Dear Alan.

I have experience with animals only, hovewer I hope my answer could help.

1/ impossible to say, in animal species there is huge lack of studies investigating long term effect of different extenders (if you mean by "solution" the extedner). in my experience you need to test several extenders to find out the best one for individual animal (I think this might be the same in humans). 

2/ I would suggest to remove "everything" before cryopreservation (better for penetration of cryoprotectants to cells)

3/ hard to say...I am working with stallions now and regarding our results (which will be published soon) larger volume (5 ml aluminium tubes) insemination doses were sig. better, among others, in total and progressive motility and viability. 

If you think you would like to discuss this topic more, don't hesitate to send me a message.

I hope my answers could help you a bit.

Jiri

Thanks Jiri, yes I meant extender - went with cryopreservation solution as I'm not 100% if extender is widely used across all species. 

I should have mentioned that the samples will not be used for fertilization upon thawing, they are going to be used for extraction of DNA and RNA. Therefore, do you think it is still required to purify the samples prior to freezing? 

Interesting point that samples stored in larger volumes have better progressive motility and viability - as my cryo storage option may not facilitate the use of straws (sarstedt boxes only) I am looking for any information that will give me confidence to store samples in 2ml cryo vials. 

Thanks again! 

Hi Alan,

we are using origio, cook  ivf media for sperm storage we get good post thaw survival rate.

Hi Alan,

only from experience with non-human material - there are definitely individual differences in tolerance of storage media. Sometimes you get higher motility to start with, but then decreased long-term viability, so definitely validate your storage media with the specific individual over time.

Concerning viability and motility after thawing I would rather go for straws, as the smaller volume is more likely to achieve consistent temperature gradients within the sample. Of course for subsequent insemination a larger volume would be preferable, but I suppose that's a different story.

Hi Alan.

I thought you are asking about removing somatic cells before freezing the semen to prepare normal insemination dose. I am not sure about DNA and RNA extraction problematic, sorry.

I think you can freeze your samples to 2 ml cryovials. There are several studies (I work with equine species) that supports larger volume. In attached files you can find your support and also some references  from those studies might help. 

I would like to respond also to PA answer about consistent temperature gradients. I found two studies:

1/ http://www.ncbi.nlm.nih.gov/pubmed/19700703

Meta analysis of pregnancy outcome in cows with 0.25 and 0.5 ml straws...generally no difference

2/ http://bibliotecadigital.uca.edu.ar/repositorio/investigacion/how-important-are-internal-temperature.pdf

Internal temperature gradients in Frech straws.

I did not read the second study thoroughly, there might be some information which might suggest how temperature gradients will look like in larger volumes. However I did not find any similar study directly comparing gradients in 0.25 or 0.5 and larger volumes. This will be very interesting to do as many people say without any relevant knowledge that smaller volumes are better...

One more thing I would like to add. We are using 5 ml aluminium tubes and one huge advantage is that when you manipulate (taking out and putting back in to LN2 to choose the right sample) such volume will not thaw immediately as 0.25 and 0.5 ml volumes so you will not put thawed sample back to the LN2.

Jiri

Hi Trupti (and anyone else who might have info), 

Do you think it is possible to make a simplified version of the Origio sperm freezing medium? As I'm restricted by costs buying it is not an option, however I'm certain we already have most of the components/reagents in the lab. 

human sperm can be frozen with no other preservative than 10% glycerol, unlike most other species, many of which cannot be frozen with maintained fertility.  It appears that the ability for sperm to penetrate and fertilize the ovum is much more difficult than to preserve the DNA and RNA.  As a former faculty member at the U. of Minnesota, I was involved in developing methods for freezing  boar semen in 1969 where glycerol proved to be detrimental to fertility despite fantastic motility after thawing. i futhermore worked with  stallion semen in Sweden 1988 and water buffalo semen for FAO in Pakistan.  I  would not expect that you need to be particularly careful with semen frozen for extraction of DNA and RNA  since maintaining the ability for the sperm to penentrate in vivohas been the problem.

Hi 

I used to fix semen with ethanol for DNA analysis or simply keep the sample in -80C for 1 week. I analysis the sample by flowcytometry and it gives excellent results for ploidy, fragmentation and maturation.

http://www.ncbi.nlm.nih.gov/m/pubmed/23525372/

Dear Alan, I have experiences of fish sperm cryopreservation, specially sturgeon fishes.

1.Related to your questions,in my field, there are so many different extenders with different cryoprotectants, dilution rations, working volume,... but it is said that the simpler extender with the same results should be considered as the best. Also I have read that glycerol is the best cryopretectant of mamalian sperm because of the least harmful effects than methanol or DMSO, noting that it may be used for internal fertilization.

2. Why it is so important f Dose it make deleterious effect on human spermatozoa?In fishes, when we aspirate the milt from male, it is consumed that it is mostly spermatozoa, with low amount of other epithelial cells which have no effect on further fertilization. By the way, separation of any non desirable cell from the solution could be beneficial, but remember, getting rid of the other cells, may makes the spermatozoa exposure to a new BAD biochemical or physical condition to be more weak or expend their own ATP already.

3. Its up to the aim of study. Normally it begins with low volumes to set the conditions, afterward, finding new protocols for larger volume. Again be careful, result from low volumes might be different from other volumes.Low volumes in straws make it more quickly and precise temperature exchange during cooling rates than the other volumes.

Truly, Shahrouz

hi Alan see some suggestions below:

(1) cryopreservation solution, which is best for long term storage?

depends on the clinic and what they prefer and what works for them. many products on the market.

have attached package insert for the one I use. had excellent results with this.

(2) removal of contaminating somatic cells, before or after freezing?

all somatic cells should be removed prior to freezing especially if contaminated to prevent cross contamination when sample is used. ie PCR Wash and freeze removes HIV Virus, we also advise patients to freeze prior to chemo.

(3) cryo tubes or straws, which is best?

high security straws are the safest in terms of breakage, integrity and protection from cross-contamination by viruses. they are also available in many different sizes so you should ideally choose a size depending on the amount of sperm required and the type of procedure it will be used for.

you should also take into consideration the type of cryopreservation tanks you have...straws much easier to work with.

Thanks everyone for the informative feedback. 

To refocus the question - I will be freezing samples from normozoosperic patients, therefore there should be no difficulties regarding low cell number and the problems associated with freezing those type of samples. 

I will proceed with a glycerol based freezing media but what I'm trying to find out is what is the best method for preparing this. i.e. the minimal number of components required. 

Thanks again for your input. 

Dear Alan,

Years ago, when comercial media to freeze semen wasn't available, I used to prepare a home-made recipe based on glycerol and egg yolk. 

Please find attached. If you need something more, let me know

Good luck!

Hi Maraia

if we spoke about old method coconut fresh was used as preservative for sperms before  And still used it for animals

http://www.cbra.org.br/pages/publicacoes/animalreproduction/issues/download/v13/v13n2/p057-062%20(AR724).pdf