Table 1. The optical sensitivity for the cones of the human eye and the horse eye at intensities corresponding to moonlight. Absorption coefficient Pupil diameter Focal length Cone diameter Length of cone outer segment Double length with tapetum Optical sensitivity for white light Symbols k A (mm) f (mm) d (mm) l (mm) l (mm) Sw(mm2 sr) Human (Homo sapiens) A 0.035 B 7000 A 16700 B 1.5 C 30 0.08 Horse (Equus caballus) A 0.035 30000 25000 1 (4) D 8 D 0.1 A[18] B[19] An average of the cone diameter in parafovea and perifovea is used. C[20] DThe tapetum is the reason for the double effective length of the cone outer segments. doi:10.1371/journal.pone.0003711.t001 of human colour vision. The experimenter (AB), who managed to discriminate colours even at an intensity of 0.007 cd/m2, seemed to have coarser spatial resolution at low light intensities than the other subjects since she needed help to write down her choices. These individual differences among humans may deserve further attention and studies. The low light intensities that we used in the behavioural experiment are likely to be mesopic to horses. For humans, this means that colours appear less and less saturated, which indicates that rod signals become stronger while cone signals become weaker. A similar process could influence horse colour vision even more than it does for us since horses do not have a rod-free area in the retina as we do in the centre of our fovea. On the contrary, rods dominate most of the horse visual streak and with a smaller pupil, the rod signals would probably take over at even higher light intensity than in humans. The large eye of the horse is not adapted to nocturnal colour vision. Instead it probably favours achromatic rod vision in dim light where the large eyes and pupils are exceptionally good at catching light and where signal summation presumably enhances sensitivity without too much loss in spatial resolution. With the now confirmed colour vision ability in moonlight intensities, horse colour vision still functions over the largest changes in illumination colour that occur during the sunset and twilight period [7]. At dimmer light intensities the ability to detect movements from possible predators may be the most important visual task, and achromatic vision may therefore be favoured. Methods Animals We trained three horses with normal vision in a behavioural experiment; Chap, a 14 year old half blood gelding, Rex, a 11 year old thoroughbred gelding and Rosett, a Shetland pony mare of an honourable age of 33. Eyes from a one-year-old half blood mare and a twelve-year-old draft mare, obtained directly after their death from a veterinary hospital in Helsingborg, Sweden, were used to obtain measurements for the calculation of the sensitivity of the eye. Both mares were put down after decision of their owners only, and even though the horses were unwell their Colour Vision Threshold eyes were still fine and valuable for our study. All experiments were approved by the Swedish animal welfare agency (M148-05) and the Swedish board of Agriculture (83-5942/08). Stimuli and apparatus The horses were trained to discriminate between blue and green stimuli. To make intensity an unreliable cue five different brightness versions of blue and seven different brightness versions of green were used as stimuli. All stimulus colours were printed on a printer (Canon Pro 9000) on white Munken Pure Copy paper (Artic Paper Munkedals AB, Munkedal, Sweden) in the size 15621 cm. The stimuli were kept in transparent plastic cases that did not influence the reflectance of the stimuli but kept them clean. The reflectance S(l) of all stimuli and the spectrum of the illumination I(l) used in experiment (halogen spotlights) were measured using a spectroradiometer (S2000, Ocean Optics Inc., Dunedin, FL, USA). The spectral sensitivity Ri(l) of photoreceptor i was calculated from the horse cone sensitivity peaks (428 and 539 nm) [22] and a typical mammal rod peak sensitivity (500 nm) [23] using the Stavenga-Smits-Hoenders rhodopsin template [24]. The relative number of quanta Qi absorbed by the horse’s cone type i looking at a stimulus was calculated using Equation 1. ð Qi~ S(l)I(l)Ri(l)dl 1 ð Þ Both the quantum catch for the long wavelength-sensitive cone and the quantum catch for the rods (Table 2) were taken into consideration when combining the different ‘‘brightness versions’’ of blue and green for the training sessions. An equal number of times, the positive stimulus was brighter or darker than the negative stimulus and the stimulus pairs were presented in a pseudo-random order. The results were examined to make sure there was no correlation between stimulus brightness and choice Table 2. Relative quantum catches for horse photoreceptors Colours Q SWS Q LWS Q Rod Colours Q SWS Q LWS Q Rod Green 99 1 Green 38 2 Green 57 3 Green 34 4 Green 27 5 Green 17 6 Green 15 7 857 747 683 472 582 339 306 543 411 434 315 333 219 202 Blue 1 Blue 2 Blue 3 Blue 4 Blue 5 151 131 119 106 80 622 463 378 322 235 475 382 318 267 197 Seven shades of green and five shades of blue were tested and the relative quantum catches for the long wavelength-sensitive (LWS) cone type and rods were calculated according to Eqn. 1. These values were used to make sure that brightness of the colour stimuli were comparable for the horse. Relative quantum catches of the short wavelength-sensitive (SWS) cone type is included for comparison. doi:10.1371/journal.pone.0003711.t002 PLoS ONE | www.plosone.org 4 November 2008 | Volume 3 | Issue 11 | e3711