10 INOCULATION OF CULTURE-NEGATIVE PORCINE SEMEN WITH NOVEL BIOFILM-FORMING BACTERIA
Authors: Clark S. , Ness A. , Baldrighi J. , Borst L. , Maddox C. , Payne B. 
Published: January 2008

With the growing number of boar studs having semen analysis performed by reproductive specialists, a growing number of diagnostic challenges are encountered. Semen analysis classically involves evaluation of sperm cell motility, morphology, and concentration; however, culture of the extended semen sample for bacterial contamination has become routine. Two isolates, Achromobacter xylosoxidans and Ralstonia pickettii, have recently been identified in the water distillation system of a boar stud facility that uses this water to extend the raw semen in various semen extenders. Insemination of sows with contaminated semen has resulted in severe pyometras diagnosed on necropsy. The effect of these bacteria on sperm motility has not been examined in a controlled setting. The objective of this study was to determine the effects of A. xylosoxidans (AX) and R. pickettii (RP) on pH and motility in culture-negative semen samples over a 7-day period at 16�C. Banked clinical isolates of AX and RP were plated on Columbia blood agar and incubated for 48 h at 37�C. For each isolate, a single colony was selected and transferred to 10 mL of Luria broth. The broth was then incubated for 24 h at 37�C. Optical density measurements were performed at 24 and 48 h of growth, followed by quantification of bacteria by plate counts of serially diluted broth cultures (colony forming units). At 24 h, AX and RP reached levels of 1 � 108 and 1 � 107 [colony-forming units (cfu) mL–1], respectively. Concentration of bacteria in clinical infection was determined to be approximately 1 � 104 and 102 for AX and RP, respectively. In order to attain concentrations similar to those in clinical infection, dilution of the bacteria was necessary. Centrifugation of broth culture at 4000 rpm for 5 min was performed and the bacterial pellet was re-suspended in culture-negative semen in Modena (SGI, LTD, Cambridge, IA, USA) extender to concentrations mimicking those in clinical infection. The samples were then incubated at 16�C and rotated once daily. Motility and morphology, viewed using computer-assisted sperm analysis (CASA: Spermvision; Minitube of America, Verona, WI, USA), and pH (Accumet AB15, Fisher Scientific, Hanover Park, IL, USA) were measured daily for each sample at 25�C. Data from 4 replicates were used in the analysis. For motility, ANOVA revealed no significant differences (P < 0.05) between the control and inoculated samples. A PROX MIXED analysis (SAS, SAS Institute, Inc., Cary, NC, USA) revealed no treatment-by-time interaction with sperm motility after inoculation. For sample pH, statistically significant differences (P < 0.05) were noted between all of the samples, primarily contributed by a treatment-by-time effect. The pH of the control sample became more basic over the 7-day period (from 6.94 to 7.32). This phenomenon was also observed in all of samples; however, semen inoculated with AX appeared to remain closer to neutral pH than did the RP samples. Although statistically significant differences were noted in pH, the addition of biofilm bacteria did not negatively affect the motility of extended porcine semen during this time period. Further experiments need to be performed in relation to different concentrations, time period of bacterial growth, and determination of final cfu mL–1.