**3.2 Vitamin D3 at different doses reverses anxiety-like profile of the middle-aged and old OVX and OVX rats given with 17β-estradiol after long-term absence of estrogen as measured in light: dark box**

Two-way ANOVA statistical test revealed a significant interaction between hormone condition and treatments ([F(5,44) = 11.52, *p* < 0.01) and [F(5,44) = 16.75, *p* < 0.05], respectively), with significant effects of hormone conditions ([F(5,44) = 19.22, *p* < 0.05 and [F(5,44) = 9.56, *p* < 0.001], respectively) and treatment ([F(5,44) = 9.88, *p* < 0.01] and [F(5,32) = 7.26, *p* < 0.05], respectively) in the time spent and number of entries in the light compartment of the middle-aged OVX rats. The *post-hoc* test demonstrated significant differences for these groups (*p* < 0.05).

Vitamin D3 supplementation at all doses did not alter the time spent and number of entries in the light compartment in the intact middle-aged rats as compared to the control rats (**Figure 3a, b**, *p* > 0.05). Following 12 weeks of post-ovariectomy period, the middle-aged female rats showed a profound decrease of the time spent and number of entries in the light compartment as compared to the control females (**Figure 3a, b**, *p* < 0.05). The 17β-E2 injection (0.5 μg/kg, s.c.) resulted in an increase of the time spent and number of entries in the light compartment in the middleaged OVX rats as compared to the middle-aged OVX rats administered with solvent (**Figure 3a, b**, *p* < 0.05), but did not reach the values of control rats (**Figure 3a, b**, *p* < 0.05).

Vitamin D3 treatment at dose of 5.0 mg/kg markedly elevated time spent and number of entries in the light compartment of the middle-aged OVX rats as compared to the middle-aged OVX rats given with solvent (**Figure 3a, b**, *p* > 0.05). Co-administration of vitamin D3 at dose of 5.0 mg/kg and 17β-E2 to the middle-aged OVX rats produced greater increase of time spent and number of entries in the light compartment than that of the middle-aged OVX rats given 17β-E2 or solvent (**Figure 3a, b**, *p* > 0.05). Vitamin D3 treatment at doses of 1.0 mg/kg and 2.5 mg/kg individually or plus 17β-E2 did not change time spent and number of entries in the light compartment of the middle-aged OVX as compared to the middle-aged OVX rats treated with 17β-E2 (**Figure 3a, b**, *p* > 0.05). The time spent and number of entries in the light compartment in the middle-aged OVX rats administered vitamin D3 at these doses in combination with 17β-E2 were lower than that for middle-aged control rats and were higher than for middle-aged OVX rats (**Figure 3a, b**, *p* > 0.05).

Two-way analysis of variance showed a significant hormone condition × treatment interaction ([F(5,44) = 11.52, *p* < 0.01] and [F(5,44) = 12.74, *p* < 0.05], respectively), significant effect of hormone conditions ([F(5,44) = 11.22, *p* < 0.05] and [F(5,44) = 11.56, *p* < 0.001], respectively) and significant effect for treatment ([F(5,44) = 10.08, *p* < 0.01] and [F(5,32) = 12.26, *p* < 0.05)], respectively) for time spent and number of entries in the light compartment of the old OVX rats. *Post-hoc* analyses revealed differences among the groups for the anxiety-like state during experimental sessions (*p* < 0.05).

**43**

**Figure 3.**

(**Figure 4a, b**, *p* > 0.05).

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age*

The old intact rats treated with vitamin D3 at doses of 1.0, 2.5 or 5.0 mg/kg failed to modify time spent and number of entries in the light compartment as compared to the old control rats (**Figure 4a, b**, *p* > 0.05). The old OVX rats given with solvent displayed a significant decrease of time spent and number of entries in the light compartment as compared to the old control rats (**Figure 4a, b**, *p* > 0.05). Vitamin D3 supplementation administered in all doses to the old OVX rats induced increase of the time spent and number of entries in the light compartment as compared to the old OVX rats treated with solvent (**Figure 4a, b**, *p* > 0.05). Combined administration of cholecalciferol at dose of 1.0 mg/kg with 17β-E2 to the old OVX rats more significantly increased time spent and number of entries in the light compartment as compared to the old OVX rats treated with solvent or 17β-E2 (**Figure 4a, b**, *p* > 0.05). The old OVX rats administered with vitamin D3 at doses of 2.5 mg/kg and 5.0 mg/kg plus 17β-E2 showed identical parameters of time spent and number of entries in the light compartment like as old OVX rats treated with 17β-E2

*administered dose of 17β-estradiol (17β-E2) was 0.5 μg/rat s.c., once daily, for 14 days.*

*Effects of cholecalciferol administration on anxiety-like behavior of the middle-aged OVX rats following longterm estrogen deficiency in the light/dark test. (a) Time spent in the light box, sec; (b) The number of entrances in the light box. The obtained results show the mean ± standard error of the mean (SEM). \*—p < 0.05 as compared to the control group of sham-operated rats, #—p < 0.05 as compared to the old OVX rats treated with solvent, ##—p < 0.05 as compared to the old OVX rats treated with 17β-estradiol. Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day s.c., once daily, for 14 days. The* 

**3.3 Vitamin D3 administration changes behavioral reactivity of the middle-aged** 

Accordingly to the two-way ANOVA test, there were significant differences for the grooming behavior between hormone conditions ([F(5,44) = 8.12, *p* < 0.05] and [F(5,44) = 9.22, *p* < 0.05], respectively) between drug treatment ([F(5,44) = 12.51,

**and old OVX and OVX rats treated with 17β-estradiol**

*DOI: http://dx.doi.org/10.5772/intechopen.82596*

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age DOI: http://dx.doi.org/10.5772/intechopen.82596*

#### **Figure 3.**

*Fads and Facts about Vitamin D*

Vitamin D3 supplementation administered in all doses to the old OVX rats resulted in increase of the time spent into the open arms and the number of entries into the open arms as compared to the old OVX rats treated with solvent (**Figure 2a, b**, *p* > 0.05). Combined administration of cholecalciferol at dose of 1.0 mg/kg with 17β-E2 to the old OVX rats more significantly increased the time spent into the open arms and the number of entries into the open arms as compared to the old OVX rats treated with solvent or 17β-E2 (**Figure 2a, b**, *p* > 0.05). The old OVX rats administered with cholecalciferol at doses of 2.5 mg/kg and 5.0 mg/kg plus 17β-E2 showed similar values of the time spent into the open arms and the number of entries into the open arms like as old OVX rats treated with 17β-E2 (**Figure 2a, b**, *p* > 0.05).

**3.2 Vitamin D3 at different doses reverses anxiety-like profile of the middle-aged and old OVX and OVX rats given with 17β-estradiol after long-term absence** 

Two-way ANOVA statistical test revealed a significant interaction between hormone condition and treatments ([F(5,44) = 11.52, *p* < 0.01) and [F(5,44) = 16.75,

([F(5,44) = 19.22, *p* < 0.05 and [F(5,44) = 9.56, *p* < 0.001], respectively) and treatment ([F(5,44) = 9.88, *p* < 0.01] and [F(5,32) = 7.26, *p* < 0.05], respectively) in the time spent and number of entries in the light compartment of the middle-aged OVX rats. The *post-hoc* test demonstrated significant differences for these groups (*p* < 0.05). Vitamin D3 supplementation at all doses did not alter the time spent and number of entries in the light compartment in the intact middle-aged rats as compared to the control rats (**Figure 3a, b**, *p* > 0.05). Following 12 weeks of post-ovariectomy period, the middle-aged female rats showed a profound decrease of the time spent and number of entries in the light compartment as compared to the control females (**Figure 3a, b**, *p* < 0.05). The 17β-E2 injection (0.5 μg/kg, s.c.) resulted in an increase of the time spent and number of entries in the light compartment in the middleaged OVX rats as compared to the middle-aged OVX rats administered with solvent (**Figure 3a, b**, *p* < 0.05), but did not reach the values of control rats (**Figure 3a, b**,

Vitamin D3 treatment at dose of 5.0 mg/kg markedly elevated time spent and number of entries in the light compartment of the middle-aged OVX rats as compared to the middle-aged OVX rats given with solvent (**Figure 3a, b**, *p* > 0.05). Co-administration of vitamin D3 at dose of 5.0 mg/kg and 17β-E2 to the middle-aged OVX rats produced greater increase of time spent and number of entries in the light compartment than that of the middle-aged OVX rats given 17β-E2 or solvent (**Figure 3a, b**, *p* > 0.05). Vitamin D3 treatment at doses of 1.0 mg/kg and 2.5 mg/kg individually or plus 17β-E2 did not change time spent and number of entries in the light compartment of the middle-aged OVX as compared to the middle-aged OVX rats treated with 17β-E2 (**Figure 3a, b**, *p* > 0.05). The time spent and number of entries in the light compartment in the middle-aged OVX rats administered vitamin D3 at these doses in combination with 17β-E2 were lower than that for middle-aged control rats

and were higher than for middle-aged OVX rats (**Figure 3a, b**, *p* > 0.05).

ment interaction ([F(5,44) = 11.52, *p* < 0.01] and [F(5,44) = 12.74, *p* < 0.05], respectively), significant effect of hormone conditions ([F(5,44) = 11.22, *p* < 0.05] and [F(5,44) = 11.56, *p* < 0.001], respectively) and significant effect for treatment ([F(5,44) = 10.08, *p* < 0.01] and [F(5,32) = 12.26, *p* < 0.05)], respectively) for time spent and number of entries in the light compartment of the old OVX rats. *Post-hoc* analyses revealed differences among the groups for the anxiety-like state during

Two-way analysis of variance showed a significant hormone condition × treat-

*p* < 0.05], respectively), with significant effects of hormone conditions

**of estrogen as measured in light: dark box**

**42**

experimental sessions (*p* < 0.05).

*p* < 0.05).

*Effects of cholecalciferol administration on anxiety-like behavior of the middle-aged OVX rats following longterm estrogen deficiency in the light/dark test. (a) Time spent in the light box, sec; (b) The number of entrances in the light box. The obtained results show the mean ± standard error of the mean (SEM). \*—p < 0.05 as compared to the control group of sham-operated rats, #—p < 0.05 as compared to the old OVX rats treated with solvent, ##—p < 0.05 as compared to the old OVX rats treated with 17β-estradiol. Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day s.c., once daily, for 14 days. The administered dose of 17β-estradiol (17β-E2) was 0.5 μg/rat s.c., once daily, for 14 days.*

The old intact rats treated with vitamin D3 at doses of 1.0, 2.5 or 5.0 mg/kg failed to modify time spent and number of entries in the light compartment as compared to the old control rats (**Figure 4a, b**, *p* > 0.05). The old OVX rats given with solvent displayed a significant decrease of time spent and number of entries in the light compartment as compared to the old control rats (**Figure 4a, b**, *p* > 0.05).

Vitamin D3 supplementation administered in all doses to the old OVX rats induced increase of the time spent and number of entries in the light compartment as compared to the old OVX rats treated with solvent (**Figure 4a, b**, *p* > 0.05). Combined administration of cholecalciferol at dose of 1.0 mg/kg with 17β-E2 to the old OVX rats more significantly increased time spent and number of entries in the light compartment as compared to the old OVX rats treated with solvent or 17β-E2 (**Figure 4a, b**, *p* > 0.05). The old OVX rats administered with vitamin D3 at doses of 2.5 mg/kg and 5.0 mg/kg plus 17β-E2 showed identical parameters of time spent and number of entries in the light compartment like as old OVX rats treated with 17β-E2 (**Figure 4a, b**, *p* > 0.05).

## **3.3 Vitamin D3 administration changes behavioral reactivity of the middle-aged and old OVX and OVX rats treated with 17β-estradiol**

Accordingly to the two-way ANOVA test, there were significant differences for the grooming behavior between hormone conditions ([F(5,44) = 8.12, *p* < 0.05] and [F(5,44) = 9.22, *p* < 0.05], respectively) between drug treatment ([F(5,44) = 12.51,

#### **Figure 4.**

*Effects of cholecalciferol administration on anxiety-like behavior of the old OVX rats following long-term estrogen deficiency in the light/dark test. (a) Time spent in the light box, sec; (b) The number of entrances in the light box. The obtained results show the mean ± standard error of the mean (SEM). \*—p < 0.05 as compared to the control group of sham-operated rats, #—p < 0.05 as compared to the old OVX rats treated with solvent, ##—p < 0.05 as compared to the old OVX rats treated with 17β-estradiol. Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day s.c., once daily, for 14 days. The administered dose of 17β-estradiol (17β-E2) was 0.5 μg/rat s.c., once daily, for 14 days.*

*p* < 0.01] and [F(5,44) = 12.56, *p* < 0.01], respectively) and an interaction between hormone condition and treatments ([F(5,44) = 7.16, *p* < 0.01] and [F(5,44) = 9.26, *p* < 0.01], respectively) in the middle-aged and old OVX rats. Further *post-hoc* test revealed differences for grooming between experimental groups of the OVX rats with different age (*p* < 0.05).

Vitamin D3 injected at several doses failed to demonstrate any changes of behavioral reactivity of the middle-aged and old intact females in the OFT as compared to the middle-aged control rats (**Tables 1** and **2**, *p* > 0.05).

A significant decrease of grooming behavior was registered in the middle-aged and old OVX rats given with solvent as compared to the control (**Tables 1** and **2**, *p* < 0.05). 17β-E2 significantly reduced grooming reactions in the middle-aged and old OVX rats as compared to the middle-aged OVX rats (**Tables 1** and **2**, *p* < 0.05). The middle-aged and old OVX rats treated with vitamin D3 in all tested doses alone or in a combination with 17β-E2 did not demonstrate any modifications of motor and rearing activities as compared to the middle-aged OVX rats given with solvent (**Tables 1** and **2**, *p* < 0.05).

However, the middle-aged and old OVX rats treated with vitamin D3 at doses of 1.0, 2.5 and 5.0 mg/kg demonstrated an increase of grooming behavior as compared to the middle-aged OVX rats. A co-administration of vitamin D3 at these doses with 17β-E2 decreased grooming behavior as compared to both middle-aged, as well as old intact and OVX rats received with solvent or 17β-E2 (**Tables 1** and **2**, *p* < 0.05).

**45**

**Table 1.**

*\**

*#*

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age*

**OVX and OVX females treated with 17β-estradiol**

**3.4 Modifications of 25-hydroxyvitamin D3, estradiol and calcium levels in the blood serum following vitamin D3 administration in the middle-aged and old** 

The middle-aged intact rats treated with cholecalciferol at doses of 1.0, 2.5 and 5.0 mg/kg increased 25-OH-VD3 levels (**Figure 5**, *p* < 0.05) and failed to alter estradiol levels in the serum blood as compared to the control rats (**Figure 6**, *p* > 0.05). Long-term ovariectomy in the middle-aged female rats resulted in a significant decrease of estradiol and 25-OH-VD3 levels in the blood as compared to the middleaged control females (**Figures 5** and **6**, *p* < 0.05). The 17β-E2 supplementation (0.5 μg/kg, SC) failed to modify 25-OH-VD3 levels in the blood of the middleaged OVX rats as compared to the middle-aged OVX rats administered with solvent (**Figure 5**, *p* > 0.05), and the value of this parameter in the middle-aged OVX/17β-E2 females were lower than that of the value of middle-aged control rats. However, 17β-E2 supplementation significantly increased estradiol levels in the blood of the middle-aged OVX rats as compared to the middle-aged OVX rats given

The middle-aged OVX rats treated with cholecalciferol at all tested doses significantly increased estradiol levels in the serum blood as compared to the middle-aged OVX rats treated with solvent (**Figure 6**, *p* < 0.05). However, the value of estradiol levels in the middle-aged OVX rats treated with cholecalciferol at these doses were lower than that of the value of middle-aged control rats. The middle-aged OVX rats treated with cholecalciferol at doses of 2.5 and 5.0 mg/kg significantly increased 25-OH-VD3 levels in the serum blood as compared to the middle-aged OVX rats

**Groups Crossing Rearing Grooming** Middle-aged control rats + solvent 73.3 ± 4.2 12.1 ± 0.8 3.0 ± 0.2 Middle-aged intact rats + cholecalciferol 1.0 mg/kg 68.0 ± 2.4 12.0 ± .05 3.2 ± 0.2 Middle-aged intact rats + cholecalciferol 2.5 mg/kg 59.3 ± 5.6 10.5 ± 0.8 3.2 ± 0.2 Middle-aged intact rats + cholecalciferol 5.0 mg/kg 69.5 ± 4.2 11.7 ± 0.8 3.5 ± 0.2 Middle-aged OVX rats + solvent (OVX/solvent rats) 62.4 ± 2.3 12.3 ± 0.6 1.2 ± 0.5\* Middle-aged OVX rats +17β-E2 (OVX/17β-E2 rats) 60.3 ± 2.6 13.2 ± 0.3 3.3 ± 0.4# Middle-aged OVX rats + cholecalciferol 1.0 mg/kg 65.2 ± 2.5 12.1 ± 0.6 4.0 ± 0.2# Middle-aged OVX rats + cholecalciferol 2.5 mg/kg 76.9 ± 4.2 13.2 ± 0.8 3.8 ± 0.4# Middle-aged OVX rats + cholecalciferol 5.0 mg/kg 72.3 ± 4.4 11.0 ± 0.5 4.1 ± 0.6#

*The obtained results show the mean ± S.E.M. Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day, s.c., once daily, for 14 days. 17β-Estradiol (17β-E2) was given at 0.5 μg/rat, s.c.,* 

*Cholecalciferol influences on behavioral parameters of the middle-aged OVX rats following long-term estrogen* 

62.1 ± 2.8 12.6 ± 0.6 0.6 ± 0.2\*,#,##

73.2 ± 2.4 10.2 ± 0.4 0.5 ± 0.2\*,#,##

65.4 ± 5.6 12.5 ± 0.8 0.8 ± 0.2\*,#,##

*DOI: http://dx.doi.org/10.5772/intechopen.82596*

with solvent (**Figure 6**, *p* < 0.05).

Middle-aged OVX rats + cholecalciferol 1.0 mg/

Middle-aged OVX rats + cholecalciferol 2.5 mg/

Middle-aged OVX rats + cholecalciferol 5.0 mg/

*p < 0.05 as compared to the control group of the old sham-operated rats.*

*p < 0.05 as compared to the old OVX rats treated with solvent. ##p < 0.05 as compared to the old OVX rats treated with 17β-estradiol.*

kg + 17β-E2

kg + 17β-E2

kg + 17β-E2

*once daily, during 14 days.*

*deficiency in the open field test for 5 min.*
