Mouse taste preference tests: why only two bottles?

Two-bottle tests have been used extensively to measure the preference for taste and nutrient solutions but there has been little work with tests involving more than two bottles. Here, we compare the results obtained in two-bottle tests with those obtained in three- and six-bottle tests. In Experiment 1, we measured the preferences for 2 mM saccharin, 50 mM citric acid, 0.3 mM quinine hydrochloride and 75 mM NaCl displayed by 129X1/SvJ (129) and C57BL/6J (B6) mice. Mice drank more taste solution when they received two bottles providing taste solution and one providing water than when they received either a standard two-bottle test or two bottles providing water and one providing taste solution. The three-bottle tests also revealed the left spout side preferences of the 129 strain and were generally better at distinguishing between the 129 and B6 strains (i.e. were more sensitive) than were the two-bottle tests. In Experiment 2, we measured intakes and preferences in tests with six bottles, with one, two, three, four or five containing 75 mM NaCl and the rest containing water. NaCl preferences were monotonically related to the number of NaCl spouts available. A follow-up experiment found similar results whether the index of ingestion was volume intakes or licks. This argues that spillage cannot account for the effect of spout number on taste solution intake. Together, the results suggest that (i) the number of bottles of taste solution and water has a profound influence on taste solution intake and preference, and (ii) three-bottle tests may be more sensitive than two-bottle tests in many circumstances.