Effect of washing stallion's penis prior to semen collection on bacterial load of the distal genital tract and semen

Abstract

The external genital tract of stallions is physiologically colonized by a variety of bacteria, most of which are considered non-pathogenic, with a small proportion classified as pathogenic or potentially pathogenic. When collecting stallion semen, proper hygiene is essential because bacterial contamination can affect semen quality and the reproductive health of mares. Currently, there is no consensus regarding the best practice for washing the stallion's penis before semen collection. The most common approaches are (1) no washing, (2) washing with warm water or a mild soap, and (3) washing with chlorhexidine soap before semen collection for export purposes. Our aim was to determine effects of washing the stallion's penis before semen collection on the bacterial load of the genital tract and semen and on semen characteristics. Five stallions were included and randomly assigned to one of three treatments: control (NW), washing with warm water (WW), and washing with chlorhexidine soap and warm water (WC). Treatments lasted for two consecutive weeks, with three semen collections per week. After each treatment period, stallions underwent a two-week rest interval before switching to the next treatment in a crossover design. On the first day of each treatment period, semen and swabs from the urethral fossa were collected for bacteriological analysis without prior cleaning procedure. Sampling was repeated after the two week-washing protocols. Samples were serially diluted up to 1 × 10^-8 and plated on Columbia Agar with 5% sheep blood and Schaedler Agar with vitamin K 1% and 5% sheep blood. Colony forming units (CFU) were counted after 72 hours of aerobic and anaerobic incubation at 37°C. Native semen was analyzed for pH, sperm concentration, motility and membrane integrity (CASA), and morphology. Semen was subsequently diluted with Equiplus extender (Minitube) without antibiotics and an aliquot of 2 ml was stored at 5°C for 72 hours and analyzed every 24 hours for motility and membrane integrity. For statistical analysis, CFU and sperm characteristics were compared among treatments by a general linear model for repeated measures. Total bacterial load decreased over time (P<0.01) and an effect of treatment (P< 0.05) and a treatment x time interaction was observed (P<0.05). The number of CFU/ml after treatment was already decreased in WW and even more in WC compared to NW (CFU/ml for aerobic and anaerobic culture, respectively, WW: 5.2 × 10⁵ and 5.8 × 10⁵, WC: 3.0 × 10⁴ and 6-8 × 10⁴, NW: 4.5 × 10⁶ and 6.2 × 10⁶). Total motility and percentage of membrane-intact sperm decreased over time (P<0.001), but there was no treatment effect. In conclusion, washing the stallion's penis with warm water or chlorhexidine soap before semen collection reduces the total bacterial count in semen without compromising semen characteristics.