Plasma metabolomic profiling of healthy pregnant mares and mares with experimentally induced placentitis.

BACKGROUND: Metabolomics may represent an avenue for diagnosis of equine ascending placentitis. OBJECTIVES: To characterise the plasma metabolomic profile in healthy mares and mares with induced ascending placentitis, with the goal of identifying metabolites with potential clinical value for early diagnosis of placentitis. STUDY DESIGN: Controlled in vivo experiment. METHODS: Placentitis was induced in 10 late-term pregnant pony mares via Streptococcal equi subsp. zooepidemicus inoculation in five mares between days 285 and 290 of gestation, while five mares served as healthy controls. Repeated ultrasound examinations and jugular venipuncture were performed to obtain combined thickness of the uterus and placenta (CTUP) and plasma for NMR spectroscopy. Mares with increased CTUP were diagnosed with placentitis and treated in accordance with published therapeutic recommendations. NMR metabolomic analysis was performed to identify and quantify plasma metabolites at each time point. Concentrations were compared using ANOVA with repeated-measures and PLS-DA analysis. RESULTS: Four hours post-inoculation, a significant increase was detected in the metabolites alanine, phenylalanine, histidine, pyruvate, citrate, glucose, creatine, glycolate, lactate and 3-hydroxyisobutyrate that returned to baseline by 12 hours. On day 4, a significant reduction in the metabolites alanine, phenylalanine, histidine, tyrosine, pyruvate, citrate, glycolate, lactate and dimethylsulfone was seen in infected mares compared with controls. MAIN LIMITATIONS: There were small numbers of mares within groups. In addition, this work compares healthy animals with animals treated with multimodal therapeutics following diagnosis of placentitis without an untreated cohort. CONCLUSIONS: Two phases of metabolite changes were noted after experimental infection: An immediate rise in metabolite concentration involved in energy, nitrogen, hydrogen and oxygen metabolism within 4 hours after inoculation that was followed by a decrease in metabolite concentrations involved in energy and nitrogen metabolism at 4 days, coinciding with ultrasonographic diagnosis of placentitis.

Metabolomic Profile of Allantoic and Amniotic Fluid in Late-term Gestational Mares Characterized by 1H-nuclear Magnetic Resonance Spectroscopy.

The amniotic and allantoic fluid compartments in the mare serve essential roles throughout pregnancy and parturition. Although the global metabolomic profile of amniotic fluid in women has been extensively characterized, current data for equine fetal fluids are limited. Therefore, the goal of this study was to characterize the global metabolomic profile of equine allantoic and amniotic fluid through nuclear magnetic resonance spectroscopy. Fetal fluids were collected between 270 and 295 days of gestation from 12 pregnancies through ultrasound-guided transabdominal puncture. A total of 24 samples (n = 10 allantoic fluid; n = 9 amniotic fluid; n = 5 admixed fluid) were analyzed by one-dimensional proton (1H) and two-dimensional (1H-13 C) nuclear magnetic resonance spectroscopy. Metabolites were integrated and compared between fluid types using a Kruskal-Wallis test at P < .05 significance. A total of 28 distinct metabolites were found in allantoic and admixed fluid, whereas 23 metabolites were identified in amniotic fluid. Allantoic fluid contained significant elevations (P < .05) in the metabolites betaine, creatine, creatinine, citrate, histidine, nitrophenol, tryptophan, π-methylhistidine, and unknown metabolite #1 compared with amniotic fluid, whereas amniotic fluid contained statistically increased concentrations of the metabolite lactate compared with allantoic fluid (P = .003).

Transforming growth factor-beta (TGFbeta) and its signaling components in peri-ovulatory pig follicles.

The present studies investigated the hypothesis that TGFbeta plays a role in mediating LH/hCG-induced maturation, ovulation and/or luteinization of follicles in the pig. In Experiment 1, the temporal and spatial gene expression patterns of TGFbeta signaling components were examined in pig follicles which had been induced to ovulate and luteinize in vivo by hCG treatment, or by the LH-surge. Pre-pubertal pigs were injected with PG-600 followed by hCG, and ovaries were collected surgically at 0, 1, 12, 24 and 48h post-hCG. Post-ovulatory follicles were also collected from cycling gilts on Day 4 (D4) of the estrous cycle. Pre- and post-ovulatory follicles were used for the measurement of mRNA (PCR) and protein (Western blots) abundance and for protein localization by immunohistochemistry (IHC). Steady state amounts of mRNA for TGFbeta3 and TGFbetaR2 were increased (P<0.05, as compared to 0h) at 12h and on D4, respectively, while TGFbeta2 protein showed a tendency to increase on D4. TGFbeta signaling components did not change significantly. By IHC, the localization of TGFbeta components was as follows: pre-ovulatory follicles; TGFbeta1 - granulosa cells (GC), TGFbeta2 - theca cells (TC), TGFbetaR1 and 2 - GC and TC: post-ovulatory follicles; TGFbeta1 and 2 and TGFbetaR1 and 2 - luteinizing TC and GC. In Experiment 2, TGFbeta1 (1-100ng/ml) alone had no significant effect on progesterone (P4) secretion by pig GC in culture. Furthermore, while LH+IGF-1 (positive control) stimulated P4 approximately 10-fold, TGFbeta at 10 and 100ng/ml added in combination with LH+IGF-1, had no effect on P4 accumulation. In conclusion, data from the present study on temporal and spatial patterns of expression of the TGFbeta-system suggest that TGFbeta may play a role in the overall process of luteinization, but it appears not to influence steroidogenesis in luteinizing pig follicles.

Expression and localization of hypoxia inducible factor-1alpha mRNA in the porcine ovary.

Angiogenesis is an essential process during follicular development and corpora lutea (CL) formation. Recent studies have shown that vascular endothelial growth factor (VEGF) is an essential regulator of ovarian angiogenesis. Several lines of evidence have indicated that the production of VEGF is regulated by hypoxia inducible factor-1alpha (HIF-1alpha), especially under hypoxic conditions, but the expression of HIF-1alpha has not been well characterized in the porcine ovary. The present study examined the expression of HIF-1alpha mRNA and its localization in porcine ovaries at different stages of the estrous cycle. Northern blot analyses of total CL RNA indicated hybridization of the porcine HIF-1alpha probe to transcripts of approximately 3.8 kb. The mRNA expression of HIF-1alpha was highest in CL during the early luteal phase, followed by a decrease during the mid- and late-luteal phases. Using in situ hybridization, abundant HIF-1alpha mRNA was evident in follicles and CL. Within non-atretic follicles, HIF-1alpha mRNA was highly expressed in the granulosa cell layer, while weaker labeling was evident in the theca interna. These results suggest that HIF-1alpha may play a role in the regulation of cellular metabolism and blood supply during follicular growth and CL formation.