I wouldn't think so, chances are, they won't even notice. I have two broodies who are missing from the flock right now. The other ladies couldn't care less. They are still laying just fine. I think it would be the same for you with the roo. Just try not to stress them when you remove him as scaring them too badly could interrupt them laying for a little bit. I had a rogue cat around here, and she had my flock so stressed that until she was relocated, i was getting about 1/4 of the eggs i normally got. It took about two days for them to resume production.