Many factors influence bone development directly or indirectly in birds, among which nutritional factors, genetics, gender, and the absolute growth rate. On the other hand,   bone tissue is complex and composed of inorganic substances such as calcium and phosphorus , which provide hardness and strength and organic substances, which give elasticity to bone (Adebiyi et al., 2009).

As is known, grapes represent an important source of bioavailable polyphenols such as flavonoids (Yammine et al., 2018). Also, the compound  proanthocyanidins extracted from the grape seed is one of the bioactive compounds that possesses a wide range of pharmacological and biochemical properties (Katiyar et al., 2017). Phenolic compounds extracted from grapes have many benefits in maintaining bone health in animals. In this regard, Holman and Weaver (2014) showed that feeding rats on dried grapes increased bone calcium content as well as bone structure and strength. As noted by Shen et al., (2012), dried fruits and bioactive compounds, including phenolics, flavonoids and resveratrol extracted from fruits and grapes are important in maintaining the health and durability of bones and thus increasing their strength. These effects are attributed to the role of antioxidants in fruits in reducing the effects of oxidative stress. In the bones, resveratrol grape possesses important anti-inflammatory and antioxidant properties, which benefit bone health.

In addition, increased fruit intake has favorable effects on bone mineral concentration (Prynne et al., 2006), as well as, a positive relationship between fruit consumption level and bone mineral density (Li et al., 2013). According to Manach et al., (2004), these phenolic compounds have attracted great interest as a nutritional intervention against degenerative diseases, including osteoporosis. On other hand, grape intake is thought to protect bone by blocking inflammatory molecules, that stimulate osteoclasts (Chu et al., 2015).

As well as, grape seeds also contain many minerals, including calcium (0.56%), phosphorous (0.31%) and total ash (2.68%) (Hajati et al., 2015), which are important in the formation of the skeleton of chickens. In the background of the above details, this research was conducted to determine the efficacy of grape seeds, grape leaves powder, or their extracts on some broiler femur bone characteristics, the serum and bone content of calcium and phosphorous and the total ash content at 35 days of age.

This work was carried out at Poultry Field, College of Agriculture, University of Basra for a period of 5 weeks (35 days) from 8th October 2018 to 12^th November 2018. Three hundred and twenty four day-old broiler chicks (Ross- 308) were randomly distributed among nine treatment groups containing three replicates of 12 birds each. Every group was fed on one of the following experimental diets. The first treatment was the control group, the birds in 2^nd and 3^rd treatments were given grape seed (GS) powder at 15 and 30 g/kg feed respectively, the 4^th and 5^th groups of birds were given grape leaves powder at 15 and 30 g/kg feed respectively, the 6^th and 7^th groups of birds were given grape seed extract (GSE) at 2 and 4 ml/l with drinking water, while the 8^th and 9^th groups of birds were given grape leaf extract (GLE) at 2 and 4 ml/l with drinking water, respectively. The basic diets were prepared according to the recommended nutritional needs of the broiler. The composition of the basal diet is presented in (Table 1). Chemical analysis of grape seeds and leaves were carried out according to AOAC (2016) (Table 2). The birds were fed a starter diet until 21days of age, followed by a finisher diet from 22 to 35 days. All birds had ad libitum access to feed with the 35 day’s period.




 
At the end of the 35 days experiment, three birds of similar body weight from each treatment were sacrificed to study thigh bone (Femur) characteristics. To preparing the bone, medical scissors were used to rid the thigh bone of flesh and connective tissues according to Hall et al., (2003) method. Where the thigh bones were labeled and immersed in boiling water (100°C) for ten minutes and the remnants of soft tissues were removed by hand. They were then oven-dried for 6 h at 105°C. The femur length and weight were determined. Bone Index (BI) was calculated according to the equation of Seedor et al., (1991).  
Femur ash content was determined by ashing the bone in a Muffle furnace for 6 h at 600°C according to the procedure of AOAC (2016), the percent of total ash was determined relative to the dry weight of the bone. Calcium percentage in the femur was calculated or analysed according to Cresser and Parsons (1979) and phosphorous according to Harner and Wilson, (1986). The predicted skeletal weight (g) was calculated according to the equation of Taylor et al., (1965):
 
Predicted skeletal weight (g) =  
Serum calcium (Ca) and Phosphorous (P) were measured using commercial kits (Randox), then the Ca: P ratio in bone and serum was calculated.
 
Statistical analysis
 
The data were subjected to analysis of variance (One -way ANOVA) in accordance with completely randomized design (CRD) using SPSS software (2015) to analyze the results. Duncan’s new multiple range test (Duncan, 1955) was applied to the separated means at a significant level of 0.05 (SPSS, 2015).

There were no effects of GSP, GLP, GSE and GLE supplementation on bone weight, bone index and total ash except for bone length and predicted skeletal weight (Table 3). A significant improvement (P≤0.05) was recorded in the bone length in the GSP (30 g/kg), GLP (15 g/kg), GSE, GLE groups as compared with control.



Also, the supplementation of grape seed extract (T6aT7) improved significantly (P≤0.05) predictive value for skeletal weight compared to the other treatments. While a significant decrease was observed when using the lower level of grape seed or leaf powder (15 g/Kg) and grape leaf extract (2ml/l) compared to the control.

The improvement in bone length and predicted skeletal weight (Table 3) may be attributed to the presence of the proanthocyanidin compound, which is extracted from grape seeds. A previous study concluded that feeding rats on proanthocyanidin led to an increase in bone calcium content (Yahara et al., 2005). Also, rats fed on dried grapes increased bone calcium content, bone structure and strength (Holman and Weaver, (2014). In this context, Shen et al., (2012) indicated the effect of dried fruits and bioactive compounds, including phenolic, flavonoids and resveratrol extracted from fruits and grapes, indicating their importance in maintaining the health and durability of the bones and thus increasing their strength, attributing these effects to the role of antioxidants in fruits in reducing the effects of oxidative stress in the bones and that resveratrol possesses important anti-inflammatory and antioxidant properties, which benefit bone health. Additionally, a recent study, suggests that orally administered proanthocyanidin-rich grape seed extract improved implant osseointegration by alleviating the impaired bone health induced by ovariectomy as a model of estrogen hormone deficiency in mice (Tenkumo  et al., 2020).

The calcium and phosphorus in blood sera, bone ash, and the Ca to P ratio were not significantly affected (P≥0.05) by dietary GSP, GLP, GSE and GLE except for calcium percentage in bone ash (Table 4). Calcium percent increased (P≤0.05) in grape leaf extract groups (T8,T9) as compared to control, which reached 32.28 and 31.23%, respectively, while the remaining treatments did not differ in the level of calcium in bone ash.



The improvement in calcium percent of the grape leaf extract treatments may be due to the presence of resveratrol compound in grape leaves, which has many properties in preserving bone health (Shen et al., 2012). According to Holman and Weaver (2014), rats fed a diet enriched in grapes retained 44% more net calcium in the bones than rats fed a control diet. So, the researchers suggest that consuming grape products may improve calcium utilization and suppress bone turnover in ovariectomized rats, leading to improved bone quality.  In contrast with our results, Kaya et al., (2014) reported that serum calcium and phosphorus concentration were decreased in layer hens that were fed grape seed extract, at levels 20-25 mg/kg diets.