Low density Porcicoll separates spermatozoa from bacteria and retains sperm quality

[EN] Antibiotics are added to semen extenders to control the growth of bacteria contaminating semen during collection but may contribute to the development of antibiotic resistance. An alternative would be physical separation of spermatozoa from bacteria. The objective of the present study was to evaluate two low densities of Porcicoll for removal of bacteria, and for their effect on sperm recovery and sperm quality. Semen was collected from boars at a commercial station. Aliquots of 8 extended ejaculates were subjected to colloid centrifugation through 20% Porcicoll (P20) and 30% Porcicoll (P30) in 500 mL tubes and then stored at 17 °C. Microbiological examination and sperm quality evaluation (computer assisted sperm analysis and flow cytometry) were carried out on controls and all colloid-selected samples immediately after preparation and again after storage for 3 and 7 days. The microorganisms found were mainly bacteria from the environment, gut or skin. There was a considerable reduction or complete removal of some bacteria by both colloids. Recovery rates were 86% for P20 and 81% for P30. Sperm quality was not adversely affected by colloid centrifugation on day 0, and thereafter showed a more gradual deterioration in colloid centrifuged samples than in controls, possibly due to lower bacterial contamination. There were no differences in sperm quality between the two colloid treatments. Thus, these results show that contaminating bacteria in semen can be controlled by centrifugation through low density colloidsSIThis work was funded by a pump grant from the Society for Reproduction and Fertility, UK, to JMM and by grants to FMP (RTI2018-095183-B-I00, MCI/AEI/FEDER, EU, and LE023P20, Junta de Castilla y León/Consejería de Educación/FEDER, EU

Removal of bacteria from boar semen using a low-density colloid

P. 272-278Antibiotics are added to semen extenders when preparing commercial semen doses for artificial insemination according to national and international guidelines. However, this addition of antibiotics represents non-therapeutic usage and could be contributing to the development of antibiotic resistance. Colloid centrifugation was shown to reduce the load of bacteria present in boar semen and was capable of removing all bacteria if performed directly after semen collection, albeit with some loss of spermatozoa. The present experiment was conducted with a low density colloid to investigate whether it was possible to separate all of the spermatozoa from seminal plasma i.e. without selection for robust spermatozoa, or whether this would have a detrimental effect on sperm quality. Ejaculates from nine boars were extended in Beltsville Thawing Solution without antibiotics and were transported to the laboratory for Single Layer Centrifugation (SLC) on modified Porcicoll i.e. at a low density (S). A further modification was that a sterile inner tube was included inside some of the 50 mL centrifuge tubes to facilitate harvesting of the sperm pellet (M). Aliquots of all samples (control, S and M) were cultured for bacterial quantification and identification using standard microbiological methods. Sperm quality was evaluated daily. Three of the C and M samples and five of the S samples did not contain any bacteria. Mean bacterial counts for the remaining samples (colony forming units/mL) were as follows: C 259 ± 216; S 30 ± 22; M 33 ± 15 (P < 0.01). Citrobacter spp., Staphylococcus simulans, Klebsiella variicola, Escherichia coli, Myroides odoratimimus, Proteus spp. and Enterococcus faecalis were identified in the control samples. There were marginal differences in sperm quality among treatments, with sperm velocity and linearity being higher in S and M samples than in C at all time points. However, sperm viability, capacitation and acrosome status were marginally better in controls than in S or M on day 0, but these differences disappeared during storage. Conclusions: centrifugation through a low density colloid can remove or reduce bacterial contamination in boar ejaculates without using antibiotics. Furthermore, it is possible to collect boar ejaculates without bacterial contamination by paying strict attention to hygiene.S