Sule, you may be the first, or may have an opportunity to validate this assay for the bull. I assume this is the same or similar assay to Halosperm, marketed for human sperm. Is it based on DNA migration from digested sperm into agarose gels?

If you are going to try to establish a cut-off (minimal acceptable score), you will need sperm from bulls which are not good sires. Even these may show no difference from good breeders, since DNA fragmentation is only one of many possible reasons for poor fertility. Alternatively, you can induce DNA fragmentation in sperm by exposing the sperm to UV, peroxide, X-ray, etc at different levels. For some of these, this exposure may have to be after digestion on the gel, but before incubation. By generating a dose-response curve, you can at least determine if the sperm are easily damaged.

Another possibility, if other DNA fragmentation methods do show differences, is that Halomax is not sensitive enough. For example, we use the Comet assay which is more sensitive than SCSA; both are more sensitive than Halosperm.

Good luck!

Dr. Muller,

I appreciated your precious comments and suggestions. You are right that Halomax is produced by the same company that is also produces Halosperm. The technique is the same with the minor changes. I have performed Halosperm assay in human sperm before; indeed, there are a lot of published work on this assay. However, Halomax is specific to bull sperm and I have not seen any cut-off of DNA fragmentation for this assay yet.

You are correct again that Halomax should be used as a complementary assay where DNA fragmentation should be tested by another assay. I have also done that.

Unfortunately, I will not be able to redo some of my experiments where I can use spermatozoa from a random sire. However, I may try to induce DNA fragmentation from spermatozoa from my experimental population. In this case, the challenge will be to use bull spermatozoa whose plasma membranes are thicker than those in human spermatozoa. Do you think that the peroxide and X-ray can induce DNA fragmentation in bull spermatozoa?

Thank you so much your thoughts, I will keep working on the improvements!!

Sule

The peroxide should be added after digestion, but the X-rays should damage DNA even in live sperm.

Hi, maybe a bit late, but we tried Halomax in bull several years ago. It worked well, although it produced different results than SCSA (somewhat expected). We produced a paper (I think it can be downloaded from my profile):

http://www.ncbi.nlm.nih.gov/pubmed/18992075

You might try to incubate the samples at 37 °C and measure fragmentation before and after. It might disclose between-bull differences (we did it in that paper). Anyway, you must remember that cattle has been selected for decades for good-quality spermatozoa, so it is not unusual not finding differences (although I suppose you are using males with different fertility records).

As Dr. Muller says, comet seems more sensitive, some of my colleagues are using it for analysing damage in fish spermatozoa. However, SCSA is very repeatable and pretty fast when you have it well standardized. We tried TUNEL with good results, but it is somewhat more variable than SCSA and more expensive.

By the way, you always can contact a colleague that is using the SCSA and carry out a collaboration. The SCSA allows you to freeze and store the samples, so you can send them to the colleague for analysis.

Here is the paper I was talking about in the other post:

https://www.researchgate.net/publication/23459268_DNA_status_on_thawed_semen_from_fighting_bull_a_comparison_between_the_SCD_and_the_SCSA_tests?ev=prf_pub

Article DNA status on thawed semen from fighting bull: A comparison ...

Thanks a lot for your response.