Three 3" nails every 16" is good. Wedge the old ones up a little with a couple 2x4s to the floor before nailing. You can drill a 2 1/2"
hole in the center for the plumbing without loosing strength, if you can't just lower it. The one cut out for vent pipe should be hangered from a piece that is hangered off the two full length joists. I would also use a couple tubes of flooring glue, glue between old and new and between new and floor boards.