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

SIMPLE SUMMARY: Laboratory mice often look identical, so they are commonly marked by cutting the ear via ear-punching, or marking the tail with permanent marker. Ear-punching is permanent but could be painful, and mice could become stressed by weekly tail-marking, so we compared impacts on mice over 5 weeks. We also explored whether local anaesthetic cream could reduce any ear-punching effects. We found that ear-punching, even with anaesthetic, caused mice to be sniffed and groomed by their cagemates for at least 5 min after marking. Mice ear-punched with anaesthetic also groomed themselves and their ears ~5 times more than tail-marked and control mice. Facial grimacing was most common in the unmarked cagemates of tail-marked mice, and possibly in mice ear-punched with anaesthetic. The next day, mice ear-punched with anaesthetic were significantly less likely to eat unfamiliar food in an anxiety test than tail-marked or control mice. Over 5 weeks, ear-punched mice approached the handler significantly less than unmarked mice did, and tail-marked mice showed reduced defecation during re-marking. Other behaviour, bodyweight, and corticosterone showed no treatment effects. This suggests ear-punching caused some signs of pain and anxiety, and anaesthetic did not help. Tail-marking appeared more humane, showing no differences from the controls. ABSTRACT: 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 < 0.001) and sniffed (p < 0.001) by their cagemates more than MP and control mice were. EP+A mice groomed themselves (p < 0.001) and their ears (p < 0.001) ~5 times more than most other mice; their cagemates also increased self-grooming (p < 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 < 0.001). Across weeks, defecation during marking of MP mice decreased (p < 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.