Ayr can't include Kenny Sinclair and Jon Steel, while Malleny Park coach Andy Nicol's request for Moffat">Stuart Moffat has also been rejected.
Instead, all three players will be at Murrayfield with the Glasgow squad to watch the Celtic Cup Final between Edinburgh and Ulster.
Ayr spokesman Forbes Watson said: "We have to question if this is the sort of message pro sides should be giving out to the clubs. Surely they should be encouraging our support, particularly as some of the allocated players are short of game time."
Andy Nicol said: "We will just have to crack on but it is a disappointment."