I need to remove the extender from the straws without lost too many cells. I don't need the spermatozoa viable because after the separation I will remove the tail and plasma membrane and perform protein extraction. I've already tried two washes with Sp-Talp (200G/5 min), and Percoll 45% only (6000G/5 min), but the best result I had with Percoll gradient 45 and 90%, normally used to process the semen for IVP. However, this way I lost too many cells.
Does anyone have a better and efficient way to separe the cells and the extender?
dear Leticia, the method that you are using is to get clean motile spermatozoa. If just want to collect sperm cell regardless their quality, just centrifuged in TALP with slow speed (200 g) will do for longer time (ie., 30 minutes or more) to obtain sperm pellet. The lipid from extender will stay on the top layer. Remove supernatant then resuspend in another TALP and centrifuge again at the same speed. Centrifuge at high speed may damage the sperm cell.
Dear Leticia,
when I need to remove extender from buck semen, i usually centrifuge it at 1000g for 5 minutes, remove supernatant carefully, resuspend in plain PBS and centrifuge again. Hope it works well with you.
Dear Leticia
Just cut 2-4 straws and dispense the contents in a 1 ml microcentrifuge tube and then centrifuge at 4000 rpm for 10 minutes. Discard the supernatant and resuspension the pellet in 0.5 ml PBS and again centrifuge at the same rpm but for only 5 minutes. Discard supernatant and resuspend the pellet in new PBS. I think you will get required sperm.
Thank you all!!!
I got it! I reduced the semen volume that I was washing per tube. I was putting too much semen (about 250 ul) for a small amount of Sp-TALP, and the sample was not clean enough. I centrifuged 125 ul of semen in 1 ml of Sp-TALP (200G/5 min), recovered the pellet (about 150 ul), centrifuged again in 1,2 ml of Sp-TALP (200G/5 min), recovered 100 ul of the pellet.
Hi Leticia,
If you want ultra pure motile sperm cells. You can use swim-up method for sperm-separation. In this method, you have to use alot of straws from each group. Briefly, 200 ul semen from straws is put in the botton of the tube containing 1,5 ml SP-TALP. Be careful about dont distrube semen and give 45 angle in a tube bord and wait in a waterbath or incubator in 37C for 1 hour. And you have to collect SP-TALP until sperm line of tube and put to a centrifuge tube and centrifuge 400-600 G for 10 min. And you can use ultrapure pelet contains only sperm cells.
You can use this method. It is warranty ultra pure sperm cells without any extender.
My best wishes,
Umut
You don't need to remove cryoprotectants from thawed semen during insemination, because sperms are motile and they leave the deposited extended semen. For ivf, two to three times washing is required to remove toxic cryoprotectants.
Regards
For removing semen diluents/glycerol from thawed semen, you may try following:
1. Carefully planned centrifugation and pellet re-suspension 2-3 times in suitable media like PBS (1000 to 1,500g for 10 min)
2. Micro-dialysis: using semi-micro dialysis tubing (2.55 mm diameter, molecular weight cutoff of 12,000 - 14,000) and 200 capillary pipette, a capillary suction apparatus, preferably at lower temp (4°C to 8°C)
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