Table 2.

Relevance, specificity and item-total correlation of each item and categories of the scale in horses submitted to castration or only anaesthesia

Variable	Criteria	Specificity (%) 1	Relevance (confidence interval) 2			Item-total correlation 3
			GA x GC	GAA X GC	GCA x GC
Positioning in the stall	The horse’s head is at the outside door	78.3	18.6 (4.4-78.9)*	48.3 (10.0-233.1)*	0.1 (0–0.4)	0.4
	The horse is inside the stall, but looking at the outside door, observing the environment	19.5	0.6 (0.2-2.0)	0.1 (0.0-0.7)	1.9 (0.6-6.16)
	The horse is eating	0.9	NE	NE	NE
	The horse is not close to the outside stall door and does not look interested in the environment	0.9	NE	NE	NE
Head position	Above the withers or grazing	95.0	NE	NE	NE	0.2
	At the level of the withers	3.3	NE	NE	NE
	Below the withers but not eating	0.0	NE	NE	NE
Response to opening the door	The horse moves towards the door or is already positioned at the outside door	15.8	1.0 (0.2-3.7)	1.5 (0.4-6.0)	1.9 (0.6-6.5)	0.3
	The horse looks at the door, but does not move towards the door	15.8	0.7 (0.2-3.3)	0.3 (0–1.7)	0.5 (0.1-1.7)
	The horse does not respond to opening the door	1.7	NE	NE	NE
Response to approach and presence of the observer	Moves towards or looks to the observer	84.7	0.3 (0.1-1.5)	0.2 (0.1-0.9)	1.4 (0.3-6.1)	0.2
	Moves away from the observer	2.5	NE	NE	NE
	Does not move	12.7	3.8 (0.8-18.8)	4.6 (1.0-21.2)*	0.7 (0.1-3.7)
Appetite for hay	The horse eats hay	99.1	NE	NE	NE	0.2
	The horse does not eat hay	0.8	NE	NE	NE
Response to concentrate food	Moves to the food and eats	90.7	NE	NE	NE	0.2
	Hesitates to move towards the food, but eats	8.0	NE	NE	NE
	Shows no interest in food, not eating	0	NE	NE	NE
Locomotion	The horse moves freely alone	84.2	47.6 (4.9-464.0)*	5.8 (1.6-21.3)*	0.2 (0–0.6)	0.4
	The horse does not move, or is reluctant to move	10.0	0 (0–0.2)	0.2 (0.1-0.7)	6.2 (1.6-23.2)*
	The horse is agitated, restless	5.8	NE	NE	NE
Locomotion when led by the evaluator	The horse moves freely when led	86.5	108.2 (4.4- > 999.9)*	68.1 (4.9-946.5)*	0 (<0.001-0.1)	0.4
	The horse does not move, or is reluctant to move when led	12.2	NE	NE	NE
	The horse is agitated, restless	0.0	NE	NE	NE
Variable	Criteria	Specificity (%) 1	Relevance (confidence interval) 2			Item-total correlation 3
			GA x GC	GAA X GC	GCA x GC
Response to palpation of the painful area (approximately 3 cm besides the wound)	No response or change in relation to pre-procedure palpation response of the surgical wound	57.1	4.1 (1.1-15.)*	18.8 (4.6-76.0)*	0.3 (0.1-1.1)*	0.4
	Mild reaction to palpation of the surgical wound	29.4	0.1 (0–0.5)	0.1 (0–0.5)	1.9 (0.6-5.9)*
	Violent reaction to palpation of the surgical wound	13.4	2.1 (0.6-7.8)*	0.1 (0–1.3)*	1.7 (0.4-7.5)*
Response to an auditory stimulus (clap hands)	Moves and/or pays attention with ears or head movements	80.0	6.8 (0.5-84.0)	4.9 (0.7-33.6)	0.5 (0.1-2.0)	0.3
	Calm, no response	18.6	NE	NE	NE
	No response to auditory stimulus due to prostration	0.0	NE	NE	NE
Looking at the flank	The horse does not look at the flank	84.6	46.7 (9.9-219.0)*	128.9 (21.2-782.7)*	0.1 (0–0.3)*	0.4
	The horse looks at the flank	15.4	0 (0–0.1)	0 (0–0)	11.7 (3.1-43.7)*
Kicking at the abdomen	The horse does not kick the abdomen	95.8	NE	NE	NE	0.3
	The horse kicks at the abdomen	2.5	NE	NE	NE
Lifting hind limbs	No lifting of hind limbs	74.6	18.0 (3.9-81.9)*	96.5 (16.4-566.4)*	0.1 (0–0.4)	0.6
	Lifting hind limbs	22.0	0.2 (0.1-0.8)	0 (0–0.2)	4.6 (1.3-16.1)*
	Lifting one of the hind limbs and extending the head	3.4	NE	NE	NE
Head movement	Head straight ahead most of the time	87.1	91.2 (10.1-822.7)*	32.4 (6.7-155.7)*	0.1 (0–0.3)	0.4
	Lateral and/or vertical occasional head movements	12.1	NE	NE	NE
	Lateral and/or vertical continuous head movements	0.9	NE	NE	NE
Pawing on the floor (fore limbs)	Quietly standing, no pawing	89.7	17.5 (3.6-84.9)*	38.9 (6.6-230.1)*	0.1 (0–0.5)	0.4
	Pawing	9.4	0.1 (0–0.4)	0 (0–0.2)	4.1 (1.12-14.1)*
Others	Moving the tail sharply and repeatedly	21.7	0.9 (0.2-3.5)	0.5 (0.1-2.1)	1.8 (0.5-6.7)	0.5
	Moving the tail sharply and repeatedly and lifting the hind limbs	9.2	0 (0–0.2)	0 (0–0.2)	2.4 (0.9-6.9)
	Partial penis protrusion	23.2	2.3 (0.4-12.5)	8.5 (1.6-4.2)	0.4 (0–2.0)	0.2
	Penis protrusion	47.4	NE	NE	NE
			GA x GC	GAA X GC	GCA x GC
Heart rate		-	-	-	-	0.6
Respiratory rate		-	-	-	-	0.5
Systolic blood pressure		-	-	-	-	0.4
Digestive sounds		-	-	-	-	0.3

The categories written in bold letters were used for the sum of the total score. NE – not evaluated as there were not sufficient data for statistical analysis (the behaviour was either infrequently or not observed). GA – Anaesthesia only (n = 6). GAA – Pre-emptive analgesia followed by anaesthesia (n = 6). GC – Anaesthesia, castration and postoperative analgesia administered four hours after surgery (n = 6). GCA – Pre-emptive analgesia, followed by anaesthesia and castration (n = 6).

¹The specificity of each item was evaluated by investigating if that particular behaviour was present or not at TC, moment without pain, in all observations from all evaluators, in all animals from all groups. Specificity was classified as excellent (0–4.9%), good (5–14.9%), moderate (15–29.9%), or nonspecific (≥30%).

²Relevance was tested based on the possibility of distinguishing the behaviour in T4 in GC compared to the other groups. The item was considered relevant when that item differentiated GC from the other three groups, or when GC was different from animals without pain (GA and GAA). The item was considered irrelevant when there were no differences between GC and the other groups. Asterisks (*) indicate differences between groups.

³Criterion validity for item-total correlation with Pearson correlation test (weak < 0.30; moderate 0.31 – 0.60; strong 0.61 – 0.9; very strong 0.91 -1.0) [8].