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

Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm

[EN] Background: Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. Results: The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. Conclusions: We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the use of Androcoll-Bear 65% since the yield is better than Androcoll-Bear 80%. Our findings pave the way for further research on application of sperm selection techniques to sperm banking in the brown bear.SIThis work was supported in part by MINECO (CGL2013–48255-R) and Cantur S.A. Luis Anel-Lopez was supported by MINECO (CGL2013–48255-R). C ortega-Ferrusola is supported by a postdoctoral grant from “Ministerio de Economía y Competitividad “Juan de la Cierva” IJCI-2014-21671

Single layer centrifugation (SLC) for bacterial removal with Porcicoll positively modifies chromatin structure in boar spermatozoa

[EN] The storage of boar semen samples at 17 °C for artificial insemination (AI) doses enables the proliferation of the bacteria, making antibiotics necessary. This can contribute to the development of antimicrobial resistance (AMR). This study tested bacterial presence and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were split as control and low-density colloid centrifugation (single layer centrifugation, SLC, 20%, and 30% Porcicoll) into 500 ml tubes. Analyses were carried out at days 0, 3, and 7 (17 °C) for microbial presence and sperm chromatin structure analysis: %DFI (DNA fragmentation) and %HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges), and chromomycin A3 (CMA3; chromatin compaction). Besides comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these sets of variables were described by canonical correlation analysis (CCA). Results showed a significant decrease of some bacteria or a complete removal after SLC (especially for P30). SLC also caused a decrease of %HDS and an increase of disulfide bridges and low and medium mBBr populations, suggesting the removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, especially Enterobacteria, P. aeuriginosa, and K. variicola. In conclusion, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low-density colloid not only removes bacteria from boar semen, but also chromatin structure is enhanced after selectionSIThis work was funded by a pump grant from Society for Reproduction and Fertility, UK, to JMM and by the Ministry of Science and Innovation (Spain), grant number RTI2018-095183-B-I00 (MCI/AEI/FEDER, EU) and LE023P20, Junta de Castilla y León/Consejería de Educación/FEDER, EU). The authors thank I. Quintela, B. de Arriba, L. Tejerina, M. Pérez-Luengo, N. Sorarrain, and B. Martín for technical assistance, and all the personnel at Topigs Norsvin España SLU (AIM Iberica) for their help in providing sample

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

Prescribing Data in General Practice Demonstration (PDGPD) project - a cluster randomised controlled trial of a quality improvement intervention to achieve better prescribing for chronic heart failure and hypertension

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1472-6963/12/273 Extent: 11p.Background: Research literature consistently documents that scientifically based therapeutic recommendations are not always followed in the hospital or in the primary care setting. Currently, there is evidence that some general practitioners in Australia are not prescribing appropriately for patients diagnosed with 1) hypertension (HT) and 2) chronic heart failure (CHF). The objectives of this study were to improve general practitioner’s drug treatment management of these patients through feedback on their own prescribing and small group discussions with peers and a trained group facilitator. The impact evaluation includes quantitative assessment of prescribing changes at 6, 9, 12 and 18 months after the intervention. Methods: A pragmatic multi site cluster RCT began recruiting practices in October 2009 to evaluate the effects of a multi-faceted quality improvement (QI) intervention on prescribing practice among Australian general practitioners (GP) in relation to patients with CHF and HT. General practices were recruited nationally through General Practice Networks across Australia. Participating practices were randomly allocated to one of three groups: two groups received the QI intervention (the prescribing indicator feedback reports and small group discussion) with each group undertaking the clinical topics (CHF and HT) in reverse order to the other. The third group was waitlisted to receive the intervention 6 months later and acted as a "control" for the other two groups. De-identified data on practice, doctor and patient characteristics and their treatment for CHF and HT are extracted at six-monthly intervals before and after the intervention. Post-test comparisons will be conducted between the intervention and control arms using intention to treat analysis and models that account for clustering of practices in a Network and clustering of patients within practices and GPs. Discussion: This paper describes the study protocol for a project that will contribute to the development of acceptable and sustainable methods to promote QI activities within routine general practice, enhance prescribing practices and improve patient outcomes in the context of CHF and HT. Trial registration: Australian New Zealand Clinical Trials Registry (ANZCTR), Trial # 320870.Margaret Williamson, Magnolia Cardona-Morrell, Jeffrey D Elliott, James F Reeve, Nigel P Stocks, Jon Emery, Judith M Mackson and Jane M Gun

Sperm quality in frozen beef and dairy bull semen

Abstract Background There is speculation that beef bull semen quality is inferior to that of dairy bulls although few scientific studies are available in the literature. The aim of this study was to evaluate sperm quality in beef bull semen and to determine which parameters could be indicative of fertility after insemination. Sperm quality, assessed by computer assisted sperm motility analysis and flow cytometric evaluation of membrane integrity, levels of reactive oxygen species, mitochondrial membrane potential, acrosome status and DNA fragmentation index, was evaluated in beef and dairy bull semen. Results For beef bulls, normal morphology (r = 0.62, P < 0.05) and WOBBLE (r = 0.57, P < 0.05) were significantly correlated with 56-day non-return rate, whereas sperm quality was not significantly correlated with the fertility index score for dairy bulls. Membrane integrity (46 ± 8.0% versus 40 ± 11%, P < 0.05), normal morphology (87 ± 6% versus 76 ± 8%; P < 0.05), and high respiratory activity (52 ± 13 versus 12 ± 4%; P < 0.001) were higher for dairy bulls than for beef bulls. The DNA fragmentation index was lower for dairy bull spermatozoa than beef (3.8 ± 1.1% versus 6.1 ± 2.9%; P < 0.01), whereas some sperm kinematics were higher. Multivariate analysis indicated that type of bull (beef versus dairy) had an impact on sperm quality. Conclusions Different assays of sperm quality may be needed for appropriate analysis of beef and dairy bull semen. These finding could be important for cattle breeding stations when evaluating semen quality

Single layer centrifugation-selected boar spermatozoa are capable of fertilization in vitro

BACKGROUND: Good quality spermatozoa are important to achieve fertilization, viable embryos and offspring. Single Layer Centrifugation (SLC) through a colloid (Androcoll-P) selects good quality spermatozoa. However, it has not been established previously whether porcine spermatozoa selected by this method maintain their fertility. METHODS: The semen was prepared either by SLC or by standard centrifugation (control) and used for in vitro fertilization (IVF) at oocyte:spermatozoa ratios of 1:50; 1:100 and 1:300 (or 4 x 103, 8 x 103 and 24 x 103 spermatozoa/ml) to evaluate their subsequent ability to generate blastocysts. In addition, sperm motility was assessed by computer assisted sperm motility analysis. RESULTS: Total and progressive motility were significantly higher in sperm samples prepared by SLC compared to uncentrifuged samples. Sperm binding ability, polyspermy, cleavage and blastocyst rates were affected by the oocyte:sperm ratio, but not by sperm treatment. CONCLUSION: The use of SLC does not adversely affect the in vitro fertilizing and embryo-generating ability of the selected spermatozoa compared to their unselected counterparts, but further modifications in the IVF conditions would be needed to improve the monospermy in IVF systems. Since SLC did not appear to have a negative effect on sperm fertilizing ability, and may in fact select for spermatozoa with a greater potential for fertilization, an in vivo trial to determine the usefulness of this sperm preparation technique prior to artificial insemination is warranted

Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm

P. 167-173Selecting the optimal sperm population is essential for success with reproductive techniques. Porcicoll (formerly Androcoll-P) is a colloid formulation for selection of high-quality boar spermatozoa by single layer centrifugation (SLC). To date, most studies have been carried out with fresh semen and large volumes. We carried out 2 experiments to test the use of Porcicoll for thawed boar semen in small volumes. In Experiment 1, cryopreserved semen doses were thawed, split in 200-μL aliquots and layered on 1 mL of Porcicoll 70%, 80% or 90%, or buffer without colloid. We assessed sperm recovery (the proportion of the loading dose that appeared in the pellet, %), and the physiology of the selected spermatozoa (flow cytometry: Viability, apoptotic changes, capacitation, mitochondrial activity, intracellular reactive oxygen species). The most suitable proportion was Porcicoll 80%, allowing acceptable sperm recovery (16.9 ± 4.2%, compared to 70% (35.4% ± 3.0, p < 0.001) and 90% (8.2% ± 3.0, P = 0.001), and improved quality (mitochondrial activity: Porcicoll 80%: 77.7 ± 1% vs Control: 60.3 ± 0.7%, P < 0.05). In Experiment 2, we compared 3 supplements to Porcicoll 80%: 500 mM reduced glutathione (GSH), 20% seminal plasma (SP) and 0.5% bovine serum albumin (BSA). Supplementation with GSH or BSA did not cause relevant changes relative to Control. In contrast, SP induced membrane and acrosomal changes resembling capacitation, which might preclude its use in some applications, and decreased recovery (5.5% ± 1.9 vs. 24.3% ± 1.2 Control; P < 0.001). However, it could be useful prior to other applications such as in vitro fertilisation. Overall, Porcicoll is an effective colloid for isolating a high-quality population from thawed boar sperm, 80% being a balanced option for good recovery and high quality. Supplements could be useful depending on the proposed use of the spermatozoa.S