Effect of Freeze – Thaw Process on Sperm Morphology
Author – Frozen Cell
This study was performed to evaluate the effect of Freeze – Thaw (Cryo-preservation – Thawing ) process on sperm morphology, motility and viability in post thaw human semen sample. This article is elaboration of our previous article posted on Linkedin — https://www.linkedin.com/in/suman-kumar-7b6b09119/detail/recent-activity/shares/
Key words – Human sperm, Human Sperm morphology, Sperm cryo-preservation
Method – Semen of 16 numbers of healthy donors (with normal sperm count & motility) ware selected for this study. Semen sample was collected by masturbation after three to five days of abstinence. Samples of all the donors was collected on the same day.
They are allowed to liquefy for 30 to 60 minutes at 340 C.
Morphology, motility and viability of each sample was checked after proper mixing of each sample separately. All other parameter from collection to cryo-preservation and post thaw assessment was kept common for all sample.
Approx 2 ml of washing media was added with each sample and mixed properly. Semen sample + washing media mixture were layered over 2 ml of 80% of density gradient in a conical centrifuged test tube – centrifuged at 350 G for 20 minutes. Supernatant removed and pellet was mixed with 2 ml of washing media, again centrifuged at 350 G for 5 minutes. Again formed supernatant removed and pellet was mixed with Cryo-preservative media to make a final solution of sperm count 100 million per ml approx. Sperm with cryo-preservative media was kept in cryovials with proper labeling. All cryovials was freezed according to standard freezing protocol of Frozen Cell. Thawing of cryovials was done after 48 to 72 hours at 340 C for 45 minutes.
Morphology, motility and viability of post thaw sample was carried on as routine method.
- A noticeable change of approx 28% in morphological form was observed following freeze – thaw We checked the following parameters of morphology in human sperm.
- Shape of sperm head.
- Sperm tail shape / coiling of tail
- Sperm head – flagellar angle
- A reduction of 15 to 40 % in motility was noticed.
- To check the viability equal quantity of washing media with 6 m Mol of L-Arginine with thawed sample was added to make a final concentration of 3 m Mol of L-Arginine. It was noted that viability also reduced in same ratio as motility (15 to 40%).
Conclusion — It was concluded that human sperm morphology, motility and viability are being reduced in a specific pattern after freeze – thawing process