These are what we call the 'YMMV items.' Things that some people find in this work. We call them 'your mileage might vary' because not everyone sees these things in the same way. This starts discussions in the trope lists, a thing we don't want. Please use the discussion page if you'd like to discuss any of these items.
Alternate Character Interpretation: Provided courtesy of the fic's very premise — simply changing the genders of the characters but leaving their personalities and appearances the same results in some very different outlooks on the originals. On the one hoof, Rainbow Dash's Ladette athlete seem perfectly stock for the male Rainbow Blitz. On the other hoof, Rarity's FashionistaLarge Ham makes the male Elusive Camp Straight.
Fanon: The names of the gender bent characters, at least the Mane Six.
Fridge Brilliance: Applebuck wearing a red baseball cap makes more sense when you remember that Apple Bloom was based on Blossom, who also has a gender flipped version of her with a red baseball cap.
The gender-flipped counterpart of Rarity is named Elusive; both of their names mean "hard to get", the former also meaning "hard to acquire" and the latter "hard to catch".
Fridge Horror: If you've read this fic, you'll know that everything that has happened in Canon!Ponyville has happened in Alternate!Ponyville. Which means…
That also means that a female version of Discord has most likely been there…
And may be a male version of the Changeling Queen — if the episodes of the series is out of order.
A throwaway line by Dusk Shine implies that he has no biological sisters, which means that unless either Continuity Drift comes into place (or the line is edited), Shining Armor (and probably Princess Cadence) don't exist. Of course, this story waswritten before Season 2 had even begun.
Not to mention the Crystal Empire and its dark tyrannical ruler.
Fridge Logic: The story admits that there are some inherent differences between males and females in terms of physical capabilities, and yet despite that the world somehow managed to develop to be exactly the same as the normal world with the exception of genders, which implies that nothing in all of the world's history was actually affected by the gender differences.
Could be a blow for gender equality, or a sign that there's no such thing as free will.
It's not as simple as gender equality or free will as much as physical impossibility. As an example, take pregnancy. Pregnancy would significantly hamper the pregnant party, which would already change things pretty radically, and that's not even going into the other issue with pregnancy (namely, the fact that a male can impregnate multiple females in a relatively short time span, and if you switch that you end up with a bunch of unborn foals, which would cause an even bigger alteration of history).
Then again, perhaps in a world with more males than females, reproduction adapted to accommodate. Maybe mares can carry more foals at once, and from multiple stallions.
Except that's implying that the Rule63 universe has high gender imbalance, which is not the case, because both universes have an equal ration of male and female ponies among the populace; the original simply puts more focus on mares while the fanfic's alternate universe puts more focus on the stallions.
It's explicitly stated the worlds aren't 100% mirror to each other; there are a couple subtle differences, as shown with the fact Dusk Shine and Twilight Sparkle didn't end up just switching universes. Instead, Dusk's intended trip to Manehattan would have been scheduled for several hours after Twilight's intended trip, resulting in him being interrupted by Twilight's arrival.
Tear Jerker: Rarity and Applejack's reaction's to seeing their gender flipped siblings and not being able to reveal who they really are to them is just heartbreaking.
Applebuck (Apple Bloom's Gender Flip) running in just as the Mane 6 are ready to go back to their own dimension, and then he starts hurling himself against the shield that they put up because he wants Applejack to stay.