for illegal drug abuse.In two separate cell cultures, human HFDPC (hair follicle dermal papilla cells) from a female and a male donor were treated with stanozolol. In the female cell culture treatment concentrations of 0 nM (control), 1 nM, 10 nM and 100 nM were chosen. Cells were taken 0 h, 6 h, 24 h and 48 h after stimulation and totalRNA was extracted. Learning from the results of the pilot experiment, the male cell culture was treated in 10 nM and 100 nM concentrations and taken after 0 h, 6 h, 24 h and 72 h. Using quantitative real-time RT-PCR expression of characteristics of different target genes were analysed.Totally 13 genes were selected according to their functionality by screening the actual literature and composed to functional groups: factors of apoptosis regulation were Fas Ligand (FasL), its receptor (FasR), Caspase 8 and Bcl-2. Androgen receptor (AR) and both estrogen receptors (ERalpha, ERbeta) were summarized in the steroid receptor group. The growth factor group included the insulin like growth factor receptor (IGF1R) and growth hormone receptor (GHR). Fibroblast growth factor 2 (FGF2) and keratinocyte growth factor (FGF7) were summarized in the hair cycle factor group. 5alpha-Steroidreductases (SRD5A1, SRD5A2) represented the enzyme group. Three reference genes were taken for relative quantification: ubiquitin (UBQ), glycerinaldehyde-3-phsophate-dehydrogenase (GAPDH), and beta-actin (ACTB).In cell culture 1 AR, FasR, FGF2 showed significant regulations within one treatment time, significant gene expressions over time were analysed for Caspase 8. In cell culture 2 AR, FasR and SRD5A2 were significantly regulated within one treatment time.In this feasibility study first biomarker for a screening pattern of anabolic agents could be identified providing the rationality to investigate modified, metabolic pathways in the whole hair follicle.

The influence of polyphenol rich apple pomace or red-wine pomace diet on the gut morphology in weaning piglets.

Alternative food ingredients, e.g. secondary plant compounds, are discussed to have beneficial effects and improve gut health. In this study, the effect of three different diets - normal piglets starter without additives, with apple pomace or with red-wine pomace - on the intestinal morphology was investigated from 3 days prior to weaning to 4 weeks post-weaning. At five time points, six piglets from each treatment group were slaughtered; at first time point only six animals from control group were slaughtered. Villus height, crypt depth and breadth of villi and crypts were determined in the jejunum, ileum and colon in 78 piglets. Additionally, the area of the Peyer's patches in the ileum was measured. In jejunum (p < 0.01) and ileum (p < 0.001) the villus length in the control group decreased after weaning but increased over the entire feeding experiment (p < 0.001). In the two-pomace groups, no decrease was measured after weaning. In jejunum, an increase in villi breadth occurred, 73% in the control group and approximately 10% in both treatment groups. A 35% increase was found in the ileum in all groups. Peyer's patches area increased approximately 21% in the control group over 26 days of treatment, whereas in other groups no significant differences were found. Different polyphenol rich pomaces have diverse effects in the gastrointestinal tract. Red-wine pomace has an inhibitory effect on the jejunum villi growth, whereas apple and red-wine pomace have stimulating effect on crypt size in piglet colon. Apple and red-wine pomace can reduce the GALT activation via the Peyer's patches in the ileum. In conclusion, the flavanoids rich feeding regimen showed positive effects on villi morphology, GALT activation and can improve pig health.

Reference histology of veal calf genital and endocrine tissues - an update for screening on hormonal growth promoters.

An animal experiment has been performed with 42 veal calves, 21 males and 21 females, which were fed and housed according to European regulations for veal calves. The animals were kept in six groups of seven animals and fed milk replacer supplemented with three different levels of lactulose (0%, 1% and 3%) and some roughage. At the start of the experiment the animals were 1-3 weeks of age and they were slaughtered at 26 weeks. From male animals prostate, bulbo-urethal gland and testes were sampled, from female animals Bartholin's gland, uterus, cervix and ovaries were sampled. From all animals thyroid, thymus, adrenals, liver and kidneys were sampled. Histological investigation of the prostates and bulbo-urethral glands showed normal histology. This means that dilated tubules, strong secretion, increased mucinous glandular tissue and severe hyperplasia and squamous metaplasia, as is regularly observed in practice in the Netherlands, were not present in these animals. None of these prostates would be judged as positive in the screening for hormones as is performed by the Dutch Food and Consumer Product Safety Authority (VWA). The female calves also showed normal histology of Bartholin's gland except for three animals that appeared to be in oestrus and showed some metaplasie of the ducts but with a normal gland to duct ratio. These animals would be judged as suspect. The liver and kidney showed minor alterations due to slight infections during the experimental period. This experiment showed that it is possible to raise veal calves according to the practice without getting positive histology in the prostate or Bartholin's gland.

Effect of lactoferrin on selected immune system parameters and the gastrointestinal morphology in growing calves.

Lactoferrin (LF) is a cationic iron-binding glycoprotein that is abundantly expressed and secreted from glandular epithelial cells and a prominent component of the secondary granules of polymorphonuclear neutrophils. Various in vitro and in vivo experiments demonstrate anti-microbial, -viral, -mycotic and -inflammatory effects of LF, associated with modulations of the immune system. Effects of oral administered LF on selected immune system parameters were studied in calves. Five calves were fed LF beginning on day 3 of life with colostral milk and starting on day 6 of life milk replacer enriched with 0.16% LF was fed. The average daily intake of LF per calf was 1.5-1.6 g/day. Additional five calves served as control group with identical treatment except for the LF supplementation. At the end of the study (day 61 of life), all calves were slaughtered and various tissues were sampled for histological and gene-expression studies. LF given orally was shown to act as an immunomodulatory agent by enhancing the size of Peyer's patches in the ileum and increasing blood serum immunoglobulin G levels. In addition, the number of peripheral blood leucocytes increased and mRNA levels of various interleukins (IL) such as IL-1beta, IL-8, IL-10 and interferon gamma (IFNgamma) in those cells in response to LF treatment were enhanced. In blood, the mRNA expression of the pro-inflammatory marker genes IL-1beta and IFNgamma decreased over 10-week treatment. Additionally, LF feeding decreased villus sizes in the jejunum. Together these findings emphasize the ability of LF to stimulate prominent immune system parameters and that it has the capacity to modulate the immune responses in a positive way.

Effect of lactulose on growth performance and intestinal morphology of pre-ruminant calves using a milk replacer containing Enterococcus faecium.

The synthetic disaccharide lactulose is known to improve the intestinal microflora by stimulating the growth of selected probiotic bacteria in the gut. In our experiment the effects of lactulose in combination with the probiotic bacteria Enterococcus faecium on growth performance and morphology of the bovine intestine were examined. Calves aged 39 ± 2 days were randomised to three feeding groups (no. = 14 each group): control (L0), fed milk replacer (MR) containing E. faecium; a lactulose group (L1) containing additional 1% lactulose and a second lactulose group (L3) containing 3% lactulose dry matter. The calves were weighed weekly. After 19 weeks the calves were slaughtered and tissues were collected for histological studies. The average daily live weight gain tended to be higher (P < 0.1) for L3 (1350 g/day) than L0 (1288 g/day). Compared with L0, a reduction (P < 0.001) of ileal villus height due to lactulose treatment of approximately 14% in group L1 and 20% in L3 was determined. A significant decrease in the depth of the crypts about 12% in L1 and 8% in L3 was detected in the caecum. The surface area of lymph follicles from Peyer's patches was decreased by lactulose treatment. Results show that lactulose has an effect on the morphology of intestine. A significant effect on growth performance can not be confirmed. However, results permit the conclusion that lactulose feeding has the tendency to increase growth performance.