Influence of intrauterine administration of Lactobacillus buchneri on reproductive performance and pro-inflammatory endometrial mRNA expression of cows with subclinical endometritis.

Potential beneficial effects of lactic acid bacteria on the genital health of cows become of particular interest when considering the importance of an optimal uterine health status for the success of breeding in dairy farming. Therefore, the aim of the present study was to analyse the influence of an intrauterine administration of the Lactobacillus buchneri DSM 32407 on reproductive performance, uterine health status, endometrial mRNA expression of pro-inflammatory factors of cows with signs of subclinical endometritis (SCE). L. buchneri DSM 32407 (n = 56; [LAC]) or a placebo (n = 60; [PLA]) was administered on day 24-30 postpartum. Endometrial cytobrush samples of cows with SCE were taken before the administration and at three following weeks (n = 16 cows each for LAC/SCE and PLA/SCE). A higher proportion of cows of the LAC and LAC/SCE group was pregnant after the first service and median days to conception for cows pregnant on day 200 pp were shorter. Three weeks after the administration, the endometrial mRNA expression of CXCL1/2, CXCL3, CXCR2, IL1B, IL8 and PTPRC was lower in the LAC/SCE group compared with the PLA/SCE group. These findings suggest that the presence of L. buchneri DSM 32407 contributes to a uterine environment that results in a better reproductive performance.

Different inflammatory responses of bovine oviductal epithelial cells in vitro to bacterial species with distinct pathogenicity characteristics and passage number.

The bovine oviduct provides the site for fertilization and early embryonic development. Modifications to this physiological environment, for instance the presence of pathogenic bacterial species, could diminish reproductive success at early stages of pregnancy. The aim of this study was to elucidate the inflammatory responses of bovine oviductal epithelial cells (BOEC) to a pathogenic bacterial species (Trueperella pyogenes) and a potentially pathogenic bacterium (Bacillus pumilus). BOEC from four healthy animals were isolated, cultured in passage 0 (P0) and passaged until P3. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicities of infection (MOI) of T. pyogenes (MOI 0.01, 0.05, 0.1 and 1) or B. pumilus (MOI 1 and 10). BOEC remained viable when co-cultured with T. pyogenes at MOI 0.01 and with B. pumilus at MOI 1 and 10. Extracted total RNA from control and bacteria co-cultured samples was subjected to reverse transcription-quantitative polymerase chain reaction (RTq-PCR) to determine mRNA expression of various studied genes. The rate of release of interleukin 8 (IL8) and prostaglandin E2 (PGE2) from BOEC was measured by ELISA after 24 h co-culture with bacteria. RT-qPCR of various selected pro-inflammatory factors revealed similar mRNA expression of pro-inflammatory factors in BOEC co-cultured with T. pyogenes and in the controls. Higher mRNA expression of IL 1A, -1B, tumor necrosis factor alpha and CXC ligand (CXCL) 1/2, -3, -5 and IL8 and PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. In the presence of B. pumilus a higher amount of IL8 and PGE2 was released from BOEC than from controls. The viability and pro-inflammatory response of P3 BOEC incubated with bacteria was lower than in P0 BOEC. These findings illustrate the pathogenicity of T. pyogenes towards BOEC in detail and the potential role of B. pumilus in generating inflammation in oviductal cells. Culturing conditions influenced the pro-inflammatory responses of BOEC towards bacteria. Therefore, researchers conducting epithelial-bacterial in vitro co-culture should not underestimate the effects of these parameters.

In silico cytotoxicity assessment on cultured rat intestinal cells deduced from cellular impedance measurements.

Early and reliable identification of chemical toxicity is of utmost importance. At the same time, reduction of animal testing is paramount. Therefore, methods that improve the interpretability and usability of in vitro assays are essential. xCELLigence's real-time cell analyzer (RTCA) provides a novel, fast and cost effective in vitro method to probe compound toxicity. We developed a simple mathematical framework for the qualitative and quantitative assessment of toxicity for RTCA measurements. Compound toxicity, in terms of its 50% inhibitory concentration IC50 on cell growth, and parameters related to cell turnover were estimated on cultured IEC-6 cells exposed to 10 chemicals at varying concentrations. Our method estimated IC50 values of 113.05, 7.16, 28.69 and 725.15 µM for the apparently toxic compounds 2-acetylamino-fluorene, aflatoxin B1, benzo-[a]-pyrene and chloramphenicol in the tested cell line, in agreement with literature knowledge. IC50 values of all apparent in vivo non-toxic compounds were estimated to be non-toxic by our method. Corresponding estimates from RTCA's in-built model gave false positive (toxicity) predictions in 5/10 cases. Taken together, our proposed method reduces false positive predictions and reliably identifies chemical toxicity based on impedance measurements. The source code for the developed method including instructions is available at https://git.zib.de/bzfgupta/toxfit/tree/master.

Increased mRNA expression of selected antimicrobial peptides around ovulation and during inflammatory processes in the bovine endometrium postpartum.

In the uterus, the first pathogen confrontations take place at the luminal endometrial epithelium. Therefore, it is required that these cells have the potential to recognize and respond to a bacterial infection. Antimicrobial peptides (AMP), part of the innate immune system in addition to cytokines, are principal effector molecules of mucosal immunity against pathogens. One important family of AMP that can permeabilize bacterial membranes is the beta-defensin (DEFB) family, which includes the following members: DEFB1, DEFB4A, and DEFB5, lingual AMP, and tracheal AMP. The bactericidal/permeability-increasing protein is also a cationic AMP that results in the death of bacteria. Another AMP family is the S100 calcium-binding protein (S100A) family including the following members: S100A8, S100A9, S100A11, and S100A12. These AMP exert their antimicrobial action through chelation of several ions. The aim of the present study was to evaluate mRNA expression patterns of selected AMP in bovine endometrial cells collected (1) at different stages of the estrous cycle (postovulatory, early-to-mid luteal, late luteal, and pre-ovulatory phase); (2) during the puerperium depending on uterine health status (healthy, subclinical, or clinical endometritis) starting on Day 24 to 30 postpartum for 3 weeks on a weekly basis; and (3) in vitro after co-culturing with Bacillus pumilus at three different multiplicities of infection (MOI 1, 5, and 10) up to 6 hours. The results reported that the mRNA expression of all candidate AMP, except DEFB1, S100A8, and S100A9, was estrous cycle dependent. In particular, around the time of ovulation, the transcription level of most AMP was higher (P < 0.05) compared with the luteal phase. Almost all candidate AMP mRNA expression was dependent on uterine health status, with a higher transcription level (P < 0.05) in inflamed endometrial tissues, especially during the late stage of the puerperium (Day 45-51 postpartum). Members of the DEFB family were nearly unaffected in their mRNA expression in primary endometrial cells co-incubated with B. pumilus. However, S100A8 and S100A9 mRNA contents were higher after 4 and 6 hours of co-incubation with B. pumilus compared with untreated controls. In conclusion, higher mRNA expression of the candidate AMP around ovulation or in inflamed endometrial tissue during the puerperium suggests their crucial role in uterine innate immunity in the defense against invading bacteria.

Characterization of key genes of the renin-angiotensin system in mature feline adipocytes and during in vitro adipogenesis.

Obesity is a growing health problem in humans as well as companion animals. In the development and progression of obesity-associated diseases, the members of the renin-angiotensin system (RAS) are proposed to be involved. Particularly, the prevalence of type 2 diabetes mellitus in cats has increased enormously which is often been linked to obesity as well as to RAS. So far, reports about the expression of a local RAS in cat adipocytes are missing. Therefore, we investigated the mRNA expression of various RAS genes as well as the adipocyte marker genes adiponectin, leptin and PPAR-γ in feline adipocytes using quantitative PCR. To characterize the gene expression during adipogenesis, feline pre-adipocytes were differentiated into adipocytes in a primary cell culture and the expression of RAS key genes measured. All major RAS components were expressed in feline cells, but obvious differences in the expression between pre-adipocytes and the various differentiation stages were found. Interestingly, the two enzymes ACE and ACE2 showed an opposite expression course. In addition to the in vitro experiments, mature adipocytes were isolated from subcutaneous and visceral adipose tissue. Significant differences between both fat depots were found for ACE as well as AT1 receptor with greater expression in subcutaneous than in visceral adipocytes. Visceral adipocytes had significantly higher adiponectin and PPAR-γ mRNA level compared to the subcutaneous fat cells. Concerning the nutritional status, a significant lower expression of ACE2 was measured in subcutaneous adipocytes of overweight cats. In summary, the results show the existence of a potentially functional local RAS in feline adipose tissue which is differentially regulated during adipogenesis and dependent on the fat tissue depot and nutritional status. These findings are relevant for understanding the development of obesity-associated diseases in cats such as diabetes mellitus.

Identification of miRNAs in Bovine Endometrium through RNAseq and Prediction of Regulated Pathways.

Detection of miRNAs in reproductive tissues is a key step to understand their role in fertility. We hypothesize that miRNAs must be involved in pathways controlling endometrial physiology and defense against pathogens. In this study, we aimed to characterize miRNAs present in bovine endometrium and to predict regulated pathways. Cytobrush endometrial samples from four cows were collected at oestrous cycle days 1-5, 6-12, 13-18 and 19-21. RNA was extracted and sequenced using Ion Torrent (®) technology. After mapping of the reads to miRNA stem loops, rRNAs and tRNAs, data were normalized and analysed using DESeq2. Targets and pathways were predicted with miRmap and KEGG, respectively. Validation of miRNAs in tissue was done by RT-qPCR (miR-Q). A total of 221 identities were common among groups, accumulating more than 99% of miRNA expression. MiRNAs were predicted to regulate MAPK signalling pathway, lysosome and extracellular matrix (ECM)-receptor interaction. Eight miRNAs were validated by miR-Q, showing that let-7a-5p and let-7b were regulated across the oestrous cycle. This study demonstrated a high similarity in miRNA expression profile across the oestrous cycles in bovine endometrium. These miRNAs were predicted to regulate pathways involved in cell proliferation, differentiation, transport and catabolism. The number of pathways shared by different miRNAs indicates the broad range of regulation these molecules exhibit in the endometrium.

Puerperal influence of bovine uterine health status on the mRNA expression of pro-inflammatory factors.

After parturition, uterine bacterial infections lead to inflammatory processes such as subclinical/clinical endometritis with high prevalence in dairy cows. Endometrial epithelial cells participate in this immune response with the production of pro-inflammatory factors. The objective of the present study was to evaluate the endometrial mRNA expression pattern of pro-inflammatory factors during a selected postpartum (pp) period. Dairy cows with three different uterine health conditions on days 24-30 pp (healthy: n = 11, subclinical endometritis: n = 10, clinical endometritis: n = 10) were sampled using the cytobrush technique. Subsequently, each cow was sampled 3 more times in weekly intervals (days 31-37 pp; days 38-44 pp; days 45-51 pp). Samples were subjected to mRNA analysis performed by RT-qPCR. Additionally, an analysis of cultivable bacteria was performed at the early/late stage of the selected puerperal period. mRNA expression of 16 candidate genes was analyzed by using two different approaches. The first approach referred to the initial grouping on days 24-30 pp to reveal long-term effects of the uterine health on the subsequent puerperal period. The second approach considered the current uterine health status at each sampling to elucidate the impact of different points in time. Long-term effects seem to appear for chemokines, prostacyclin synthase and prostaglandin D2 synthase. If related to the current uterine health, the majority of candidate genes were significantly higher expressed in endometritic cows on days 45-51 pp in contrast to earlier stages of the puerperium. Microbiological analysis revealed the significantly higher prevalence of Trueperella pyogenes findings in cows with clinical endometritis on days 24-30 pp, but no correlations were found on days 45-51 pp. In conclusion, a strong immune response to subclinical/clinical endometritis in the late puerperium may be related to the negative impact of these conditions on reproductive performance in dairy cows.

Impact of high dietary zinc on zinc accumulation, enzyme activity and proteomic profiles in the pancreas of piglets.

The exocrine pancreas plays an important role in zinc homeostasis. Feeding very high (2000-3000mgzinc/kg diet) levels of zinc oxide to piglets for short periods is a common practice in the swine industry to improve performance and prevent diseases. The impact on pancreatic function and possible side effects during long-term feeding of high dietary zinc levels are still poorly understood. A total of 54 weaned piglets were either fed with low (57mg/kg, LZn), normal (164mg/kg, NZn) or high (2425mg/kg, HZn) zinc concentration in the diets. After 4 weeks of feeding, ten piglets per treatment were euthanized and pancreas samples were taken. Tissue zinc concentration and metallothionein abundance was greater with HZn compared with NZn and LZn (P<0.05). Similarly, activity of α-amylase, lipase, trypsin and chymotrypsin was higher with HZn as compared with NZn and LZn diets (P<0.05), whereas elastase activity was unchanged. Total trolox equivalent antioxidative capacity of pancreas tissue was higher with HZn diets compared with the other treatments (P<0.05). Pancreatic protein profiles of NZn and HZn fed piglets were obtained by 2D-DIGE technique and revealed 15 differentially expressed proteins out of 2100 detected spots (P<0.05). The differentially expressed proteins aldose reductase, eukaryotic elongation factor II and peroxiredoxin III were confirmed by immunoblotting. Identified proteins include zinc finger-containing transcription factors and proteins mainly associated with oxidative stress response and signal transduction in HZn compared with NZn pigs. Histologic examination however showed no morphologic changes. The results suggest that long-term supply of very high dietary zinc increases zinc and metallothionein concentration, and digestive enzyme activity, but also triggers oxidative stress reactions in the pancreas of young pigs. The data provide new insights into pancreatic function under outbalanced zinc homeostasis.

Bovine oviductal epithelial cells: long term culture characterization and impact of insulin on cell morphology.

In vitro models that resemble cell function in vivo are needed to understand oviduct physiology. This study aimed to assess cell functions and insulin effects on bovine oviductal epithelial cells (BOECs) cultured in an air-liquid interface. BOECs (n=6) were grown in conditioned Ham's F12, DMEM or Ham's F12/DMEM with 10% fetal calf serum (FCS) for 3 weeks. After selecting the most suitable medium (Ham's F12), increasing insulin concentrations (1 ng/mL, 20 ng/mL and 5 µg/mL) were applied, and cell morphology and trans-epithelial electrical resistance (TEER; n=4) were evaluated after 3 and 6 weeks. Keratin immunohistochemistry and mRNA expression of oviductal glycoprotein 1 (OVGP1) and progesterone receptor (PGR) were conducted (n=4) to assess cell differentiation. BOECs grown without insulin supplementation or with 1 ng/mL of insulin displayed polarization and secretory activity. However, cells exhibited only 50% of the height of their in vivo counterparts. Cultures supplemented with 20 ng/mL insulin showed the highest quality, but the 5 µg/mL concentration induced massive growth. TEER correlated negatively with insulin concentration (r=-0.459; p=0.009). OVGP1 and PGR transcripts were still detectable after 3 and 6 weeks. Cellular localization of keratins closely resembled that of BOECs in vivo. Cultures showed heterogeneous expression of PGR and OVGP1 in response to estradiol (10 pg/mL). In summary, BOECs grown for long term in an air-liquid interface expressed markers of cell differentiation. Additionally, insulin supplementation (20 ng/mL) improved the cell morphology in vitro.

Characterization of the native C-reactive protein (cCRP) and the corresponding liver mRNA in dogs.

C-reactive protein (CRP) plays an important role in the acute phase reaction in humans and dogs. For the canine CRP (cCRP) only an in silico deduced preliminary transcript and amino acid sequence is available. The objective of this study was to further characterize the native cCRP protein and its corresponding liver mRNA. Furthermore, immunological similarities of serum CRP in related animal species were investigated. Native cCRP protein was isolated from dog-sera by affinity chromatography and further analyzed by immunodetection, protein sequencing (mass spectrometry and N-terminal Edman sequencing), 2D-gel electrophoresis, and glycoprotein analysis. Furthermore, cCRP cDNA sequence was determined from dog liver total RNA by RT-PCR. Gel electrophoresis, immunodetection and glycoprotein detection revealed two cCRP isotypes with different molecular weights (22 and 25kDa) with the upper band being glycosylated. Selective glycoprotein analysis showed sialic acid terminally linked (2-6) to galactose or N-acetylgalactosamine and subsequent PNGase F treatment identified N-terminal linkage. Mass spectrometry confirmed approximately 45% of the cCRP predicted amino acid sequence and N-terminal amino acid sequencing revealed a shorter native cCRP than expected (204 amino acids). The new canine CRP mRNA sequence confirms 100% of the formerly deduced sequence. Immunological homologies to the canine CRP protein were found in selected dog-related species. This study contributes major molecular details to the knowledge about canine CRP. Such structural information may assist in developing new diagnostic tools for inflammatory-based diseases in dogs as well as other dog-related species.

Evaluation of biomarkers for osteoarthritis caused by fragmented medial coronoid process in dogs.

The aim of the present work was to evaluate whether concentrations of the carboxy-terminal cross-linked fragment of type II collagen (CTX-II), the activities of matrix metalloproteinase-2 and -9 (MMP-2/-9) and Myeloperoxidase (MPO) in canine synovial fluids (SF) can reflect structural alterations of articular cartilage in dogs with fragmented medial coronoid process (FMCP). Elbow joints with FMCP underwent radiographic and arthroscopic examination. Commercially available assays were used to analyze SF for CTX-II concentration and MMP-2/-9 activity. MPO activity was measured by o-dianisidine-assay. The MMPs were further evaluated by zymography. CTX-II concentration and MMP-2 activity showed age-dependent trends in controls. Increased enzyme activities of MPO and MMP-2/-9 were found in diseased dogs. MMP-9activity seems suitable to underline the subjective assessment of the degree of cartilage damage. These initial data of the study suggest that MPO and MMP-2/9 may be used as objective biomarkers in the diagnosis of canine osteoarthritis due to FMCP.

Effects of zinc on epithelial barrier properties and viability in a human and a porcine intestinal cell culture model.

Zinc is an essential trace element with a variety of physiological and biochemical functions. Piglets are commonly supplemented, during the weaning period, with doses of zinc above dietary requirements with positive effects on health and performance that might be attributed to anti-secretory and barrier-enhancing effects in the intestine. For a better understanding of these observations increasing zinc sulfate (ZnSO4; 0-200µM) concentrations were used in an in vitro culture model of porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells and effects on barrier function, viability, and the mRNA expression of one selected heat shock protein (Hsp) were assessed. When treated apically with zinc sulfate, the transepithelial electrical resistance (TEER) did not change significantly. In contrast, cell viability measured by lactate dehydrogenase (LDH) leakage, by ATP and by WST-1 conversion in postconfluent IPEC-J2 monolayers was affected after a 24-h treatment with 200µM ZnSO4. Caco-2 cells were more resistant to Zn. ZnSO4 did not induce any effect on viability, except when it was used at the highest concentration (200µM), and only in preconfluent cells. Furthermore, ZnSO4 induced Hsp70 mRNA expression at 200µM and was more pronounced in preconfluent cells. The observed dose-related effects of zinc are cell-line specific and depended on the differentiation status of the cells. The IPEC-J2 cell line appears to be a suitable in vitro model to characterize specific effects on porcine intestinal cells.

Hyaluronic acid concentrations in synovial fluid of dogs with different stages of osteoarthritis.

To compare hyaluronic acid (HA) concentrations measured in synovial fluid (SF) of joints with different stages of canine secondary osteoarthritis (OA), clinical-orthopedic, radiographic, macroscopic intra-operative and SF findings of 49 joints were assessed. The sum of single findings was correlated to HA concentrations measured by a commercially available ELISA. Joints were categorized into three OA-groups: non-osteoarthritic, mildly osteoarthritic, and severely osteoarthritic. A significant negative correlation was found between severity of OA and HA concentrations (r=-0.696; P<0.001). Median values of HA concentrations decreased with increasing severity of the disease. Statistically significant differences in HA concentrations were observed between the OA-groups (P<0.001). Due to overlapping values between groups, it was concluded that synovial HA concentrations may only indicate a trend of osteoarthritic disease activity, but is not suitable for staging the disease.

Differences in the proteome of high-grade versus low-grade canine cutaneous mast cell tumours.

Cutaneous mast cell tumours (MCTs) are the most common skin tumours in dogs. However, the molecular differences between benign tumours with a good prognosis and highly malignant, invasive and metastatic tumours with short survival times are for the most part unclear. In the present study the proteome of low-grade MCTs with a good prognosis was compared with that of poor-prognosis high-grade tumours independent of their mutational status of exon 11 of the KIT gene. Using two-dimensional difference gel electrophoresis and mass spectrometry, 13 proteins with a significant differential expression between the two groups were identified. Four stress response proteins (HSPA9, PDIA3, TCP1A and TCP1E) were significantly up-regulated in high-grade tumours, while proteins mainly associated with cell motility and metastasis had either increased (WDR1, ACTR3, ANXA6) or decreased (ANXA2, ACTB) expression levels. High-grade tumours also had a paradox down-regulation of transferrin, a protein that is usually up-regulated in neoplastic cells. The histologically observable dedifferentiation of high-grade tumours was reflected by decreased tryptase protein expression levels. Results of quantitative real-time RT-PCR analysis indicated that the differences in protein expression levels of most proteins were regulated at the transcript level. Based on these findings, it is hypothesized that high-grade MCT cells have a higher resistance to cellular stress and thus are able to better cope with the adverse environment in highly proliferating tumours independent of increased KIT signalling. It is noteworthy that some of the proteins identified have been proposed as therapeutic targets for human oncology and it will be interesting to evaluate their therapeutic and diagnostic potential for canine MCTs.

Quantification of ZP1, ZP2 and ZP3 mRNA of marmoset monkey (Callithrix jacchus) oocytes from periantral and antral follicles.

In mammals, the oocyte and preimplantation embryo are protected by the zona pellucida (ZP) consisting mainly of ZP glycoproteins, which are responsible for sperm binding, induction of the acrosome reaction and zona pellucida hardening to prevent polyspermia. The ZP proteins become increasingly important as possible predictors for in vitro cultured oocytes competence. As little is known about the stage-dependent expression of ZP1, ZP2 and ZP3 in marmoset monkey (Callithrix jacchus) oocytes, mRNA expression was investigated with real-time RT-PCR. Total-RNA was isolated from three different classes of marmoset oocytes; Class 1 oocytes from periantral follicles (<600 µm, n = 10), Class 2 oocytes from small antral follicles (600-1000 µm, n = 10) and Class 3 oocytes from large antral follicles (>1000 µm, n = 9). Compared with Class 1 oocytes mRNA expression of ZP1, ZP2 and ZP3 in Class 2 oocytes was significantly decreased. In Class 3 oocytes, the transcription of ZP1, ZP2 and ZP3 genes showed also a significant decrease compared with Class 1 oocytes. In this study a differently regulated expression of the ZP genes during late folliculogenesis with an obvious downregulation of ZP1, ZP2 and ZP3 could be demonstrated for the first time in the marmoset monkey.

Modelling the porcine oviduct epithelium: a polarized in vitro system suitable for long-term cultivation.

For exploring the processes leading to successful reproduction, differentiated long-term in vitro systems modelling the mammalian oviduct are needed. Therefore, in the present study culture conditions for primary porcine oviductal epithelial cells were optimized with regard to morphological differentiation and usability for extended cultivation periods. To evaluate different growth media for the primary cells, we used morphological criteria as well as real-time impedance measurement. After an initial media testing, the cells were grown on hanging membranes and the culture settings (conventionally cultured, serum gradient over the membrane and air-liquid interface) were assessed by histology and electron microscopy. We proved long-term expression of an oviduct specific marker (oviductal glycoprotein 1) and showed a hormone responsiveness of the culture system by means of quantitative reverse transcription-PCR. Differentiated epithelial cells could reproducibly be cultured up to 6 weeks in an air-liquid interface. After 3 weeks of culturing, the cells were clearly polarized and exhibited cilia. The model maintains physiological properties such as morphological features (mixed cell population of ciliated and secretory cells, apical cell-cell contacts typical for columnar epithelial cells) and oviduct-specific markers showing hormone responsiveness. We established a polarized long-term in vitro-system of the porcine oviductal epithelium preserving detailed features of the porcine oviduct. Therefore, we provide a useful tool to elucidate unsolved scientific questions concerning reproductive physiology.

MicroRNA expression profiling of elongated cloned and in vitro-fertilized bovine embryos.

The objective of this study was to identify microRNAs (miRNAs) expressed in bovine (Bos Taurus) cloned embryos at Day 17 of development (Day 0=day of nucleus transfer or in vitro fertilization) during elongation. Day 7 bovine expanded blastocysts produced by hand made cloning (HMC) or in vitro fertilization were bulk-transferred to synchronized recipient cattle (48 HMC embryos to 10 recipients and 28 in vitro-produced embryos to four recipients). Elongated embryos were retrieved at Day 17; miRNAs were isolated and subjected to microarray screening using custom composite slides spotted with human, mouse, and rat and in silico-predicted miRNAs. An initial profile of expressed miRNAs was determined in cloned embryos and somatic donor cells; this profile changed after somatic cell nucleus transfer, identifying differentially expressed miRNAs between cloned and in vitro-produced bovine embryos. Furthermore, microarray data were validated using a miRNA-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) approach (miR-Q). There was an 83% correlation (P=0.01) between microarray and qPCR data. Based on qRT-PCR, correct reprogramming of some miRNAs from the donor cells was confirmed in cloned bovine embryos, whereas other somatic miRNAs were not appropriately reprogrammed. Some of the miRNAs that were equally reprogrammed clustered on the same chromosomal location in the bovine genome. In conclusion, reprogramming of miRNAs seemed to occur in cloned bovine embryos. This could have profound implications for elucidating nuclear reprogramming in somatic cloning, as well as for the role of miRNAs in preimplantation mammalian development.

Endometrial expression of selected transcripts involved in prostaglandin synthesis in cows with endometritis.

Several cytokines and prostaglandins play an important role in preparing the endometrium for implantation and mediating pro-inflammatory events. The aim of the present study was to examine mRNA expression of interleukin 1alpha (IL-1alpha), interleukin receptor antagonist (IL-1-RN), cytosolic prostaglandin E synthase (cPGES), microsomal PGES (mPGES-1 and mPGES-2) and lipocalin-type PGDS (L-PGDS) in the bovine endometrium. Endometrial epithelium samples were collected ex vivo from cows with different status of health at day 21-27 postpartum on a dairy farm. Three groups (n=9 animals each) were defined: (1) healthy cows with no signs of endometritis (control group), (2) cows with subclinical endometritis, and (3) cows with signs of clinical endometritis. Oestrous cycle-dependent mRNA expression pattern was investigated using bovine endometrial epithelial cells from healthy uteri collected at the abattoir. These uteri were classified into post-ovulatory, early-to-mid luteal, late luteal or pre-ovulatory phase (n=8 animals for each cycle phase). After collecting endometrial epithelium using the cytobrush-method, mRNA analysis was performed by real-time RT-PCR. L-PGDS, IL-1alpha and IL-1-RN mRNA were expressed significantly higher (P<0.05) in the endometrium of cows with subclinical or clinical endometritis compared with healthy cows. A twofold lower cPGES mRNA expression (P<0.05) was detected in cows with subclinical endometritis compared to healthy cows. L-PGDS and IL-1-RN mRNA expression was increased (P<0.05) after ovulation compared with the pre-ovulatory or luteal phase, respectively. These results support the hypothesis that a dys-regulated cytokine and/or prostaglandin profile in the uterus could be induced by subclinical endometritis or clinical endometritis.

Establishment and characterization of an adherent pure epithelial cell line derived from the bovine oviduct.

The oviduct in vivo has to perform various tasks: maturation and transport of the gametes, milieu preparation for fertilization and embryonic development, and transport of the embryo. The complex arrangement of endocrine and paracrine signals being exchanged between the early embryo and the inner cell layers of the oviduct is barely understood. Therefore, a reproducible, well-characterized oviduct epithelial cell line as well as an optimized transfection protocol for DNA vectors and siRNA for this cell line has been established. A bovine oviduct primary cell culture system has been optimized using a selection medium permitting the survival of only epithelial cells. From this we established an adherent bovine oviduct pure epithelial cell line (aBOPEC-1). This cell line maintains some important characteristics of the primary cells such as the expression of estrogen receptors and p450 aromatase but it lacks some characteristics due to the selection and dedifferentiation processes (cilia, expression of progesterone receptor and oviduct specific glycoprotein-1). Optimization of the transfection protocols finally revealed a suitable DNA-transfection procedure yielding transfection efficiencies of over 50%. Additionally, siRNA transfection efficiency reached more than 90%. This new cell line builds an essential basis especially for future functional studies in the oviduct epithelium using distinct knock down experiments.

Exploring cumulus-oocyte-complex-oviductal cell interactions: gene profiling in the bovine oviduct.

Prostaglandin E(2) (PGE(2)) is present in the bovine oviduct and may play an important role in muscle contraction or as survival factor providing the optimal environment for fertilization and the early embryo. The aim of the present study was to investigate the estrous cycle-dependent changes and local distributions of PGE(2) receptors (EP) and members of the trefoil factor (TFF)-family in the bovine oviduct using real-time RT-PCR. A co-cultivation system of cumulus-oocyte-complexes (COC) with primary oviductal cells was screened for the mRNA expression pattern of selected factors. An oviductal primary cell culture was used for investigating effects of estradiol on signal transduction pathways. Quantitative RT-PCR revealed significant higher expression of EP2 and EP4 in the pre-ovulatory phase compared with the luteal phase. TFF3 mRNAwas expressed during the estrous cycle with highest level in the post-ovulatory phase showing higher expression levels in the isthmus compared with the ampulla. A different mRNA expression pattern was observed for factors involved in extracellular matrix formation in co-cultured oviductal cells compared to untreated controls. In vitro, NF-kappaB was found activated after estradiol treatment. These results suggest that a fine-tuned PGE(2) signal transduction pathway may support fertilization, early embryonic development and gamete transport in the bovine oviduct.