Investigations of Achromobacter Xylosoxidans and Ralstonia Pickettii in Porcine Semen Extension Systems By

Dilution and extension of semen longevity has been widely used in the swine industry to improve reproductive efficiency and genetic progress through artificial insemination. Through the dilution and extension of the life of the sperm cell, other organisms such as bacteria are also maintained and can produce a negative impact on sperm cell motility and potential reproductive capacity. Bacterial contamination in porcine semen is a widespread problem in semen collection facilities and routinely testing is essential to determine which organisms are present and the effectiveness of the antimicrobial in the semen diluents (extender). The antibiotics used in porcine semen extenders are generally chosen to be effective against the most common bacterial contaminants, gram-negative bacteria. Two isolates, Achromobacter xylosoxidans (AX) and Ralstonia pickettii (RP), were identified in the water distillation system of a boar stud facility that uses this water to extend the raw semen and found to produce pyometras in sows post-insemination. The effects of these bacteria have not been investigated in porcine semen diluents in long term storage (14 days). The objective of this study was to determine the effects of AX and RP on pH, motility, and progressive motility in culture negative semen samples over a 14 day period in 3 different diluents (grown on Columbia blood agar for 48 h at 37ºC. For each isolate, a single colony was selected and transferred to 10 ml of Luria broth and then incubated for 24 h at 37ºC in 5% CO 2. The broth cultures were centrifuged at 4000 rpm for 5 min. and used to make the final concentrations of approximately 2.5 x 10 7 CFUs/ml (AX) and/or 2 x 10 6 CFUs/ml (RP). There were four treatment groups per extender: AX, RP, AX + RP, and Control (no bacteria added). All samples were incubated at 16ºC and rotated once daily. Motility and morphology of all samples were iii viewed using the Computer Automated Semen Analysis Program (SpermVision ® , Minitube of America, Verona, WI), and pH were measured daily for each sample. Data from 6 replicates were was analyzed using PROC MIXED (SAS Institute, Inc., Cary, NC) with repeated measurements divided into 5 time periods (1-2 d; 3-5 d; 6-8 d; 9-11 d; and 12-14 d) post-inoculation. Overall, sample pH did not significantly increase over time, but was found to be the highest (p<0.0001) in BTS compared to XC and TXC and lower (p=0.02) in samples containing …