We are in the continuous process to get best post-thaw motility of sperm after freezing by our newly developed vitrification method. Our main focus is on semen vitrification without using any animal component. This is my first attempt at sperm preparation following vitrification-thawing in boiling water. We decided to opt for single layer density gradient centrifugation (65 % v/v) to select the best quality sperm. The pellet was re-suspended in 1 ml of Enhance-F (sperm washing medium) to remove the density gradient medium.
For the first experiment of sperm preparation after Vitrification-thawing in boiling water we selected good quality semen with the following parameters:
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Volume — 2.7 ml
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Liquefaction time – 20 minutes
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pH — 7.2 (by using pH paper)
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Sperm count per ml — 75 millions
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Total sperm count — 202.5 millions
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Motility % —- 70%
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Total Motile sperm — 142 millions
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Morphology — Approx 60% sperms have normal morphology.
Materials & Method
Materials required
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Pre-wash Vitri Mrdia
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Single layer density gradient (65%)
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Enhance-F (Sperm washing medium)
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Liquid nitrogen
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Boiling water apparatus (an electric kettle)
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A pair of ling forceps
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Incubator
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Centrifuge machine
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Microscope
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Plastic disposables: centrifuge test tube, pasture pipette, cryovials etc.
Method / Procedure performed:
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Semen collected by masturbation after an abstinence of 4 days.
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Semen kept in incubator at 32 degree C for 30 minutes to liquefy completely..
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Semen was mixed properly
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6 ml of semen taken in a test tube – slowly we added 0.35 ml of VITRI MEDIA with continuous mixing by a pasture pipette.
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Taken the whole content in a 1.6 ml cryovial.
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Kept at room temperature for 15 minutes.
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Cryovial directly plunged in liquid nitrogen. Stored in liquid nitrogen for 24 hours.
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Cryovial taken out from liquid nitrogen and directly plunged in boiling water with the help of a long
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Cryovial kept in boiling water for 50 seconds .
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After 50 seconds cryovial taken out from boiling water to dip in room temperature water for a while.
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Cryovial kept in incubator at 32 degree C for 45 minutes.
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A drop of content was taken to check post thaw motility & recovery.
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In a test tube we took 1 ml of density gradient – 65% (silane coated colloidal silica stabilized with Hepes buffer). Cryovial content was gently layered over density gradient.
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Centrifuged at 350 G for 15 minutes.
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Supernatant discarded without disturbing pellet.
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1 ml of Enhance-F (Sperm washing Medium) added over pellet. And mixed
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Centrifuged at 350 G for 5 minutes.
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Supernatant discarded.
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Added 0.5 ml of Enhance-F to pellet – Mixed gently.
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A drop of 10 micron Liter taken on slide + cover-slip to examine under microscope for motility and morphology.
Please Note — At this point it is worth to mention again that our newly developed Pre-wash Vitri Media do not have any component of animal or human origin like H.S.A., F.B.S., B.S.A. or egg yolk.
Result obtained:
We obtained a very encouraging result in term of post thaw motility, progressive motility and morphology.. More than 80% sperms have very good progressive motility. Even the number of sperm recovered was higher in comparison to conventional freeze – thaw method. Most important post thaw morphology was good enough; which usually deteriorate after conventional freezing.
Conclusion:
It is well proven that good progressive motility and morphology of sperm is very important for a successful outcome. This is more important with IUI. We observed with our initial experiment That Sperm Preparation after vitrification-thawing in boiling water gives a better result in comparison to conventional semen freezing. Moreover the process of semen vitrification is easier and cost effective.
Certainly more experiment is awaited with sperm preparation after vitrification-thawing in boiling water.
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