Sorvall st 40r centrifuge

Only ejaculates meeting the quality standard limits for the preparation of semen AI-doses were used in this study (Supplementary Table S1). Ejaculates were collected using the gloved-hand method, allowing separate collection of three different ejaculate portions as follows: the first 10 mL-SRF (sperm-peak portion), the remaining SRF and the post-SRF. These 3 portions correspond with the 3 analysed samples. A fourth sample, mimicking the EE, was generated by the proportional mixture of the other three portions 10 mL-SRF, rest of SRF and post-SRF as described previously by Perez-Patiño et al.⁴⁵ . Each one of four samples were separately centrifuged at 800 × g for 8 min (Eba 20, Hettich Zentrifugen, Germany) immediately after collection to separate spermatozoa from the SP. Both, spermatozoa and SP samples were placed in sterile tubes and transported in cooled box (4 °C) to the Veterinary Andrology Laboratory of the University of Murcia. Once in the laboratory, each sperm sample was split into two aliquots and whereas one was stored at − 80 °C until use for WB analysis, the other was immediately processed for ICC as described below. The SP samples were twice centrifuged at 1,500 × g for 10 min (Sorvall ST 40R Centrifuge, Thermo Scientific, MA, USA) to remove any remaining spermatozoa or cell debris and, then, were stored at − 80 °C until use for WB analysis.

Samples from ELS of nondistended and distended bladder preparations were prepared as described previously [9 (link)]. Briefly, the 2.9 mL ELS collected at the end of time equivalent to the time for bladder filling (nondistended condition) or at the end of filling (distended condition) were placed in 4 mL Amicon Ultra centrifugal filter units with 10 kDa molecular weight cut-off (MWCO) pore size (Millipore Sigma, Burlington, MA, USA) to be concentrated by centrifugation. The ELS samples were concentrated to approximately 80–100 µL volume at 4000× g for 25 min using SorvallST 40R centrifuge (Thermofisher, Langenselbold, Germany). The concentrated ELS (cELS) were used to detect the degradation of eATP substrate by enzymes that were released in the ELS at rest or during bladder filling.