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Identification marking mice commonly involves ear-punching with or without anaesthetic, or tail-marking with ink. To identify which is most humane, we marked weanling male BALB/c mice using ear-punching (EP), ear-punching with anaesthetic EMLA TM cream (EP+A), or permanent marker pen (MP). We compared marked mice, unmarked cagemates, and control mice ( n = 12-13/group) for 5 weeks, reapplying MP weekly. Treatment-blind observations following marking showed that EP and EP+A mice were allogroomed ( p &lt; 0.001) and sniffed ( p &lt; 0.001) by their cagemates more than MP and control mice were. EP+A mice groomed themselves ( p &lt; 0.001) and their ears ( p &lt; 0.001) ~5 times more than most other mice; their cagemates also increased self-grooming ( p &lt; 0.001). Unmarked MP cagemates ( p = 0.001), and possibly EP+A mice ( p = 0.034; a nonsignificant trend), grimaced the most. The following day, half the EP+A mice showed hyponeophagia versus no MP and control mice ( p = 0.001). Over the 5 weeks, EP mice approached the handler significantly less than unmarked cagemates did ( p &lt; 0.001). Across weeks, defecation during marking of MP mice decreased ( p &lt; 0.001). Treatment showed no effects on immediate responses during marking, aggression, bodyweight, plus-maze behaviour or corticosterone. MP mice showed no differences from controls, whilst EP and EP+A mice showed altered behaviour, so ink-marking may be the more humane identification method.

The Pen Is Milder Than the Blade: Identification Marking Mice Using Ink on the Tail Appears More Humane Than Ear-Punching Even with Local Anaesthetic