Concordokken said:

Tags male and gay suggest otherwise... Do Owls happen to have a cloaca? Otherwise I got no explanation for you.

Like all birds, owls have a cloaca - a single opening through which they excrete, mate and lay eggs. Most birds do not have a penis (ostriches and ducks are a couple that do), and mate simply by pressing their cloacas together in what is known as a 'cloacal kiss'. A few species mate in flight, but in owls, and many other species, the male climbs on top of the female in order to do this, and squirts his sperm from his cloaca into hers. It then swims up her oviduct to her eggs, fertilizing them, after which they are coated in shell and laid, and will hatch after an incubation period.

In other word, it could be either with Owlowiscious, depending on whether or not twilight sparkle got "his" gender correct at a glance.