Cryopreservation of human sperm with H.S.A. is a standard method for preserving human sperm with various indications. We will discuss a exceptionally straightforward procedure in “Novel Technique to Cryopreserve Human Sperm without H.S.A. Which do not need any modern / expensive equipment. By The technique “Novel Technique to Cryopreserve Human Sperm without H.S.A.” our aim is to give opportunity even for a basic IUI / IVF center to preserve human sperm with all safety and security. Moreover, this technique (Novel Technique to Cryopreserve Human Sperm without H.S.A.) will even help the cancer patient. Who is hoping to preserve their fertility later on in the wake of completing their treatment. This cryopreservative media does not contain Human Serum Albumin (H.S.A.) or any crude protein from animal or avian source. Accordingly even there is no possibility to infection transmission through any far off source.
H.S.A. == Human serum albumin
Sperm preservation – an introduction
Cryopreservation of human sperm began decades & decades back. Many type of cryopreservative media and technique has been used with subsequent improvement in technique and result. But one thing remain persistent that some form of crude protein is being used either as nutrition or upgrade factor for human sperm. We will discuss all basic phenomena to make cryopreservation of human sperm easy and affordable without using any crude protein.
In “Novel Technique to Cryopreserve Human Sperm without H.S.A.”, we will discuss how to preserve human sperm with 100% chemically defined cryopreservative compound. Here a cocktail of amino acid has been used to provide nutrition and as enhancement factor to human sperm.
Side by side our emphasis will be to describe an easy and cost effective method to freeze human sperm
Conventional technique of Human sperm cryopreservation
In conventional technique of human sperm cryopreservation we use permeable and non-permeable cryo-protectant along with Human serum albumin. At the same time different salts are being added to make cryopreservative media sperm friendly.
Material and method
Below mentioned chemicals was purchased from SRL , Himedia and Sigma-Aldrich. All chemicals was of AR grade. Only L-isomer of all amino acids (Essential and non-essential) selected for media preparation.
|(L- Isolucine, L-leucine, L-lycine, L-methionine, L-phenylalanine, L-tryptophan, L-tyrosine, L-valine, L-proline, L-aspartate, L-asparginine, L-glutamate, L-serine, L-cystine, L-histidine, L-threonine, L-arginine, Alanyl glutamine, NaCl, KCl, Lactic acid, MgSO4, Pot. Phosphate, Sodium bicab, Sucrose, glycine, Hepes, Gentamicin, Taurine, EDTA, KH2PO4, CaCl2, Sodium pyruvate, Glycerol in double distilled water)|
Glycerol concentration was kept at 6 % (vol / vol). An invented cocktail of amino acid was used instead of human serum albumin. Different salts was added in a predefined ratio. We set the pH of solution at 7.35 approx.
Six donors were selected for sperm preservation with Novel Technique to Cryopreserve Human Sperm without H.S.A.. All of them belong to B+ blood group. The donor’s ejaculate was with 50-60 million sperm per ml, more than 50% progressive motility and 40% or more morphological typical sperm. Donors were informed and consent taken. Semen was collected after 48 to 72 hour of abstinence in the morning hour of the by masturbation. Each ejaculate divided in two part. Part-A – only 1 /3 of semen was taken, Part-B – rest of the semen.
Both parts was prepared by swim down technique. In short, Part-A and Part-B of each ejaculate was washed twice , first time by density gradient 75% at 380 G for 15 minutes. Prepared pellet was mixed with 2 ml oh washing media and centrifuged again at 380 G for 4 minutes. Used swim down media(discontinuous gradient & washing media) was Den-1 of Frozen Cell.
After second centrifugation in Part -A appropriate quantity of our newly invented Novel cryopreservative without H.S.A. was added to make sperm concentration 30 to 50 million of each ml.
In Part-B we used conventional cryopreservative with H.S.A.. Samples was filled in cryo-vials and kept at room temperature for 10 to 20 minutes. Cryovials of Part-A and Part-B (appropriately labeled) was freezed together according as per our standard freezing protocol.
Our standard freezing protocol —
- Take approx 2 inch of liquid nitrogen in a thermocol insulating box.
- Set a plastic mesh approx 3 inch above liquid nitrogen.
- Place the cryovials over plastic mesh.
- Place the censor of temperature meter above plastic mesh along with cryovials .
- Reduce the temperature of plastic mesh at the rate of 3 to 5 degree per minute ( it can be done adding or removing the liquid nitrogen. Also slow blowing of air is very effective in maintaining the temperature reduction.
- Once temperature is less than (-) 130 degree C. Plunge all cryovial in liquid nitrogen.
- Store in liquid nitrogen for 24 hours to check post thaw recovery at different parameter.
Thawing process takes 45 to 50 minutes. First temperature of incubator was set at 32 to 340C. Cryovials taken out from liquid nitrogen and kept in incubator for 45 minutes. Content of cryovial was mixed properly after thawing.
We checked following parameters after complete thawing of each cryovials independently.
- Post thaw sperm motility.
- Progressive motility.
- Motility after 24 hour.
- Sperm morphology.
POST THAW SPERM MOTILITY
In general we noticed post thaw motility was somewhere less than conventional cryopreservative with H.S.A. As an average post thaw recovery was 10 to 20% inferior to conventional cryopreservative with H.S.A.
Surprisingly sperm preserved with cryopreservative without H.S.A. had a very good progressive motility in comparison to conventional cryopreservative with H.S.A. . Most of the motile sperm was moving in a straight line.
One negative point – speed of sperm even in forward direction was less in compare to conventional cryopreservative with H.S.A..
MOTILITY AFTER 24 HOUR
To check the motility after 24 hours, we kept the firmly closed cryovials at room temperature (15 to 25 degree C). Before checking the motility all cryovials was kept in incubator for 40 minutes at 33 degree C.
Motility after 24 hour was distinctly less with Part-A (Novel cryopreservative without H.S.A.)
In comparison to Part-B (conventional cryopreservative with H.S.A.), The difference was 20% to 50%.
Sperm morphology following freezing and thawing was noted better in case of Part-A (Novel cryopreservative without H.S.A.). That might be the reason of better post thaw progressive motility when preserved with Part-A (Novel cryopreservative without H.S.A.).
Till date the majority of the cryobiological work on sperm were based on study of various type of conventional cryopreservative with H.S.A. or different type of animal or avian serum. In the meantime extraordinary sort of modern freezer (programmable and non-programmable) has been upheld to utilize. Which might be hard to manage.
Besides, this novel cryopreservative without H.S.A., as name recommend is without H.S.A.. Human serum albumin is a potential source of disease . Consequently it must be kept away from at whatever point conceivable.
We finished up following focuses with the theme “Novel Technique to Cryopreserve Human Sperm without H.S.A.”.
- Human sperm can be effectively preserved without Human Serum Albumin (H.S.A.)
- Costly freezer (programmable or non-programmable ) isn’t compulsory to freeze human sperm.
- We assume that carbohydrate and amino acid can give nourishment to sperm.
- More investigation is required to build up an ideal media for human sperm preservation.
Conflict of interest —- Till now we did not notice any conflict on the topic “Novel Technique to Cryopreserve Human Sperm without H.S.A.”.
Acknowledgements — Frozen Cell Team is in a nonstop procedure to find out an ideal media to preserve human sperm. We are in an undertaking to find a better strategy to preserve human sperm by various procedure.
Funding — Frozen Cell do not accept any fund from any outside source. 100% funded by Frozen Cell—
Limitations — We work with besic equipments. Therefore not able to give figures regarding osmolarity, DNA fragmentation, Acrosomal status, Sperm viability etc.