Effects of administration of four different doses of Escherichia coli phytase on femur properties of 16-week-old turkeys

Numerous techniques may be used for skeletal system quality evaluation in poultry species. Postmortal isolation of bone samples enables macro-morphological evaluation in terms of bone weight, length, thickness of compact and spongy layers, and perimeters and diameters of diaphysis, epiphyses and metaphyses [16]. Microscopic evaluation of histological preparations may be used for investigation of endochondral ossification process in growth plates [17]. Microradiographic imaging of transverse sections of bone shaft is utilized for cortical porosity evaluation and standard radiographic mineral density measurement [18]. Dual-energy X-ray absorptiometry (DEXA) method and digital analysis of radiograms using precise software such as Trabecula® may be applied for both in vivo and ex vivo assessment of bone mineral density, bone mineral content and microarchitectural organization of bone samples [19,20]. As opposed to the mentioned above diagnostic techniques which provide two-dimensional analysis of bone samples, quantitative computed tomography enabling three-dimensional morphological and densitometric analysis of bones was used in this study [21].

Pelvic limb long bones such as femur, tibia and tibiotarsus in bipedal domestic meat-type birds are subjected to significant bending forces in order to withstand loading by body tissues [22-24]. It was reported that incidence of clinical manifestations of metabolic bone disorders such as leg deformations, lameness and bone fractures increases together with higher body weight reaching significant percentage in the breeding flock at final stages of the production cycle [25-30]. Experimental data from morphometric and mechanical studies on turkeys suffering from femoral fractures indicated an insufficient skeletal adaptation to heavy body weight as a causative factor responsible for locomotory function impairments [30]. Radiological analysis of tibiotarsal bone in six-week-old Pekin domestic duck has shown that lowered bone mass was associated with bone deformities and fractures [31,32]. Studies on broiler chickens from 15 different flocks revealed the presence of gross lesions in epiphysis and metaphysis of the proximal and distal femur. Moreover, bone fractures and separation of articular cartilage of the femoral bone head with progressive erosions of the subchondral bone were diagnosed confirming impaired bone formation and mineralization processes during skeletal development [33]. Decreased vBMD in proximal metaphysis between 2^nd and 4^th week, and in the diaphysis between 2^nd and 6^th week of posthatching development was observed in male Ross broiler chickens. These observations were associated with decreasing relative bone weight from 1.03% to 0.79% and tibia deformations and fractures occurrence [34]. Negative correlation between body weight and trabeculae number in tibiotarsal bone was found in growing domestic geese together with decreasing relative bone mass between 4^th and 16^th week of age. The observed negative changes of the trabecular structure combined with intensive body weight gain were suggested to be associated with bone deformities and locomotory impairments in those geese [35]. Femoral fracture incidence was also reported in heavy male turkey breeders at the age of 32–35 weeks [29]. Experimental studies on meat-type turkeys have shown that leg deformities are observed in birds characterized by decreased mechanical endurance and volumetric bone mineral density of tibia and femur [36]. In other studies on turkey model, it was shown that increased volumetric bone mineral density of femur and tibia contributes to higher values of maximum elastic strength and ultimate strength – the parameters representing mechanical endurance of bones [37].

Considering importance of intestinal bioavailability and absorption of phosphorus and its contribution to hydroxyapatite structure within bone tissue, the current study was performed to evaluate effects of administration with four different levels of Escherichia coli phytase on mineral density, morphology and mechanical endurance of femur in 16-week old turkeys. The results obtained in this study have shown that the most beneficial effects of phytase administration to growing turkeys was observed in group III receiving 125 FTU/kg of the diet. In this group, femur was characterized by increased geometric parameters such as cross-sectional area, second moment of inertia, mean relative wall thickness and cortical index when compared to the positive and negative control groups. The observed 5% insignificant increase of the volumetric bone mineral density in cortical bone of the midshaft in these birds was associated with significantly higher maximum elastic strength increased by 36% but cortical bone area decreased by 18.4% when compared to the positive control group. Similar effects of phytase administration on Cd, Wy and CBA of femur was observed in group IV that received 250 FTU/kg of the diet; however, all the differences of these parameters were significant in comparison to the positive control group and reached 9%, 44.6% and 21%, respectively. The effects of phytase administration in the dosage of 500 FTU/kg of the diet improved Cd, length and volume of femur with simultaneous reduction of CBA when compared to the positive control group. It is surprising that the analysis of femur obtained from the turkeys receiving the highest phytase dosage in this experiment has shown increased Ix value only in comparison to the positive control group, with concurrently lower CBA. In the negative control group, excluding significantly decreased value of CBA, the values of the analyzed parameters of femur were not significantly different from those obtained in the positive control group. Thus, simultaneous reduction of calcium and available phosphorus content in the turkey diet has not shown significant effects on morphological, densitometric and mechanical properties of femur. However, reduced dietary calcium and phosphorus content induced a significant decrease of final body weight of turkeys by 6.5%. It is worth to underline that both the initial and final body weights of turkeys administered with Escherichia coli phytase (Groups III – VI) were not significantly different from these parameters determined in the positive control group.

The results obtained from femur analyses show similarities with previous report describing effects of Escherichia coli phytase administration in the same experimental design on tibia properties [38]. In the previous report, ash content and specific gravity of whole tibia, as well as its maximum bone breaking force were not influenced by phytase administration at four different dosages. These observations correspond with the parameters analyzed for whole femur such as weight, length, total volume, MvBMD and Wf. However, contrary to the data from tibia obtained in the previous study, more detailed analyses of femur have shown positive effects of phytase administration in turkeys on geometric parameters and Cd assessed at the midshaft, and maximum elastic strength [38]. The results obtained in the current study are in accordance with previous experiments on male turkeys where Escherichia coli phytase administration at 250, 500, 750 and 1000 U/kg improved ash percentage and percentage of non-phytate phosphorus in middle toe and tibia in comparison to the control group. There were no statistically confirmed differentiated effects of the dosage applied on tibia and toe ash in these birds. However, Escherichia coli phytase was more effective when compared to two other commercially available phytases which were administered to turkeys [39]. The differences between the current and the previous study on turkeys results from its diverse phytase administration period and the applied dosages, as well as different age of birds and methodological approach. In the study by Applegate et al. (2003) the phytase administration was performed between 10^th and 21^st day of life when the experiment was terminated. Furthermore, neither morphometric and densitometric parameters nor mechanical endurance of the bones were analyzed in that experiment [39]. In studies on broiler chickens growing up to 49^th day of life, the inclusion of phytase at the dosage of 600 U/kg of diet has not induced significant effects on body, femur and tibia weights, as well as ash content in these bones. Moreover, densitometric analysis of whole body and tibia using DEXA method has not proven any effects of phytase administration on bone mineral density and bone mineral content [40]. The discrepancy of the results obtained in the experiment performed by Angel and colleagues (2006) and in the current study may be explained by different origin, treatment duration and dosage of the administered phytase, as well as age- and species-related differences. Among the mechanisms responsible for the observed beneficial effects of phytase administration on femur properties in this study, the improved health status of intestine and digestion processes, and better mineral absorption may be postulated. This hypothesis was confirmed in studies on turkeys in which phytase administration was associated with more efficient mineral, nitrogen and amino acid metabolism [5,41].