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A PCR detection method for discerning Serratia marcescens in extended boar semen
1Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, 17071 Girona, Catalonia, Spain.
Abstract:
Serratia marcescens is a bacterial contaminant that can be spermicidal when present in extended boar semen that is typically stored prior to breeding use at 15 to 18 °C for several days. This particular contaminant appears to originate from carrier boars, where it resides in the preputial cavity, but has also been shown to then easily contaminate the semen-processing laboratory. Screening for carrier boars to date has been performed through detection of S.marcescens in ejaculates using traditional agar plate culture techniques. These agar growth techniques are labor and time consuming due to the need for sample °titration and temporal growth followed by isolation, leading to delays in identification. The aim of this study was to develop a rapid, sensitive traditional PCR technique that can detect the presence of S.marcescens in extended boar semen. Primers for the detection of S. marcescens 16S rRNA were designed and specificity tested. After PCR optimization, assay sensitivity was evaluated using extended boar semen that was inoculated with various physiological ratios of spermatozoa: S.marcescens (100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1. 1:1 and 1:10). Samples, held at 16 °C, were tested every 24 h over a 96 h period, with bacterial DNA extraction performed at each time point using a commercial kit. As a final step, the developed technique was used to screen random samples of extended boar semen for S. marcescens contamination. Results showed that this PCR technique had a sensitivity (90%) and specificity (100%) at detecting S.marcescens in the different inoculated ratios as well as in random, naturally contaminated samples of extended boar semen. In conclusion, this study reports a traditional PCR technique that is effective at rapidly and accurately detecting the presence of S.marcescens in boar extended semen.
Insights
Serratia marcescens contamination in boar semen can be detected rapidly using a new PCR technique. This method offers high sensitivity and specificity, improving semen quality control for efficient breeding programs.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Animal Reproduction
Background:
- Serratia marcescens is a bacterial contaminant found in extended boar semen, potentially causing spermicidal effects during storage.
- Current screening methods using agar cultures are time-consuming and labor-intensive, delaying identification.
- Contamination often originates from carrier boars and can spread to semen-processing laboratories.
Purpose of the Study:
- To develop a rapid, sensitive, and specific traditional Polymerase Chain Reaction (PCR) technique for detecting Serratia marcescens in extended boar semen.
- To optimize PCR conditions and evaluate its sensitivity and specificity using spiked semen samples.
- To validate the PCR assay's effectiveness in detecting natural S. marcescens contamination in boar semen.
Main Methods:
- Design and specificity testing of primers targeting the 16S rRNA gene of Serratia marcescens.
- Optimization of PCR assay parameters.
- Evaluation of assay sensitivity using extended boar semen inoculated with varying ratios of spermatozoa to S. marcescens.
- Testing of samples stored at 16°C over a 96-hour period with DNA extraction at 24-hour intervals.
- Screening of naturally contaminated boar semen samples using the developed PCR technique.
Main Results:
- The developed PCR technique demonstrated high sensitivity (90%) and perfect specificity (100%) for detecting Serratia marcescens.
- The assay effectively identified S. marcescens across various inoculated ratios of spermatozoa to bacteria.
- The PCR method successfully detected natural contamination in random samples of extended boar semen.
- Bacterial DNA extraction was performed using a commercial kit at each time point.
Conclusions:
- A rapid and accurate traditional PCR technique for detecting Serratia marcescens in extended boar semen has been successfully developed.
- This PCR assay offers a significant improvement over traditional culture methods, enabling faster and more reliable identification of contamination.
- The validated technique can be instrumental in enhancing biosecurity and quality control in boar stud operations, ensuring the integrity of semen for breeding purposes.
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