Model of Chronic Equine Endometritis Involving a Pseudomonas aeruginosa Biofilm.

Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with lux-engineered Pseudomonas aeruginosa strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent lux-labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria (P < 0.05). A greater incidence (P < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences (P > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased (P < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly (P < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that P. aeruginosa produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.

In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus.

In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of E. coli isolates, K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed for P. aeruginosa isolates. Chelating agents (EDTA formulations) reduced E. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of P. aeruginosa and K. pneumoniae within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. An in vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine P. aeruginosa isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent "biofilm-like" matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilm in vitro, and P. aeruginosa is capable of producing biofilm-like material in vivo.

Follicular fluid hydrogen peroxide and lipid hydroperoxide in bovine antral follicles of various size, atresia, and dominance status.

PURPOSE: To avoid inducing a state of oxidative stress (OS), assisted reproductive technologies (ART) must maintain a balance of reactive oxygen species (ROS) and antioxidants during the in vitro culture of oocytes. However, oocyte requirements and tolerance thresholds for ROS during in vivo development are still unclear. Previous studies have examined ROS levels in follicular fluid (FF) using pooled samples or according to follicle size. This study sought to examine two OS markers, lipid hydroperoxides (LPO) and hydrogen peroxide (H2O2), in FF of individually sampled follicles from bovine ovary pairs according to follicle size, atresia, and dominance status. METHODS: TUNEL and cleaved Caspase-3 labeling were used to identify apoptotic granulosa cells and determine follicle atresia status. LPO were measured directly for the first time in FF. RESULTS: Non-atretic follicles and dominant follicles contained more FF H2O2 than atretic follicles and corresponding subordinate follicles, respectively. FF LPO did not vary in relation to atretic status, and no difference existed between dominant and subordinate follicles. However, FF LPO was significantly lower in first subordinate follicles than in the second subordinate follicles from each ovary pair. Neither H2O2 nor LPO levels correlated with follicle size. CONCLUSIONS: These data provide clear evidence that the events of antral folliculogenesis are relevant to ROS dynamics in vivo. Furthermore, such studies will help to optimize in vitro conditions for oocyte culture protocols, particularly when combined with a comparison of oocyte quality with respect to source follicle characteristics.

Media composition: antioxidants/chelators and cellular function.

Protection of embryos against oxidative insults during culture is necessary to maintain viability. Generation of excessive levels of reactive oxygen species (ROS) is triggered by various components of the in vitro environment, most of which embryos do not normally encounter in vivo. To compensate for these deficiencies in the culture environment, antioxidants and chelators are often used to control or suppress ROS levels as embryos develop. However, there is no consensus regarding dosage, time of exposure, or appropriate combinations of antioxidants and chelators in embryo culture. In order to elucidate this aspect of an embryo's chemical surroundings in vitro, we present the current knowledge on the function and effect of each antioxidant or chelator that is often included in an embryo culture medium.

The antral follicle: a microenvironment for oocyte differentiation.

Mammalian reproduction hinges upon the timely ovulation of a fully differentiated oocyte. This event is the culmination of a complex and dynamic developmental relationship between the oocyte and the antral follicle housing it; the antral follicle constitutes a specialized microenvironment or niche, uniquely suited to the needs of the oocyte as it approaches ovulation. During this time, the oocyte must complete its final growth, capacitation, and nuclear and cytoplasmic maturation. Its microenvironment--the antral follicle--is in turn responsible for the integrity of these processes and the production of a high quality oocyte. Components of the antral follicle, including three distinct somatic cell types (theca, granulosa and cumulus), the basal lamina, and follicular fluid, each have active and regulatory roles in oocyte differentiation. Several milestones in antral folliculogenesis also have an influence on oocyte development. This review will discuss the antral follicle microenvironment with specific attention to its importance in oocyte differentiation. As assisted reproductive technologies (ART) often require stages of oocyte differentiation to occur in vitro rather than in vivo, current knowledge of the antral follicle microenvironment will also be discussed with respect to its clinical applications.