In vitro effects of Type I interferons (IFNτ and IFNα) on bovine hepatocytes cultured with or without Kupffer cells.

In cattle, maternal recognition of early pregnancy depends on the effects of the embryonic signal interferon (IFN)-τ. IFN-stimulated genes have been upregulated in the maternal liver during early pregnancy. In this study, primary hepatocyte cell culture models were evaluated for their suitability to test Type I IFN effects invitro. The expression of target genes (interferon-stimulated gene 15 (ISG-15), interferon-induced GTP-binding protein (MX-1), C-X-C motif chemokine 10 (CXCL-10), CXCL-5, insulin-like growth factor 1 (IGF-1), IGF binding protein 2 (IGFBP-2)) was measured using reverse transcription-quantitative polymerase chain reaction in hepatocytes from monoculture or in indirect coculture with Kupffer cells (HKCid) on Days 1, 2, 3 and 4 of culture (n=21 donor cows). Gene expression was also measured on Day 4 after challenging the cultures with recombinant IFNτ, IFNα, progesterone (P4), IFNτ+IFNα or IFNτ+P4 for 6h. A significant increase in the mRNA expression of target genes in hepatocytes was shown in response to stimulation with IFNτ. The Kupffer cells in coculture did not influence the effects of IFNτ in hepatocytes. In conclusion, primary bovine hepatocyte cultures are suitable for stimulation experiments with Type I IFNs and as an extrauterine model for embryo-maternal communication. The proposed endocrine action of IFNτ in the liver may affect maternal metabolism and immune function in the liver.

Maternal Heat Stress Alters Expression of Genes Associated with Nutrient Transport Activity and Metabolism in Female Placentae from Mid-Gestating Pigs.

Placental insufficiency is a known consequence of maternal heat stress during gestation in farm animals. The molecular regulation of placentae during the stress response is little known in pigs. This study aims to identify differential gene expression in pig placentae caused by maternal heat exposure during early to mid-gestation. RNA sequencing (RNA-seq) was performed on female placental samples from pregnant pigs exposed to thermoneutral control (CON; constant 20 °C; n = 5) or cyclic heat stress (HS; cyclic 28 to 33 °C; n = 5) conditions between d40 and d60 of gestation. On d60 of gestation, placental efficiency (fetal/placental weight) was decreased (p = 0.023) by maternal HS. A total of 169 genes were differentially expressed (FDR ≤ 0.1) between CON and HS placentae of female fetuses, of which 35 genes were upregulated and 134 genes were downregulated by maternal HS. The current data revealed transport activity (FDR = 0.027), glycoprotein biosynthetic process (FDR = 0.044), and carbohydrate metabolic process (FDR = 0.049) among the terms enriched by the downregulated genes (HS vs. CON). In addition, solute carrier (SLC)-mediated transmembrane transport (FDR = 0.008) and glycosaminoglycan biosynthesis (FDR = 0.027), which modulates placental stroma synthesis, were identified among the pathways enriched by the downregulated genes. These findings provide evidence that heat-stress induced placental inefficiency may be underpinned by altered expression of genes associated with placental nutrient transport capacity and metabolism. A further understanding of the molecular mechanism contributes to the identification of placental gene signatures of summer infertility in pigs.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF1 influences its growth characteristics.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF1 (oligopeptide permease transporter) which results in expression of a truncated OppF1. The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF1 gene in the MS-H genome. Wild-type oppF1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF1-tetM or with a similar vector lacking oppF1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF1-tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF1 detected bands of expected size for full-length OppF1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF1-tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF1-tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF1-tetM (passage 27) showed that the chromosomal copy of the mutated oppF1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species.RESEARCH HIGHLIGHTS The full-length OppF1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF1 in MS-H was replaced with wild-type oppF1.

Innate immune genes in persistent mating-induced endometritis in horses.

Persistent mating-induced endometritis (PMIE) severely decreases fertility in horses. The aim of the present study was to evaluate differences between horses susceptible to PMIE and a control group in terms of the expression of selected immune response and effector genes, and the effects of oestrous cycle stage on this expression. Endometrial biopsies from 18 uterine samples of mares in the control group (eight in dioestrus, 10 in oestrus) and 16 PMIE-susceptible mares (four in dioestrus, 12 in oestrus) were analysed by quantitative real-time reverse transcription-polymerase chain reaction. Genes for pathogen recognition receptors Toll-like receptor 2 (TLR2) and NLR family CARD domain containing 5 (NLRC5), as well as tissue-specific inhibitor of metalloproteinase 1 (TIMP1), C-X-C motif chemokine ligand (CXCL) 9, CXCL10 and CXCL11 and uteroferrin were expressed at similar levels in the control group and in susceptible mares. Genes for C-C motif chemokine ligand 2 (CCL2) and the antimicrobial peptides secreted phospholipase A2 (sPLA2), lipocalin 2 and lactoferrin were all expressed at higher levels in susceptible compared with control mares. The expression of genes for the antimicrobial peptides equine ß-defensin 1 (EBD1), lysozyme (LYZ) and secretory leukoprotease inhibitor (SLPI) was also higher in susceptible than control mares. The diagnostic sensitivity of assays for EBD1, LYZ and SLP1 gene expression to detect susceptibility to PMIE was estimated to be 100%, 94% and 100% respectively, with specificities of 83%, 78% and 78% respectively. When all three tests were positive, the specificity increased to 94%, with an overall sensitivity of 94%. The present study has yielded insights into pathophysiological changes in mares susceptible to PMIE and identified robust diagnostic markers (EBD1, LYZ and SLPI) for susceptibility to this disease.

Oestrous cycle-dependent equine uterine immune response to induced infectious endometritis.

Infectious endometritis is a major cause of reduced pregnancy rates in horses. The objectives of this study were to establish a timeline of the innate immune response in the uterus of healthy horses and to investigate the oestrous cycle effect on this. Endometrial biopsies were collected from five horses before and at 3, 12, 24, 48 and 72 h after inoculation of Escherichia coli, once in oestrus and once in dioestrus. They were analysed by quantitative real-time PCR, microbiology and histology. Neutrophil numbers increased from very low levels in the absence of inflammation to severe neutrophilia 3 h after inoculation. The concentrations of mRNAs for Toll-like receptor (TLR)2, TLR4, NOD-like receptor NLRC5, tissue inhibitor of metallopeptidases 1 (TIMP1) and chemokines CCL2, CXCL9, CXCL10 and CXCL11 were all increased 3 h after inoculation of E. coli compared to levels detected prior to inoculation. Chemokine mRNA levels remained elevated for 48 h. Concentrations of mRNAs for the antimicrobial peptides equine ß-defensin 1 (EBD1), lysozyme, secretory leukoprotease inhibitor (SLPI), lipocalin 2 (LCN2), lactoferrin and uteroferrin were increased between 3 and 12 h post inoculation. The gene for secreted phospholipase A2 (sPLA2) was expressed constitutively. P19 uterocalin mRNA levels were higher in dioestrus than in oestrus over the first 24 h of inflammation. Neutrophils and many innate immune genes responded rapidly to the introduction of E. coli into the uterus, while the oestrous cycle stage had only a relatively minor effect on the response to E. coli. This study has delineated a useful model of innate immunity in infectious endometritis of healthy animals.

Deep sequencing of the uterine immune response to bacteria during the equine oestrous cycle.

BACKGROUND: The steroid hormone environment in healthy horses seems to have a significant impact on the efficiency of their uterine immune response. The objective of this study was to characterize the changes in gene expression in the equine endometrium in response to the introduction of bacterial pathogens and the influence of steroid hormone concentrations on this expression. METHODS: Endometrial biopsies were collected from five horses before and 3 h after the inoculation of Escherichia coli once in oestrus (follicle >35 mm in diameter) and once in dioestrus (5 days after ovulation) and analysed using high-throughput RNA sequencing techniques (RNA-Seq). RESULTS: Comparison between time points revealed that 2422 genes were expressed at significantly higher levels and 2191 genes at significantly lower levels 3 h post inoculation in oestrus in comparison to pre-inoculation levels. In dioestrus, the expression of 1476 genes was up-regulated and 383 genes were down-regulated post inoculation. Many immune related genes were found to be up-regulated after the introduction of E. coli. These include pathogen recognition receptors, particularly toll-like receptors TLR2 and 4 and NOD-like receptor NLRC5. In addition, several interleukins including IL1B, IL6, IL8 and IL1ra were significantly up-regulated. Genes for chemokines, including CCL 2, CXCL 6, 9, 10, 11 and 16 and those for antimicrobial peptides, including secretory phospholipase sPLA 2, lipocalin 2, lysozyme and equine ß-defensin 1, as well as the gene for tissue inhibitor for metalloproteinases TIMP-1 were also up-regulated post inoculation. CONCLUSION: The results of this study emphasize the complexity of an effective uterine immune response during acute endometritis and the tight balance between pro- and anti-inflammatory factors required for efficient elimination of bacteria. It is one of the first high-throughput analyses of the uterine inflammatory response in any species and several new potential targets for treatment of inflammatory diseases of the equine uterus have been identified.

Effect of ovarian hormones on the healthy equine uterus: a global gene expression analysis.

The physiological changes associated with the varying hormonal environment throughout the oestrous cycle are linked to the different functions the uterus needs to fulfil. The aim of the present study was to generate global gene expression profiles for the equine uterus during oestrus and Day 5 of dioestrus. To achieve this, samples were collected from five horses during oestrus (follicle >35 mm in diameter) and dioestrus (5 days after ovulation) and analysed using high-throughput RNA sequencing techniques (RNA-Seq). Differentially expressed genes between the two cycle stages were further investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The expression of 1577 genes was found to be significantly upregulated during oestrus, whereas 1864 genes were expressed at significantly higher levels in dioestrus. Most genes upregulated during oestrus were associated with the extracellular matrix, signal interaction and transduction, cell communication or immune function, whereas genes expressed at higher levels in early dioestrus were most commonly associated with metabolic or transport functions, correlating well with the physiological functions of the uterus. These results allow for a more complete understanding of the hormonal influence on gene expression in the equine uterus by functional analysis of up- and downregulated genes in oestrus and dioestrus, respectively. In addition, a valuable baseline is provided for further research, including analyses of changes associated with uterine inflammation.

High ambient temperature and humidity associated with early embryonic loss after embryo transfer in mares.

Increased ambient temperatures has been identified to contribute to reproductive outcomes for several domesticated species, but its impact on equine reproduction has not been previously investigated. Therefore, this study aimed to examine the relationship between ambient climatic conditions, as measured by temperature-humidity index (THI) between day 7 and day 14 of gestation, and early embryonic loss (EEL) in recipient mares undergoing embryo transfer. The study examined records from 834 embryo transfers at an equine breeding facility in Victoria, Australia. Early embryonic loss was defined as a negative transrectal ultrasound on day 14 of gestation after an embryo was transferred on day 7. Both maximum THI on the day of transfer (day 7) and mean THI between day 7 and day 14 were investigated for association with the outcome of EEL using multivariable logistic regression, controlling for confounders including embryo age and quality, recipient mare quality and embryo transfer quality. EEL was observed in 21% of embryo transfers. A five-unit increase in maximum THI on the day of transfer was associated with an 18% increase in the odds of EEL (p = 0.01). Similarly, the odds of EEL increased by 25% for each five-unit increase in mean THI between day 7 and 14 (p = 0.003). As both single and cumulative episodes of high THI were associated with increased EEL in embryo transfer mares from this equine breeding facility, further studies are warranted to identify similar effects in a broader population, establish causality and evaluate possible mitigation strategies in anticipation of heat waves.

Is the IL1RA/IL1B Ratio a Suitable Biomarker for Subclinical Endometritis in Dairy Cows?

The adequate expression of cytokines is essential for the prevention and healing of bovine endometrial inflammation. This study investigated the intra-uterine concentration of the proinflammatory cytokine interleukin (IL)1B and its antagonist IL1RA in cows with and without subclinical endometritis (SE). Samples were taken from 37 uteri at the abattoir and 26 uteri in vivo. Uterine secretion samples were classified as showing no signs of SE (SEneg; polymorphonuclear neutrophil granulocyte (PMN) < 5%) or showing signs of SE (SEpos; PMN ≥ 5%). Concentrations and ratios for IL1B and IL1RA were measured using a commercial and a newly established AlphaLISA kit, respectively. In both groups, a higher concentration of IL1B was detected in the SEpos group compared with the SEneg group (abattoir: p = 0.027; in vivo p < 0.001). No significant differences were observed in the concentration of IL1RA (p > 0.05). In uterine secretion samples retrieved in vivo, a lower IL1RA/IL1B ratio was detected in the SEpos group compared with the SEneg group (p = 0.002). The results of this study highlight the important role of IL1B and IL1RA during endometritis and the potential of the IL1RA/IL1B ratio as a possible biomarker for SE.

Gestational heat stress alters skeletal muscle gene expression profiles and vascularity in fetal pigs in a sexually dimorphic manner.

BACKGROUND: There is evidence that sow heat stress (HS) during gestation affects fetal development with implications for impaired muscle growth. We have previously demonstrated that maternal HS during early to mid-gestation compromised muscle fibre hyperplasia in developing fetal pigs. Thus, we hypothesised these phenotypic changes are associated with a change in expression of genes regulating fetal skeletal muscle development and metabolism. To test this, at d 60 of gestation, RNA sequencing and immunohistochemistry were performed on fetal longissimus dorsi (LD) muscle biopsies collected from pregnant gilts that had experienced either thermoneutral control (CON, 20 °C, n = 7 gilts, 18 LD samples) or controlled HS (cyclic 28 to 33 °C, n = 8 gilts, 23 LD samples) conditions for 3 weeks. RESULTS: A total of 282 genes were differentially expressed between the HS and CON groups in female LD muscles (false discovery rate (FDR) ≤ 0.05), whereas no differentially expressed genes were detected in male LD muscles between the two groups (FDR > 0.05). Gestational HS increased the expression of genes associated with transcription corepressor activity, adipogenesis cascades, negative regulation of angiogenesis and pro-inflammatory signalling in female LD muscles. Immunohistochemical analyses revealed a decreased muscle vascularity density in fetuses from HS group for both sexes compared to those from the CON group (P = 0.004). CONCLUSIONS: These results reveal gilt HS during early to mid-gestation altered gene expression profiles in fetal LD muscles in a sexually dimorphic manner. The molecular responses, including transcription and angiogenesis repressions and enhanced adipogenesis cascades, were exclusively observed in females. However, the associated reductions in muscle vascularity were observed independently of sexes. Collectively this may indicate female fetal pigs are more adaptive to gestational HS in terms of gene expression changes, and/or there may be sexually dimorphic differences with respect to the timing of muscle molecular responses to gestational HS.

Gene expression in bovine endometrial cells and blood-derived neutrophils stimulated by uterine secretions.

Uterine epithelial cells (UEC) and migrated polymorphonuclear cells (PMN) play important roles in the uterine defence against microbial infection. The aims of the present study were to investigate i) whether undiluted uterine secretions modulate the expression of genes associated with the innate immune response in UEC and PMN in vitro, ii) whether these changes differ between the two cell populations and iii) whether uterine secretions from cows with subclinical endometritis produce a different response to those from unaffected cows. Therefore, undiluted uterine secretions, cytobrush and biopsy samples were collected from bovine uteri at a local abattoir. All cows had calved at least 3 months prior to sample collection. Subclinical endometritis was diagnosed by cytology (≥5% polymorphonuclear neutrophils) and histology. The uteri were thereby retrospectively categorised as endometritis-positive (E-pos; n = 14), if either the cytology or the histology results were positive, or endometritis-negative (E-neg; n = 17), if both diagnostics were negative. Cultured UEC responded to secretions from E-pos and E-neg cows with an increased gene expression of CXC ligand (CXCL) 8 and interleukin (IL) 6 compared to incubation with control medium alone. PMN expressed significantly higher mRNA levels of CXCL5, CXCL8 and IL1B in response to supernatant from UEC incubated with secretions from both groups (E-pos and E-neg) compared to those incubated with control medium alone. Gene expression of IL10 in uterine epithelial cells remained comparable to the control in cells exposed to E-pos secretions and was 3.6 times lower in those exposed to E-neg secretions. These results demonstrate that the expression of genes associated with the innate immune response in UEC and indirectly also PMN is affected by uterine secretions in vitro. Depending on the target gene, these changes differ between the two cell populations. UEC exposed to uterine secretions from cows without subclinical endometritis produce lower levels of IL10 compared to those exposed to secretions from affected cows or control medium alone. Therefore, the model established in this study can be used as a valuable tool to further understand the contributions of the two cell populations to the coordinated immune response in the uterus.

Treatment of Retained Fetal Membranes in the Mare-A Practitioner Survey.

Retained fetal membranes (RFM) is a common post-partum problem in mares for which the treatment is highly variable. The aim of this study was (i) to investigate the different treatments used by equine practitioners for RFM and (ii) to determine if there is a difference between treatments used by reproductive specialists and general equine practitioners. Information regarding treatment of RFM was sought from veterinary practitioners via a survey and this was compared to recommendations in the current literature. The survey was sent out to equine veterinarians and mixed practitioners with a high equine case load. Most treatments of RFM were in line with current recommendations, while some obsolete practices are still routinely performed by a small number of practitioners. Treatment recommendations for RFM have changed over the last few decades, but there are no universally accepted guidelines. The vast variety of treatments reported by practitioners in the present survey reflect this lack of guidance. More extensive research is needed in this area to establish evidence-based, uniformly agreed upon protocols.