Astolfo sure has a hard time of in the fandom.
(pun intended)
It's morally hard for me to comment on another culture's view of "third gender" especially considering the weird side effects western/christian morality has had on Japan over several hundred years.
In any case it's a slightly homophobic joke by western standards, but also in the gay community, it just be like that sometimes, and in cultures where the morals around sex are very different, it just be that way sometimes.
There's also just the fact that this just how alot of the Japanese fanworks approach the character as well.
Kinda like how when Deathnote was still being released, and the anime adaptation came out the doujin community did alot of yaoi of the period where Light and L where handcuffed at all times.
It's a good chuckle, you timed out the joke well, it could certainly be better, but serves a good little joke.
Thanks for making this.