Persistent Mating-induced Endometritis Research Articles

General and comparative aspects of endometritis in domestic species: a review.

Endometritis is a leading cause of sub- and infertility in domestic animal species. The healthy uterus is colonized by commensal bacteria, viruses, and yeast/fungi that represent the non-pathogenic microbiota. A shift in the number or type of organisms accompanied by immune dysfunction, however, may trigger uterine infection and inflammation. Metritis is associated with inflammation of all uterine layers (endometrium, myometrium, and perimetrium) whereas endometritis is a more superficial inflammation involving solely the endometrium. Endometritis generally occurs at two time points in domestic animal species, post-partum and post-mating. Post-partum endometritis may chronically persist, either as a low-grade disease that often manifests as a vaginal discharge but not a systemic illness (in some species termed clinical endometritis) or sometimes subclinical where features are only detected by endometrial sampling. Contamination of the uterus at the time of mating occurs by direct deposition of semen (ejaculated or artificially inseminated) into the uterus. Improper drainage of the ejaculatory fluid or an inadequate immune response may result in persistent mating-induced endometritis. Both post-partum and post-mating endometritis interferes with fertility by creating a suboptimal environment for embryo development and placentation and chronic endometritis may have an impact on sperm survival and fertilisation ability. In the post-partum animal, there may also be changes in milk production and maternal behaviour which can affect offspring health and survival. Preventive strategies for endometritis largely depend on monitoring their known risk factors, which are sometimes specific with regard to the species. Effective, non-antibiotic therapy for endometritis is not available to date. Overall, extensive research has been performed in cattle and horses to unravel key aspects of endometritis, but in sows and bitches, the available literature is scant. Thus, the need and opportunity to investigate the condition vary considerably among domestic species and necessitate their comparative assessment. This article reviews general and comparative aspects of the diagnosis and classification, pathogenesis, preventive strategies, and therapeutics of endometritis in domestic species with a specific focus on cows, mares, sows, and bitches.

The effect of dexamethasone and flunixin-meglumine on ovulation, endometrial oedema, and inter-ovulatory interval length in the mare

The use of flunixin-meglumine (a potent non-steroidal anti-inflammatory drug) during the critical period of intrafollicular prostaglandin production before ovulation (24 and 36 h after hCG treatment) results in a high rate of ovulatory failure and formation of haemorrhagic anovulatory follicles (HAF) in the mare. Dexamethasone is commonly used to prevent persistent mating-induced endometritis in susceptible mares, but the effect on ovulation blockage within the pre-ovulatory critical window of intrafollicular prostaglandins production following hCG administration has not been determined. Six mares were followed during four consecutive cycles in a crossover design; once in oestrus with a follicle of >32 mm in diameter, mares were treated with hCG (Hour 0) and assigned to one of 4 groups randomly: 1) FM, mares received 1.7 mg/kg flunixin-meglumine at Hour 24 and 36; 2) CON, mares received no further treatment. 3) DEX1, mares received 0.1 mg/kg dexamethasone at Hour 24, and 4) DEX2, mares received 0.1 mg/kg dexamethasone at Hour 24 and 36. For all groups, ovulation and HAF rates, endometrial oedema profiles and the inter-ovulatory intervals (IOI) were determined and compared statistically. All CON and DEX mares ovulated normally and did not form any HAF. On the contrary, FM mares developed a HAF in 83% of cycles (P < 0.01). The endometrial oedema score was lower following DEX administration than FM (P < 0.05). The mean IOI was longer (P < 0.05) in DEX1 and DEX2 groups (26.5 and 26 days, respectively) than in CON and FM groups (21.5 and 22 days, respectively). In conclusion, dexamethasone treatment given either once or twice during the critical window of hCG-induced ovulation did not block or delay ovulation, but had a similar ovulation rate than untreated control mares. However, the inter-ovulatory intervals of dexamethasone treated mares was longer than control and FM treated mares. Finally, dexamethasone treatment was more effective in reducing endometrial oedema than FM.

Effect of platelet lysate on uterine response of mares susceptible to persistent mating-induced endometritis

Many mares are susceptible to persistent mating-induced endometritis (PMIE), an important cause of reduced fertility. Platelet lysate (PL) derives from freeze-thawing platelets after concentration, so that growth factors are released from the platelets. Among the advantages of PL compared to platelet-rich plasma (PRP), it can be frozen stored and allogenic use for PL might also be conceivable. Platelet-rich plasma beneficially reduced inflammatory response in PMIE mares when administered 24 h pre- or 4 h post-AI. The aim of this study was to test the effect of PL on inflammatory uterine response in mares susceptible to PMIE. A total of 14 mares susceptible to PMIE (based on presence of fluid or inflammatory cells 24 h after AI) underwent an untreated (Ctr) cycle followed by a treated (PL) cycle. From each mare, 100 mL of citrated whole blood was obtained for PRP production by centrifugation. The resultant PRP was brought to a final volume of 10 mL with platelet poor plasma and frozen at −80 °C to obtain PL. On untreated cycles, mares were inseminated with frozen-thawed semen 36 h after ovulation induction. On treated cycles, PL was thawed, infused into the uterus 12 h after ovulation induction, and AIs were performed 24 h later. The number of neutrophils in uterine cytology (score 1(normal)-3(severe inflammation)) evaluated by optical microscopy, uterine fluid accumulation (height x width) and uterine edema (score 0–3) observed in ultrasonography, were analysed. Pregnancy was evaluated by ultrasonography 14 days after ovulation. A significant decrease (P < 0.05) was observed on cytology score (PL 1.3 ± 0.1 vs Ctr 2.0 ± 0.1), fluid accumulation (PL 79.5 ± 30.1 mm2 vs Ctr 342.7 ± 52.9 mm2) and edema score (PL 1.8 ± 0.2 vs Ctr 2.3 ± 0.2) in treated mares. Pregnancy rate in PL-treated cycles (3/12) and control cycles (2/14), were not significantly different (P > 0.05). According to the results, we conclude that treatment with PL in mares classified as susceptible to PMIE appears to reduce the inflammatory response after breeding, based on clinical signs of uterine edema, IUF accumulation and PMNs migration.

Periovulatory administration of firocoxib did not alter ovulation rates and mitigated post-breeding inflammatory response in mares

Non-steroidal anti-inflammatory drugs (NSAIDs) are a therapeutic option for the treatment of inflammation. However, negative effects of non-selective NSAIDs for treatment of mares with endometritis have been described, including delayed uterine clearance and impairment of ovulations. Firocoxib is a specific cyclooxygenase-2 (COX-2) inhibitor and has the ability to act in the uterus of mares. We investigated the effects of firocoxib on ovulation rate, numbers of polymorphonuclear neutrophils (PMNs), and COX-2 protein levels in the endometrial tissue of susceptible mares after insemination. Two experiments were conducted. In experiment 1, twenty mares were evaluated in two consecutive estrous cycles broken into the following groups: Control - no pharmacological interference; Treatment - mares were treated with 0.2 mg/kg of firocoxib orally. The treatment began on the day of ovulation induction, and firocoxib was administered until one day after artificial insemination (AI). Ovulation was induced with 1 mg of deslorelin acetate and the mares were inseminated 24 h after the injection. Ovulation was confirmed 48 h after induction, and embryos were collected eight days after ovulation. Experiment 2: Nine mares susceptible to persistent mating-induced endometritis (PMIE) were artificially inseminated. The mares were examined with ultrasound and inseminated with fresh semen in two consecutive cycles, control and treated, in a cross-over study design. The amount of intrauterine fluid was measured, and endometrial samples were collected 24 h after AI. The number of PMNs was determined by endometrial cytology and biopsy, and COX-2 labeling in endometrial samples was evaluated by immunohistochemistry. Firocoxib treatment did not induce ovulatory failure or affect embryo recovery rate in Experiment 1. In Experiment 2, firocoxib treatment reduced inflammation after AI in mares as evidenced with results regarding PMN numbers/percentage and endometrial COX-2 staining. In conclusion, the proposed treatment with firocoxib reduced endometrial inflammation in mares susceptible to PMIE after breeding, with no adverse effects.

Preimplantation factor modulates acute inflammatory responses of equine endometrium.

Persistent mating‐induced endometritis (PMIE) is a significant cause of mare infertility hence its treatment would advance the management of susceptible mares. Preimplantation factor (PIF) is secreted by viable embryos, including human, mouse and cattle, and is essential for maternal immune‐tolerance without immune‐suppression by modulating inflammation. This preliminary study aimed to test whether PIF exerts inflammatory‐modulating properties upon equine endometrium challenged with Escherichia coli‐derived lipopolysaccharide (LPS) using endometrial explant culture. Follicular (n = 3), luteal (n = 4), anoestrous (n = 4) and transitional (n = 4) stage endometrial explants were established and cultured in triplicate in either serum‐free medium alone (control) or medium with; 50 or 100 nmol/L synthetic PIF (sPIF); 3 μg/mL LPS; LPS and 50 or 100 nmol/L sPIF; or scrambled PIF (PIFscr; same amino acid composition arranged in a different order). Media samples were collected at 24 and 72 h, representing acute and chronic inflammatory response. Radioimmunoassay determined Prostaglandin F2α (PGF 2α) as an inflammatory marker. The only significant observation was the abrogation of PGF 2α response to LPS challenge by 100 nmol/L PIF for follicular stage tissue, 24 h after treatment. Further studies are therefore, warranted to realise PIF potential in managing PMIE.

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 β-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.

Uterine clinical findings, fertility rate, leucocyte migration, and COX-2 protein levels in the endometrial tissue of susceptible mares treated with platelet-rich plasma before and after AI

Persistent mating-induced endometritis (PMIE) results in decreased fertility in horses, thereby causing a significant impact in the horse market. Platelet-rich plasma (PRP), a modulator of the inflammatory response, has been largely used in veterinary medicine. Here, we investigated the effects of PRP on uterine inflammation, conception rate, endometrial polymorphonuclear neutrophil (PMN) migration, and COX-2 protein levels in the endometrial tissue. Thirteen PMIE-susceptible mares were used for artificial insemination (AI). The mares were inseminated with fresh semen in three consecutive cycles in a cross-over study design. The following cycle classifications were used: control cycle, no pharmacological interference; pre-AI, 20 mL of PRP was infused 24 h before AI; and post-AI, 20 mL of PRP was infused four h after AI. Follicular dynamics were monitored daily by transrectal ultrasound. When a follicle larger than 35 mm was detected, ovulation was induced with deslorelin acetate (1 mg, im). AI was performed 24 h after ovulation induction. Intrauterine fluid (FLU) was evaluated by ultrasonography before and 24 h after AI. PMNs in uterine cytology (CYT) and biopsy (HIS) were also observed before and 24 h after AI. Pregnancy was determined within 14 days after ovulation. Number of COX-2 positive cells was evaluated by immunohistochemistry. Both PRP treatments resulted in a decrease of PMNs in the CYT after breeding when compared to controls. FLU did not differ between cycles; however, the conception rates were significantly higher in the PRP mares. Mares positive for endometritis decreased in both treatment groups, and a more intense positive COX-2 labeling was observed in the control group when compared to the two treatment groups. In conclusion, PRP beneficially reduces inflammatory response in PMIE mares independent of when treatments were administered, thus increasing chances of successful pregnancy.

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: 1mg/ml CRISP-3, 150μg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10ml 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.

Exogenous lactoferrin suppresses the mRNA expression of the pro-inflammatory cytokine tumor necrosis factor (TNF) in mares susceptible to persistent mating-induced endometritis

Exogenous lactoferrin suppresses the mRNA expression of the pro-inflammatory cytokine tumor necrosis factor (TNF) in mares susceptible to persistent mating-induced endometritis

Non-traditional treatments for endometritis in mares

Routine treatment for persistent mating-induced endometritis is directed at enhancing the clearance of accumulated fluid from the uterus and includes the use of ecbolic agents (oxytocin, prostaglandin F2a) which may be used alone or in combination with large-volume uterine lavage and administering antimicrobials if infection is diagnosed. However, traditional therapies are not always effective in resolving chronic uterine inflammation or infections. Treatment failure may be caused by uterine exudates, inspissated mucus or biofilm produced by some bacteria and yeast. Exudate can interfere with antibiotic penetration, whereas biofilm can confer antibiotic resistance. The endometrium of mares with delayed uterine clearance or chronic endometritis produces more mucus than reproductively healthy mares. Loss of cilia and abnormal mucus blanket provide areas for bacterial attachment. This review describes new strategies of endometritis treatment that may be added to typical therapy and includes intrauterine mucolytics, chelators, immunomodulators, corticosteroids, nonsteroidal antiinflammatory drugs and others.

Use of mesenchymal stem cells or autologous conditioned serum to modulate the inflammatory response to spermatozoa in mares

Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were performed with six normal mares in each study to evaluate the effects of intrauterine administration of ACS, dexamethasone, or a placebo (experiment 1), or allogeneic MSCs or a placebo (experiment 2) on the inflammatory response to spermatozoa using clinical and biochemical endpoints. Treatment with ACS and MSCs significantly (P < 0.05) reduced the number of neutrophils in the uterine lumen 6 hours after the sperm challenge. An increase (P < 0.05) in the anti-inflammatory cytokine IL-1Ra was observed after treatment with MSCs before exposure to spermatozoa. There was no difference in IL-1Ra concentration in mares treated with ACS, dexamethasone, or a placebo. Mesenchymal stem cells and ACS were able to modulate the immune response to spermatozoa in normal mares. The effect may be due to an increase in IL-1Ra in MSCs-treated mares, but other bioactive molecules may be responsible for the decrease in neutrophils in ACS-treated mares. Autologous conditioned serum and bone-derived culture expanded MSCs were able to modulate the uterine inflammatory response to spermatozoa in normal mares. Treatment with allogeneic stem cells may be beneficial if a similar modulation in inflammatory cytokines occurs in mares affected by persistent mating–induced endometritis.

The effect of Platelet-Rich Plasma (PRP) on intraluminal fluid and pregnancy rates in mares susceptible to Persistent Mating-Induced Endometritis (PMIE)

The effect of Platelet-Rich Plasma (PRP) on intraluminal fluid and pregnancy rates in mares susceptible to Persistent Mating-Induced Endometritis (PMIE)

Effect of contextually congruent stallion vocalization playback on intrauterine pressure in the mare

Although oxytocin can be an effective treatment for persistent mating-induced endometritis (PMIE), frequent administration is required due to the short half-life of oxytocin and exogenous oxytocin may produce tetanic spasms of the myometrium. Teasing with a stallion during estrus elicits endogenous oxytocin secretion and uterine contractions, which may result in a more optimum physiologic response. Recently, it has been shown that mares will respond to contextually congruent stallion vocalization recordings with teasing behavior. The aim of this study was to determine if contextually congruent stallion vocalization would induce uterine contractions and increased uterine pressure in the mare. We hypothesized that teasing, contextually congruent stallion vocalization (with or without stallion scent), and exogenous oxytocin would increase intrauterine pressure in estrous pony mares, but that oxytocin would cause the greatest increases in intrauterine pressure. Changes in uterine pressure were recorded using a wireless telemetry capsule inserted into the uterine lumen (SmartPill pH.p Capsule), a novel method of instrumentation for uterine pressure studies. Twentyfour hours after administration of human chorionic gonadotropin, five reproductively normal pony mares were instrumented with an intrauterine telemetry pressure capsule during estrus. Baseline pressure was recorded for

Effect of ketoprofen treatment on the uterine inflammatory response after AI of jennies with frozen semen

Artificial insemination (AI) involving the placing of frozen-thawed semen directly into the jenny uterine body is associated with very low pregnancy rates. This might be because of an exacerbation of the acute response of the endometrium to sperm, as seen in mares with persistent induced mating endometritis. Pregnancy rates can be increased in such mares, however, by including anti-inflammatory treatments in the insemination protocol (Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. Theriogenology 2008;70:1093–100; Rojer H, Aurich C. Treatment of persistent mating-induced endometritis in mares with the non-steroid anti-inflammatory drug vedaprofen. Reprod Domest Anim 2010;45:e458–60). To investigate the endometritis caused by the use of frozen-thawed semen in jennies, and to assess the response to ketoprofen treatment, endometrial cytological samples and biopsies from six healthy jennies were examined in a crossover design experiment. Samples were taken from jennies in estrus (E; control) and at 6 hours after AI with or without ketoprofen (+K and −K, respectively). Ketoprofen was administered iv 24 hours before and for 4 days after insemination (total = 2.2 mg/kg/24 hours for 5 days). All animals showed a severe inflammatory response to semen deposition. Polymorphonuclear neutrophil numbers in the cytological smears and biopsies differed significantly between the +K and E animals. No significant differences were recorded, however, between the +K and −K treatments. Eosinophils were observed in all sample types from all groups; these cells appear to be a feature of the normal jenny endometrium. Slight fibrosis was observed in some biopsies, but no significant relationship with inflammation was found. Intense cyclooxygenase-2 (COX-2) immunohistochemical labeling was detected in the −K biopsies. Less intense labeling was seen in those of the +K animals, and mainly localized in the stratum compactum. No differences in COX-2 labeling were observed between the +K and E animals. Plasma concentrations of ketoprofen remained detectable until 2 hours after administration, after which the compound was rapidly eliminated. In summary, jennies are susceptible to endometritis after insemination with frozen-thawed semen. Ketoprofen reduces this inflammation by inhibiting COX-2; no reduction in the number of polymorphonuclear neutrophils occurs. The physiological and pharmacological characteristics of jennies should be taken into account when designing treatments for acute endometritis aimed at enhancing pregnancy rates after insemination with frozen-thawed sperm.

The effect of platelet-rich plasma on endometrial pro-inflammatory cytokines in susceptible mares following semen deposition

Mares susceptible to persistent mating-induced endometritis (PMIE) are a source of substantial economic inefficiency. Recent studies report a significant post insemination up-regulation of endometrial anti-inflammatory cytokines IL-10 and IL-Ra in normal versus susceptible mares, as well as significantly increased and prolonged expression of IL-1b, IL-6, IL-8, and IL1Ra in susceptible mares following intrauterine inoculation with E.coli [1]. Furthermore, nitric oxide is suspected to play a significant role in uterine clearance, because increased endometrial expression of inducible nitric oxide synthetase (iNOS) has been reported in susceptible mares following insemination [2]. This project was developed to investigate the expression of inflammatory biomarkers of uterine biopsies obtained from susceptible mares following intrauterine PRP (platelet-rich plasma) infusion. Platelets contain alpha-granules that, upon activation, secrete bioactive molecules with regenerative capacity in the form of growth factors, cytokines, and chemokines. Barrenmares with a history of PMIE (n 1⁄4 9) underwent daily ultrasonographic examination of the reproductive tract per rectum while in estrus. All mares underwent an untreated (no PRP) cycle followed by a treated (PRP) cycle. When a dominant follicle >35 mm in diameter and evidence of endometrial edema were detected, 1.0 mg deslorelin acetate (SucromateR Bioniche Animal Health Pullman, WA) was administered intramuscularly. On the treatment cycle, 180 ml of citrated whole blood was obtained for PRP processing in a specialized blood fraction separating centrifuge system (Angel Cytomedix, Inc. Gaithersburg, MD). The resultant PRP was brought to a final volume of 10 ml with plateletpoor plasma and infused into the uterus. Mares were inseminated with >200 million motile sperm 24-36 hours following deslorelin administration. An endometrial biopsy obtained the following day was preserved in RNAlaterR (Sigma Inc., St Louis, MO) for reverse transcription polymerase chain reaction (RT–PCR) analysis [1] for mRNA expression of inflammatory biomarkers IL-1b, IL1-Ra, TNFa, IL-6, IL-8, IL-10, and iNOS. Data were analyzed using SAS (SAS Institute, Cory NC) with Wilcoxon signed-rank test (non-parametric). Comparisons were made between pretreatment and post-treatment groups. Significance was set to P < 0.05. In susceptible mares treated with an intrauterine infusion of PRP, the mRNA expression of IL-1b, IL-6, IL-8 and iNOS was significantly down-regulated (P < 0.05) when compared with untreated mares. All other cytokines were similar in treated and untreated mares. Therefore, following semen deposition, PRP influences the mRNA expression of endometrial pro-inflammatory cytokines and iNOS in mares susceptible to PMIE. References

Efficacy of human chorionic gonadotropin to induce ovulation in the mare, when associated with a single dose of dexamethasone administered at breeding time

Human chorionic gonadotropin (hCG) and dexamethasone have a role in the reproductive management protocols of mares susceptible to persistent mating-induced endometritis (PMIE). However, it is possible that there is interference between these 2 drugs. The objective of this retrospective study was to determine the efficacy of hCG to induce ovulation in mares treated with dexamethasone at breeding time. Medical records from 152 mares and 223 cycles were analysed. In 113 cycles, mares susceptible to PMIE were treated with 1500 iu hCG and a single dose of 50 mg dexamethasone, 110 cycles were used as controls and subsequent ovulation was assessed ultrasonographically and compared. Dexamethasone did not inhibit ovulation in mares susceptible to PMIE. Multiple administrations of dexamethasone to mares in early oestrus have induced ovulation failure. However, a single dose of dexamethasone, administered at breeding time, has been used as an effective modulator of PMIE in susceptible mares and does not interfere with efficacy of hCG to induce ovulation.

Treatment of Persistent Mating‐Induced Endometritis in Mares with the Non‐Steroid Anti‐Inflammatory Drug Vedaprofen

Recently, successful treatment of mares with a history of persistent mating-induced endometritis (PMIE) with dexamethasone has been reported. As systemic treatment of horses with glucocorticoids should be handled with caution, we tested the hypothesis that treatment with the non-steroid anti-inflammatory drug (NSAID) vedaprofen, an inhibitor of cyclooxygenase-2, may have comparative, positive effects on fertility. Barren mares with a history of repeated PMIE were treated with vedaprofen (n = 8; initially 2 mg/kg bodyweight followed by 1 mg/kg orally twice daily) from 1 day before the first insemination to 1 day after ovulation or left untreated (n = 9). All mares received oxytocin (20 I.E. s.c.) thrice daily. Uterine swabs were collected for bacteriology and cytology. The day after ovulation, fluid accumulation was detected in three control mares and four treated mares (n.s.). The percentage of neutrophils in uterine cytology was significantly increased in comparison to the day before ovulation irrespective of treatment. Pregnancy was confirmed in two of nine mares in the control group and seven of eight mares in the treatment group (p < 0.05). NSAIDs may positively affect fertility in mares with a history of PMIE.

Effect of seminal plasma on uterine inflammation, contractility and pregnancy rates in mares

There is conflicting evidence over the role seminal plasma plays in sperm transport and inflammation within the uterus of mares. In in vitro studies, seminal plasma has been shown to reduce polymorphonuclear neutrophil (PMN) function, but the opposite effect on uterine inflammation has been reported in vivo. To study the effect of seminal plasma on uterine contractility, inflammation and pregnancy rates by inseminating mares with low doses of sperm free from seminal plasma (Group 1) and containing seminal plasma (Group 2). Synchronised mares were inseminated with 50 x 10(6) sperm in either skim milk extender or seminal plasma. Uterine lavage was performed 6 h after insemination to assess the inflammatory response. The contraction frequency of the uterus was measured over a 4 min period 10 mins and 6 h after insemination, using B-mode ultrasonography. Pregnancy rates were assessed 16 days after insemination. Uterine contractions were less frequent in Group 1 mares inseminated with seminal plasma and significantly more PMNs were found in the lavage fluid of those mares. Pregnancy rates were identical in both groups (62%). This study provides evidence that seminal plasma decreases uterine contractility and increases the inflammatory response of the uterus to semen. No effect of seminal plasma on pregnancy rates was demonstrated. Mares that develop persistent mating-induced endometritis may have inherently poor uterine contractility and impaired uterine clearance. The presence of seminal plasma during breeding may not be desirable in these mares. The role of seminal plasma in problem mares warrants additional study.

Development and clinical application of a broad range 16S quantitative PCR assay for detection of bacteria in the uterus of the mare

Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were performed with six normal mares in each study to evaluate the effects of intrauterine administration of ACS, dexamethasone, or a placebo (experiment 1), or allogeneic MSCs or a placebo (experiment 2) on the inflammatory response to spermatozoa using clinical and biochemical endpoints. Treatment with ACS and MSCs significantly (P < 0.05) reduced the number of neutrophils in the uterine lumen 6 hours after the sperm challenge. An increase (P < 0.05) in the anti-inflammatory cytokine IL-1Ra was observed after treatment with MSCs before exposure to spermatozoa. There was no difference in IL-1Ra concentration in mares treated with ACS, dexamethasone, or a placebo. Mesenchymal stem cells and ACS were able to modulate the immune response to spermatozoa in normal mares. The effect may be due to an increase in IL-1Ra in MSCs-treated mares, but other bioactive molecules may be responsible for the decrease in neutrophils in ACS-treated mares. Autologous conditioned serum and bone-derived culture expanded MSCs were able to modulate the uterine inflammatory response to spermatozoa in normal mares. Treatment with allogeneic stem cells may be beneficial if a similar modulation in inflammatory cytokines occurs in mares affected by persistent mating–induced endometritis.

Markers of the uterine innate immune response of the mare

Reproductive efficiency in mares is low and persistent mating-induced endometritis (PMIE) is an important cause of subfertility. Mating-induced endometritis (MIE) an obligate precursor to PMIE, is a ubiquitous, transient inflammatory response to the presence of sperm, seminal components and pathogens. However, the specific inflammatory pathways that derive from MIE and that may also be precursors to PMIE are not clear. The ability to identify and measure robust, repeatable markers of inflammation integral to MIE may be key to understanding the progression to PMIE. The aim of the study was to (i) refine a protocol for inducing MIE and in doing so test a range of cellular and molecular parameters as valid markers of MIE to facilitate future studies of mares susceptible to PMIE (ii) concurrently identify those parameters with potential as inflammatory indicators during MIE to inform and enhance early treatment regimens in practice. Mating-induced endometritis was induced in pony mares using a stringent protocol; mares were treated intrauterine with frozen/thawed semen ( n = 5; FTS) or frozen/thawed extender ( n = 6: FTEx). The parameters tested were measured before treatment were compared to samples collected at strategic time points after treatment: uterine cytology using cytological (at 8, 16, 24, 48 and 72 h after treatment) or histological analysis (at 24 and 72 h); uterine bacteriology (at 24 and 72 h); secretion of prostaglandin F 2α (PGF 2α; at 8, 16, 24, 48 and 72 h); peripheral concentrations of serum amyloid A (SAA; at 24 h); endometrial mRNA gene expression, focussing upon IL8 and TLR4, as examples of genes pertinent to inflammation (at 24 h). Uterine neutrophil cell numbers in both treatment groups increased at 8 ( P < 0.001), 16 ( P < 0.01) and 24 ( P < 0.01) h after insemination, indicative of MIE and distinguished between different treatments because neutrophil numbers were greater from FTS mares than FTEx mares 8 h after challenge. Uterine neutrophil cell numbers, assessed by histology, increased ( P < 0.001) 24 and 72 h after treatment. Prostaglandin F 2α concentrations increased ( P < 0.05) 16 h after treatments, while SAA concentrations and bacterial growth scores were not significantly different after treatment. Endometrium from pony mares expressed mRNA for IL8 and TLR4 but expression was not altered after insemination. The protocol induced MIE, as confirmed by uterine cytology and maybe used hereafter as a repeatable and robust method for studying immune mechanisms that underlie MIE and so may aid the understanding of progression to persistent inflammation. It can be concluded that of the range of parameters tested, neutrophil cell numbers by cytological analysis and PGF 2α were regarded as the most accurate markers of inflammation during MIE and important for use in practice.