**1. Introduction**

Anxiety disorders are twice common in women than in men and the risk increased more during the menopausal transition [1, 2]. Menopause indicates the termination of a woman's reproductive life. It is defined as the permanent cessation of menstruation induced by the loss of ovarian follicular activity [2, 3]. Menopause represents an important stage in female lives, which is often associated with a plethora of complaints and sufferings. Mood disturbances, especially anxiety and depression, are commonly associated with menopause [3, 4]. For women, data suggest that estrogens are strongly implicated in the regulation of mood and behavior, as well as in the pathophysiology of mood disorders [5–7]. Nowadays, there is a strong tendency to increase the duration of living in the whole world, however, the median age of programmed termination of a woman's reproductive life failed to alter [5–7]. Thus, majority of women in the aging population lives significant period of their lifetime in a postmenopausal state which is clearly associated with very low

estrogen levels that could be one of the marked trigger factors for development of affective-related disorders [5, 7].

A strategy to alleviate the mood disorders associated with menopause is hormonal replacement therapy (HRT) [8]. However, controversial results related to the effectiveness of such treatment have been frequently reported [9]. These discrepancies could be associated to various factors, one of them being the time when estrogen restitution is initiated after the beginning of menopause [10, 11].

There is a growing interest about the potential of diet and nutrients to improve the mental health of the women population and for the treatment of psychiatric disorders [10, 11]. In the case of mood disorders, the limitations of psychotropic drugs to achieve adequate rates of clinical remission and functional recovery have promoted the search for complementary approaches [12–15]. Menopausal women are now choosing to take alternate and complementary therapies marketed as "natural" treatments that offer the positive health effects of estrogens without the unwanted side effects [12, 13]. Among other nutraceuticals, one of such "natural" substances for treatment of affective-related diseases could be vitamin D (VD) [14, 15].

VD is a neuroactive secosteroid with well-known skeletal physiological role and diverse "non-skeletal" functions in the human body [16, 17]. "Non-skeletal" functions of VD are connected with its different range of outcomes in the central nervous system, such as neuroplasticity, apoptosis, cell proliferation and differentiation [18, 19]. All functions of VD in the body (classical functions, i.e., effect upon calcium-phosphate management and the non-classical ones) are imposed by the nuclear VD receptor (VDR), regulating directly the gene expression [20, 21]. Nuclear VDR are member of receptors family for transcription factors which are activated by numerous ligands [20, 21]. VDR are present in most tissues and cells in the body, and within the brain it shows some specificity to the prefrontal cortex, hippocampus, cingulate gyrus, thalamus, hypothalamus and substantia nigra [22]. This is of relevance as many of those brain regions have been implicated in the physiology of affective-related disorders.

Estrogen deficiency effects on affective-related behavior are restricted to certain periods of age after ovary removal [23, 24]. Preclinical data suggest that onset age of menopause can be important to obtain behavioral positive or negative results. Thus, it is of great interest to evaluate the effects of repeated cholecalciferol administration on anxiety-related behavior in the middle-aged and old female rats with long-term estrogen deficiency.

The aim of the present study was to determine if repeated systemic treatment with cholecalciferol affects anxiety-like behavior in the middle-aged and old female rats after long-term ovariectomy.

### **2. Materials and methods**

#### **2.1 Animals**

Female albino Wistar rats (12–14 months, middle-aged rats or 16–18 months, old rats, weighing 230–240 or 260–270 g, respectively) from the special biocollection of Koltushi vivarium (St. Petersburg, Russia) were used in the present study. All rats were allocated in groups and were allowed to accommodate for 1 week in the animal house at I.P. Pavlov Institute of Physiology, of the Russian Academy of Sciences, before subjecting them to behavioral testing and pharmacological treatments. They were provided with a standard pellet diet and were given water ad libitum. The

**37**

**2.4 Animal groups**

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

animals were kept at a temperature of 23 ± 2°C and a 12 h light/dark cycle as well as a constant relative humidity (50 ± 10%) during all experimental sessions. Female rats of different age were randomly separated into experimental groups accordingly

Vitamin D3 and 17β-E2 treatments, as well as anxiety-related tests were carried out by double-blind method by using rules of the Health guide for the care and use of Laboratory animals (1978) formulated by the National Institute of Health. Females of different age were placed in the special room for behavioral trials at least 1 h prior to the beginning of the experimental sessions which were performed from 09:00 am to 12:00 am. The experimental protocols of this study were approved by the Institutional Animal Ethics Committee of I.P. Pavlov Institute of Physiology,

Long-term ovariectomy surgery was performed as previously described [25]. Briefly, middle-aged and old female rats were anesthetized with ketamine (70 mg/ kg b.w.) mixed with xylazine (10 mg/kg b.w.). To avoid inflammation, the rats were administered with meloxicam (1 mg/kg b.w.). The fallopian tube was crushed and the ovary was removed by cutting. The effectiveness of long-term ovariectomy or 17β-estradiol (17β-E2) application was assessed by vaginal smears. The ovariectomized (OVX) females of different age were housed in groups of five in cages separated by groups. To assure the long-term absence of estrogens, all rats after

17β-estradiol, 17β-E2 (Sigma, USA) at low dose of 5.0 μg/rat [7, 26] and vitamin D3 as cholecalcirefol (Sigma, USA) at several doses (1.0, 2.5 or 5.0 mg/kg) [27] were subcutaneously (s.c.) administered once daily starting 14 days prior to the cognitive experiments. 17β-E2 was dissolved in sterile sesame oil, VD3was dissolved in 95% ethanol solvent, aliquoted and stored at −80°C. The stock of VD3 was dissolved in sterile water, resulting in a solution of cholecalciferol with 2% ethanol. All drug solutions were freshly prepared before each behavioral testing. 17β-E2 and cholecalcirefol were injected in a volume of 0.1 ml. The middle-aged OVX females were

The estrogen, 17β-E2 (E-8875, Sigma Chemical Co., St. Louis, MO, USA) was dissolved in sterile sesame oil. Cholecalcirefol (C-9756, Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 95% ethanol, aliquoted and stored at −80°C. The stock of cholecalciferol was diluted in sterile water, resulting in a solution of cholecalciferol with

The low dose of 17β-E2 (5.0 μg/rat subcutaneously, s.c.) was chosen from the studies performed by Estrada-Camarena and co-workers [26, 28]. Three doses of cholecalciferol (1.0, 2.5 or 5.0 mg/kg, s.c.) were chosen from the behavioral study performed by Idrus and co-workers [27]. All solutions were freshly prepared before each experimental series. All preparations were administered in a volume of 0.1 ml. Following 12 weeks after ovariectomy, cholecalciferol, 17β-E2 and oil solvent were injected once daily for 14 days.

Female rats (middle-aged or old, intact and OVX) were randomly divided into

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

to their age, including the control groups.

Russia (protocol 1095/1 from June 25, 2012).

surgery were remained to the housing facilities for 12 weeks.

15.5–17.5 months middle-aged at the onset for drug treatments.

24 groups, accordingly to their age, with 8 rats in each group.

2% ethanol. 17β-E2 was injected subcutaneously (s.c. at a dose of 0.5 μg/rat).

**2.2 Surgery**

**2.3 Drug treatments**

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

animals were kept at a temperature of 23 ± 2°C and a 12 h light/dark cycle as well as a constant relative humidity (50 ± 10%) during all experimental sessions. Female rats of different age were randomly separated into experimental groups accordingly to their age, including the control groups.

Vitamin D3 and 17β-E2 treatments, as well as anxiety-related tests were carried out by double-blind method by using rules of the Health guide for the care and use of Laboratory animals (1978) formulated by the National Institute of Health. Females of different age were placed in the special room for behavioral trials at least 1 h prior to the beginning of the experimental sessions which were performed from 09:00 am to 12:00 am. The experimental protocols of this study were approved by the Institutional Animal Ethics Committee of I.P. Pavlov Institute of Physiology, Russia (protocol 1095/1 from June 25, 2012).

#### **2.2 Surgery**

*Fads and Facts about Vitamin D*

affective-related disorders [5, 7].

could be vitamin D (VD) [14, 15].

physiology of affective-related disorders.

long-term estrogen deficiency.

rats after long-term ovariectomy.

**2. Materials and methods**

**2.1 Animals**

estrogen levels that could be one of the marked trigger factors for development of

A strategy to alleviate the mood disorders associated with menopause is hormonal replacement therapy (HRT) [8]. However, controversial results related to the effectiveness of such treatment have been frequently reported [9]. These discrepancies could be associated to various factors, one of them being the time when estro-

There is a growing interest about the potential of diet and nutrients to improve the mental health of the women population and for the treatment of psychiatric disorders [10, 11]. In the case of mood disorders, the limitations of psychotropic drugs to achieve adequate rates of clinical remission and functional recovery have promoted the search for complementary approaches [12–15]. Menopausal women are now choosing to take alternate and complementary therapies marketed as "natural" treatments that offer the positive health effects of estrogens without the unwanted side effects [12, 13]. Among other nutraceuticals, one of such "natural" substances for treatment of affective-related diseases

VD is a neuroactive secosteroid with well-known skeletal physiological role and diverse "non-skeletal" functions in the human body [16, 17]. "Non-skeletal" functions of VD are connected with its different range of outcomes in the central nervous system, such as neuroplasticity, apoptosis, cell proliferation and differentiation [18, 19]. All functions of VD in the body (classical functions, i.e., effect upon calcium-phosphate management and the non-classical ones) are imposed by the nuclear VD receptor (VDR), regulating directly the gene expression [20, 21]. Nuclear VDR are member of receptors family for transcription factors which are activated by numerous ligands [20, 21]. VDR are present in most tissues and cells in the body, and within the brain it shows some specificity to the prefrontal cortex, hippocampus, cingulate gyrus, thalamus, hypothalamus and substantia nigra [22]. This is of relevance as many of those brain regions have been implicated in the

Estrogen deficiency effects on affective-related behavior are restricted to certain periods of age after ovary removal [23, 24]. Preclinical data suggest that onset age of menopause can be important to obtain behavioral positive or negative results. Thus, it is of great interest to evaluate the effects of repeated cholecalciferol administration on anxiety-related behavior in the middle-aged and old female rats with

The aim of the present study was to determine if repeated systemic treatment with cholecalciferol affects anxiety-like behavior in the middle-aged and old female

Female albino Wistar rats (12–14 months, middle-aged rats or 16–18 months, old rats, weighing 230–240 or 260–270 g, respectively) from the special biocollection of Koltushi vivarium (St. Petersburg, Russia) were used in the present study. All rats were allocated in groups and were allowed to accommodate for 1 week in the animal house at I.P. Pavlov Institute of Physiology, of the Russian Academy of Sciences, before subjecting them to behavioral testing and pharmacological treatments. They were provided with a standard pellet diet and were given water ad libitum. The

gen restitution is initiated after the beginning of menopause [10, 11].

**36**

Long-term ovariectomy surgery was performed as previously described [25]. Briefly, middle-aged and old female rats were anesthetized with ketamine (70 mg/ kg b.w.) mixed with xylazine (10 mg/kg b.w.). To avoid inflammation, the rats were administered with meloxicam (1 mg/kg b.w.). The fallopian tube was crushed and the ovary was removed by cutting. The effectiveness of long-term ovariectomy or 17β-estradiol (17β-E2) application was assessed by vaginal smears. The ovariectomized (OVX) females of different age were housed in groups of five in cages separated by groups. To assure the long-term absence of estrogens, all rats after surgery were remained to the housing facilities for 12 weeks.

#### **2.3 Drug treatments**

17β-estradiol, 17β-E2 (Sigma, USA) at low dose of 5.0 μg/rat [7, 26] and vitamin D3 as cholecalcirefol (Sigma, USA) at several doses (1.0, 2.5 or 5.0 mg/kg) [27] were subcutaneously (s.c.) administered once daily starting 14 days prior to the cognitive experiments. 17β-E2 was dissolved in sterile sesame oil, VD3was dissolved in 95% ethanol solvent, aliquoted and stored at −80°C. The stock of VD3 was dissolved in sterile water, resulting in a solution of cholecalciferol with 2% ethanol. All drug solutions were freshly prepared before each behavioral testing. 17β-E2 and cholecalcirefol were injected in a volume of 0.1 ml. The middle-aged OVX females were 15.5–17.5 months middle-aged at the onset for drug treatments.

The estrogen, 17β-E2 (E-8875, Sigma Chemical Co., St. Louis, MO, USA) was dissolved in sterile sesame oil. Cholecalcirefol (C-9756, Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 95% ethanol, aliquoted and stored at −80°C. The stock of cholecalciferol was diluted in sterile water, resulting in a solution of cholecalciferol with 2% ethanol. 17β-E2 was injected subcutaneously (s.c. at a dose of 0.5 μg/rat). The low dose of 17β-E2 (5.0 μg/rat subcutaneously, s.c.) was chosen from the studies performed by Estrada-Camarena and co-workers [26, 28]. Three doses of cholecalciferol (1.0, 2.5 or 5.0 mg/kg, s.c.) were chosen from the behavioral study performed by Idrus and co-workers [27]. All solutions were freshly prepared before each experimental series. All preparations were administered in a volume of 0.1 ml. Following 12 weeks after ovariectomy, cholecalciferol, 17β-E2 and oil solvent were injected once daily for 14 days.

#### **2.4 Animal groups**

Female rats (middle-aged or old, intact and OVX) were randomly divided into 24 groups, accordingly to their age, with 8 rats in each group.

The following experimental groups for the middle-aged and old female rats were created in the present study:

1 and 2—middle-aged or old intact female rats + solvent.

3 and 4—middle-aged or old intact female rats + cholecalciferol 1.0 mg/kg (middle-aged or old intact − vitamin D3 1.0).

5 and 6—middle-aged or old intact female rats + cholecalciferol 2.5 mg/kg (middle-aged or old intact − vitamin D3 2.5).

7 and 8—middle-aged or old intact female rats + cholecalciferol 5.0 mg/kg (middle-aged or old intact − vitamin D3 5.0).

9 and 10—middle-aged or old OVX + solvent (middle-aged or old OVX − Sol).

11 and 12—middle-aged or old OVX rats + 17β-E2 (middle-aged or old OVX − 17β-E2). 13 and 14—middle-aged or old OVX rats + cholecalciferol 1.0 mg/kg (middleaged or old OVX − vitamin D3 1.0).

15 and 16—middle-aged or old OVX rats + cholecalciferol 2.5 mg/kg (middleaged or old OVX − vitamin D3 2.5).

17 and 18—middle-aged or old OVX rats + vitamin D3 5.0 mg/kg (middle-aged or old OVX − vitamin D3 5.0).

19 and 20—middle-aged or old OVX rats + cholecalciferol 1.0 mg/kg + 17β-E2 (middle-aged or old OVX − vitamin D3 1.0 − 17β-E2).

21 and 22—middle-aged or old OVX rats + cholecalciferol 2.5 mg/kg + 17β-E2 (middle-aged or old OVX − vitamin D3 2.5 − 17β-E2).

23 and 24—middle-aged or old OVX rats + cholecalciferol 5.0 mg/kg + 17β-E2 (middle-aged or old OVX − vitamin D3 5.0 − 17β-E2).

The treatment period for animals was 14 days, and at the end of the treatment period (1 h after the last dose of solvent, vitamin D3 or 17β-E2), all animals were subjected to the elevated plus maze (EPM), light-dark test (LDT) and the open field test. During testing sessions in all behavioral tests, the control and experimental groups of rats were also given with solvent, vitamin D3 or 17β-E2.

## **2.5 Behavioral tests**

### *2.5.1 Elevated plus maze test*

EPM is commonly accepted as standard test of anxiety-like behavior and was used to assess anxiety-like behavioral responses [29, 30]. This test is sensitive to putative anxiogenic-like and anxiolytic-like drugs [31]. EPM consist of two open arms (50 × 10 cm2 ) and two closed arms (40 × 10 cm2 ) with a central platform (10 × 10 cm2 ) and elevated 50 cm above the floor level. All female rats from control and experimental groups were randomly placed at the center of the EPM and allowed them to freely move in the apparatus for 5 min. The number of entries and total time spent in open arms were accepted as parameters of anxiolytic-like effects of treatments. The apparatus was cleaned with damp cloth after each trial to avoid place preference and the influence of olfactory stimuli.

#### *2.5.2 Light/dark test*

The apparatus consist of two identical boxes (30 × 40 × 40 cm), one of which with white walls and floor and illuminated by a 60 Watt light from above, while the other of the box was painted black and had a lid so it was not illuminated [32, 33]. The number of entrance and the total time in the light box were registered for 5 min [7]. The increase in the number of entrances and the total time in the light box were postulated as manifestation of anxiolytic-like effects of treatments. The apparatus was cleaned with damp cloth after each trial to avoid place preference and the influence of olfactory stimuli.

**39**

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

**2.6 Determination of estradiol, 25-OH-VD3 and calcium levels in the** 

serum was separated, frozen and stored at −20°C until biochemical assessment. Estradiol levels were assessed using commercial available ELISA kit (DRG Diagnostics, Marburg, Germany). The sensitivity of the estradiol ELISA kit was 1.0 pg/ml. Measurement of 25-hydroxyvitamin D3 (25-OH-VD3) levels was performed by ELISA kit (CSB-E08098r, Cusabio Biotech Co., Ltd., Wuhan, P.R. China). Technical variability for 25-OH-VD3 ELISA kit was low with coefficients of variation of <10% intra-assay and < 15% inter-assay. Detection range of 25-OH-VD3 levels was 20–100 μg/L. The sensitivity of the 25-OH-VD3 using ELISA kit was 5.0 μg/L. Calcium concentrations were detected by spectrophotometric method using calcium assay colorimetric kit (ab102505, Abcam, France). The sensitivity of the calcium kit was 0.1 m/M. All the procedures of estradiol, 25-OH-VD3 and calcium kits were conducted following the manufacturer's instruction manual.

Blood samples were collected in tubes and centrifuged. After centrifugation,

Data were expressed as means ± standard error (SEM). Differences among means were postulated as significant at р ≤ 0.05. Behavioral and biochemical data were analyzed using a two-way ANOVA and subsequent *post-hoc* analysis was conducted with Dunnett's multiple comparison test. Statistical calculation was carried

out using SPSS software 19 version (SPSS Inc., Chicago, IL., USA).

**3.1 Vitamin D3 in different doses decreases anxiety-like profile of the** 

**long-term absence of estrogen as measured in the EPM test**

differences for these groups (*p* < 0.05).

**middle-aged and old OVX and OVX rats given with 17β-estradiol after** 

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

The effect of cholecalciferol on locomotor, rearing and grooming activities was evaluated automatically using an open-field computer-aided controlling system as described previously [34]. The apparatus consists of a square platform (80.0 cm × 80.0 cm; wall height 36.0 cm). The floor of the platform was divided into 16 equal squares of 19.5 cm × 19.5 cm. A video camera fixed at the top, and the apparatus was illuminated by a light source of 120 Lux on the ceiling. Each rat was placed at the center of the apparatus and allowed to explore freely for 5 min. Total number of central and peripheral square crossings were recorded for each animal. The apparatus was cleaned with damp cloth after each trial to avoid place prefer-

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

ence and the influence of olfactory stimuli.

*2.5.3 Open field test*

**blood serum**

**2.7 Statistical analysis**

**3. Results**

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

#### *2.5.3 Open field test*

*Fads and Facts about Vitamin D*

created in the present study:

(middle-aged or old intact − vitamin D3 1.0).

(middle-aged or old intact − vitamin D3 2.5).

(middle-aged or old intact − vitamin D3 5.0).

(middle-aged or old OVX − vitamin D3 1.0 − 17β-E2).

(middle-aged or old OVX − vitamin D3 2.5 − 17β-E2).

(middle-aged or old OVX − vitamin D3 5.0 − 17β-E2).

groups of rats were also given with solvent, vitamin D3 or 17β-E2.

aged or old OVX − vitamin D3 1.0).

aged or old OVX − vitamin D3 2.5).

or old OVX − vitamin D3 5.0).

**2.5 Behavioral tests**

arms (50 × 10 cm2

*2.5.2 Light/dark test*

(10 × 10 cm2

*2.5.1 Elevated plus maze test*

The following experimental groups for the middle-aged and old female rats were

3 and 4—middle-aged or old intact female rats + cholecalciferol 1.0 mg/kg

5 and 6—middle-aged or old intact female rats + cholecalciferol 2.5 mg/kg

7 and 8—middle-aged or old intact female rats + cholecalciferol 5.0 mg/kg

9 and 10—middle-aged or old OVX + solvent (middle-aged or old OVX − Sol). 11 and 12—middle-aged or old OVX rats + 17β-E2 (middle-aged or old OVX − 17β-E2). 13 and 14—middle-aged or old OVX rats + cholecalciferol 1.0 mg/kg (middle-

15 and 16—middle-aged or old OVX rats + cholecalciferol 2.5 mg/kg (middle-

17 and 18—middle-aged or old OVX rats + vitamin D3 5.0 mg/kg (middle-aged

19 and 20—middle-aged or old OVX rats + cholecalciferol 1.0 mg/kg + 17β-E2

21 and 22—middle-aged or old OVX rats + cholecalciferol 2.5 mg/kg + 17β-E2

23 and 24—middle-aged or old OVX rats + cholecalciferol 5.0 mg/kg + 17β-E2

The treatment period for animals was 14 days, and at the end of the treatment period (1 h after the last dose of solvent, vitamin D3 or 17β-E2), all animals were subjected to the elevated plus maze (EPM), light-dark test (LDT) and the open field test. During testing sessions in all behavioral tests, the control and experimental

EPM is commonly accepted as standard test of anxiety-like behavior and was used to assess anxiety-like behavioral responses [29, 30]. This test is sensitive to putative anxiogenic-like and anxiolytic-like drugs [31]. EPM consist of two open

The apparatus consist of two identical boxes (30 × 40 × 40 cm), one of which with white walls and floor and illuminated by a 60 Watt light from above, while the other of the box was painted black and had a lid so it was not illuminated [32, 33]. The number of entrance and the total time in the light box were registered for 5 min [7]. The increase in the number of entrances and the total time in the light box were postulated as manifestation of anxiolytic-like effects of treatments. The apparatus was cleaned with damp cloth after each trial to avoid place preference and the influence of olfactory stimuli.

) and elevated 50 cm above the floor level. All female rats from control

) with a central platform

) and two closed arms (40 × 10 cm2

place preference and the influence of olfactory stimuli.

and experimental groups were randomly placed at the center of the EPM and allowed them to freely move in the apparatus for 5 min. The number of entries and total time spent in open arms were accepted as parameters of anxiolytic-like effects of treatments. The apparatus was cleaned with damp cloth after each trial to avoid

1 and 2—middle-aged or old intact female rats + solvent.

**38**

The effect of cholecalciferol on locomotor, rearing and grooming activities was evaluated automatically using an open-field computer-aided controlling system as described previously [34]. The apparatus consists of a square platform (80.0 cm × 80.0 cm; wall height 36.0 cm). The floor of the platform was divided into 16 equal squares of 19.5 cm × 19.5 cm. A video camera fixed at the top, and the apparatus was illuminated by a light source of 120 Lux on the ceiling. Each rat was placed at the center of the apparatus and allowed to explore freely for 5 min. Total number of central and peripheral square crossings were recorded for each animal. The apparatus was cleaned with damp cloth after each trial to avoid place preference and the influence of olfactory stimuli.

## **2.6 Determination of estradiol, 25-OH-VD3 and calcium levels in the blood serum**

Blood samples were collected in tubes and centrifuged. After centrifugation, serum was separated, frozen and stored at −20°C until biochemical assessment. Estradiol levels were assessed using commercial available ELISA kit (DRG Diagnostics, Marburg, Germany). The sensitivity of the estradiol ELISA kit was 1.0 pg/ml. Measurement of 25-hydroxyvitamin D3 (25-OH-VD3) levels was performed by ELISA kit (CSB-E08098r, Cusabio Biotech Co., Ltd., Wuhan, P.R. China). Technical variability for 25-OH-VD3 ELISA kit was low with coefficients of variation of <10% intra-assay and < 15% inter-assay. Detection range of 25-OH-VD3 levels was 20–100 μg/L. The sensitivity of the 25-OH-VD3 using ELISA kit was 5.0 μg/L. Calcium concentrations were detected by spectrophotometric method using calcium assay colorimetric kit (ab102505, Abcam, France). The sensitivity of the calcium kit was 0.1 m/M. All the procedures of estradiol, 25-OH-VD3 and calcium kits were conducted following the manufacturer's instruction manual.

#### **2.7 Statistical analysis**

Data were expressed as means ± standard error (SEM). Differences among means were postulated as significant at р ≤ 0.05. Behavioral and biochemical data were analyzed using a two-way ANOVA and subsequent *post-hoc* analysis was conducted with Dunnett's multiple comparison test. Statistical calculation was carried out using SPSS software 19 version (SPSS Inc., Chicago, IL., USA).
