Dear Mohammad.

In my opinion the biggest challenge is to perform some selection (either by breeding or in laboratory) of males whose semen should be processed (chilled or cryopreserved).

I think there are protocols (for production of chilled or frozen-thawed semen) that provide excellent results. There is very little area for some dramatic increase of quality by e.g. modification of extenders, sperm selection prior processing or manipulation of sperms pre- and post-thaw. I had many dogs in my lab that provided ejaculate of excellent quality (pre and post-thaw) but there were also dogs that were impossible to freeze. I was trying to change some steps in freezing protocols in these males but without greater impact. Therefore I think that when the results of chilling and freezing protocols are poor in some males and excellent in others, don’t chill or freeze the bad ones.

The question is how to select these poor freezers. I think that this may be a good field to study. Development of some diagnostic tools (markers) which could help us to select dogs with ejaculate of good and poor quality for further processing is a good way how to produce chilled or frozen-thawed semen for insemination with good results.

Best regards,

Jiri

dear Jiří Šichtař

thank you for replying.

you mentioned appropriate notes.

WE have been depleting DNA fragmented sperm in Human cells and some animal sperm. If interested I could provide a protocol.

Dear Henry Clemente

I am grateful for your attention. please send mentioned protocol if it is possible

thanks

Dear Thara K M

thanks for your reply.

good luck

I am attaching a protocol for the depleion of DNA fragmented sperm. Let me know if you have any questions

dear Henry Clemente

thank you

Dear Mohammad,

Back in 2000, I had a PhD degree on "Cryopreservation and Acrosome Reaction in Dog Spermatozoa" in Bristol Univ., UK. For further details, please see below:

https://www.researchgate.net/profile/Omer_Ucar/publications

as given under titles of both "Thesis" and "Data".

Briefly, I have few tings to say herein for quick commenting on "canine sperm cooling and freezing":

1- "Oxidative stress vulnerability" of dog sperm (due mainly to high unsaturated:saturated fatty acid ratio and low cholesterol content)

2- Diluent type (milk-, TRIS-based, etc.)

3- Dilution rate (1:4 to 1:8, as sperm-diluent),

4- Antioxidant usage, if any (e.g. SOD, Threhalose, Vit E, Orvus STM paste)

4- Centrifugation used or not (for removal of seminal plasma or even its addition after post-thaw)

5- Glycerol concentration (2-8%, v/v),

6- Sugar type (Fructose, Lactose, etc)

7- Cooling and freezing rates (varying rates depending mostly on diluent type, centrifugation, glycerol concentration and packaging type).

Above all, I strongly recommend you to use both step-wise freezing (using programmable freezer) and to check sperm parameters not only the classical ones (such as motility, morphology, acrosomal integrity) but also, if possible, other functional quality parameters (such as acrosome reaction, HOS test, thermoresistance, DNA integrity, Zona-binding or even in vivo fertility) at pre- and post- cooling/freeze-thawing stages. As appreciated mostly, just simple checks of quality parameters of sperm cannot provide convincing outcomes of any scientific trial concerned.

Good luck, Omer

Our efforts to deplete DNA fragmented canine sperm has required us to use lectin based nanoparticles. www.clemente-ass0ciates.com

dear Omer Ucar

I really appreciate your comprehensive and beneficial explanation.

wish u the best