Table 1 Effects of TFRD on production performance of caged laying hens. Data are presented as means (SD)a
Items
|
CONb
|
LCD
|
TFRD1
|
TFRD2
|
Laying rate, %
|
93.68 ± 2.01
|
66.79 ± 3.13**
|
71.55 ± 3.11
|
79.98 ± 5.27#
|
AEW, g
|
61.51 ± 0.96
|
57.81 ± 1.22*
|
59.26 ± 0.50
|
57.35 ± 0.63
|
ADFI, g
|
119.52 ± 0.30
|
119.22 ± 0.19
|
119.12 ± 0.48
|
119.32 ± 0.28
|
FCR
|
2.08 ± 0.01
|
3.10 ± 0.20**
|
2.81 ± 0.14
|
2.64 ± 0.16#
|
aMeans were calculated on n = 6 replicates (6 hens per replicate) per treatment.
bTFRD, 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.
Table 2 Effects of TFRD on egg quality of caged laying hens. Data are presented as means (SD)a
Items
|
CONb
|
LCD
|
TFRD1
|
TFRD2
|
Egg shape index
|
|
|
|
|
3 wk
|
1.29 ± 0.03
|
1.30 ± 0.04
|
1.31 ± 0.03
|
1.31 ± 0.06
|
6 wk
|
1.29 ± 0.05
|
1.29 ± 0.04
|
1.29 ± 0.04
|
1.33 ± 0.04
|
9 wk
|
1.30 ± 0.02
|
1.29 ± 0.04
|
1.30 ± 0.02
|
1.33 ± 0.04
|
Eggshell strength, N
|
|
|
|
|
3 wk
|
45.15 ± 8.22
|
32.67 ± 8.64*
|
32.83 ± 5.30
|
32.23 ± 5.52
|
6 wk
|
43.21 ± 7.57
|
25.74 ± 8.98**
|
23.76 ± 5.47
|
24.19 ± 6.69
|
9 wk
|
49.90 ± 8.27
|
23.63 ± 3.93**
|
21.14 ± 2.19
|
24.06 ± 3.93
|
Eggshell thickness, mm
|
|
|
|
|
3 wk
|
0.34 ± 0.02
|
0.31 ± 0.02*
|
0.30 ± 0.04
|
0.29 ± 0.02
|
6 wk
|
0.36 ± 0.03
|
0.27 ± 0.05**
|
0.25 ±0.02
|
0.26 ± 0.04
|
9 wk
|
0.35 ± 0.02
|
0.24 ±0.03**
|
0.22 ± 0.01
|
0.24 ± 0.02
|
Yolk color
|
|
|
|
|
3 wk
|
14.59 ± 0.57
|
7.51 ± 0.77**
|
7.64 ± 0.66
|
7.59 ± 0.54
|
6 wk
|
14.58 ± 0.43
|
7.85 ± 0.53**
|
7.80 ± 0.84
|
8.19 ±0.79
|
9 wk
|
13.96 ± 0.36
|
7.98 ± 0.41**
|
7.98 ± 0.54
|
8.49 ± 0.72
|
Albumen height, mm
|
|
|
|
|
3 wk
|
8.16 ± 1.23
|
7.46 ± 1.00
|
8.33 ± 1.61
|
7.51 ± 0.78
|
6 wk
|
8.29 ± 1.50
|
7.80 ± 1.22
|
7.69 ± 1.10
|
7.69 ± 1.20
|
9 wk
|
8.44 ±1.78
|
7.79 ± 0.97
|
7.61 ± 0.87
|
8.09 ± 1.59
|
Haugh unit
|
|
|
|
|
3 wk
|
89.06 ± 7.36
|
86.27 ±5.15
|
90.67 ± 8.83
|
87.50 ± 4.29
|
6 wk
|
89.86 ± 7.39
|
89.31 ± 6.36
|
87.78 ± 6.34
|
88.72 ± 6.68
|
9 wk
|
90.24 ± 8.66
|
88.35 ± 3.62
|
86.54 ± 4.42
|
89.42 ± 8.36
|
aMeans were calculated on n = 8 eggs per treatment.
bTFRD, 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.
*P < 0.05, **P < 0.01 vs. CON; #P < 0.05, ##P < 0.01 vs. LCD.
Table 3 Effects of TFRD on serum antioxidant indicators of caged laying hens. Data are presented as means (SD)a
Items
|
CONb
|
LCD
|
TFRD1
|
TFRD2
|
MDA, nmol/mL
|
3.52 ± 0.29
|
5.17 ± 0.46**
|
4.52 ± 0.39
|
4.21 ± 0.56##
|
T-AOC, U/mL
|
9.44 ± 1.28
|
5.62 ± 0.77**
|
6.63 ± 0.99
|
7.27 ± 1.28#
|
T-SOD, U/mL
|
267.46 ± 9.32
|
218.17 ± 14.56**
|
226.47 ± 14.82
|
224.23 ± 13.23
|
GSH-Px, U/mL
|
949.41 ± 88.30
|
784.99 ± 76.03**
|
808.74 ± 108.56
|
918.67 ± 75.88#
|
aMeans were calculated on n = 6 hens per treatment.
bTFRD, 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; MDA, malondialdehyd; T-AOC, total antioxidant capacity; T-SOD, total superoxide dismutase; GSH-Px, glutathione peroxidase.
*P < 0.05, **P < 0.01 vs. CON; #P < 0.05, ##P < 0.01 vs. LCD.
Table 4 Effects of TFRD on bone mineral density of caged laying hens. Data are presented as means (SD)a
Items
|
CONb
|
LCD
|
TFRD1
|
TFRD2
|
Femur BMD, g/cm2
|
0.36 ± 0.03
|
0.27 ± 0.02**
|
0.28 ± 0.02
|
0.30 ± 0.02#
|
Tibia BMD, g/cm2
|
0.35 ± 0.04
|
0.28 ± 0.02*
|
0.29 ±0.04
|
0.31 ± 0.02
|
aMeans were calculated on n = 5 hens per treatment.
bTFRD, 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; BMD, bone mineral density.
*P < 0.05, **P < 0.01 vs. CON; #P < 0.05, ##P < 0.01 vs. LCD.
Table 5 Effects of TFRD on bone histomorphometry parameters of caged laying hens. Data are presented as means (SD)a
Items
|
CONb
|
LCD
|
TFRD1
|
TFRD2
|
Ct.Ar, %
|
20.20 ± 2.78
|
13.21 ± 1.11*
|
16.95 ± 1.08
|
18.08 ± 2.10#
|
CW, mm
|
0.43 ± 0.03
|
0.39 ± 0.04
|
0.39 ± 0.06
|
0.44 ± 0.04
|
Tb.Ar, %
|
40.67 ± 2.20
|
32.30 ± 2.71*
|
39.54 ± 2.46
|
40.72 ± 3.51#
|
Tb.Th, um
|
29.08 ± 2.50
|
20.02 ± 2.57*
|
27.37 ± 2.22#
|
26.57 ± 1.61#
|
Tb.Sp, um
|
32.57 ± 1.16
|
40.38 ± 2.86*
|
35.20 ± 1.71
|
34.19 ± 2.17#
|
Tb.N, #/mm
|
16.28 ± 1.25
|
15.80 ± 1.56
|
15.76 ± 1.43
|
16.75 ± 1.44
|
aMeans were calculated on n = 3 hens per treatment.
bTFRD, 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; Ct.Ar, cortical area ration; CW, cortical width; Tb.Ar, percent trabecular area; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Tb.N, trabecular number.
*P < 0.05, **P < 0.01 vs. CON; #P < 0.05, ##P < 0.01 vs. LCD.
Table 6 Effects of TFRD on serum bone metabolism biomarkers of caged laying hens. Data are presented as means (SD)a
Items
|
CONb
|
LCD
|
TFRD1
|
TFRD2
|
ALP, 100U/L
|
0.43 ± 0.10
|
1.33 ± 0.19**
|
1.26 ± 0.23
|
0.98 ± 0.12#
|
OCN, ng/mL
|
17.41 ± 5.25
|
40.27 ± 8.26**
|
29.88 ± 5.28
|
28.81 ± 6.46#
|
TRACP, U/L
|
56.93 ± 7.17
|
73.71 ± 9.38**
|
63.88 ± 7.64
|
59.45 ± 8.26#
|
Ca, mmol/L
|
2.44 ± 0.10
|
2.49 ± 0.06
|
2.43 ± 0.05
|
2.41 ± 0.08
|
aMeans were calculated on n = 6 hens per treatment.
bTFRD, 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; ALP, alkaline phosphatase; OCN, osteocalcin; TRACP, tartrate resistant acid phosphatase.
*P < 0.05, **P < 0.01 vs. CON; #P < 0.05, ##P < 0.01 vs. LCD.
Table 7 Basal diet and low calcium diet formulation and nutrient levels
Dietary ingredient
|
Content (%)
|
Basal diet
|
Low calcium diet
|
Corn
|
61.86
|
60.97
|
Soybean meal
|
25.84
|
24.05
|
Bran
|
0.50
|
8.00
|
Limestone
|
9.50
|
4.95
|
Calcium hydrogen phosphate
|
1.0
|
0.73
|
NaCl
|
0.30
|
0.30
|
Premixa
|
1.0
|
1.00
|
Total
|
100
|
100
|
Nutrient levels
|
|
|
Metabolic energyb, MJ/kg
|
11.77
|
11.93
|
Crude protein, %
|
17.25
|
17.58
|
Calcium, %
|
3.75
|
2.00
|
Available phosphorus, %
|
0.427
|
0.459
|
aPremix provided per kilogram of diet: vitamin A, 10,000 IU; vitamin D, 2,000 IU; vitamin E, 10mg; vitamin K3, 2.0 mg; vitamin B1, 0.5 mg; vitamin B2, 4.0 mg; vitamin B6, 2.0 mg; vitamin B12, 0.01 mg; choline chloride, 400 mg; nicotinic acid, 30 mg; pantothenic acid, 8.0 mg; folic acid, 0.5 mg; iron, 40 mg; copper, 6.0 mg; zinc, 50 mg; magnesium, 70 mg; iodine, 0.3 mg; and selenium, 0.1 mg.
bThe metabolic energy is the calculated value, and the rest is the measured value.
Table 8 Primers used for the quantitative polymerase chain reaction
Genes
|
GenBank ID
|
Primers sequence (5’ to 3’)
|
Products (bp)
|
RUNX2a
|
NM_204128.1
|
F: GATTACAGACCCCAGGCAGG
|
75
|
|
|
R: TGGCTCAAGTAGGACGGGTA
|
|
OPG
|
NM_001033641.1
|
F: GTTCCTACTCGTTCCACACC
|
115
|
|
|
R: GCTCTTGTGAACTGTGCCTTTG
|
|
RANKL
|
NM_001083361.1
|
F: AGGAGAAATAAGCCCGAGAA
|
108
|
|
|
R: TTTGTTATGATGCCAGGATGTA
|
|
β-actin
|
NM_205518.1
|
F: CACGATCATGTTTGAGACCTT
|
100
|
|
|
R: CATCACAATACCAGTGGTACG
|
|
aRUNX2, runt related transcription factor 2; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor kappa-B ligand.