mated and unmated females

[hwei]

Hello everybody!

We’re trying to measure males’ fertility (we use C. remanei), and we’re wondering - is there any quick way to score the number of mated and unmated females? Can we count by the presence of mating plugs? We’ll pair one male with five virgin females, and we give him 2~3 hours and see how many he can mate. Of course we can just isolate these females in separate plates and check if they lay eggs, but that involves a lot of picking.

Any suggestions are welcome!

Thank you!!

/Hwei-yen

[HillelSchwartz]

I’ve never worked with remanei, but if looking for mating plugs turns out not to be good enough, a couple of suggestions:

1. You could pick off the male, and see how many females contain eggs (as opposed to oocytes) an appropriate time later, without picking the females to other plates. Still a little picking, but much less than by the method you propose, and far fewer plates used. If you have trouble telling the difference between animals containing eggs and animals containing oocytes, you could pick them into bleach, although then the advantage in reduced picking, while still real, is greatly reduced.
2. Could you grow the males in the presence of dye, and look for dye transfer to the females?

[hwei]

Hej! Thank you!! Then we would count females with eggs instead, just that we probably need to train our eyes a bit more… And just curious - how does the dye work? You add it in E. coli?

Many thanks again!

/Hwei-yen

@HillelSchwartz post:2

I’ve never worked with remanei, but if looking for mating plugs turns out not to be good enough, a couple of suggestions:

1. You could pick off the male, and see how many females contain eggs (as opposed to oocytes) an appropriate time later, without picking the females to other plates. Still a little picking, but much less than by the method you propose, and far fewer plates used. If you have trouble telling the difference between animals containing eggs and animals containing oocytes, you could pick them into bleach, although then the advantage in reduced picking, while still real, is greatly reduced.
2. Could you grow the males in the presence of dye, and look for dye transfer to the females?

[HillelSchwartz]

The reports I can recall used dyes to visualize sperm transfer. Singson, Hill, and L’Hernault describe using SYTO 17 and cites a previous paper using Nile Blue A. You might try asking the authors for detailed protocols and for recommendations of other dyes you might try.

[medawg]

Mike Miller’s lab has used a dye called mitotracker red that stains the mitochondria in sperm and can be used as well. You basically just soak the males in the dye for a few hours and then do the mating and can see the labelled sperm in the hermaphrodites/females. They have more details in their papers.

[hwei]

many many thanks!! ;D

[Marc_Perry]

I tried using Nile Blue (as well as every other viable dye on the lab shelf) when I was a post-doc, and in my experience, scoring the staining (of Nile Blue) under a dissecting scope took a trained eye (i.e., it was not glaringly obvious). For me, scoring fertilized eggs would be easier in this instance.

[Sperm_or_Egg]

I second Marc’s point. None of the sperm dyes that I’ve tested are easily visible by dissecting microscope, whereas embryos vs. oocytes are obvious with very little practice.

-Harold