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.

Result —-

  1. 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
  1. A reduction  of  15 to 40 % in  motility  was noticed.
  1. 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
References
  1. https://academic.oup.com/humrep/article/17/3/704/642300
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044926/
  3. http://www.scielo.org.za/scielo.php?script=sci_arttext&pid=S1019-91282014000100006

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