Total flavonoids of Chinese herb Rhizoma Drynariae alleviates bone loss in caged laying hens

Background: Caged layer osteoporosis (CLO) is a common bone metabolic disease and is threatening the modern poultry industry. The objective of this study was to evaluate effects of total flavonoids of Rhizoma Drynariae (TFRD), a Chinese herbal extract, on bone health, egg quality and production performance in caged laying hens. Totals of 144 Lohmann pink-shell laying hens of 32-wk-old were randomly allocated to 4 groups (CON, LCD, TFRD1 and TFRD2, respectively). The CON group was fed a basal diet, the LCD group was fed a low calcium diet (LCD) and the TFRD1 and TFRD2 groups were fed LCD supplemented with 0.5 and 2.0 g/kg TFRD, respectively. Results: Supplying 2.0 g/kg TFRD produced protective effects on calcium deficiency-induced bone loss in caged laying hens, including reduced serum levels of alkaline phosphatase and tartrate resistant acid phosphatase activities (both P < 0.05) and osteocalcin content (P < 0.05), enhanced femur bone mineral density (P < 0.05), improved bone microarchitecture deterioration, down-regulated mRNA expressions of RANKL (P < 0.01) and up-regulated RUNX2 and OPG levels (both P < 0.01). Furthermore, compared to LCD group, 2.0 g/kg TFRD treatment had lower malondialdehyd levels (P < 0.01), and higher total antioxidant capacity (P < 0.05) and glutathione peroxidase (P < 0.05) activities, which contributed to the protective effects of bone loss. TFRD supplementation did not affect egg quality while 2.0 g/kg TFRD treatment improved laying rate (P < 0.05) and decreased feed conversion ratio (P < 0.05). Conclusions: These findings suggested that TFRD has beneficial effects on bone health and production performance in caged laying hens, which can be used for the peroxidase; ALP:

pathways, such as OPG/RANKL/RANK, Wnt/β-catenin and BMP pathways [14][15][16], and finally achieve the goal of prevention and treatment of osteoporosis. However, it is unclear whether TFRD also has ideal preventive effects on CLO. Thus, this study aimed to evaluate effects of TFRD on bone health, egg quality and production performance in caged laying hens.

Performance of caged laying hens
As shown in Table 1, low calcium diet (LCD) significantly decreased laying rate (P < 0.01) and average egg weight (AEW) (P < 0.05), and increased feed conversion ratio (FCR) (P < 0.01). Supplying 2.0 g/kg TFRD in LCD improved laying rate (P < 0.05) and reduced FCR (P < 0.05). Average daily feed intake (ADFI) among groups did not differ in the study. Table 1 Effects of TFRD on production performance of caged laying hens Egg quality Table 2 shows that, the eggshell strength and eggshell thickness of caged laying hens fed LCD gradually decreased during the experiment period. At 9 wk, the eggshell strength decreased by 52.6% (P < 0.01) and eggshell thickness decreased by 31.4% (P < 0.01) compared to the CON group. In addition, yolk color also dropped significantly (P < 0.01). Compared to the LCD group, the supplementation of TFRD did not affect egg quality. Table 2 Effects of TFRD on egg quality of caged laying hens

Serum redox of caged laying hens
The serum redox indicators of the LCD group was significantly affected (Table 3), as shown by the malondialdehyde (MDA) (P < 0.01) levels was enhanced, and the total antioxidant capacity (T-AOC) (P < 0.01), total superoxide dismutase (T-SOD) (P < 0.01) and glutathione peroxidase (GSH-Px) (P < 0.01) activities were reduced. 0.5 g/kg TFRD treatment did not have a significant impact on these indicators.

Femur and tibia bone mineral density (BMD)
As shown in Table 4, LCD group had lower BMD of femur (P < 0.01) and tibia (P < 0.05) compared with the CON group. TFRD supplementation did not affect tibia BMD, while 2.0 g/kg TFRD treatment significantly increased the femur BMD (P < 0.05). Table 4 Effects of TFRD on bone mineral density of caged laying hens Bone histomorphometry Fig. 1 shows that effects of TFRD supplementation on the microstructure of the tibia tissue. In the LCD group, many large absorption cavities was appeared on the cortical bone and the cortical bone area was reduced. The trabecular bone became loose or broken with decreased area. With TFRD treatment, the absorption cavity was reduced and the trabecular bone structure was more complete. In addition, the static parameters of bone histomorphometry were analyzed (Table 5). Compared with the CON group, LCD group had significantly lower cortical area ration (P < 0.05), percent trabecular area (P < 0.05) and trabecular thickness (P < 0.05), and higher trabecular separation (P < 0.05). 2.0 g/kg TFRD treatment enhanced cortical area ration (P < 0.05), percent trabecular area (P < 0.05) and trabecular thickness (P < 0.05), and had a lower trabecular separation (P < 0.05) than that of LCD hens. Trabecular number and cortical width among groups did not differ in the study.

Discussion
In the present study, LCD resulted in a significant reduction in laying rate, average egg weight and eggshell quality in caged laying hens. Ca is a critical nutrients and laying hens need to mobilize about 2.2 g of Ca daily for eggshell formation [7].
Long-term Ca deficiency in laying hens will limit the secretion of hormones, leading to reduced or even stopped egg production [17]. Supplying TFRD improved production performance reduction of caged laying hens caused by LCD. It has been reported that TFRD has estrogen-like effects [18], which may benefit for the reproductive system of laying hens. However, supplementation with TFRD did not affect the egg quality, which is consistent with the results of previous studies [19,20].
LCD also resulted in elevated serum MDA levels and a significant decrease in antioxidase activity of caged laying hens. Ca is a key second messenger involved in intra-and extracellular signaling pathways [21]. Ca homeostasis in the body is closely related to oxidative stress and dietary Ca modulates oxidative and inflammatory stress in mice and humans [22]. Our results showed that supplementation with TFRD decreased the MDA levels and increased antioxidase activity, which indicated that TFRD can alleviate oxidative stress caused by LCD in caged laying hens. Furthermore, total oxidative/anti-oxidative status are close related to BMD in osteoporosis [23,24]. The femur and tibia BMD of caged laying hens fed a LCD was significantly reduced. BMD still is the gold standard for the osteoporosis diagnosis [25]. TFRD treatment increased femur BMD in caged laying hens, which indicated that TFRD have a therapeutic effect on low BMD. Furthermore, bone histomorphology showed that TFRD improved the microstructure of tibia tissue, as evidenced by raised cortical area, percent trabecular area and trabecular thickness. Bone histomorphometry is particularly valuable in analyzing the pathology of different forms of bone diseases and in defining the mechanisms by which drugs affect bone [26]. It provides information that is not available from BMD analysis.
Bone metabolism biomarkers, also known as bone turnover markers (BTMs), are a useful adjunct for the diagnosis and therapeutic monitoring of bone metabolic disorders [27]. BTMs are divided into markers of bone formation or resorption and can provide information that is useful for the management of osteoporosis

Conclusions
In conclusions, TFRD treatment produced protective effects on Ca deficiencyinduced bone loss in caged laying hens, including decreased levels of bone matabolism biomarkers, enhanced femur bone mineral density, improved bone microarchitecture deterioration, and regulated the key gene expressions of bone formation and bone resorption. In addition, supplying TFRD improved production performance reduction and alleviated oxidative stress of caged laying hens caused by low Ca diets. These findings suggested that TFRD has beneficial effects on bone health in caged laying hens, which can be used for the prevention of CLO.

Experimental design and diets
A total of 144 Lohmann pink-shell laying hens of 32-wk-old from a commercial farm in the Hubei province of China were randomly allocated to 4 groups (CON, LCD, TFRD1 and TFRD2) with 6 replications of 6 hens per replication. The CON group was fed a corn-soybean basal diet (Table 7), the LCD group was fed a low calcium diet (LCD) ( Table 1) and the TFRD1 and TFRD2 groups were fed LCD supplemented with 0.5 and 2.0 g/kg TFRD (Beijing Qihuang Pharmaceutical Manufacturing Co., Ltd), respectively. Table 7 Basal diet and low calcium diet formulation and nutrient levels

Management and sample collections
The hens were randomly assigned to cages (80 cm-width × 80 cm-length × 40 cmheight) of 6 hens per cage. The hens were kept in an environmentally controlled room with ad libitum feeding and watering and with the temperature controlled at 22°C and 16 h/d of illumination throughout the entire experimental period. The experiment lasted 9 wks. At the end of the experiment, 6 hens from each group were randomly selected, and blood samples were individually collected from the wing vein and then were centrifuged at 3,000 r/min for 10 min at 4 °C to obtain serum. In addition, hens were euthanized with intravenous sodium pentobarbital at a dose of 100 mg/kg. The criteria for euthanasia were somnolence, akinesia and dyspnea. Then, the femur and tibia were collected from the hens.

Production performance
The number of eggs and egg weight were recorded daily (at 13:00) throughout experiment on a replication basis and hen-day laying rate was calculated. Average egg weight (AEW) was calculated as the mean weight of all eggs from each replicate. Feed consumption was measured weekly on a replication basis. Average daily feed intake (ADFI) was calculated using the following equation: ADFI = feed consumption (g) / (hen number × time (d)) and feed conversion ratio (FCR) was calculated as (feed intake / (egg weight × egg production)).

Egg quality determination
At 3, 6 and 9 wk, 8 eggs were randomly selected from each group for egg quality determination. Egg shape index was calculated by diameter/height × 100. The eggs were weighed prior to being cracked. Eggshell strength was evaluated using an egg shell force gauge model II (Robotmation Co., Ltd., Tokyo, Japan). Eggshell thickness was measured on the large end, equatorial region, and small end, respectively, using an eggshell thickness gauge (Robotmation Co., Ltd., Tokyo, Japan). Egg weight, albumen height, yolk color, and haugh unit were measured by using a digital egg tester (DET-6000, Nabel Co., Ltd, Kyoto, Japan).

Serum redox assessment
The activities of total antioxidant capacity (T-AOC) contents, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and the contents of malondialdehyde (MDA) in serum were analyzed using analysis kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Bone mineral density (BMD) measurement
The BMD of femur and tibia was measured with dual energy X-ray absorptiometry (InAlyzer, MEDIKORS Inc., Korea). The detection sensitivity of the instrument was 0.001 g/cm 2 . A standard calibration block was used to calibrate the device before measurements were made, according to the operator's manual.

Bone histomorphology
The left tibias were removed from hens and fixed overnight at 4°C in 4% paraformaldehyde, then decalcified and embedded into paraffin. Serial sections (5 μm) were cut and stained with Goldner Trichrome stain. In addition, the static parameters of bone histomorphometry were analyzed.

Bone metabolism biomarkers analysis
The activities of alkaline phosphatase (ALP), tartrate resistant acid phosphatase

Total RNA extraction and real-time quantitative PCR of bone related genes
According to the manufacturer's protocol, the total RNA was extracted from the tissues of the femur by adding TRIzol Reagent (Invitrogen, USA). After extraction with chloroform, isopropanol was added to make the RNA precipitate. After washing with 75% ethanol, the RNA was eluted in ribonuclease-free water. The cDNA was synthesized using ABScript II RT Master Mix (ABclonal Technology, Wuhan, China).
The forward and reverse primer sequences for runt related transcription factor 2 (RUNX2), osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were designed based on available sequences on the NCBI GenBank and are listed in Table 8. β-actin was chosen as an internal standard to control for normalization purposes. Quantitation of the mRNA level by QPCR was performed on a real-time PCR system using iTaq Universal SYBR Green Supermix (Bio-Rad, Richmond, CA, USA). The threshold cycle (C T ) indicated the fractional cycle number at which the amount of amplified target reached a fixed threshold, so we can obtain the relative gene expression level by the 2 − ΔΔC T method for fold induction. All PCR operations were performed in triplicate.   b TFRD, total flavonoids of Rhizoma Drynariae; CON, basal diet; LCD, low calcium diet; TFRD1, low calcium diet supplemented with 0.5 g/kg TFRD; TFRD2, low calcium diet supplemented with 2.0 g/kg TFRD; AEW, average egg weight; ADFI, average daily feed intake; FCR, feed conversion ratio.
* P < 0.05, ** P < 0.01 vs. CON; # P < 0.05, # # P < 0.01 vs. LCD. b The metabolic energy is the calculated value, and the rest is the measured value.  Figure 1 Effects of TFRD on microstructure of tibia tissue of caged laying hens. Representative image Checklist.pdf