**3.1 Vitamin D3 in different doses decreases 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 the EPM test**

For vitamin D3 supplementation, two-way ANOVA analysis revealed a significant interaction between hormone condition and treatments ([F(5,44) = 12.83, *p* < 0.05] and [F(5,44) = 9.47, *p* < 0.01], respectively), with significant effects of hormone conditions ([F(5,44) = 9.47, *p* < 0.01 and [F(5,44) = 7.88, *p* < 0.01], respectively) and treatment ([F(5,44) = 15.24, *p* < 0.05] and [F(5,44) = 11.02, *p* < 0.05], respectively) in the time spent into the open arms or the number of entries into the open arms of the middle-aged OVX rats. The *post-hoc* test demonstrated significant differences for these groups (*p* < 0.05).

Vitamin D3 application at all tested doses did not modify the time spent into the open arms and the number of entries into the open arms in the intact middle-aged rats as compared to the control rats (**Figure 1a, b**, *p* > 0.05). Following 12 weeks of post-surgery period, the middle-aged female rats showed a profound decrease of the time spent in the open arms and the number of entries into the open arms as compared to the control females (**Figure 1a, b**, *p* < 0.05). The 17β-E2 injection (0.5 μg/kg, s.c.) resulted in an increase of the time spent in the open arms and the number of entries into the open arms in the middle-aged OVX rats as compared to the middle-aged OVX rats administered with solvent (**Figure 1a, b**, *p* < 0.05), but did not reach the values of control rats (**Figure 1a, b**, *p* < 0.05).

Vitamin D3 treatment at dose of 5.0 mg/kg significantly increased the time spent in the open arms and the number of entries into the open arms of the middle-aged OVX rats as compared to the middle-aged OVX rats given with solvent (**Figure 1a, 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 a more greater increase of the time spent in the open arms and the number of entries into the open arms than that of the middle-aged OVX rats given 17β-E2 or solvent (**Figure 1a, b**, *p* > 0.05). However, vitamin D3 treatment at doses of 1.0 and 2.5 mg/kg individually or plus 17β-E2 failed to alter the time spent in the open arms and the number of entries

#### **Figure 1.**

*Effects of cholecalciferol administration on anxiety-like behavior of the middle-aged ovariectomized (OVX) rats following long-term estrogen deficiency in the elevated plus maze. (a) Time spent into the open arms, sec; (b) The number of entries into the open arms. The obtained results show the mean ± standard error of the mean (SEM). \*—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 (17β-E2). Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.), once daily, for 14 days. The administered dose of 17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days.*

**41**

**Figure 2.**

*0.5 μg/rat s.c., once daily, for 14 days.*

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

into the open arms of the middle-aged OVX as compared to the middle-aged OVX rats treated with 17β-E2 **Figure 1a, b**, *p* > 0.05). The time spent in the open arms and the number of entries into the open arms in the middle-aged OVX rats administered with 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

Two-way ANOVA test demonstrated a significant hormone condition × treatment interaction ([F(5,44) = 11.44, *p* < 0.05] and [F(5,44) = 11.12, *p* < 0.01], respectively), significant effect of hormone conditions ([F(5,44) = 9.22, *p* < 0.01] and [F(5,44) = 16.88, *p* < 0.01], respectively) and significant effect for treatment ([F(5,44) = 11.56, *p* < 0.05], and [F(5,44) = 12.56, *p* < 0.05], respectively) for the time spent into the open arms or the number of entries into the open arms of the old OVX rats. *Post-hoc* analyses revealed differences among the groups for the anxiety-

The old intact rats treated with vitamin D3 at doses of 1.0, 2.5 or 5.0 mg/kg failed to alter the time spent into the open arms and the number of entries into the open arms as compared to the old control rats (**Figure 2a, b**, *p* > 0.05). The old OVX rats given with solvent displayed a significant decrease of the time spent into the open arms and the number of entries into the open arms as compared to the old control

*Effects of cholecalciferol administration on anxiety-like behavior of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the elevated plus maze. (a) Time spent into the open arms, sec; (b) The number of entries into the open arms. The obtained results show the mean ± standard error of the mean (SEM). \*—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 (17β-E2). Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.), once daily, for 14 days. The administered dose of 17β-estradiol was* 

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

like state during experimental sessions (*p* < 0.05).

(**Figure 1a, b**, *p* > 0.05).

rats (**Figure 2a, b**, *p* > 0.05).

into the open arms of the middle-aged OVX as compared to the middle-aged OVX rats treated with 17β-E2 **Figure 1a, b**, *p* > 0.05). The time spent in the open arms and the number of entries into the open arms in the middle-aged OVX rats administered with 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 1a, b**, *p* > 0.05).

Two-way ANOVA test demonstrated a significant hormone condition × treatment interaction ([F(5,44) = 11.44, *p* < 0.05] and [F(5,44) = 11.12, *p* < 0.01], respectively), significant effect of hormone conditions ([F(5,44) = 9.22, *p* < 0.01] and [F(5,44) = 16.88, *p* < 0.01], respectively) and significant effect for treatment ([F(5,44) = 11.56, *p* < 0.05], and [F(5,44) = 12.56, *p* < 0.05], respectively) for the time spent into the open arms or the number of entries into the open arms of the old OVX rats. *Post-hoc* analyses revealed differences among the groups for the anxietylike state during experimental sessions (*p* < 0.05).

The old intact rats treated with vitamin D3 at doses of 1.0, 2.5 or 5.0 mg/kg failed to alter the time spent into the open arms and the number of entries into the open arms as compared to the old control rats (**Figure 2a, b**, *p* > 0.05). The old OVX rats given with solvent displayed a significant decrease of the time spent into the open arms and the number of entries into the open arms as compared to the old control rats (**Figure 2a, b**, *p* > 0.05).

#### **Figure 2.**

*Fads and Facts about Vitamin D*

Vitamin D3 application at all tested doses did not modify the time spent into the open arms and the number of entries into the open arms in the intact middle-aged rats as compared to the control rats (**Figure 1a, b**, *p* > 0.05). Following 12 weeks of post-surgery period, the middle-aged female rats showed a profound decrease of the time spent in the open arms and the number of entries into the open arms as compared to the control females (**Figure 1a, b**, *p* < 0.05). The 17β-E2 injection (0.5 μg/kg, s.c.) resulted in an increase of the time spent in the open arms and the number of entries into the open arms in the middle-aged OVX rats as compared to the middle-aged OVX rats administered with solvent (**Figure 1a, b**, *p* < 0.05), but

Vitamin D3 treatment at dose of 5.0 mg/kg significantly increased the time spent in the open arms and the number of entries into the open arms of the middle-aged OVX rats as compared to the middle-aged OVX rats given with solvent (**Figure 1a, 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 a more greater increase of the time spent in the open arms and the number of entries into the open arms than that of the middle-aged OVX rats given 17β-E2 or solvent (**Figure 1a, b**, *p* > 0.05). However, vitamin D3 treatment at doses of 1.0 and 2.5 mg/kg individually or plus 17β-E2 failed to alter the time spent in the open arms and the number of entries

*Effects of cholecalciferol administration on anxiety-like behavior of the middle-aged ovariectomized (OVX) rats following long-term estrogen deficiency in the elevated plus maze. (a) Time spent into the open arms, sec; (b) The number of entries into the open arms. The obtained results show the mean ± standard error of the mean (SEM). \*—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 (17β-E2). Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.), once daily, for 14 days. The administered dose of 17β-estradiol was* 

did not reach the values of control rats (**Figure 1a, b**, *p* < 0.05).

**40**

**Figure 1.**

*0.5 μg/rat s.c., once daily, for 14 days.*

*Effects of cholecalciferol administration on anxiety-like behavior of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the elevated plus maze. (a) Time spent into the open arms, sec; (b) The number of entries into the open arms. The obtained results show the mean ± standard error of the mean (SEM). \*—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 (17β-E2). Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.), once daily, for 14 days. The administered dose of 17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days.*

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).
