Dear Researchers,
I am working on a protocol to study the effects of a compound on sperm motility.
1) I will be collecting the Cauda epididymis at the animal facility and will be taking the tissue to lab which takes approximately an hour. I am wondering if there is any special media or methods to carry Cauda epididymis without affecting the sperm vitality.
2) Could someone please share a protocol on how to isolate/prepare the sperms for in vitro studies?
3) After isolation, I need to keep those sperms alive for 6hrs to see the effect of a compound at every hour interval. What is the best medium for this kind of study?
Any information you can provide me would be greatly appreciated.
Thanks,
Nimmi
Hi Nirmala,
A very well organized protocol for collection of cauda epididymis (and their transport at cold temperature) is available at http://www.cosmobio.co.jp/export_e/products/detail/collection_and_transport_of_cauda_epididymis.asp?entry_id=12585
Moreover, videos on YouTube could help you:
https://youtu.be/HZQ-zs0I3y8
https://youtu.be/-urHJhj9Gkw
https://youtu.be/AVdq1umcetQ
In order to have controls or different times (two) for your experiments, keep in mind that you could collect one testis at a time if you don't euthanize the mouse.
However, if you want serial samples from the same male, I think that the best thing is the technique for in vivo sperm retrieval from the epididymis who was assessed in mice (Del Val GM and Robledano PM, 2013) at http://www.ncbi.nlm.nih.gov/pubmed/23760960
Ludovic
http://www.cosmobio.co.jp/export_e/products/detail/collection_and_transport_of_cauda_epididymis.asp?entry_id=12585
https://youtu.be/HZQ-zs0I3y8
https://youtu.be/-urHJhj9Gkw
https://youtu.be/AVdq1umcetQ
http://www.ncbi.nlm.nih.gov/pubmed/23760960
Hi Ludovic
Thanks for taking your time to answer my questions and sharing the links. The videos are amazing.
After reading and watching the videos I assume that transporting them in cold pack shouldn't affect the sperm viability. Once they are brought to the lab, spermatozoa can be isolated under liquid paraffin and from here onward the temperature should be maintained 37oC.
Is Biggers, Whitten and Whittingham (BWW) medium with the following recipe a good medium or any other suggestions?
NaCl 94.7 mmol/ L, KCl 4.8 mmol/L, CaCl2 1.71 mmol/L, KH2PO4 1.2 mmol/L, MgSO4·7H2O 1.2 mmol/L, NaHCO3 25 mmol/L, glucose 5.6 mmol/L, HEPES 20 mmol/L, penicillin 60 mg/L, streptomycin 100 mg/L, 0.6% fatty acid free BSA, pH 7.6 (WHO, 1999).
Nimmi
I think this type of medium will be sufficient to perform an "in vitro culture" of several hours.
Feel free to get back in Research Gate after your experiments.
Dear Nirmala;
In attached files, you can find the protocol has been used in our lab for spermatological analyses.
Kind Regards,
Ali
Hi Ali,
Thanks for sharing the papers which contain very useful information. Where can I get this Rat 1-cell embryo culture (mR1ECM) medium or the recipe for preparing the same?
Nimmi
44
Dear Nirmala;
You can find useful information in the following links:
http://www.bioone.org/doi/abs/10.1274/032.033.0104
http://www.ncbi.nlm.nih.gov/pubmed/8182591
Regards,
Ali
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models