Artificial insemination of queen bee problems with laying eggs

i have problem and i hope i will get the answer from you
some of my queens after i inseminating them. the queens didn’t start laying eggs. i waited more than 40 days without result
some of them took 5 min Co2 the day before and the others 5 min co2 the next day of the insemination
i dont know what is the problem with them ?

How long between emergence and insemination?

Usually between 8-9 days old

That sounds about right. I asked because if you wait too long that will result in queens who can’t lay fertilized eggs and if the bees keep up they will remove the drone eggs in worker brood for a while. There are a lot of tricks to II on queens. CO2 of course is one.

i checked the spermatheca of some of them look perfect full creamed color
should i give them third Co2 treatment

I can’t say if it will help, but it probably won’t hurt.