Effect of strontium ranelate and cerium oxide addition in the diet on bone quality and expression level of osteocalcin and alkaline phosphatase genes in broiler chicken

Abstract Background In the modern broiler industry, leg and gait disorders are considerable problems. Fast‐growing broilers are especially susceptible to bone abnormalities, causing major problems for broiler producers. Strontium ranelate (SrR) has been used successfully for the treatment of osteoporosis in humans. In addition, cerium oxide (CeO) is an anti‐stress agent applied in the biological system. Methods This study was conducted to investigate the effect of SrR, CeO, and their combinations on tibia quality in broilers. A total of 384 one‐day‐old Ross chicks were divided into six treatments, with four replicates per treatment (16 birds per replicate). The control group was fed a standard diet, and other groups were fed SrR at levels 450, 900 mg/kg feed, CeO at levels 300 and 600 mg/kg feed and a combination of 450 SrR + 300 CeO mg/kg feed. Bone mineral density (BMD), bone mineral content (BMC), bone strength (BS), tibia area, tibia weight, bone Length, bone diameter, minerals in tibia bone of male broilers, alkaline phosphatase gene (ALP) and osteocalcin gene (OC) in male broilers were analysed. Results The results showed that the addition of SrR and CeO had no significant influence (p > 0.01) on BMD, BMC, BS, bone weight, bone length and bone diameter. While there was a significant interaction between sex and treatments, especially in the combination group, BS in females significantly (p < 0.01) increased compared to the control group. Generally, females were found to be more responsive to treatments than males. Significant increases in gene expression were noticed in OC with the addition of low levels of SrR and CeO and mixed group compared to the control. The gene expression of ALP was increased significantly only in a combination group compared to the control group. Conclusion It is concluded that SrR and CeO can be used as beneficial additives in the feed to improve the tibia quality of broilers.


INTRODUCTION
Modern broilers grow three to four times as quickly as earlier generations (Browning & Cowieson, 2013). Leg disorders, bone weakness, and broken bones are common problems in broilers that are attributed to poor growth, downgrading at slaughter, and reducing the bird's routine activities, such as walking, standing, eating, and drinking subsequently causing economic loss at the end (Bell & Weaver, 2002;Manohar et al., 2015;Meseret, 2016;Kierończyk et al., 2017).
The dietary elements have the greatest impact on poultry bone strength (BS) (Adu & Olarotimi, 2020;Ammann, 2006;Bell & Weaver, 2002; Thorp et al., 1991). Although several additional inorganic elements are present in the bone that may be crucial for bone health and strength, calcium and phosphor are major inorganic nutrients because they make up 95% of the mineral matrices (Council, 1994). To promote BS in chickens, researchers have traditionally focused on calcium and phosphorus minerals and vitamin D (Fleming et al., 1998).
Inorganic (mineral) ingredients in bone tissue determine bone density and mechanical strength, whereas organic components ensure bone flexibility (Council, 1994). Bone mineral density (BMD) is a biophysical metric that is used to assess bone structure (Urist & Deutsch, 1960). A pathological mineralization deficiency is responsible for the decline in bone density that contributes to osteoporosis (Ensrud & Crandall, 2017).
Strontium (Sr) is an alkaline element and rare earth metal (Browning & Cowieson, 2013). The physical and chemical properties are similar to that of calcium (Skoryna, 1981). In addition, stable Sr isotopes do not have any significant health threat (Gulson & Wong, 2006). Sr functions as a moderately effective antiresorptive agent in humans (Tamba & Alexa-Stratulat, 2017). In the clinical setting, therapeutic uses of Sr salts such as strontium ranelate (SrR) are involved in preventing osteoporotic fractures (Meunier et al., 2004). Sr appeared to stimulate bone formation and inhibit bone resorption in rodents (Tamba & Alexa-Stratulat, 2017). In poultry, the average intake of Sr is about 19 mg/kg of feed (Doberenz et al., 1969).
SrR consists of two strontium atoms coupled by ranelic acid. It is used to treat osteoporosis (Pilmane et al., 2017). SrR improved bone mass/quality and increased BS in osteoporotic patients through changes in bone matrix characteristics and BMD in clinical trials (Bakhit et al., 2018). In addition, the use of SrR is approved in the United States, Europe and Australia to stimulate bone formation in humans (Suzuki et al., 2008).
Cerium oxide (CeO) is an oxide of the rare-earth element consisting of the metal cerium with the chemical formula CeO 2 . CeO has many uses either commercially or biologically, and its impact on animals and poultry is studied by several workers (Adu & Olarotimi, 2020;Bölükbaşı et al., 2016;Rajeshkumar 2017;Reka et al., 2019). Numerous studies showed that Ce, as nanoparticles, gave a potential therapeutic tool in preventing and treating oxidative stress in animals and poultry (CórdobaJover 2019). In biological contexts, it has been reported that CeO nanoparticles can mimic enzymatic antioxidants such as superoxide dismutase (Heckert et al., 2008).
The distribution of enzymes and proteins involved in bone production and resorption can influence the process of bone remodelling. Furthermore, osteoporosis is caused by an imbalance in bone growth and resorption (Ahmadi & Ashrafizadeh, 2018). Various scientific evidence indicated the role of nutrition on specific gene expression, among those genes responsible for bone formation, alkaline phosphatase (ALP) and osteocalcin (OC) genes. ALP is an important component in the creation of hard tissue and is strongly expressed in mineralized tissue cells (Golub et al., 2007), which is involved in bone mineralization (Nelson et al., 2020). In addition, OC (bone γ-carboxyglutamic acid protein) regulates bone metabolism, in which a decrease in the amount of the OC gene diminishes bone metabolism and predisposes bone to diseases (Ahmadi & Ashrafizadeh, 2018), OC is required for BS in the longitudinal direction of the long bone by regulating the alignment of biological apatite parallel to collagen fibrils and plays an important function in bone structure (Komori, 2020).
The objectives of this study were to examine the effect of SrR and CeO supplementations in the diet on the bone quality of broilers to improve the welfare of birds.

Primer design
Each of the forward and reverse primers was designed by the National Center for Biotechnology Information (NCBI) bioinformatics TA B L E 2 Gene bank reference accession number.

RNA extraction and complementary DNA synthesis
At the end of the experiment, one male bird from each replicate was slaughtered and samples of bone marrow were taken from the tibia.
RNA was extracted from fresh tibia bone marrow using an extraction kit for tissue obtained from Favorgene RNA (Korea).
Five micrograms of total RNA was converted to cDNA by the Beta Script Kit for cDNA synthesis from (Beta Bayern GMbH, Nurnberg, Germany) following the manufacturer's instructions.

2.3.3
Real-time polymerase chain reaction amplification Polymerase chain reaction(PCR) amplification of genes was done in 20 μL of reaction mixture containing 2× SYBER Green Master(Addbio, Korea), 10 pmol of forwards, 10 pmol reverse primer, DNase-free water, and a template of cDNA (Applied Bio-system Model 7500 Real-Time thermos-cycler) was run. The temperature profile included the following steps: step one was an initial denaturation at 95 • C for 5 min, ΔΔCT values were calculated as powers of (2 −ΔΔCT ) to account for the exponential doubling of the PCR (Livak & Schmittgen, 2001). Then, the geometric mean was calculated for the (2 −ΔΔCT ) of each replicate in the groups and then the standard (SE) error was calculated to draw the figure.

Statistical analysis
Statistical analysis of the data (bone area, BMC, BMD, BS, bone weight, bone length, and bone diameter) is presented as means ± SE. One-way analysis of variance (ANOVA) followed by a factorial test to evaluate the effect of additives and sexes on treats, was performed using SAS program version 9.3. Differences were reported as significant in all instances with a p < 0.05. Statistical analysis of the data of tibia (mineral content) is presented as means ± SE. One-way ANOVA followed by a Completely Randomized Design test in order to determine the effect of additives on treats was performed using SAS program version 9.
Differences were reported as significant in all instances with a p < 0.05.
To evaluate the homogeneity of variances and normality of data, as well as to determine the significance and interactions of main effects, the SAS statistical software (PROC GLM) was utilized (SAS, 9.3, 2013).
Duncan's multiple range test was employed to discover differences between individual treatment means and interactions were observed in p < 0.05.

Body weight
The addition of SrR, CeO and their combination had no significant (p > 0.01) effect on body weight (Table 4).

Bone quality
Rapid growth in birds immediately post-hatches imposes a high mechanical load on the skeleton, which is still underdeveloped TA B L E 3 Represents the effects of SrR, CeO and their combination on Bone quality. interaction between sex and treatments especially in females in the group fed 900 mg/kg SrR; the area of the tibia was significantly more than in females in the group fed 450 mg/kg SrR and females that fed 300 mg/kg CeO. This significant increase in the area related to the increase in the diameter of the tibia in this group is shown in Table 4; in addition to the increase in the cortical and medullary area of the tibia, the use of Sr resulted in an increase in the total area of the tibia (Shahnazari et al., 2007) The impact of additives on BMC in treatments was not significant. While in females, a significant (p < 0.01) increase was observed between a group fed 900 mg/kg SrR compared to groups fed 450 mg/kg SrR and 300 mg/kg CeO; this difference was related to increasing the area of the tibia. Marie et al. (1993) obtained a similar result with ovariectomized osteopenic rats when fed strontium salt. Additionally, Ammann et al. (2004) noted that Sr therapy increased BMC in female rats. Similarly, Shahnazari et al. (2007) reported that BMC increased significantly with Sr addition to the fed in broilers.
The addition of SrR, CeO and their combination had no significant (p > 0.01) impact on the BMD. While BMD in males is significantly TA B L E 4 Represents the effects of SrR, CeO and their combination on Bone measurements.

Treatments Tibia weight (g) Tibia length (mm) Tibia diameter (mm)
Additive supplementations (p < 0.01) higher than BMD in females as well as there is a significant (p < 0.01) interaction between treatments and sex in BMD of the tibia. BMD in females significantly (p < 0.01) increased in the group that received SrR (900 mg/kg) compared with both 300 mg/kg CeO and 600 mg/kg CeO. Aveline et al. (2021) found a significant increase in BMD of ovariectomized rats that received 625 mg/kg/day of SrR. Ammann et al. (2004) also revealed that Sr has a substantial impact on BMD. This variation with previous data may be due to the animal species used in the research.
The addition of SrR, CeO, and their combination has no significant effect on BS in treatments. The results showed a significant difference between males and females, BS in males was significantly (p < 0.01) higher than BS in females. While there was a significant (p < 0.01) interaction between treatments and sex. Sex is a factor that influences bone growth. Size differences, as well as hormonal differences, can account for the differences in growth and BS between males and females (Rath et al., 1999). In the combination group (450 SrR + 300 CeO mg/kg feed), females' BS increased significantly more than in the control group. That means dietary SrR and CeO supplementation has the ability to reduce lameness and osteoporosis in broiler chickens.
Bone problems have been a key issue that perilously affects broilers' health and welfare, resulting in severe economic loss (Nakhon et al., 2019). Bone structural and metabolic disturbances (like tibial dyschondroplasia, osteoporosis, and osteoarthrosis) are common in broiler chickens (Khan et al., 2021). In a study on females, Meunier et al. (2009) revealed a significant increase in BS in the group that was administered Sr. Similarly, Ammann et al. (2004) stated that treated rats with 900 mg/kg/day had a stronger bone in comparison to the other groups.
Also in a study with rats, when fed a diet containing 625 mg/kg/day, SrR showed anti-fracture efficacy by influencing the determinants of BS TA B L E 5 Effects of SrR, CeO and their mixture on the tibia mineral content in the male chicken broiler.  (Bain et al., 2009). In addition, Ferraro et al. (1983), Dahl et al. (2001), Grynpas and Marie (1990), Marie et al. (2001), and Marie and Hott (1986) observed that supplementing (316-634 mg/kg/day Sr 2+ ) inhibited bone resorption by inhibiting the action of bone osteoclast cells, resulting in an increase in bone volume with no detrimental effect on bone mineralization. Table 4 shows that no significant (p > 0.01) difference was observed in the weight, length and diameter of bones among treatments. While there was a significant (p < 0.01) difference in all parameters between males and females, this difference was related to the weight because the weight of males was more than females. There was a significant (p < 0.01) interaction between treatments and sex. Results showed that males and females have different responses to the addition of SrR, CeO and their combination in the diet. As shown in Table 4, the length of the tibia in males was significantly (p < 0.01) different from females in all groups except in the group that was fed 600 mg/kg CeO, which showed an insignificant difference between females and males at the same time there is no difference with males in each of control, groups fed 450 mg/kg SrR and the combination.

Tibia mineral content in male chicken
Tibia minerals content in male birds showed significant (p < 0.01) differences among groups for Ca and Sr content, while phosphorus and magnesium content was not affected significantly ( Sr to be absorbed in the intestine in which both are found in divalent charges and these two elements are chemically very close to each other thus the body absorbs Sr in the same way as Ca (Browning & Cowieson, 2013;Rajeshkumar et al., 2017;Wadkins & Peng, 1981).
Also, Gad (2014) (Shahnazari et al., 2006). A significant (p < 0.01) highest value of Ca content was found in the combinations group (SrR + CeO mixture) in the tibia compared to 450 and 900 mg/kg). This may be due to the synergistic effects of both additives. Insignificant effects were observed with the addition of CeO to the diet. The phosphorus and magnesium level in the tibia was not affected significantly. Similar outcomes were found by Browning and Cowieson (2015) in broiler chicken at 28 days old by adding 1200 mg Sr/kg to the diet.

OC gene expression
The effect of SrR and CeO and their combinations on the OC gene expression in males of broiler chickens is demonstrated in Figure 1.

ALP gene expression
ALP is highly expressed in mineralized tissue cells and plays a critical role in the formation of hard tissue by increasing the inorganic phosphate local portion and facilitating mineralization while lowering extracellular pyrophosphate concentration, which is a mineral formation inhibitor (Vimalraj, 2020). Figure

CONCLUSION
The present study concludes that the addition of SrR, CeO and their combinations in the diet of broilers had no significant influence on live weight, BMC, BMD, BS, bone weight, length, and diameter. Moreover, the intake of combination minerals significantly increased BS in the females compared with the control group. There were significant interactions between treatments and sex for all parameters. Also, Sr can be replaced with Ca in the mineral content of the growing tibia. Higher gene expression of OC significantly was noticed in all treated groups compared to the control, except the group fed 900 mg/kg SrR. ALP gene expression was increased significantly in the combination group (450 SrR + 300 CeO mg/kg feed) compared to the control group. The SrR and CeO can be used as beneficial additives in the feed to improve the tibia quality of broilers.

AUTHOR CONTRIBUTIONS
KAA is the project leader who is also the main supervisor to PSS who contributed to the idea, design and execution of the study. PSS performed the bone quality and measurements. KAA and PSS determined the expression level of genes. KAA and PSS were responsible for the statistical analysis. Both authors contributed equally to the write-up of the final manuscript.