It could be that he heard that they fly at night? You never know what little details small children hear and remember and you can't predict what they will make of the information they glean from a trip.
Would it help to tell him more about owls and present them as families (mummy, daddy, and baby)? Sometimes presenting scary entities as families makes them more domestic, familiar and approachable. You could tell him that they are birds who build nests in trees just like other birds, that they lay eggs and have baby owls (what are baby owls called anyway?), that they find food for their baby and feed him in the nest, that the baby owl will one day (night?) teeter to the edge of the nest and jump out and hopefully fly, just as he also learns to do all sorts of big boy things.
DD3 has an early summer birthday, and I think her learning to sleep through the night between 2.5 and 3 coincided with the autumn equinox and the arrival of darker and earlier winter nights. Maybe blackout blinds/curtains might help your DS? By the same token, she loved a night light. I got one that glowed green because that was her favourite colour. Her bedroom window was at the side of the house, but lights from cars could illuminate the room, or flash across the walls or her closed eyes when they went past on the road outside, and I think the heavy curtains and night light kept the light steady and predictable in her room.
When DD3 was about 4 I started playing classical music CDs at night and this seemed to help her relax enough to drift off. She liked all sorts of pieces, from Arvo Part to Beethoven to Chopin, and on and on.. She still does - the Bach cello suites are her current favourites. I stuck to classical because DD3 was always very sensitive to sound (she would wake at the drop of a pin when napping as a baby), and muzak and lights and smells in supermarkets used to set her screaming. She also hated going in the car unless I had classical music playing. (DD4 liked Lenny Kravitz, by contrast). I thought classical music offered more soothing choices, longer pieces that she could concentrate on, sometimes a beginning, middle and end to a piece, so to speak, and repeated motifs so maybe a better intellectual experience than nursery rhymes set to music for instance, or even Baby Einstein toddler music selections. This isn't music snobbery here I listen to anything except C&W and especially Irish C&W -- but a practical choice, and I figured if she had any sort of an ear for music, classical music would enhance it.
DD3 also had a lot of allergies, it turned out. She developed hay fever quite young, and has food allergies - dairy being the most likely to cause problems. I also found she is allergic to aspirin and has problems with latex, and there are food allergies and sensitivities that are related to latex allergy that I avoided too, for her -- avocado, kiwi, banana, apples, carrots, celery, potato and tomato. She was always a terribly picky eater so many of these weren't an issue, but she complained about bananas making her mouth very itchy, and pizza gave her hives on her chin and neck. Daily Clarityn almost year round sorted out the hay fever to a great extent but she went on to develop mild asthma nevertheless.