Project protocol — Contents

Equipment and consumables

Procedures

Data

Refs

Workflow

Day Procedure performedSucrose position% SucroseAge (wk)Data collected -7 Mice arrived at the animal facility, housed 5 per cage, separated according to sex and strain - 0 12 - -7 to -1 Mice are allowed to acclimate to general housing conditions - 0 12-13 - 1Mice are housed individually with continuous access to 2 calibrated pre-weighed sippers tubes, each containing 40 mL water and ~ 20 g rodent chow - 0 13 - 1-4 Each mouse is assessed for chow and water intakes every 24 h for 4 days - 0 13 baseline chow0, water0 intakes 5-8 Half of the mice (ascending group) within a strain are given chow and the lowest concentration of sucrose (0.0001%) in one bottle and water in the other bottle positioned accordingly and then changed every 24 h L-R (d2), R-L (d3), L-R (d4) 0.0001^{a} 13 chow1, sucrose1, water1 intakes 5-8 The other half of the mice (descending group) within a strain are also given chow and the highest concentration of sucrose (20%) in one bottle and water in the other bottle positioned accordingly and then changed every 24 h R-L (d2), L-R (d3), R-L (d4) 20^{b} 13 chow9, sucrose9, water9 intakes 9-12 As in day 5-8, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 0.001^{a} 13-14 chow2, sucrose2, water2 intakes 9-12As in day 5-8, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4) 10^{b} 13-14 chow8, sucrose8, water8 intakes 13-16 As in day 9-12, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 0.01^{a} 14 chow3, sucrose3, water3 intakes 13-16As in day 9-12, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4)5

^{b} 14 chow7, sucrose7, water7 intakes 17-20 As in day 13-16, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 0.1^{a} 14-15 chow4, sucrose4, water4 intakes 17-20As in day 13-16, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4) 2.5^{b} 14-15 chow6, sucrose6, water6 intakes 21-24 As in day 17-20, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 1^{a} 15 chow5, sucrose5, wate5r intakes 21-24As in day 17-20, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4) 1^{b} 15 chow5, sucrose5, wate5r intakes 25-28 As in day 21-24, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 2.5^{a} 15-16 chow6, sucrose6, water6 intakes 25-28As in day 21-24, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4) 0.1^{b} 15-16 chow4, sucrose4, water4 intakes 29-32 As in day 25-28, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 5^{a} 16 chow7, sucrose7, water7 intakes 29-32As in day 25-28, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4) 0.01^{b} 16 chow3, sucrose3, water3 intakes 33-36 As in day 29-32, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 10^{a} 16-17 chow8, sucrose8, water8 intakes 33-36As in day 29-32, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4) .001^{b} 16-17 chow2, sucrose2, water2 intakes 37-40 As in day 33-36, ascending group of mice are given chow and the next higher concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr L-R (d2), R-L (d3), L-R (d4) 20^{a} 17 chow9, sucrose9, water9 intakes 37-40As in day33-36, descending group of mice are given chow and the next lower concentration of sucrose in one bottle and water in the other bottle whose positions are altered every 24 hr R-L (d2), L-R (d3), R-L (d4) 0.0001^{b} 17 chow1, sucrose1, water1 intakes

L =left; R =right;d =day;a.ascending group;b.descending group.• Pair of 2 calibrated (100 mL capacity, + 0.1 g = + 0.1 mL; Lab Products, Seaford, DE)

sipper tubes

• 9 various sucrose solution concentrations: 0% (baseline, water), 0.0001%, 0.001%, 0.01%, 1%, 2.5%, 5%, 10%, 20%

• Purina Mouse chow (5.3 kcal/g))

a.Initially, each animal is provided with a pre-weighed ration (~20 g, 5.3 kcal/g) of chow and two calibrated and pre-weighed sipper tubes each filled with water (40 mL each, + 0.1 g = + 0.1 mL).

b.Each mouse of each strain is assessed for chow and water intakes from each individual bottle every 24 h over 4 days.

Investigator's note: "Whereas chow spillage was measured and adjustments of intake were made accordingly, it should be noted that there was no systematic measurement of water or sucrose spillage, although very infrequent malfunction of a sipper tube (resulting in large spillage) was noted, and the data discarded." See Lewis et al. 2008.

Body weights of the mice are periodically measured throughout the study, and a mean body weight is chosen for each animal of each strain for further analysis.c.

To minimize potential bottle position preference effects, the position of the two water bottles are switched across animals and across strains every 24 h according to a left (L)–right (R)–R–L and R–L–L–R position respectively.d.

Following baseline, each mouse of each strain is given chow, one bottle of water and one bottle of sucrose each day.e.

In these two-bottle preference tests, 9 sucrose concentrations are tested: 0.0001%, 0.001%, 0.01%, 0.1%, 1.0%, 2.5%, 5.0%, 10% and 20%.f.

Half of the mice of each strain are tested in an ascending sucrose concentration order, and the remaining half are tested in a descending order with sucrose bottle position regularly altered every 24 h.g.

Chow, sucrose, and water intakes (+0.1 g) are measured daily for each concentration of sucrose given.h.

Data collected by investigator•

daily chowintakewith different sucrose concentration ( 0% (baseline), 0.0001%, 0.001%, 0.01%, 1%, 2.5%, 5%, 10%, or 20%) in the drinking water.

•daily sucroseintakeat different sucrose concentration (0% (baseline), 0.0001%, 0.001%, 0.01%, 1%, 2.5%, 5%, 10%, or 20%) in the drinking water.

•daily waterintakewith different sucrose concentration (0% (baseline), 0.0001%, 0.001%, 0.01%, 1%, 2.5%, 5%, 10%, or 20%) in the drinking water.

Definitions and calculations

fluid= sucrose solution (mL) + water (mL)

percent sucrose= (sucrose solution/fluid) * 100

chow calories= chow intake (g) * 5.3 (kcal/g)

sucrose calories= sucrose solution intake (mL) * 0.0004 (kcal/mL)

total calories= chow calories + sucrose calories

percent sucrose calories= (sucrose calories/(total calories)) * 100

sucrose solution concentrations:1= 0.0001%,2= 0.001%,3= 0.0%,4= 0.1%,5= 1%,6= 2.5%,7= 5%,8= 10%,9= 20%