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• W2135544331 abstract "Solea senegalensis is a promising species for European Aquaculture, however individuals born in captivity display a reproductive failure that hinders the sustainable culture of this species. The reproductive problem has been focused on males reared in captivity. It has been demonstrated that wild males held with captivity breed females produced viable spawns, but non viable spawns were obtained in the opposite situation (Mananos et al., 2007, Carazo 2013). The absence of courtship is not the only reproductive problem in this species. Sperm quality is in general low, and spermatozoa show low resistance to hyperosmotic shock, high levels of DNA fragmentation, high levels of apoptotic cells and also display different and heterogeneous sperm subpopulations within the same seminal sample (Beirao et al 2009; Beirao et al 2011). The aim of this study is to implement a sperm selection method for optimal sperm subpopulation recovery. In particular, a method to eliminate apoptotic spermatozoa has been used. Annexin V binds to phosphatidylserine, which is externalized to the outer surface of the sperm membrane in early apoptosis. In this study Annexin V-MACS beads have been used for the separation of apoptotic spermatozoa. This technique has been used in sperm samples from wild and F1 individuals. In order to confirm the efficiency of the technique, apoptotic cell population was studied by flow cytometry using YOPRO-1 and a caspases detection kit. MATERIALS AND METHOS Sperm samples from adult Senegalese sole males (wild and F1 broodstocks from el Bocal, IEO, Santander) were obtained by gently pressing the testes on the fish pigmented side. Ejaculates were pooled (3-5 males/pool) attending to similar motility parameters to avoid the influence of the sperm quality, to get volume enough to perform the study and following the routine in Aquaculture companies prior to artificial fertilisation. Each pooled sample was split into two aliquots. One of them was directly cryopreserved following the only published protocol for this species so far (Rasines et al 2013). Sperm was diluted (1:2 ratio) in Mounib extender with cryoprotectants (10% BSA and 10% DMSO), loaded into 0.5 mL French straws, exposed to liquid nitrogen vapour during 7 min and rinsed into liquid nitrogen until used. This aliquot was considered as control in the experiment.Magnetic activated cell sorting (MACS) was performed with the other aliquot using a MiniMACS separation unit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following manufacturer’s instructions. Magnetically labelled cells (apoptotic cells) flushed from the MS columns were discarded and the apopototic-reduced elution was cryopreserved following the same protocol as the control. Samples were thawed (37 oC, 7 s) and washed prior to cytometer analysis. Two apoptosis detection protocols were carried out: 1) YOPRO-1 (150 nM) (Invitrogen, Leiden, The Netherlands) and 2) CaspGLOW Fluorescein Active Caspase Staining Kit (eBioscience, San Diego, USA) following manufacture’s instructions. Both of them were co-stained with PI (2 µg/mL) (Sigma, Madrid, Spain). After the incubation time samples were analyzed twice in a FACSort Plus Analyzer (Becton–Dickinson, USA) acquiring 10000 events per replicate. The red fluorescence emitted by PI was detected using a 610 nm filter and the green fluorescence emitted by the active caspase detection kit (FITC) and YOPRO-1 with a 516 nm filter.For each staining, significant differences between the apoptotic cell percentage (green positive/red negative cells for each protocol) between control and MACS samples were evaluated by ANOVA with a SNK (Student- Newman-Kleus) multiple range test (P < 0.05). All statistical analyses were conducted with SPSS software (version 20.0). RESULTS Results demonstrated that Magnetic Activated Cell sorting eliminated apoptotic cells from Solea senegalensis seminal samples. In samples obtained from captured males, apoptotic cell population significantly decreased after the process. This decrease is observed independently of the assessment method used (caspases and YO-PRO-1). However, in F1 individuals, only caspase-positive-cells significantly decreased after the selection. Moreover, the comparative study between seminal samples from captured and F1 individuals showed significant differences only in caspase positive cells, being the percentage of YO-PRO-1 positive cells similar in both cases.DISCUSSION AND CONCLUSIONS This study demonstrated that caspase determination is more specific than YO-PRO-1 in the calculation of apoptotic cells in S. senegalensis seminal samples. The percentage of apoptotic cells (caspase positive) is, as expected, significantly higher in F1 seminal samples. Magnetic activated cell sorting is applicable in this species for non-apoptotic spermatozoa recovery, but fertility trials must be done to confirm the real potential of the technique. ACKNOWLEDGEMENTS Authors would like to acknowledge AQUAGAMETE FA 1205 COST Action,Junta de Castilla y Leon (EDU1084/2012) and Fondo Social Europeo, Dr. I. Rasines, I. Martin, J.R. Gutierrez-Martin, J. Baines and M de la Hera.REFERENCES Beirao, J.; Soares, F.; Herraez, M.P.; Dinis, M.T., Cabrita, E., 2009: Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level. Theriogenology 72 (9),1251-61.Beirao, J.; Soares, F.; Herraez, M.P.; Dinis, M.T., Cabrita, E., 2011: Changes in Solea senegalensis sperm quality throughout the year. Animal Reproduction Science 126,122–129Carazo, I., 2013. Reproductive behaviour and physiology of Senegalese sole, (Solea senegalensis) broodstock in captivity. PhD Thesis. University of Barcelona, Spain, 209 Mananos, E.; Ferreiro, I.; Bolon, D.; Guzman, J.M.; Mylonas, C.C.; Riaza, A., 2007: Different responses of Senegalese sole (Solea senegalensis) broodstock to a hormonal spawning induction therapy, depending on their wild or captive-reared origin. Proceedings of Aquaculture Europe 07, Istanbul, Turkey, pp. 330-331.Rasines, I.; Gomez, M.; Martin, I.; Rodriguez, C.; Mananos, E.; Chereguini O., 2013: Artificial fertilisation of cultured Senegalese sole (Solea senegalensis): Effects of the time of day of hormonal treatment on inducing ovulation. Aquaculture, 392-395, 94-97." @default.
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• W2135544331 date "2015-06-01" @default.
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• W2135544331 title "OPTIMAL SPERM SUBPOPULATION SELECTION IN Solea senegalensis" @default.
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