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.

Characterization of an Ex Vivo Equine Endometrial Tissue Culture Model Using Next-Generation RNA-Sequencing Technology.

Simple SummaryNotwithstanding extensive research into fertility problems in mares, pregnancy rates have remained low mainly because of endometrial inflammation (endometritis). In the field of equine research, endometrial explants have been used to carry out in vitro studies of the mare’s endometrium. However, there has been no wide-ranging assessment of relative stability of this model over time. The aim of this study was to perform an in-depth transcriptomic assessment of endometrial explants over a culture period of 72 h and assess if they are representative of the whole mare. Explants at 24 h demonstrated significant changes when compared to biopsies at 0 h as expected. Even though gene expression changes were seen between 24 and 48 h of culture, prior to this window changes were dominated by the effects of explanting and culture and subsequently, transcription was generally compromised. Our results, therefore have defined the optimal period when explants can be used to study equine endometritis and how the endometrium is modulated during inflammation. It highlights the use of abattoir derived samples to understand the physiology and pathophysiology of the equine endometrium, negating the need to collect repeated uterine biopsies from living mares.Persistent mating-induced endometritis is a major cause of poor fertility rates in the mare. Endometritis can be investigated using an ex vivo equine endometrial explant system which measures uterine inflammation using prostaglandin F2α as a biomarker. However, this model has yet to undergo a wide-ranging assessment through transcriptomics. In this study, we assessed the transcriptomes of cultured endometrial explants and the optimal temporal window for their use. Endometrium harvested immediately post-mortem from native pony mares (n = 8) were sampled (0 h) and tissue explants were cultured for 24, 48 and 72 h. Tissues were stored in RNALater, total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were defined using DESeq2 (R/Bioconductor). Principal component analysis indicated that the greatest changes in expression occurred in the first 24 h of culture when compared to autologous biopsies at 0 h. Fewer DEGs were seen between 24 and 48 h of culture suggesting the system was more stable than during the first 24 h. No genes were differentially expressed between 48 and 72 h but the low number of background gene expression suggested that explant viability was compromised after 48 h. ESR1, MMP9, PTGS2, PMAIP1, TNF, GADD45B and SELE genes were used as biomarkers of endometrial function, cell death and inflammation across tissue culture timepoints. STRING assessments of gene ontology suggested that DEGs between 24 and 48 h were linked to inflammation, immune system, cellular processes, environmental information processing and signal transduction, with an upregulation of most biomarker genes at 24 h. Taken together our observations indicated that 24–48 h is the optimal temporal window when the explant model can be used, as explants restore microcirculation, perform wound healing and tackle inflammation during this period. This key observation will facilitate the appropriate use of this as a model for further research into the equine endometrium and potentially the progression of mating-induced endometritis to persistent inflammation between 24 and 48 h.

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.

Relationship between the presence of intrauterine fluid and cervical bacteria in heavy draft mares before and after mating.

ABSTRACTWhile intrauterine fluid (IUF) is observed as mucous in healthy mares during estrus, the presence of IUF during diestrus is an indicator of endometritis. Mating induces endometrial inflammation in healthy mares, called mating-induced endometritis, that disappears within 1–2 days after mating. On the other hand, it is also known that IUF remaining beyond 12 hr after mating can cause persistent inflammation, bacterial growth, and decreased fertility. This study aimed to investigate the relationship between the presence of IUF and cervical bacteria before and after mating. Mares were classified into three groups based on the degree of IUF: Grade 1 (G1), neither IUF nor echogenic lines at the inner lining of the uterus; Grade 2 (G2), small amount of IUF (<0.5 cm in diameter) or an echogenic lines in the intrauterine cavity; and Grade 3 (G3), obvious IUF (≥0.5 cm in diameter). The positive culture rates of G1, G2, and G3 mares before mating were 15.8% (n=6/38), 25.9% (n=7/27), and 31.8% (n=14/44), respectively, and did not differ significantly between the groups. After mating, the positive culture rates of G1, G2, and G3 mares were 18.2% (n=2/11), 56.1% (n=23/41), and 72.3% (n=34/47), respectively. The positive culture rates were significantly higher in G2 (P<0.05) and G3 (P<0.01) mares compared with G1 mares. These results from after mating suggest that not only mares with obvious IUF but also those with a small amount of IUF (<0.5 cm in diameter) or echogenic lines in the intrauterine cavity are at risk of mating-induced endometritis.

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

The Effect of Cysteine-Rich Secretory Protein-3 and Lactoferrin on Endometrial Cytokine mRNA Expression After Breeding in the Horse

The equine uterus undergoes a transient innate immune response after breeding, also known as mating-induced endometritis. The deposition of spermatozoa triggers the expression of pro-inflammatory cytokines, which results in the migration of polymorphonuclear neutrophils (PMNs) into the endometrium and the uterine lumen. Select seminal plasma proteins, specifically cysteine-rich secretory protein 3 (CRISP-3) and lactoferrin, have been shown to affect the activity of the PMNs, either by suppressing (CRISP-3) or promoting (lactoferrin) the phagocytosis of spermatozoa based on their viability in vitro. Conjointly, many components of inseminate, including seminal plasma, bacteria, and spermatozoa itself, have shown to have an effect on the expression of endometrial cytokines after breeding. The objective of this study was to determine if select proteins affect the mRNA expression of endometrial cytokines after insemination. Six mares were bred during four consecutive estrous cycles with treatments in randomized order of: 1mg/mL CRISP-3, 150 ug/mL lactoferrin, seminal plasma, or Lactated Ringer’s Solution (LRS) to a total volume of 10 mL combined with 1×109 progressively motile spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis. No treatment effects were found for the mRNA expression of IL-1β, IL-6, IL-8, IL-10, TNFα, and IFNγ, while lactoferrin significantly suppressed the mRNA expression of IL-1RN when compared to LRS. In conclusion, the seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on the expression of select endometrial cytokines at 6 hours post breeding.

MAIN FACTORS AFFECTING MARE INSEMINATION WITH CRYOPRESERVED DOMESTIC AND FOREIGN SPERM

Artificial insemination (AI) has the following advantages over natural mating: fast selection effect, easy transportation, wide dissemination of valuable genetic material, economic use of semen and rational use of sires, preventing sexually transmitted infections, reserving sperm in cryobanks. Among the factors influencing the result of AI in horse breeding are time, frequency rate and insemination repetition, quality and quantity of semen, the depth of insertion of semen into the uterus of a mare, the observance of temperature mode and sanitary regulations during AI procedure, the reproductive performance of mares. There are two main approaches to freezing of stallion sperm and thus two AI technologies for semen packaging and equipment for its introduction into the uterus of mares. The first approach worked out and used in Russia is based on a sparing mode of semen cryopreservation in large volumes, 20-25 ml per dose. The second method, developed abroad, provides pre-treatment of sperm by centrifugation and maximum removal of semen plasma. In another procedure currently used in Russia the small volumes (5-6 ml) of semen are cryopreserved after centrifugation, removal of 50-60 % of semen plasma and thickening semen by dialysis. In recent years frozen semen of stallions from Europe and America is being actively imported to Russia. Despite the technological differences, domestic and foreign approaches to the cryopreservation of stallion’s semen provide its similar qualitative characteristics after thawing. The aim of our research was to identify the most significant factors affecting pregnancy rates in artificially inseminated mares when domestic and foreign protocols were used to freeze sperm. Herein, an impact of five such factors (i.e. sperm packing/cryopreservation technology, time of insemination, reproductive state and gynecological soundness of the mares, the activity of sperm) on the effectiveness of AI with frozen semen was estimated in mares of various breeds (Arabian, Akhal-Teke, Trakehner, Hanoverian, Russian Riding horse, American Standardbred, Russian Trotter and Orlov Trotter). Experiments were carried out in 2012 to 2014 at Tersk stud, at a private farm (Mr. A.A. Kazakov the owner) and at experimental stable of the All-Russian Research Institute for Horse Breeding. We compared the data on 106 estrus cycles of 53 mares, artificially inseminated with domestic (granules and tubes of 5 ml and 15-25 ml) and foreign frozen semen (0.5 ml in straws). All used sperm doses in various packages were divided into two groups with the activity above and below 1.5 points (15 %). The time recommended for AI with frozen semen was divided into three intervals (12 h before ovulation, during ovulation, 6 h after ovulation). The animals were conventionally grouped as barren and maiden mares, lactating mares, mares after late (6 or more months of pregnancy) abortion, and also with regard to absence or presence of gynecological pathologies such as vaginal discharge, fluid and air in the uterus, mating-induced endometritis. The effectiveness of artificial insemination was evaluated with regard to pregnancy rate, occurred abortions number and successful foaling. So, two of five factors analyzed were found out to determine reliably a successful AI with frozen semen at high significance level (p < 0.001). They were the sperm activity of  15 % and healthy mares with no pathology observed. Cryopreservation technology, the type of sperm doses’ packaging and also the mare’s reproductive status (i.e. barren, maiden, lactating or after late abortion) are of secondary importance. It is shown that mares’ artificial insemination with one dose of semen during the 6-hour period after ovulation provides the same pregnancy rate (67.7 %) as insemination during the 12-hour period before ovulation (65.0 %) or insemination at the time when the ovum is released from a follicle.

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

Uterine fluid from bitches with mating-induced endometritis reduces the attachment of spermatozoa to the uterine epithelium

Persistence of free fluid in the uterine lumen of bitches with endometrial hyperplasia appears to be diagnostic for mating-induced endometritis and is associated with reduced chances of pregnancy. This study investigated the possibility that reduced fertility might be associated with an effect of uterine fluid on sperm. Uterine lavage fluid was collected pre- and post-insemination from normal bitches without ultrasonographically-detectable luminal fluid (n=4), and previously non-pregnant bitches with endometrial hyperplasia and luminal fluid (n=4). Concentrations of polymorphonuclear neutrophils (PMNs) were measured and the effect of the fluid on the attachment of spermatozoa to the uterine epithelium was studied using medium (M) 199 as a control. To elucidate whether any effect was accounted for by the presence of PMNs, attachment was also measured in M199 with PMNs added at the concentration found in lavage fluid.Pre-insemination lavage fluid from both groups contained low concentrations of PMNs which increased post-insemination; the increase was larger for bitches with uterine fluid. Compared with M199 controls, lavage fluid reduced the attachment of spermatozoa; fluid from bitches with endometrial hyperplasia and uterine fluid had a greater effect than normal bitches, and post-insemination fluid had a greater effect than pre-insemination fluid. Spermatozoal attachment was reduced by a similar magnitude for M199 with added PMNs, although post-insemination fluid from bitches with endometrial hyperplasia reduced attachment more than M199 with added PMNs. Poor fertility in bitches with uterine luminal fluid might be partially associated with impaired attachment of spermatozoa to uterine epithelium, mediated principally, but not solely, by PMN influx into the uterine lumen.

Prevalence and effect of uterine luminal free fluid on pregnancy and litter size in bitches

Free fluid within the uterine lumen has been identified ultrasonographically as a transient finding in normal bitches in the period immediately after mating, and delayed clearance of the fluid has been documented in bitches with endometrial hyperplasia. This study used B-mode ultrasonography to investigate the prevalence of free fluid in the uterine lumen in normal bitches (n = 53) and bitches with endometrial hyperplasia (n = 10) on Days −7, 0, +5, and +14 in relation to estimated ovulation. For normal bitches without endometrial hyperplasia, 47 (86.6%) bitches become pregnant, whereas for bitches with endometrial hyperplasia, 3 (30%) become pregnant. In both the groups, the presence of uterine fluid on Days −7 and 0 was not associated with the probability of a bitch becoming pregnant, whereas the presence of uterine fluid on either Day +5 or +14 was significantly associated with a reduced likelihood of pregnancy. In pregnant bitches, uterine luminal fluid present on Day 0 was associated with a smaller litter size. This is the first study to establish the prevalence and effect of uterine luminal free fluid in bitches. We postulate that as the uterine luminal free fluid may be detected in normal bitches before mating and has no effect on the pregnancy rate or litter size in either group, this fluid is “physiological.” However, it seems that the later in estrus the uterine fluid is present, the more likely it is to have an adverse effect on fertility. In some cases, this is manifest as a reduced litter size, while, importantly, the presence of fluid 5 to 14 days after ovulation is strongly associated with an absence of pregnancy, both in bitches with and without endometrial hyperplasia. We propose that ultrasonographic detection of uterine luminal free fluid after mating may be a useful prognostic indicator for pregnancy outcome, which may allow the targeting of treatments specifically to a population of animals that presumably develop mating-induced endometritis.

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.