95 N-Acetyl cysteine as a potential treatment for equine persistent breeding-induced endometritis

Abstract

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Transient uterine inflammation is a normal response to breeding; however, PBIE-susceptible mares do not clear this inflammation in a timely fashion. Uterine inflammation at the time of embryonic descent from the oviducts ultimately results in early embryonic death and is seen clinically as infertility. Several risk factors for PBIE have been identified and include age, parity, anatomical conformation, such as poor perineal conformation and cervical fibrosis, as well as other reproductive tract abnormalities. N-Acetyl cysteine (NAC), a mucolytic used to treat endometritis in mares, has anti-inflammatory properties, affects inflammatory cytokines, and is a mild inhibitor of nitric oxide synthase. Increased nitric oxide, causing smooth muscle relaxation and alterations in inflammatory cytokines, has been documented in PBIE mares. The objective of our study was to determine if NAC treatment would lower nitric oxide and inflammatory cytokine levels, thereby resolving PBIE. A randomised, blinded, crossover design clinical trial was performed utilising PBIE-susceptible mares (n=10). Mares were screened for bacterial endometritis before enrolment in the study and only mares that had negative bacterial cultures were used. No other treatments were given to mares while they were enrolled in the study. Intrauterine infusion of 180mL of 3.3% NAC was performed 12h before insemination, when a follicle >35mm was present. Mares were sampled for endometrial cytology and endometrial fluid to determine inflammatory cytokine (ELISA) and nitric oxide (colourimetric assay) levels at 12 and 72h post-insemination. Endometrial biopsies were taken at the same time points post-insemination to determine gene expression of inflammatory cytokines by qPCR. Clinical signs of endometrial oedema and intrauterine fluid volumes were assessed at 12 and then every 24h post-breeding. Statistical assessment of the data was performed using a repeated-measures ANOVA. Uterine inflammation, as determined by percent number of neutrophils on endometrial cytology (P=0.0006), and interleukin 6 gene expression (P=0.003) were highest at 12h. Uterine oedema was greatest at 12 and 24h (P=0.02) and uterine fluid volumes were highest at 24h (P=0.02). Interestingly, interleukin-6 protein levels were not in accordance with gene expression, and were highest at 72h. In this clinical trial, pre-breeding intrauterine treatment with NAC did not affect nitric oxide levels, cytokine gene expression, or clinical signs of PBIE. However, the pattern of inflammation noted in this study is consistent with that described in PBIE mares. Nevertheless, the assessment of inflammatory cytokines, both at the gene and protein level at different time points post-AI, in combination with clinical signs will add to the understanding of the alterations in inflammatory cytokine levels in mares susceptible to PBIE.