Nucleocounter sp 100 sperm cell counter

Males were killed by carbon dioxide asphyxiation followed by cervical dislocation. Weights for the carcass, testes, epididymides, and seminal vesicles were recorded. The right caudal epididymis was incubated in 500 μl of phosphate-buffered saline (PBS) for at least 15 min at 37° in a Petri dish. Following incubation, the vas deferens and caput epididymis were removed and the cauda was snipped and incubated again for 15 min at 37°. After the second incubation, sperm were extruded from the cauda using curved forceps. Once the sperm suspension was collected in a microcentrifuge tube, the Petri dish was rinsed with additional PBS to collect remaining suspension, bringing the final suspension volume to 1 mL. The sperm count was determined using a NucleoCounter SP-100 sperm cell counter (Chemometec).

After the maturation period, the cumulus cells were removed from the oocytes by vortexing at 1660 rounds/min for 2 min in 300 µL of TL-HEPES-PVA supplemented with 0.1 mg/mL hyaluronidase. The denuded oocytes were then washed three times in IVM medium and three times in IVF medium, which consisted of Tris-buffered medium^{51 (link)} supplemented with 2 mM caffeine and 0.2 mg/mL BSA. Groups of 40 oocytes that have been previously subjected to in vitro maturation were placed in 50-µL drops of IVF medium and coincubated with frozen-thawed spermatozoa prepared as described by Gil et al.^{52 (link)}. For each replicate, two semen straws were thawed at 37 °C for 20 s, and 200 µL of spermatozoa from both straws was pooled, washed three times in Dulbecco’s phosphate-buffered solution (PBS, Gibco, Grand Island, NY) supplemented with 4 mg/mL BSA, and pelleted at 1900×g for 3 min. The resulting pellet was resuspended in 1 mL of IVF medium, quantified with a Sperm Cell Counter NucleoCounter SP-100 (ChemoMetec, Allerød, Frederiksborg, Denmark), adequately re-suspended to 2.4 × 10⁶ spermatozoa/mL, and finally added to the IVF drop containing the oocytes. Thus, each oocyte was exposed to a total of 3000 spermatozoa in a humidified atmosphere air with 5% CO₂ at 38.5 °C under an overlay of paraffin oil, for 5 h^{53 (link)}.