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Doctoral thesis, 2019

Characterization, quantification and removal of potential pathogens from stallion semen

Al-Kass, Ziyad


In Sweden, equine artificial insemination is most frequently carried out with liquid semen rather than frozen semen. Many factors affect sperm quality during semen storage, including the presence of bacteria and addition of antibiotics. Use of Single Layer Centrifugation (SLC) or Modified SLC (MSLC) has been shown to improve sperm quality, resulting in an increase in the time for which the sperm sample retains its function during storage. These techniques can also separate spermatozoa from bacteria. The purpose of this thesis was to investigate the role of MSLC, antibiotics and bacteria on sperm quality and storage time. This thesis comprised 4 studies: Study I was a retrospective study of potential pathogenic bacteria isolated from Swedish stallion semen during the period 2007 to 2017. Study II was to identify bacteria in semen by conventional laboratory culture methods. Study III was to isolate pathogenic and nonpathogenic bacteria in stallion semen using metagenomic analysis. Study IV was to investigate the effect of MSLC and the effect of presence or absence of antibiotics in the extender on sperm quality. Our results showed that potentially pathogenic bacteria such as Taylorella equigenitalis, Klebsiella pneumoniae, beta haemolytic streptococci and Pseudomonas aeruginosa appeared infrequently in Sweden. However, many nonpathogenic bacteria were found. Metagenomic analysis enabled more bacteria to be identified than other methods. The bacterial genera identified were different between studies, animals and ejaculates, even from animals kept on the same stud. Corynebacterium spp. were the most frequently found non-pathogenic bacteria identified in all our studies. The bacterial population was decreased using MSLC, sperm quality was improved and the shelf-life of the sperm samples was increased. The presence of antibiotics in the extender did not affect sperm viability. The bacterial population was greater in samples without antibiotics than in samples with antibiotics; bacteria appeared in all samples, even those with extender containing antibiotics. There was no evidence that bacteria isolated from a stallion on one stud should also be isolated from other individuals on the same premises. More work is needed to investigate the effects of particular bacterial genera on sperm quality. In addition, it would be interesting to investigate the shape and size of bacteria in relation to spermatozoa and the proportions of different bacteria removed using MSLC. Further modifications to the SLC technique might enable the removal of more bacteria.


Modified Single Layer Centrifugation; male fertility; microorganism; liquid semen; sperm quality; 16S sequencing; pony stallions; semen evaluation

Published in

Acta Universitatis Agriculturae Sueciae
2019, number: 2019:17
ISBN: 978-91-7760-352-8, eISBN: 978-91-7760-353-5
Publisher: Department of Clinical Sciences, Swedish University of Agricultural Sciences

Authors' information

Al-Kass, Ziyad
Swedish University of Agricultural Sciences, Department of Clinical Sciences

UKÄ Subject classification

Clinical Science

URI (permanent link to this page)