Phagocytic and killing capacities of uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis

The host defense competence of uterine-derived polymorphonuclear leukocytes (PMN) from mares considered resistant (grade I uteri) and susceptible (grade III uteri) to chronic endometritis was evaluated for phagocytic and killing (bactericidal) capacities, using a fluorochrome assay. Peripheral blood PMN from noncategorized mares and from grade I and grade III mares were used as controls. Uterine-derived PMN from mares with grade I uteri were functionally competent for phagocytosis and killing of Candida albicans, whereas uterine-derived PMN from mares with grade III uteri had significantly less phagocytic and killing capacities (P greater than or equal to 0.0001). Results of the present study, together with data obtained from chemotactic responsiveness and deformability assays of a previous study, indicated an overall deficiency in the host defense mechanism of uterine-derived PMN from mares with grade III uteri obtained 12 hours after induced Streptococcus zooepidemicus infection. This deficiency may account for the susceptibility of mares with grade III uteri to chronic endometritis.     

Endometrial nitric oxide synthase activity in mares susceptible or resistant to persistent breeding‐induced endometritis and the effect of a specific iNOS inhibitor in vitro

Emerging research suggests that the nitric oxide system may play a role in persistent breeding‐induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose‐dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activity in vitro. Six susceptible and six resistant mares were selected based on preset criteria and the results of an intrauterine challenge with killed spermatozoa during oestrus. Endometrial biopsy samples were collected 24 hr post‐challenge and cultured at 37°C for 24 hr in L‐arginine supplemented minimum essential medium with or without a specific iNOS inhibitor (1,400 W dihydrochloride, 1 mM). The medium and the cultured endometrial tissue were collected after 24 hr of culture and assayed for NO and total protein, respectively. Total NO content of the medium, normalized to endometrial tissue wet weight or total protein, was used as a measure of endometrial NOS activity. Non‐parametric tests were applied for statistical analysis. Susceptible mares had significantly greater endometrial NOS activity than resistant mares. The iNOS inhibitor treatment significantly reduced NOS activity in endometrial samples derived from susceptible and resistant mares. These findings provide a basis for in vivo testing of specific iNOS inhibitors as preventative or therapeutic options for PBIE in mares.     

Opsonins of Streptococcus in uterine flushings of mares susceptible and resistant to endometritis: control of secretion and partial characterization

The release of opsonins into the uterine lumen of mares susceptible or resistant to endometritis was examined after intrauterine inoculation of a filtrate of Streptococcus culture fluid or vehicle. Uterine flushings were collected at 0.5 hour before and 2, 4, 6, 8, and 24 hours after inoculation on day 2 or 3 of estrus and on day 7 or 8 after ovulation. Amounts of opsonins in flushings were quantified as the H2O2 produced by leukocytes incubated with flushings-opsonized bacteria, compared with H2O2 produced by leukocytes incubated with nonopsonized bacteria. Opsonin values in flushings increased (P less than 0.025) in all mares after inoculation of filtrate or vehicle. For mares resistant to endometritis, opsonin values were greater at diestrus than at estrus. The opposite was true for mares susceptible to endometritis, resulting in a status (susceptible vs resistant) X stage of cycle interaction (P less than 0.025). Overall, opsonins were higher (P less than 0.05) in flushings of mares susceptible to endometritis than in flushings of mares resistant to endometritis, but this difference was only apparent at estrus. Preliminary characterization of opsonins in uterine secretions by ammonium sulfate fractionation and gel filtration indicated that opsonins were mainly associated with an ammonium sulfate-soluble fraction of high molecular weight (greater than 4 X 10(6] and an ammonium sulfate-precipitable fraction that was associated with immunoglobulin G.     

Comparison of peripheral blood and uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis: chemotactic and cell elastimetry analysis

The functional competence of peripheral blood and uterine-derived polymorphonuclear leukocytes (PMN) from 12 mares were analyzed for chemotactic responsiveness using a chemotactic chamber (filter) assay and for deformability by cell elastimetry analysis. Peripheral blood PMN obtained from control mares and from 8 mares experimentally inoculated via the uterus with 1 x 10(9) Streptococcus zooepidemicus had similar normal chemotactic responsiveness and were highly deformable before and at 12 hours after inoculation. Uterine PMN obtained 12 hours after uterine inoculation with S zooepidemicus from resistant mares were not as deformable as peripheral blood PMN, but were within normal functional limits. The chemotactic responsiveness of uterine PMN from these mares was normal. Uterine PMN obtained from mares considered susceptible to endometritis 12 hours after uterine infection did not have chemotactic responsiveness and were nondeformable. The results indicated profound differences in the functional competence of uterine PMN between mares considered resistant and susceptible to chronic endometritis.     

The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating‐induced endometritis

In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating‐induced endometritis PMIE Select seminal plasma proteins cysteine‐rich secretory protein‐3 (CRISP‐3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP‐3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1 mg/ml CRISP‐3, 150 μg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10 ml combined with 1 × 10⁹ spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro‐inflammatory cytokines interleukin (IL)‐1β, IL‐8, tumour necrosis factor (TNF)‐α, interferon (INF)‐γ, anti‐inflammatory cytokines IL‐1RN and IL‐10, and inflammatory‐modulating cytokine IL‐6). Seminal plasma treatment increased the mRNA expression of IL‐1β (p = .019) and IL‐8 (p = .0068), while suppressing the mRNA expression of TNF (p = .0013). Lactoferrin also suppressed the mRNA expression of TNF (p = .0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro‐inflammatory response seen in susceptible mares.     

Concentrations of uterine luminal prostaglandins in mares with acute and persistent endometritis

Intrauterine infusion of 1 per cent oyster glycogen solution was used to induce acute endometritis in four genitally normal mares. Numbers of viable neutrophils recovered in uterine washings had increased by 1 h after infusion and remained elevated for at least 72 h. There was a significant correlation between numbers of viable neutrophils and total protein concentrations and between prostaglandin (PG)F and PGE2 concentrations in washings. There was also a significant relationship between concentrations of 15-keto-13, 14-dihydro PGF2 alpha in plasma and PGF in washings. Intrauterine concentrations of PGF were influenced by cycle stage and in turn the induced acute endometritis interfered with normal ovarian function. Mares with persistent endometritis had significantly higher concentrations of PGF and total protein and percentage of neutrophils and mononuclear cells in washings than normal mares. White blood cells from mares were capable of producing PGF and PGE2 in vitro.     

Plasma cell numbers in uteri of mares with persistent endometritis and in ovariectomised mares treated with ovarian steroids

Immunoglobulins A, G and M were localised by immunoperoxidase staining of endometrial sections from ovariectomised mares. Treatment with progesterone or oestradiol-17 beta did not significantly affect numbers of cells secreting any of the isotypes. Mares with persistent endometritis did not have significantly greater numbers of endometrial plasma cells than genitally-normal mares.     

Enrofloxacin‐based therapeutic strategy for the prevention of endometritis in susceptible mares

González, C., Moreno, L., Fumuso, E., García, J., Rivulgo, M., Confalonieri, A., Sparo, M., Sanchez Bruni, S. Enrofloxacin‐based therapeutic strategy for the prevention of endometritis in susceptible mares. J. vet. Pharmacol. Therap. 33 , 287–294. Enrofloxacin (EFX) is often used empirically to prevent uterine infections in mares in order to improve efficiency on Commercial Embryo Transfer Farms. This study investigated the uterine distribution of EFX and its metabolite ciprofloxacin (CFX) in mares and assessed the minimal inhibitory concentrations (MIC) of EFX against various common pathogens as a basis for establishing a rational dosing schedule. Plasma and uterine pharmacokinetic (PK) studies were performed in two groups (n = 5) of healthy mares following intravenous (i.v.) administration of EFX at either 2.5 and at 5 mg/kg bodyweight. Plasma and endometrial tissue samples, taken before for up to 48 h after treatment were analysed by Reverse Phase HPLC. MIC values for wild strains of Gram‐negative (Escherichia coli, Pseudomonas aeruginosa) and Gram‐positive bacteria (β‐haemolytic streptococci) ranged from 0.25–2 and 1.5–3.0 μg/mL respectively. In terms of tissue distribution, the sum of the endometrial concentrations of the parent drug (EFX) and its active metabolite (CFX) (in terms of AUC), exceeded those in plasma by 249% and 941% following administration of EFX at 2.5 and 5 mg/kg respectively. After i.v. treatment with EFX at 5 mg/kg, endometrial concentrations of EFX and CFX above the MIC value were detected for 36–48 and 22–43 h posttreatment for Gram‐negative and ‐positive isolates respectively. Concentrations above MIC were maintained for much shorter periods at the lower (2.5 mg/kg) treatment dose. Based on these results, a conventional dose (5 mg/kg) of EFX given prebreeding followed by two further doses at 36–48 h postbreeding are proposed as a rational strategy for using of EFX as a preventative therapy against a variety of common bacterial strains associated with equine endometritis.     

Effect of adjunctive treatment with intravenously administered Propionibacterium acnes on reproductive performance in mares with persistent endometritis

OBJECTIVE: To determine whether treatment with a preparation of Propionibacterium acnes would improve pregnancy and live foal rates in mares with persistent endometritis. DESIGN: Randomized placebo-controlled clinical trial. ANIMALS: 95 mares with a cytologic diagnosis of persistent endometritis. PROCEDURES: Mares were treated with P acnes or placebo (both administered IV) on days 0, 2, and 6. No attempt was made to alter additional treatments administered by attending veterinarians. Information on breeding history, physical examination findings, results of cytologic examination and microbial culture of uterine samples, additional treatments administered, breeding dates, results of pregnancy examinations, whether a live foal was produced, and reactions to treatment was recorded. RESULTS: In multivariate logistic regression models, mare age, year of entry into the study, and first breeding within 8 days after first treatment with P acnes or placebo were significantly associated with pregnancy. Fewer number of cycles bred and younger age were significantly associated with delivery of a live foal in a separate multivariate analysis. Results of multivariate logistic regression modeling indicated that mares treated with P acnes were more likely to become pregnant and to deliver a live foal, compared with placebo-treated controls. CONCLUSIONS AND CLINICAL RELEVANCE: IV administration of P acnes as an adjunct to conventional treatments in mares with a cytologic diagnosis of persistent endometritis improved pregnancy and live foal rates. The optimal effect was detected in mares bred during the interval extending from 2 days before to 8 days after first treatment with P acnes.