For the best is the annexin V test from biolegend

Hi Massimo; please find my paper below, it will help you finding the kit . TUNEL assay has been published in Urology journal. If you need any of them, please feel free to contact me for any technical issues, okay? All the best

Journal Article: Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species.

Reda Mahfouz, Rakesh Sharma, Aparna Thiyagarajan, Vaishali Kale, Sajal Gupta, Edmund Sabanegh, Ashok Agarwal

ABSTRACT:

To examine sperm motility, total antioxidant level (TAC), DNA fragmentation, and medical history in infertile men with high seminal high reactive oxygen species (ROS). Prospective study. Tertiary care hospital. Infertile men (n=101). Group I (n=57) included men with seminal ROS (10% greater DNA fragmentation in our patients (95% confidence interval 1.01-1.53). Group II showed poor motility, a higher incidence of leukocytospermia, and higher ROS-TAC scores compared with group I. ROS was negatively correlated with sperm curvilinear velocity (r=-.24), linearity (r=-.24), and sperm motility (r=-.31). Sperm motility was correlated with %TUNEL(+ve) sperm (r=-.39). An increase in seminal ROS levels by 25% was associated with a 10% increase in sperm DNA fragmentation. Sperm motility was affected by seminal ROS and sperm DNA fragmentation.

Dear Caruso, I used the Roche kit for sperm TUNEL test, with fluoresceíne, and you can use live/dead fixable by invitrogen to label the live+tunel positive population.

If for me, I will not use TUNEL on spermatozoa. First of all, There are a lot more methods which are better and more sensitive as compare to TUNEL. You may use Annexin V to detect PS or PSA to detect intact acrosome. Those markers can labelled with fluorochrome and readily analysed by flow cytometry. And for me, acrosome damaged or PS externalization occurs before DNA fragmentation. Thanks

I used the Invitrogen kit. Not bad, maybe a bit expensive, but similar in cost to other kits. Maybe an advantage is that it uses a double-step procedure, with a fluorescent antibody to tag the Br-dUTP (instead of using fluorescent nucleotides).

By the way, you can find some preprint versions of some papers describing the methods in my profile:

https://www.researchgate.net/publication/44581014_Improving_the_effect_of_incubation_and_oxidative_stress_on_thawed_spermatozoa_from_red_deer_by_using_different_antioxidant_treatments

https://www.researchgate.net/publication/221868859_Effect_of_Several_Antioxidants_on_Thawed_Ram_Spermatozoa_Submitted_to_37C_up_to_Four_Hours

https://www.researchgate.net/publication/26792456_Washing_increases_the_susceptibility_to_exogenous_oxidative_stress_in_red_deer_spermatozoa

and some others...

Article Improving the effect of incubation and oxidative stress on t...

Article Effect of Several Antioxidants on Thawed Ram Spermatozoa Sub...

Article Washing increases the susceptibility to exogenous oxidative ...

Thanks a lot! Your suggestions are all very interesting! I work with basophils and I study the activation test. For me this is a new field.Reda, I had already read some of your work with great interest, and certainly also I read this that you advice to me! I will read certainly the work suggested Felipe. I will study the Roche and Invitrogen Kit. Joaquìn, sorry, I don't sure to clearly understand if when you say that you use Annexin V, you mean that DNA fragmentation is directly correlated to Annexin V externalization? Could you suggest any article to read? Kok I think that you have a really good experience. Could you suggest any work on use of Annexin for PS detection and PSA for intact acrosome detection? Thanks at all really! Ciao

Hi again.

We have been use PNA-FITC (green) and PNA-PE/PNA-TRITC (orange) for detection of damaged acrosomes (yes, PSA seems to be OK too). Invitrogen (Life Technologies) offers a range of Alexa dyes (violet to far red, If I remember) conjugated with PNA, which can be used similarly (at a higher price, though).

About Annexin V, there is no problem using it for assessing spermatozoa. There are many publications, and I used it myself. However, I prefer the propidium ioide/YO-PRO-1 combination, which should yield similar populations (they detect different things, though!).

I wrote a review on the subject, just if you want to take a look (there are many others):

https://www.researchgate.net/publication/51444041_Probes_and_techniques_for_sperm_evaluation_by_flow_cytometry

Article Probes and Techniques for Sperm Evaluation by Flow Cytometry

Just another thing. DNA fragmentation dynamics is very special in spermatozoa... there is no "real" apoptosis. You can get most spermatozoa "dead" or a high population of "apoptotic" ones, and still get low levels of DNA fragmentation.