Bodnar1 project protocol

Diet preference: Sucrose intake in males of 12 inbred strains of mice   (2006)

Project protocol — Contents
Equipment and consumables
Procedures
Data
Refs

Workflow and sampling

Workflow

Day	Procedure performed	Sucrose position	% Sucrose	Age (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	-
1	Mice 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)	0.0001a	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)	20b	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)	0.001a	13-14	chow2, sucrose2, water2 intakes
9-12	As 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)	10b	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)	0.01a	14	chow3, sucrose3, water3 intakes
13-16	As 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)	5b	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)	0.1a	14-15	chow4, sucrose4, water4 intakes
17-20	As 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)	2.5b	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)	1a	15	chow5, sucrose5, wate5r intakes
21-24	As 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)	1b	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)	2.5a	15-16	chow6, sucrose6, water6 intakes
25-28	As 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)	0.1b	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)	5a	16	chow7, sucrose7, water7 intakes
29-32	As 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)	0.01b	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)	10a	16-17	chow8, sucrose8, water8 intakes
33-36	As 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)	.001b	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)	20a	17	chow9, sucrose9, water9 intakes
37-40	As 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)	0.0001b	17	chow1, sucrose1, water1 intakes

L = left; R = right; d = day; a. ascending group; b. descending group.

Equipment and consumables

• 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))

Procedures

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.

c. 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.
d. 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.
e. Following baseline, each mouse of each strain is given chow, one bottle of water and one bottle of sucrose each day.
f. 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%.
g. 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.
h. Chow, sucrose, and water intakes (+0.1 g) are measured daily for each concentration of sucrose given.

Data collected by investigator

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