J Nutr Sci Vitaminol, 58, 230–239, 2012

The Interactive Effect of Dietary Water-Soluble Vitamin Levels on

the Depression of Gonadal Development in Growing Male Rats
Kept under Disturbed Daily Rhythm

Miho HANAI and Takatoshi ESASHI*

Department of Nutrition and Life Science, Kanagawa Institute of Technology, 1030 Shimo-Ogino,
Atsugi, Kanagawa 243–0292, Japan
(Received December 22, 2011)

Summary The purpose of this study was to clarify the effects of nutrients on the gonadal
development of male rats kept under constant darkness as a model of disturbed daily
rhythm. In the present study we examined the effects of nine water-soluble vitamins. We
selected 7 water-soluble vitamins (choline, nicotinic acid (NA), pantothenic acid (PA), vita-
min B6 (VB6), vitamin B1 (VB1), vitamin B2 (VB2) and folic acid (FA)) as experimental
factors for the first experiment (Ex. 1) and biotin and vitamin B12 (VB12) as experimental
factors for the second experiment (Ex. 2). The dietary content of these vitamins was normal
or six times the normal content. Lighting condition (L.C.) was also added as a factor. Four-
week-old male rats (Fischer 344 strain) were kept under constant darkness or normal light-
ing (12-h light/dark cycle) for 4 wk. The depression of gonadal development in the constant
darkness groups (D-groups) was shown. The L.C., PA, VB6 and VB1 influenced testes devel-
opment, and these three vitamins had interactions with L.C. Among the normal lighting
groups (N-groups), the highest value for testes weight was observed under the normal-PA,
high-VB6 and high-B1 diet; on the other hand, among the D-groups, it was observed under
the high-PA, normal-VB6 and normal-VB1 diet. The results showed that the depression of
gonadal development in rats kept under disturbed daily rhythm was improved by getting a
high amount of PA and normal amount of VB6 and VB1.
Key Words constant darkness, disturbed daily rhythm, gonad, water soluble vitamin, or-
thogonal array

The number of people living under disturbed daily
rhythm has been increasing due to the globalization of
business and social activities as well as the diversification
of the forms of labor. Such irregularities in daily rhythm
adversely affect bio-regulatory mechanisms, resulting in
an abnormal diurnal rhythm that can impede biological
activities structurally and functionally. However, there is
currently no source of collected basic data on the nutri-
tional aspects of maintaining or promoting health under
the condition of disturbed daily rhythm. The perspective
behind this research is as follows: as basic data on the
relationship between nutritional status and gonadal
development in rats with disturbed daily rhythm accu-
mulate, these data can be used for human research, and
finally, dietary reference intakes can be compiled for per-
sons living under disturbed daily rhythm.
Lighting is one of the key external factors for the for-
mation of daily rhythm. Rats kept under constant dark-
ness develop disturbances in their feeding and motor-
activity rhythms, and suffer from altered rhythms of
hormone secretion and enzyme activity (1, 2). Esashi
et al. (3, 4) have reported that rats kept under constant
darkness showed depressed gonadal development and a
decreased delivery rate. The depression of gonadal devel-
* Deceased; passed away on March 26, 2012.
E-mail: hanai@bio.kanagawa-it.ac.jp
opment in these rats was accelerated by a low-protein
diet (4). These findings indicate that the gonads have
high sensitivity to constant darkness and nutrients.
In previous papers, we have reported the effects on
rat gonadal development of protein, methionine, vita-
mins, minerals and oil (5), minerals (6), amino acids,
an AIN-76 diet and an AIN-93G diet (7), the interaction
between protein and vitamins (8), water- and fat-soluble
vitamins (9) and each of the fat-soluble vitamins (10).
We have demonstrated that a high level (three times
the normal (AIN-93G)) of water-soluble vitamin and a
low level (1/3.3 of normal) of fat-soluble vitamin diet
mitigated the depression of gonadal development in
rats kept under constant darkness (9). Therefore, as a
next step, we attempted to clarify the effects on gonadal
development of nine kinds of water-soluble vitamins
under a diet with a low level of fat-soluble vitamins.
In this experiment, the content of each water-soluble
vitamin was normal or six times the normal content.
This study was divided into two experiments, because
a single experiment to test all nine water-soluble vita-
mins couldn’t be done. In the first experiment (Ex. 1)
we examined seven water-soluble vitamins, and in the
second experiment (Ex. 2) we examined two water-sol-
uble vitamins. Experiment 1 was carried out using an
orthogonal array (11, 12), which is the experimental
design we applied in previous studies (5–7, 10), and Ex.

230
 Effects of Water-Soluble Vitamins on Gonadal Development in Rats with Disturbed Daily Rhythm 231

2 was based on three-way layout methods. line, nicotinic acid (NA), pantothenic acid (PA), vitamin
B6 (VB6), vitamin B1 (VB1), vitamin B2 (VB2) and folic
METHODS
acid (FA)) and the lighting condition were selected as
Animals. For each experiment, 48 Fischer strain factors, and the experimental protocol and diet compo-
(F344) male rats (purchased from Charles River Japan, sition were designed based on the L16(215)-type orthogo-
Inc., Kanagawa, Japan, at 3 wk of age) were preliminar- nal array, which can be used to examine up to eight fac-
ily maintained for a week on the AIN-93G purified diet tors (11, 12). The examined factors and their levels are
(13) and then divided into 16 experimental groups of shown in Table 1. Eight types of experimental diet were
three rats each (Ex. 1) or eight experimental groups of prepared based on the L16(215)-type orthogonal array
six rats each (Ex. 2). No differences were found among (Table 2).
the mean body weights of rats from any of these groups. In Ex. 2, two water-soluble vitamins (biotin and
The rats were kept under constant darkness (D-groups) vitamin B12 (VB12)) and the lighting condition were
or normal lighting (12-h light/dark cycle, N-groups)
for 4 wk. Food intake and body weight were recorded Table 1. Estimated factors and their levels in the
every other day. The care of rats kept under constant L16(215)-type orthogonal array (Experiment 1).
darkness included lighting a red lamp for about 2 h; the
lamp was for photographs and such lighting has been Factor Row Level 1 Level 2
demonstrated to not cause a phase variation in circa-
dian rhythms. The rats were housed in individual, stain- A: Lighting condition 1 Normal Constant
less steel, wire-mesh-bottomed cages at 22⫾1˚C and lighting darkness
55⫾5% humidity, in a room free from specific patho- B: Choline 2 Normal High (⫻6)
gens. Food and distilled water were provided to all rats C: Nicotic acid 4 Normal High (⫻6)
D: Pantothenic acid 6 Normal High (⫻6)
ad libitum.
E: Vitamin B6 8 Normal High (⫻6)
Animals were maintained in accordance with the
F: Vitamin B1 10 Normal High (⫻6)
Guidelines for the Care and Use of Laboratory Animals
G: Vitamin B2 12 Normal High (⫻6)
(Notification of the Prime Minister’s Office in Japan). H: Folic acid 14 Normal High (⫻6)
Diets. In Ex. 1, seven water-soluble vitamins (cho-

Table 2. Composition of the diets based on the L16(215)-type orthogonal array (Experiment 1).
(g/100 g diet)

Diet groups
Ingredients
1 2 3 4 5 6 7 8

Milk casein1 9 9 9 9 9 9 9 9
L-Cystine 0.135 0.135 0.135 0.135 0.135 0.135 0.135 0.135
␣-Corn starch 64.14 63.09 62.80 62.54 61.69 61.44 62.14 62.29
Sucrose 10 10 10 10 10 10 10 10
Fiber 5 5 5 5 5 5 5 5
Soybean oil 7 7 7 7 7 7 7 7
Mineral mix.2 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5
tert-Butylhydroquinone 0.0014 0.0014 0.0014 0.0014 0.0014 0.0014 0.0014 0.0014
Fat-soluble vitamin mix.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

D-Biotin4 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

Vitamin B125 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025

Choline bitartrate 0.25 0.25 0.25 0.25 1.50 1.50 1.50 1.50
Nicotinic acid6 0.03 0.03 0.18 0.18 0.03 0.03 0.18 0.18
Ca pantothenate7 0.16 0.16 0.96 0.96 0.96 0.96 0.16 0.16
Pyridoxine7 0.07 0.42 0.07 0.42 0.07 0.42 0.07 0.42
Thiamin7 0.06 0.36 0.06 0.36 0.36 0.06 0.36 0.06
Riboflavin7 0.06 0.36 0.36 0.06 0.06 0.36 0.36 0.06
Folic acid6 0.02 0.12 0.12 0.02 0.12 0.02 0.02 0.12
1
Vitamin-free milk casein.
2
AIN-93G mineral mixture containing 67.2 g/kg of phosphorus.
3
AIN-93 fat-soluble vitamin mixture.
4
Diluted with sucrose to 1,000 times.
5
Diluted with mannitol to 1,000 times.
6
Diluted with sucrose to 10 times.
7
Diluted with sucrose to 100 times.
232 HANAI M and ESASHI T

selected as factors, and the experimental protocol and
diet composition were designed based on the three way-
classification. The examined factors and their levels,
and the diet composition are shown in Tables 3 and 4,
respectively.
The water-soluble vitamin contents were normal
(AIN-93G diet) or high, six times the normal content.
In a previous experiment (9), the high water-soluble
vitamin diet, with three times the normal vitamin con-
tent, improved the depression of gonadal development
of rats kept under constant darkness. In the present
experiment, we used an even higher vitamin content,
six times the normal content. The normal and high lev-
els of vitamins are shown as level 1 and level 2, respec-
tively. The dietary protein level was low, 9% casein and
0.135% cystine, and the fat-soluble vitamin levels were
low, 1/3.3 of normal content. Other components were
based on the AIN-93G diet.
In Ex. 1, there were eight kinds of diet and 16 groups.
In Ex. 2, there were four kinds of diet and eight groups.
Analysis. After 4 wk of treatment, the rats were
decapitated, and the blood and gonadal organs (testes,
epididymides, seminal vesicles and prostate) were col-
lected. The gonadal organs were weighed. Blood was
centrifuged (3,000 rpm, 15 min, 4˚C), and the serum
obtained was stored at ⫺20˚C until analysis, when se-
rum testosterone concentration was measured by a ra-
dioimmunoassay kit (CIS Diagnostic Co., Tokyo, Japan).
Statistical analysis. Statistical analysis followed the
original method for the orthogonal array table (Ex. 1) or
three-way classification (Ex. 2) (11, 12, 14).
The effects of factors and interactions between factors
on gonadal organ weights and serum testosterone con-
centrations were determined by ANOVA. Then, in order
to get the most suitable combination of factors and their
levels, the estimated values of population mean were
calculated (14). The estimated values of gonadal organ
weight and serum testosterone concentration were cal-
culated only in accordance with the factors that showed
significant differences (p⬍0.05). Therefore, the values
in the tables are estimated values of population mean,
which have a statistical difference. For analysis, we used
the software developed by the Japan Technology Train-
ing Institute Co., Ltd. (Tokyo, Japan; JUSE-QCAS/V6.0).
RESULTS

Table 3. Estimated factors and their levels in the three- The functioning of the gonadal organ during the
way classification (Experiment 2). growing period is in direct proportion to the weight of
the organ (15). Therefore, in this study, the weights of
Factor Level 1 Level 2 the gonadal organs were compared by absolute value,
not by the value per 100 g body weight. The highest
A: Lighting condition Normal Constant recorded weight of gonadal organs was evaluated as a
lighting darkness suitable indicator of normal development of the rats.
I: Biotin Normal High(⫻6) Results of ANOVA
J: Vitamin B12 Normal High(⫻6) The effects of dietary water-soluble vitamin content

Table 4. Composition of the diets (Experiment 2).

(g/100 g diet)

Diet groups
Ingredients
1 2 3 4

Milk casein1 9 9 9 9
L-Cystine 0.135 0.135 0.135 0.135
␣-Corn starch 63.77 63.77 62.80 62.54
Sucrose 10 10 10 10
Fiber 5 5 5 5
Soybean oil 7 7 7 7
Mineral mix.2 3.5 3.5 3.5 3.5
tert-Butylhydroquinone 0.0014 0.0014 0.0014 0.0014
Fat-soluble vitamin mix.3 0.3 0.3 0.3 0.3
Choline bitartrate 0.25 0.25 0.25 0.25
Water-soluble vitamin mix.4 1 1 1 1

D-Biotin5 0.02 0.02 0.12 0.12

Vitamin B126 0.025 0.15 0.025 0.15
1
Vitamin-free milk casein.
2
AIN-93G mineral mixture containing 67.2 g/kg of phosphorus.
3
AIN-93 fat-soluble vitamin mixture.
4
AIN-93 water-soluble vitamin mixture omitted biotin and vitamin B12.
5
Diluted with sucrose to 1,000 times.
6
Diluted with mannitol to 1,000 times.
 Effects of Water-Soluble Vitamins on Gonadal Development in Rats with Disturbed Daily Rhythm 233

on gonadal organ weight, serum testosterone concen-

A⫻H
tration, body weight, total food intake and food effi-
ciency are shown in Tables 5 and 6.
The effects of the lighting condition and water-solu-

A⫻G

**
*
*
ble vitamins as well as the effects of their interaction on
gonadal organ and body weight were observed. In par-
ticular, the lighting condition and the interaction of the

A⫻F

*
*
*
*
lighting condition and PA affected many of the assessed
items.
Estimated values of population mean of body weight, total

A⫻E

**
*
*

*
food intake, food efficiency, gonadal organ weight and serum
testosterone concentration

Effects of water-soluble vitamins on reproductive organ weight, serum testosterone level and body weight: results of analysis of variance-1.

A⫻D
The estimated values of the factors (lighting condi-

**
*
*
*
*
*
*
*
*
tion and nine water-soluble vitamins) that had a sig-
nificant effect on body weight, total food intake, gonadal

A⫻C
organ weight and serum testosterone concentration are

**
shown in Tables 7 and 8.
Body weight, total food intake and food efficiency. In Ex.

A⫻B
1, the estimated values of the body weight are shown

**
*

*
twice in Table 7, because these values couldn’t be calcu-
lated using eight factors at a time by the software which FA7
we used. The estimated values of the lighting condition, (H)

**
*
*
choline, NA, PA, VB6 and VB2, which interacted with
the lighting condition, are shown as the estimated val-
VB26
(G)

**
ues (A) in Table 7, and the estimated values of VB1 and
FA, which did not interact with the lighting condition,
are shown as the estimated values (B) in Table 7. The
VB15
(F)

highest values for body weight (163.7 g and 147.2 g in

*
Table 7) were observed in rats maintained under normal
lighting and a normal-choline, normal-NA, high-PA,
VB64
(E)

*
high-VB6, normal-VB2, normal-VB1 and high-FA diet.

Nicotinic acid, 3 Pantothenic acid, 4 Vitamin B6, 5 Vitamin B1, 6 Vitamin B2, 7 Folic acid.
In the D-groups, the highest value for body weight was
observed in a normal-choline, high-NA, high-PA, nor-
PA3
(D)

**
*
*

mal-VB6, normal-VB2, normal-VB1 and high-FA diet

(Table 7). The interactions of the lighting condition and
choline, NA, PA, VB6, and VB2 showed that the effects
NA2
(C)

**
*
*

of vitamin levels in the diet differed according to the

lighting condition.
In Ex. 2, only the lighting condition was observed to
Choline

affect the body weight (Tables 6 and 7).

(B)

**
*

*
*

*
Lighting condition; Normal lighting or constant darkness.

The highest value for food intake in Ex. 1 (331.8 g)

was observed in rats maintained under normal lighting
and a normal-choline diet (Table 7). In Ex. 2, the high-
est value for food intake (338.1 g) was observed in rats
condition1 (A)

maintained under normal lighting and a high-biotin

diet (Table 7). In the N-groups, the food intake increased
8
Lighting

**
**
**
**
**
**
**
**

**
*
*

when changing to a high-biotin diet from a normal-bio-

tin diet (from 321.0 to 338.1 g, 17.1-g increase, Table
7) but decreased following this change in the D-groups
(from 303.9 to 294.8 g, 9.1-g decrease, Table 7).
* : p⬍0.05, ** : p⬍0.01.

As for the food efficiency, in both experiments, only

(g)
(g/100 g BW)
(g)
(g/100 g BW)
(mg)
(mg/100 g BW)
(mg)
(mg/100 g BW)

(ng/mL)

(g)
(g)

the effect of lighting condition was observed (Tables

5–7).
Testes weight. In Ex. 1, the highest value for testes
weight (2.200 g) was observed in rats maintained under
Seminal vesicles

Food efficiency
Epididymides

normal lighting and a normal-PA, high-VB6 and high-

Testosterone

Body weight
Food intake

VB1 diet, and the lowest value (0.907 g) was observed in

Table 5.

Prostate

rats maintained under constant darkness and a normal-

Testes

PA, high-VB6 and high-VB1 diet. In the D-groups, the

1
2
8

highest value for testes weight (1.547 g) was observed

234 HANAI M and ESASHI T

Table 6. Effects of water-soluble vitamins on reproductive organ weight, serum testosterone level and body weight: results
of analysis of variance (Experiment 2).

Lighting Biotin VB122

A⫻I A⫻J I⫻J A⫻I⫻J
condition1 (A) (I) (J)

3
Testes (g) **
(g/100 g BW)
Epididymides (g)
** *
(g/100 g BW)
**
Seminal vesicles (mg)
**
(mg/100 g BW)
**
Prostate (mg)
**
(mg/100 g BW)
**
**
Testosterone (ng/mL) ** *
Body weight (g)
Food intake (g)
**
Food efficiency
**
** *
1
Lighting condition; Normal lighting or constant darkness.
2
Vitamin B12.
3
: p⬍0.05, : p⬍0.01.
* **
in rats on a high-PA, normal-VB6 and normal-VB1 diet 19.0-mg increase, Table 8) but decreased following this
(Table 8). change in the D-groups (20.0- or 20.1-mg decrease,
In the N-groups, the testes weight decreased when Table 8).
changing to a high-PA diet from a normal-PA diet In Ex. 2, only the lighting condition was observed to
(0.053- or 0.054-g decrease, Table 8) but increased affect the epididymides weight (Tables 6 and 8).
in response to this change in the D-groups (0.232- or Seminal vesicle weight. In Ex. 1, the highest value
0.233-g increase, Table 8). In addition, the interaction for seminal vesicle weight (176.5 mg) was observed in
of the lighting condition and VB6 showed that the tes- rats maintained under normal lighting and a normal-PA
tes weight increased with the change to a high-VB6 and high-FA diet, and the lowest value (17.0 mg) was
diet from a normal-VB6 diet in the N-groups (0.023-g observed in rats maintained under constant darkness
increase, Table 8) but decreased following this change and a normal-PA and normal-FA diet. In the D-groups,
in the D-groups (0.213- or 0.214-g decrease, Table 8). the highest value for seminal vesicle weight (44.2 mg)
Moreover, the interaction of the lighting condition and was observed in rats on a high-PA and high-FA diet
VB1 showed that the testis weight increased with the (Table 8).
change to a high-VB1 diet from a normal-VB1 diet in In the N-groups, the seminal vesicle weight decreased
the N-groups (0.077- or 0.078-g increase, Table 8) but when changing to a high-PA diet from a normal-PA
decreased following this change in the D-groups (0.194- diet (11.6- or 11.7-mg decrease, Table 8) but increased
or 0.195-g decrease, Table 8). in response to this change in the D-groups (14.7-mg
In Ex. 2, only the lighting condition was observed to increase, Table 8).
affect the testes weight (Tables 6 and 8). In Ex. 2, only the lighting condition was observed to
Epididymides weight. In Ex. 1, the highest value for affect the seminal vesicle weight (Tables 6 and 8).
epididymides weight (282.3 mg) was observed in rats Prostate weight. In Ex. 1, the highest value for pros-
maintained under normal lighting and a normal-PA and tate weight (136.2 mg) was observed in rats maintained
high-VB1 diet, and the lowest value (105.7 mg) was under normal lighting and a normal-choline, normal-
observed in rats maintained under constant darkness NA, high-PA and normal-VB2 diet, and the lowest value
and a normal-PA and high-VB1 diet. In the D-groups, (33.3 mg) was observed in rats maintained under con-
the highest value for epididymides weight (155.3 mg) stant darkness and a high-choline, high-NA, normal-PA
was observed in rats on a high-PA and normal-VB1 diet and normal-VB2 diet. In the D-groups, the highest value
(Table 8). for prostate weight (68.8 mg) was observed in rats on
In the N-groups, the epididymides weight decreased a normal-choline, normal-NA, high-PA and high-VB2
when changing to a high-PA diet from a normal-PA diet diet (Table 8).
(22.9- or 23.0-mg decrease, Table 8) but increased in In the N-groups, the prostate weight increased slightly
response to this change in the D-groups (29.5- or 29.6- when changing to a high-PA diet from a normal-PA
mg increase, Table 8). In addition, the interaction of the diet (0.4-mg increase, Table 8), but it increased greatly
lighting condition and VB1 showed that the epididymi- in response to this change in the D-groups (14.7- or
des weight increased with the change to a high-VB1 14.8-mg increase, Table 8). In addition, the interaction
diet from a normal-VB1 diet in the N-groups (18.9- or of lighting condition and VB2 showed that the pros-
 Effects of Water-Soluble Vitamins on Gonadal Development in Rats with Disturbed Daily Rhythm 235

Table 7. The estimated value of the combined effect of water-soluble vitamins on body weight, food intake and food
efficiency.

Experiment 1 Experiment 2

Estimated values (A)1 Estimated values (B)***

Factor Estimated
Factor Estimated Factor Estimated Factor Estimated level value
level value level value level value A (g)
ABCDEG2 (g) ABCDEG (g) FH (g)

1111113 161.8 211111 139.5 11 143.7 1 153.6 max

111112 155.1 211112 138.0 12 147.2 max 2 135.3 min
111121 163.1 211121 137.0 21 142.4 min
111122 156.4 211122 135.5 22 145.9
111211 162.4 211211 144.6
111212 155.8 211212 143.1
111221 163.7 max4 211221 142.1
111222 157.1 211222 140.6
112111 157.7 212111 140.1
112112 151.0 212112 138.6
112121 158.9 212121 137.7
112122 152.3 212122 136.1
Body weight 112211 158.3 212211 145.2
(g) 112212 151.7 212212 143.7
112221 159.6 212221 142.7
112222 153.0 212222 141.2
121111 151.5 221111 133.4
121112 144.9 221112 131.9
121121 152.8 221121 131.0
121122 146.2 221122 129.4 min4
121211 152.2 221211 138.6
121212 145.6 221212 137.0
121221 153.5 221221 136.1
121222 146.9 221222 134.5
122111 147.4 222111 134.1
122112 140.8 222112 132.5
122121 148.7 222121 131.6
122122 142.1 222122 130.0
122211 148.1 222211 139.2
122212 141.4 222222 137.6
122221 149.4 222221 136.7
122222 142.7 222222 135.1

Factor level Estimated Factor Estimated

AB value (g) level AI value (g)
Food intake
11 331.8 max 11 321.0
(g)
12 313.2 12 338.1 max
21 305.1 21 303.9
22 286.7 min 22 294.8 min

Factor level Estimated Factor Population

A value level A value
Food
effciency
1 0.291 max 1 0.291 max
2 0.267 min 2 0.256 min
1
Estimated values (A): the combinational effects of vitamins that interacted with the lighting condition, Estimated values
(B): the combinational effects of vitamins that did not interact with the lighting condition.
2
A: lighting condition, B: choline, C: niacin, D: pantothenic acid, E: vitamin B6, F: vitamin B1, G: vitamin B2, H: folic acid,
I: biotin.
3
1: normal lighting or normal level, 2: constant darkness or high level.
4
max: maximum, min: minimum.
236 HANAI M and ESASHI T

Table 8. The estimated value of the combined effect of water-soluble vitamins on gonadal weight and serum testosterone.

Experiment 1 Experiment 2

Factor level Estimated Factor level Estimated Factor level Estimated

ADEF1 value (g) ADEF value (g) A value (g)

11112 2.009 2111 1.315 1 1.903 max

1112 2.177 2112 1.121 2 0.948 min
Testes
1121 2.122 2121 1.102
(g)
1122 2.200 max3 2122 0.907 min3
1211 2.046 2211 1.547
1212 2.123 2212 1.353
1221 2.069 2221 1.334
1222 2.146 2222 1.140

Factor level Estimated Factor level Estimated Factor level Estimated

ADF value (mg) ADF value (mg) A value (mg)
Epididymides
111 263.3 211 125.8 1 237.0 max
(mg)
112 282.3 max 212 105.7 min 2 105.6 min
121 240.4 221 155.3
122 259.3 222 135.3

Factor level Estimated Factor level Estimated Factor level Estimated

ADH value (mg) ADH value (mg) A value (mg)
Seminal
vesicles 111 163.9 211 17.0 min 1 160.4 max
(mg) 112 176.5 max 212 29.5 2 27.7 min
121 152.3 221 31.7
122 164.8 222 44.2

Factor level Estimated Factor level Estimated Factor level Estimated

ABCDG value (mg) ABCDG value (mg) A value (mg)

11111 135.8 21111 52.9 1 120.0 max

11112 124.4 21112 54.1 2 46.5 min
11121 136.2 max 21121 67.7
11122 124.8 21122 68.8
11211 124.7 21211 41.8
11212 113.3 21212 42.9
Prostate
11221 125.1 21221 56.6
(mg)
11222 113.7 21222 57.7
12111 127.3 22111 44.4
12112 115.9 22112 45.5
12121 127.7 22121 59.2
12122 116.3 22122 60.3
12211 116.2 22211 33.3 min
12212 104.8 22212 34.3
12221 116.6 22221 48.0
12222 105.2 22222 49.2

Factor Population Factor Estimated

level mean level value
AIJ (ng/mL) AIJ (ng/mL)
Testosterone
N.S.
(ng/mL) 111 1.305 211 0.844
112 1.729 max 212 0.510
121 1.580 221 0.361 min
122 1.246 222 0.785
1
A: lighting condition, B: choline, C: niacin, D: pantothenic acid, E: vitamin B6, F: vitamin B1, G: vitamin B2, H: folic acid,
I: biotin, J: vitamin B12.
2
1: normal lighting or normal level, 2: constant darkness or high level.
3
max: maximum, min: minimum.
 Effects of Water-Soluble Vitamins on Gonadal Development in Rats with Disturbed Daily Rhythm
tate weight decreased when changing to a high-VB2
diet from a normal-VB2 diet in the N-groups (11.4-mg
decrease, Table 8), but it increased in response to this
change in the D-groups (1.1- or 1.2-mg increase, Table
8).
In Ex. 2, only the lighting condition was observed to
affect the prostate weight (Tables 6 and 8).
Serum testosterone concentration. In Ex. 1, there was
no effect of lighting condition or vitamins on serum tes-
tosterone concentration (Table 5).
In Ex. 2, the effects of lighting condition and the
interaction of lighting condition, biotin and VB12 on
the serum testosterone concentration were observed
(Tables 6 and 8). The highest value for testosterone con-
centration (1.729 ng/mL) was observed in rats main-
tained under normal lighting and a normal-biotin and
high-VB12 diet, and the lowest value (0.361 ng/mL)
was observed in rats maintained under constant dark-
ness and a high-biotin and normal-VB12 diet (Table
8). In the D-groups, the highest value for testosterone
concentration (0.844 mg/mL) was observed in rats on a
normal-biotin and normal-VB12 diet (Table 8).
The interaction of three factors, the lighting condi-
tion, biotin and VB12, showed that in the N-groups, the
testosterone concentration increased with the change
to a high-VB12 diet from a normal-VB12 diet when the
diet included normal biotin (from 1.305 to 1.729 ng/
mL, Table 8), but in the D-groups, the concentration
decreased as a result of this change (from 0.844 to
0.510 ng/mL, Table 8).
DISCUSSION
The purpose of this study was to clarify the most suit-
able quantities and ratios of nutrients necessary to sup-
port gonadal development under the case of disturbed
daily rhythm. We previously reported the requirements
for various amino acids, minerals and fat-soluble vita-
mins (5–10). In a previous study (9), we observed that a
low fat-soluble vitamin and high water-soluble vitamin
diet, 1/3.3 of normal content of fat-soluble vitamins
and three times the normal content of water-soluble
vitamins, mitigated the depression of gonadal develop-
ment in rats kept under constant darkness as a model of
disturbed daily rhythm. As a next step, we attempted to
clarify which vitamins from a selection of water-soluble
vitamins are useful for the mitigation of the depres-
sion of gonadal development. In this experiment, the
water-soluble vitamin content was six times the normal
content, because our goal was to enhance the effect of
water-soluble vitamins.
The composition of the AIN-93G diet for growing
rats and mice is calculated on the basis of obtaining the
optimal body weight (16). The protein and fat contents
in the AIN-93G diet are higher than those in the AIN-
93M diet for adult rats and mice, but the vitamin con-
tent of the AIN-93G diet is the same as that of the AIN-
93M diet. In this experiment, in the case of rats kept
under normal lighting, the body weight changed from
161.8 to 163.7 g (1.2% increase) (Table 7) when the
quantity of PA and VB6 in the diet increased to six times
237
the normal, and the body weight changed from 143.7
to 147.2 g (2.4% increase) (Table 7) when the quantity
of FA in the diet increased to six times the normal. These
values were not very large, but these results showed that
the diet with more PA, VB6 and FA than is included in
the AIN-93G diet was more desirable for the increase in
body weight in growing rats from 4 to 8 wk old.
As in our previous studies (6, 8–10), the body weight
gain decreased when rats were kept under constant
darkness in the present study. Moreover, dietary choline,
NA, PA, VB6 and VB2 had interactions with the light-
ing condition, which indicates that the amount of these
vitamins for body weight gain differs based on the light-
ing condition. It is interesting that the amount of vita-
min for body weight gain differs when constant dark-
ness is maintained. Further research to interpret this
result is needed.
In the present study the lighting condition and seven
vitamins influenced gonadal development. PA, VB6,
VB1 and VB2 had interactions with the lighting condi-
tion, while choline, NA and FA did not have interaction
with the lighting condition. Biotin and VB12, whether
their levels were normal or high, did not affect gonadal
development.
It has been reported that a PA-free diet reduces body
weight gain, gonadal weight and serum testosterone
concentration, which indicates that PA is necessary
to maintain gonadal function (17). It has also been
reported that a mild PA deficiency diet (1/100 of nor-
mal level) decreases the serum triglyceride level and free
fatty acid level after a 12-h fast (18). On the other hand,
in an experiment with excess PA, a 3% PA diet (approxi-
mately 3,000 times the normal PA content) induced
reductions of food intake and body weight, diarrhea
and enlarged lungs, but a 1% PA diet (approximately
600 times the normal content) did not induce these phe-
nomena (19). In the present study, the dietary PA level
was six times the normal content, a high but not exces-
sive level, and a high-PA diet increased gonadal weight
of rats in the D-groups but decreased gonadal weight
of rats in the N-groups. It is difficult to interpret the
present results, because there is currently no report of
any study examining the relation between PA and daily
rhythm, although there are reports about the relation
between PA and the gonads. The fact that a high-PA diet
increases gonadal weight in the D-groups shows that
the high-PA diet is effective in mitigation of the depres-
sion of gonadal development in rats kept under dis-
turbed daily rhythm. A study focused on PA metabolism
and daily rhythm is needed.
More VB2 was needed for gonadal development by
the D-groups than by the N-groups, but this effect was
shown on prostate weight only and not on all gonads.
Growth failure, hair loss, skin lesions, nervous system
abnormalities and impaired fertility have been reported
as symptoms of VB2 deficiency in experimental ani-
mals (20). VB2 has been demonstrated to participate
in gonadal development (20). On the other hand, it
has been reported that VB2 excess cannot be induced,
because there is an upper limit of absorption and accu-
238 HANAI M and ESASHI T
mulation of VB2 by the body’s organs (20). In the pres-
ent study, a high-VB2 diet increased prostate weight of
rats in the D-groups but decreased prostate weight of
rats in the N-groups. It is difficult to clear up the rea-
son for the results of the present study by the references
which have been reported about the relation between
VB2 and prostate, and VB2 and daily rhythm. However,
in the D-groups, the high-VB2 diet tended to decrease
the weight of the testes, epididymides and seminal ves-
icles (p⬍0.08, p⬍0.07 and p⬍0.09 respectively, data
not shown), which may account for the difference. As
a whole, we believe the results show that a normal-
VB2 diet is better than a high-VB2 diet for gonadal
development.
Our study showed that normal levels of VB1 and
VB6, not high levels of VB1 and VB6, were needed for
gonadal development when keeping rats under constant
darkness.
It was reported that a 1% VB1 diet (about 1,700
times the normal content) did not influence body weight
or testes weight (21). In the present study, the rats kept
under normal lighting showed no adverse effects from a
high-VB1 diet, which supported the results of the previ-
ous study. The rats kept under constant darkness, in con-
trast, showed adverse effects on the testes and epididymi-
des weights from a high-VB1 diet. In a study about the
relation between dietary VB1 and daily rhythm, a VB1
deficient diet induced a low body temperature and disap-
pearance of body temperature rhythm (22), but there is
no report about the relationship between gonadal devel-
opment, dietary VB1 and daily rhythm. The results of
the present study suggest that the metabolism of VB1
might have been changed in rats kept under constant
darkness, but it is difficult to draw certain conclusions.
Further study of this issue is needed.
A high-VB6 diet increased the testes weight of rats
in the N-groups but decreased it in the D-groups. This
result shows that there was an adverse effect of the
high-VB6 diet on the D-groups. An excess-VB6 diet
induced neurological damage in an animal experiment
(23). It was also reported that the administration of
VB6 at 250 or 500 mg/kg BW intraperitoneally induced
abnormal spermatogenesis and testis atrophy after 2 or
4 wk of treatment (24, 25). On the other hand, Fuku-
watari et al. reported that a 1% VB6 diet (about 1,400
times the normal level) decreased body weight but did
not influence testes weight of rats (26). In the present
study, the rats kept under normal lighting showed no
adverse effects from a high-VB6 diet, which supported
the results of the previous study. In contrast, the rats
kept under constant darkness showed adverse effects
on the testes from a high-VB6 diet. As with VB1, the
metabolism of VB6 might have been changed in rats
kept under constant darkness. Further study is required
to clarify the meaning of this finding.
Regardless of the lighting condition, choline, NA and
FA influenced seminal vesicle and prostate weights. A
high-choline and NA diet decreased prostate weight
and a high-FA diet increased seminal vesicle weight.
These vitamins did not influence testis or epididymis
weights. We suspect there may be a priority ranking
in gonadal organs; the testes and epididymides, which
are related to spermatogenesis and sperm maturation,
are more important than the accessory sexual organs,
the seminal vesicles and prostate. So it is possible that
the testes and epidydimides would be protected from a
changing dietary vitamin level.
The serum testosterone concentration was influenced
by biotin and VB12, which did not influence the gonadal
organs. The effects of biotin and VB12 on testosterone
were different based on the lighting condition, and also,
the interaction between biotin and VB12 was opposite
between the N-groups and D-groups. In our previous
study of water-soluble vitamin mixtures we reported
that the testosterone level was not influenced by water-
soluble vitamin mixture levels that were low (1/3.3 of
the normal content), normal or high (3 times the nor-
mal content) (9). In the present study we examined
water-soluble vitamins individually, and their levels in
the diets were normal or high (6 times the normal con-
tent), and we obtained more detailed results.
It has been reported that a biotin deficiency causes
the seminiferous tubules to atrophy and decreases the
serum testosterone concentration, and these symptoms
are obtained by biotin administration (27). It has also
been reported that VB12 deficiency causes testis histo-
logical alteration (28), but there is no report about the
relation between testosterone and VB12. Furthermore,
there is no report about the interaction between biotin
and VB12 on testosterone secretion. Therefore, it is dif-
ficult to discuss the results of interaction between biotin
and VB12. Earlier studies had shown that VB12 was
related to biological rhythm and was effective in correct-
ing sleep rhythm disorder (29), but a later study showed
that VB12 does not have this effect (30). The results of
the present study, showing that the effect of VB12 on
testosterone differed according to the lighting condition,
suggest that the absorption, excretion or metabolism of
VB12 would be changed by keeping rats under constant
darkness, or that a process related to testosterone secre-
tion as part of the metabolism in which VB12 partici-
pates would be changed by keeping rats under constant
darkness. Further study of this issue is required.
The present results showed that the depression of
gonadal development of rats kept under constant dark-
ness can be diminished by feeding a high-PA diet. On the
other hand, a high-VB1 and high-VB6 diet promoted the
depression of gonadal development, and the toxicities
of B1 and VB6 on gonadal development were shown.
Usually the toxicity of water-soluble vitamins does not
appear when they are given by oral ingestion, but under
the particular situation of our experiment, the toxicity
could be shown with a high level of sensitivity, which
indicates that the particular situation of vitamin inges-
tion should be considered when studying toxicity.
In this study, the serum vitamin concentrations and
the metabolic products of vitamins in serum or urine
were not measured, because the purpose of this study
was to accumulate data about the effects of a range of
vitamins on gonadal development, mainly by using fac-
 Effects of Water-Soluble Vitamins on Gonadal Development in Rats with Disturbed Daily Rhythm
tor analysis, as in previous studies (5–10). As the next
step, we will focus on the main vitamins that influenced
the gonadal development in rats kept under constant
darkness as a model of disturbed daily rhythm. We hope
to clarify how the function and metabolism of these
vitamins will be changed by the disturbed daily rhythm,
and how these vitamins will affect the gonadal develop-
ment at the cellular or the molecular levels.
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