Assessment of the impact of mycobacterium cell wall fraction (MCWF) on sperm parameters

Abstract

Mycobacterium cell wall fraction (MCWF) is a biologic used for the treatment of endometritis. Considerable research has been performed on this therapeutic, but with no consistency regarding treatment protocol. For ease of administration, many practitioners utilize intrauterine MCWF at the time of breeding. Intrauterine treatments should be avoided within 24 h of breeding due to the risk of uterine contamination and initiation of the inflammatory cascade in mares predisposed to endometritis. Therefore, the goal of this study is to evaluate the impact of MCWF on sperm parameters within breeding doses. We hypothesize that MCWF will not detrimentally impact sperm and can be added to breeding doses. To evaluate this, 7 ejaculates (n = 7) were collected and centrifuged at 200 × g for 10 min to reduce the seminal plasma and reconstituted in BotuPharma Gold at a concentration of 20 × 10⁶ spermatozoa/mL. Doses were split into treated (1 mg/mL MCWF) and control (1.5 mL saline). Viability of sperm cells was assessed with a Nucleocounter. Additionally, total and progressive motility were assessed using computer-assisted sperm analysis (CASA). Doses were slowly cooled to a resting temperature of 4°C. Following cooling, aliquots were assessed for computer-assisted total and progressive motility, viability, and visually at 24, 48, and 72 h. Statistics were run utilizing SAS 9.4, and semen parameters were assessed using a 2-way ANOVA (MCWF vs. control) for repeated measures (T0, T24, T48, T72). Significance was set to P ≤ 0.05. When assessing viability, samples decreased with time (P < 0.01), but no effect of treatment was noted (P = 0.72). When assessing total motility on CASA, there was an overall decrease noted regarding treatment (P < 0.01) and time (P < 0.001), and when assessing individual time points, this decrease in total motility was only at T72 (P = 0.02). When assessing progressive motility on CASA, there was an overall decrease relating to time and MCWF treatment (P < 0.01), and this was noted at a decrease at all time points evaluated (P < 0.01). Due to debris within the treatment, visual assessment of motility was also analyzed, where a reduction in motility was noted at T72 regarding total motility (P < 0.01) and progressive motility (P < 0.01) when comparing MCWF treated to control. In conclusion, MCWF does not appear to impact sperm viability. CASA was found unreliable in this assessment due to debris. MCWF treatment was only found to impact visual assessment of motility at T72. This product may be considered safe for use with fresh semen, and additional work is needed to investigate its impact on long-term storage of cooled semen.