Desde la perspectiva de género hacia la construcción de un modelo de

Sodium Alendronate (supplied by Merck, Whitehouse Station, USA) was administered weekly to groups OVX ALD, GOAT OVX ALD and COW OVX ALD at a dose rate of 7mg/kg bodyweight/week. The dose was calculated weekly for each individual rat. It was dissolved in milli-Q water, then mixed into liquid raspberry jelly to make a total

5-5 volume of 3ml, (method based on Kuhn-Sherlock & Schollum, 2001) (21). Alendronate is recommended to be taken on an empty stomach, therefore rats were fed in the late afternoon at 4pm when food intake is believed to be minimal and their stomachs are most likely to be empty. Rats are feed daily between 8am and 10am at which time they generally consume all of their diet. The SHAM, OVX, GOAT OVX, AND COW OVX groups received a plain raspberry jelly placebo.

5.2.4. Bone densitometry by Duel energy X-ray Absorptiometry (DEXA)

At 20, 30 and 38 weeks of age weeks in vivo DEXA were performed to assess whole body, lumbar spine and femur measurements. The measurements were bone mineral density (BMD), bone mineral content (BMC), bone area, whole body fat mass, lean mass, bone mass, and % of body fat (PFAT). Measurements were taken using a “Hologic Discovery A” bone densitometer (Bedford, MA, USA). On each day that scans were undertaken, a quality control (QC) scan was taken to ensure that its precision met the required DEXA manufacturer’s coefficient of variation. The coefficient of variation (CV) for the QC data was 0.98 – 1.01%. Coefficient of variance for the femurs with repositioning between the scans was 1.20%, and without repositioning the scans the coefficient was 0.60%. These values ranged between 0.61% and 1.38% for the lumbar spine.

Each rat underwent three regional high-resolution scans of the spine and left and right femurs. Before scanning, the rats were anaesthetised with a drug mixture of Ketamine, Acepromazine (ACP), sterile water, and Xylazine at a ratio of (5:2:2:1). The dose was administered via intra-peritoneal injection at 0.05ml/100g of body weight. Rats were positioned supine with right angles between the spine and femur, and between femur and tibia.

5.2.5. Euthanasia and sample collections

Upon completion of the trial at 38 weeks of age the rats were euthanized by

exsanguinations under anaesthesia. The anaesthetic was administered at a dose rate of 0.1ml/100g of bodyweight, using the same mixture and ratio as described in the above section (DEXA). Both hind legs of each rat were subsequently removed by simple dissection, and placed in phosphate buffered saline (PBS) at -20° Celsius pending further analysis.

5-6

5.2.6. Femurs

Left femurs were thawed and their weight and length determined after any residual adherent soft tissue had been removed. The femurs were prepared and sliced using the method described in Chapter 3. Subsequent measurements were taken from the twelve slices (slices 5-16) that constituted the tubular portion of the femoral shaft. These slices spanned 60 % of the overall length, and extended from the upper limit of the

intercondylar ridge (slice 5) at the distal end of the femoral shaft, to the upper limit of the third trochanter (slice 16) at the proximal end of the femoral shaft.

Each of the bone cross sectional surfaces was scanned using the same method described in Chapter 2 (Fig. 5-1).

Figure 5-1. Picture describing the cross sectional profile of the femur slice.

1. Shows the area used to calculate the bone area (mm2). 2. Shows the area used to calculate

marrow cavity area (mm2_{). 3. Shows the area used to calculate the total cross sectional area}

(mm2).

5.2.7. Statistical analysis

Data were analysed in the statistical package “SAS” (version 9.3) (Sas Institute, Cary, USA). The mean bone area, mean marrow cavity area, and mean total cross sectional area of the slices taken from the femoral shaft were each normally distributed and were, therefore, amenable to parametric analysis. Uterus weights, bodyweight and diet intake data and DEXA were found for the most part to be normally distributed. Where non-

5-7 normal data were found a log transformation was required to obtain near normal

distribution on graphic analysis.

Uterus weights and diet intake were examined by one-way ANOVA. Bodyweights and DEXA measurements were analyzed using General linear model (GLM) two-way ANOVA. The effects of diet and drug on bone area (mm2), marrow cavity (mm2), and total cross sectional area (mm2) in the femoral shaft were compared by one way repeated measures ANOVA. The pattern of variation of each of these parameters between slices was explored using multivariate principal component analysis (PCA). A one way ANOVA was then conducted on the individual principal component scores in each axis of variation in order to statistically compare the effects of the diets. Data from incorrect scanning or damaged bone samples caused during preparation were removed from analysis. All tests used Tukey post-hoc testing and significance was set at (p≤0.05).

5.3.

Results

Sixteen rats died during the trial due to a vitamin A deficiency (pg. 5.2) and other complications incurred during the trial. Diet compositions including calcium,

phosphorus, percent fat and percent protein were confirmed by chemical analysis (data not shown).

Ovariectomy surgery was confirmed by extraction of the uterus post-mortem at 38 weeks of age. As expected, mean uterus weights were significantly higher in the sham rats compared to ovariectomized rats (p<0.0005). One outlier was shown in the ovariectomized rat population indicating a failed ovariectomy surgery. All data from that rat were removed from all analyses.

5.3.1. Duel energy X-ray Absorptiometry (DEXA)